Embed Size (px)
Citation preview
Seediscussions,stats,andauthorprofilesforthispublicationat:http://www.researchgate.net/publication/283182959
N-succinylchitosanpreparation,characterization,propertiesandbiomedicalapplications:Astateoftheartreview
ARTICLEinREVIEWSINCHEMICALENGINEERING·JANUARY2015
ImpactFactor:2.41·DOI:10.1515/revce-2015-0016
READS
11
5AUTHORS,INCLUDING:
ShahidBashir
UniversityofMalaya
2PUBLICATIONS0CITATIONS
SEEPROFILE
YinYinTeo
UniversityofMalaya
23PUBLICATIONS27CITATIONS
SEEPROFILE
AmirAzamKhan
UniversityMalaysiaSarawak
16PUBLICATIONS108CITATIONS
SEEPROFILE
Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,
lettingyouaccessandreadthemimmediately.
Availablefrom:ShahidBashir
Retrievedon:29December2015
Rev Chem Eng 2015; aop
*Corresponding author: S. Ramesh, Centre for Ionics, Faculty of Science, Department of Physics, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia, e-mail: [email protected] Bashir and Yin Yin Teo: Faculty of Science, Department of Chemistry, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, MalaysiaK. Ramesh: Centre for Ionics, Faculty of Science, Department of Physics, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, MalaysiaAmir Azam Khan: Faculty of Engineering, Department of Mechanical and Manufacturing Engineering, Universiti Sarawak (UNIMAS), 94300 Kota Samarahan, Sarawak, Malaysia
Shahid Bashir, Yin Yin Teo, S. Ramesh*, K. Ramesh and Amir Azam Khan
N-succinyl chitosan preparation, characterization, properties and biomedical applications: a state of the art reviewDOI 10.1515/revce-2015-0016Received March 11, 2015; accepted July 2, 2015
Abstract: N-succinyl chitosan (NSC) remains a promis-ing chitosan derivative to develop targeted drug delivery, wound dressings, and tissue engineering systems. All these systems are important in life sciences. NSC is an amphiprotic derivative obtained from the N-acylation of chitosan. NSC exhibits extraordinary biocompatibility, significantly increased aqueous solubility in acidic and basic media without affecting the biological properties, appreciable transfection efficiency, and the ability to stim-ulate osteogenesis. NSC shows enhanced bioavailability, which highlights its potential applications in the biomedi-cal field. This review briefly introduces chitosan, includ-ing its limitations as a biomaterial, and modifications of chitosan with a particular focus on acylation, along with a comprehensive overview of the synthesis, charac-terization, properties, biodistribution, and toxicological/biopharmaceutical profile of NSC. Furthermore, it exten-sively surveys current state-of-the-art NSC-based formula-tions for drug delivery with special emphasis on protein delivery, anti-cancer activity in the colon, as well as nasal and ophthalmic targeted gene/drug delivery. Moreover, it discusses NSC-based biomaterial applications in articu-lar, adipose, and bone tissue engineering. In addition, it describes recent contributions of NSC-based hydrogels in wound dressings along with a brief account of drug deliv-ery in combination with tissue engineering. Finally, it
presents potential current challenges and future perspec-tives of NSC-based formulations in the biomedical field.
Keywords: characteristics; drug delivery; N-succinyl chi-tosan; tissue engineering; wound dressings.
1 IntroductionPolymers are extensively utilized in daily life as well as in industrial applications. Recent advances in science have often involved polymers, and researchers have called this the “polymer age”. “Polymer” is derived from Greek, in which poly means “many” and mer means “unit”. Small molecules combine to form long-chain molecules called polymers or macromolecules. Polymers are classified into two types, natural and synthetic polymers. Polymers obtained from natural sources are known as natural poly-mers, and artificially produced polymers are known as synthetic polymers (Pillai and Panchagnula 2001, Ha and Gardella 2005).
Natural polymers are widely studied and utilized in daily life and in industrial applications due to their excel-lent properties. Among the natural polymers, polysaccha-rides are the most extensively studied biomaterials and are exploited in drug delivery, tissue engineering, and wound dressing applications due to their inherent properties such as biocompatibility, biodegradability, and non-tox-icity (Ramesh and Tharanathan 2003, Tharanathan 2003, Tiwari et al. 2014). Chitosan is a semisynthetic natural polysaccharide obtained from the deacetylation of chitin under alkaline conditions (Khor 2002, Tharanathan and Kittur 2003, Kurita 2006). Chitosan is the second most produced biopolymer in terms of annual production after cellulose. Chitosan exhibits excellent properties, such as biocompatibility, biodegradability, non-toxicity, non-immunogenicity, non-carcinogenicity, mucoadhesion, and antimicrobial properties. Its non-toxic degradable prod-ucts, permeation, and transfection-enhancing properties also play a significant role in its extensive exploitation in biomedical formulations (Kurita 2001, Rinaudo 2006).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
2 S. Bashir et al.: Chitosan derivatives
However, the uncontrollable degradability, low trans-fection efficiency, limited colloidal stability, and water solubility of chitosan have inhibited its development in biomedical formulations. Therefore, chemical modifica-tion of chitosan seems to be an encouraging prospect in order to improve these properties. It also extends the range of chitosan exploitation in drug delivery, tissue engineer-ing, and wound dressings (Casettari et al. 2012). Chemi-cal modification of chitosan has substantially improved these properties. Therefore, chitosan derivatives have gar-nered increased significant scientific interest due to their outstanding properties (Sashiwa et al. 2003, Mourya and Inamdar 2008, Hawary et al. 2011). The introduction of an acyl group into chitosan bestows it with marvelous physi-cal, chemical, and biological properties suitable for bio-medical applications (Hirano et al. 2002, Choi et al. 2007). N-succinyl chitosan (NSC) is an acyl derivative of chitosan that is biocompatible, biodegradable, and water soluble in acidic as well as in alkaline media (Yan et al. 2006a,b). Due to these properties, NSC has recently garnered con-siderable attention in various fields such as drug deliv-ery, tissue engineering, and wound dressing applications in vitro as well as in vivo. NSC is potentially robust and is rich in reactive functional (-NH2, -OH, and -COOH) groups. The undeniable characteristics of NSC such as excellent non-toxicity, enhanced transfection efficacy, magnificent moisture retention properties, and enzyme immobiliza-tion activity as compared to its progenitor chitosan have provided it with excellent prospects in the biomedical field. It is bioactive and promotes osteogenesis. NSC-based formulations are available as hydrogels, beads, blends, composites, matrices, films, micelles, liposomes, microspheres, and nanoparticles for drug delivery. Simi-larly, NSC-based tissue engineering (cartilage, adipose, and bone) devices like injectable gels and scaffolds have been reported in recent years. NSC-based hydrogel com-posites have been used in wound dressing applications.
Chitosan is a natural polymer with numerous interest-ing properties, which have ultimately made it an emerg-ing research topic in the biomedical field in the past few decades. A variety of studies have investigated the use of chitosan in drug delivery, tissue engineering, and wound dressings (Kumar 2000). Researchers have princi-pally investigated the chemistry and biomedical applica-tions of chitosan but have paid less attention to chitosan derivatives (Rinaudo 2006, Prashanth and Tharanathan 2007). A large number of chitosan derivatives have been reported, with properties better than those of chitosan (Alves and Mano 2008, Mourya and Inamdar 2008). NSC is one such chitosan derivative that possesses better prop-erties than chitosan. Recently, a carboxymethyl chitosan
derivative has been described (Jayakumar et al. 2010). The authors highlighted the chemistry of carboxymethyl chitosan and its biomedical applications. Therefore, it is timely to review the synthesis, physicochemical, and biological characteristics of NSC as well as its biomedi-cal applications. NSC has outstanding physicochemical and biological properties. Studies have illustrated that it is highly suitable for bioactive and therapeutic compound delivery, the repair of damaged tissue, and wound dress-ings. NSC also has excellent moisture absorption and retention abilities, superior chelating ability, significant apoptosis inhibitory activity, astonishing enzyme immo-bilization activity, strong antioxidant activity, and greater bioactivity than its parent molecule chitosan. Conse-quently, due to these properties, researchers have widely studied NSC in biomedical applications in vitro as well as in vivo. This review therefore intends to provide detailed information on formulations containing NSC along with their preparation techniques and applications in the bio-medical field. Furthermore, it also identifies the applica-tions of NSC in drug delivery in combination with tissue engineering. Moreover, the moisture absorption and retention abilities of NSC are important in wound dressing applications. To the best of our knowledge, this is the first ever extensive review to comprehensively describe the state of the art on the synthesis, characteristics, formula-tions, and biomedical applications of NSC.
2 BackgroundIn this section, a brief overview of chitosan is provided, followed by a discussion on the limitations and modifica-tion of chitosan; specifically, acylation is described.
2.1 Chitosan
Chitosan is a semisynthetic amino polysaccharide with a unique structure. It is a copolymer of 2-acetamido-2-deoxy-β-D glucopyranose and 2-amino-2-deoxy-β-D glu-copyranose. These two reiterating units are linked through (1-4)-β-glycosidic linkages. Chitosan has a rigid crystal-line structure and multi-dimensional properties with a complex structure. A large range of biomedical and indus-trial applications of chitosan are available. Chitosan has excellent biocompatibility, biodegradability, low immuno-genicity, and toxicity, as well as versatile biological activi-ties (Jayakumar et al. 2007, Mourya and Inamdar 2008, Rinaudo 2008).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 3
soluble up to pH 6.5. Furthermore, the ionization degree depends on the strength of the acid and the pH, which has been experimentally confirmed during the protonation of chitosan in the presence of aqueous acids during solubili-zation (Fan et al. 2009).
It should be highlighted that the pH of the small intes-tine is 4.5 and the pH of the ileum is 7.4; the pH varies gradually from the small intestine to the terminal ileum, so chitosan is not soluble in the lower part of the intes-tine. Therefore, chitosan cannot be used to deliver a drug in the lower part of the intestine. In order to enhance the oral bioavailability of a drug using chitosan as a drug carrier, its dissolution in the lower part of the intestine is required. Thus, although researchers have made impres-sive contributions, much work is still required to improve drug bioavailability. This can only be achieved through modification. Modification of chitosan enhances its water solubility at higher pH and can be done through the deri-vatization of the amino and hydroxyl groups of chitosan. After modification of the amino and hydroxyl groups, they are quaternized (Polnok et al. 2004) with carboxyalkyl (Wongpanit et al. 2005), hydroxyalkyl (Richardson and Gorton 2003), and acyl derivatives. Among these deriva-tives, acyl derivatives have substantial importance in bio-medical applications.
2.2 Chitosan modification
The abovementioned significant disadvantages of chitosan have motivated researchers to achieve further modifica-tions. Modification of the amino and hydroxyl groups of chi-tosan improves its solubility in acidic as well as in alkaline environments, improves its biodegradability and biocom-patibility, enhances transfection efficiency, and decreases toxicity. Modification has substantially enhanced the bio-medical applications of chitosan. Figure 2 shows several modification methods, while some examples of chitosan derivatives are listed in Table 1.
2.2.1 Acylation
The chemical reaction between chitosan and acyl halides or aliphatic carboxylic anhydrides is called acylation. There are numerous acid anhydrides, lactones, and acyl halides, which form a variety of acylated outcomes. The acylation of chitosan takes place at the amino and hydroxyl groups of chitosan. Acylation, in which substitution takes place at an amino group, is called N-acylation, and sub-stitution of a hydroxyl group is known as O-acylation.
Chitosan is obtained by the thermochemical deacety-lation of chitin in the presence of alkali (Pillai et al. 2009). Several approaches have been reported for the synthesis of chitosan. These are alkaline in nature because potassium or sodium hydroxide is used for the hydrolysis of chitin. Chitin is the second most abundant natural polysaccha-ride and is acquired from the exoskeleton of crustaceans, insects, and certain fungi. Chitin has limited applications due to its poor solubility in organic and aqueous solutions. In contrast, chitosan is a suitable alternative due to its sol-ubility in acidic media (below pH 6.0) and its vast range of practical applications as a biomaterial (Mima et al. 1983). The properties of chitosan depend on the degree of dea-cetylation and the molecular weight (Mw). The structures of chitin and chitosan are shown in Figure 1.
2.1.1 Limitations of chitosan
Chitosan has enhanced drug absorption properties that have been highly cited in the literature. Its enhanced drug absorption properties are due to the mucoadhesive nature of chitosan. However, drug absorption by chi-tosan decreases at higher pH due to its poor solubility at pH greater than 6.0. Chitosan is soluble in dilute acids. Acids used for the dissolution of chitosan include inor-ganic acids like HCl and organic acids like methanoic acid, ethanoic acid, oxalic acid, and lactic acid; chitosan is insoluble at neutral and alkaline pH. The mechanism of solubilization involves the protonation of the amino func-tional group at the C-2 position of the glucosamine units. The acetyl groups distributed over the chitosan chain are also a factor responsible for changes in solubility. Chitosan with a deacetylation degree of 86% is partially
Figure 1: Structure of chitin and chitosan.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
4 S. Bashir et al.: Chitosan derivatives
Chitosan
Sulfation
PhosphorylationCarboxyacylationHydroxyacylation
N-CarboxyacylationO-Carboxyacylation
Thio ureaderivatatives
Sugarderivatives
Alkylation Metal ion chelates
HydroxyalkylationCarboxyalkylation
N-CarboxyalkylationO-Carboxyalkylation
ThiolationAcylationQuatemization
Figure 2: Various modifications of chitosan.
Table 1: Chitosan modification methods and examples of each modified chitosan.
Modification of chitosan Examples
Quaternization N,N,N-trimethyl chitosan chloride (Thanou et al. 2000), N,N,N-trimethyl chitosan iodide (Domard et al. 1986). Quaternized derivatives of N,N,N-trimethyl carboxymethyl chitosan, N-ethyl-N,N-dimethyl carboxymethyl chitosan, N-propyl-N,N-dimethyl carboxymethyl chitosan. N-butyl-N,N-dimethyl carboxymethyl chitosan, N-isobutyl-N,N-dimethyl carboxymethyl chitosan (Guo et al. 2008).
Alkylation N-furfuryl- N, N-dimethyl chitosan, N-propoyl-N, N-dimethyl chitosan, N-diethylmethylamino chitosan.Carboxyalkylation N-carboxymethyl chitosan, N, N, N-trimethyl carboxymethyl chitosan, N-ethyl-N,N-dimethyl
carboxymethyl chitosan, N-carboxylpropyl chitosan, N-propyl-N,N-dimethyl carboxymethyl chitosan. N-butyl-N,N-dimethyl carboxymethyl chitosan, N-isobutyl-N,N-dimethyl carboxymethyl chitosan. N-carboxybenoyl chitosan.
Hydroxyaklylation Hydroxyethylchitosan, Hydroxypropyl chitosan, 3-Trimethlammonium-2-hydroxypropyl-N-chitosan, N-hydroxyethyl-O-benzyl, N-hydroxypropylyl-O-benzyl, N-hydroxybutyl-O-benzyl chitosan.
N-acylation(carboxylacylation) Maleic, succinic, (2-0cten-1-yl) succinic, thiosuccinic, acetylthiosuccinic, glutaric, phathalic and citraconic anhydrides react with citosan to form carboxyacyl chitosan derivatives (Hirano and Moriyasu 2004).
Thiolation Chitosan-thioglycolic acid, chitosan-2-iminothiolane, chitosan-cysteine conjugate.Thiolated urea derivatives Thiourea chitosan, acetyl and chloroacetyl thiourea chitosan, benzoyl thiourea (Zhong et al. 2008)
Thiourea chitosan(antibacterial), Benzoyl thiourea chitosan (Eweis et al. 2006).Sulfation or sulphonation Chitosan sulfate, N-sulfofurfuryl chitosan, O-sulfated chitosan, N-sulfated O-carboxymethyl chitosan
(Jayakumar et al. 2007), N-sulphonato-N,O-carboxymethyl chitosan, (N,O)-sulfated chitosan (Karadeniz et al. 2011).
Phosphorylation Phosphorylcholine and N-Methylenephosphonic chitosan, N-phosphonomethyl chitosan, chitosan phosphate (Matevosyan et al. 2003).
Sugar derivatives Lactosaminated NSC (Jain et al. 2014), Glactosyl conjugated NSC (Lu et al. 2010), Glactosylated chitosan.
N-acylation is the most extensively studied chitosan modification. It proceeds through the addition or elimi-nation mechanisms in acetic acid/methanol, methanol/ethanol, pyridine, dichloroethane/trichloroacetic acid, or formamide/ methanol (Kurita 1986). N-acylation typi-cally proceeds toward the formation of amide functional groups because amides are more stable.
Acid anhydrides are extensively used for the N-acylation of chitosan, but anhydrides have very low solu-bility in aqueous media. Therefore, this reaction takes place
under heterogeneous conditions, and methanol is used to solvate anhydride in the medium. The presence of aqueous acetic acid in methanol also leads to selective acylation at an amino functional group. Diverse N-acyl deriva-tives have been synthesized by researchers. For example, Hirano et al. reported N-saturated fatty acyl derivatives. These derivatives were synthesized through the reaction of chitosan with short and long chain anhydrides. These anhydrides are propionic, butyric, pentanoic, succinic, lauric, itaconic, palmitic, stearic, and myristic anhydrides.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 5
Short-chain acyl derivatives show greater water solubility, while longer chain derivatives are water insoluble regard-less of the degree of substitution. The reaction of chitosan with cyclic anhydride usually forms carboxyacyl chitosan. These derivatives exhibit water solubility in the degree of substitution range of 0.45–0.8 and in the pH range of 4.0–7.0. The introduction of lactones to the amino group of chitosan forms hydroxyacyl derivatives with improved water solubility, but lactones are much less reactive and more stable. For these reasons, few N-hydroxyacyl deriva-tives have been published (Hirano et al. 2002, Wu et al. 2006, Champagne 2008).
Chitosan is a multi-nucleophilic polymer, as can be seen from its molecular structure. Substitution can occur at an amino or hydroxyl group, but substitution at an amino group is given the priority due to the greater reactivity of this group. However, O-acylation can be accomplished
by the protection of N-amino groups and controlling the experimental conditions. Amino groups can be pro-tected by N-phthaloyl groups; this allows O-acylation in N,N-dimethylformamide (DMF) at a high temperature. In addition, methanesulfonic acid at a low temperature and H2SO4 can also be used for the O-acylation of chitosan without additional protection of the amino group. Figure 3 shows N-acylation of chitosan.
3 Succinyl chitosan
3.1 N-succinyl chitosan
N-acylation of chitosan has already been discussed in detail. Carboxyacyl formation takes place upon the
Figure 3: N-acylation of chitosan.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
6 S. Bashir et al.: Chitosan derivatives
introduction of cyclic anhydrides to chitosan. Among these anhydrides, the reaction of succinic anhydride with chitosan in dimethyl sulfoxide or acetic acid/methanol mediums produces NSC with a specific degree of succi-nylation. The mechanism of NSC synthesis is shown in Figure 4. The degree of succinylation can be measured by the ninhydrin assay. The degree of succinylation depends on the reaction conditions. NSC has excellent solubility below pH 4.5 and above pH 7. Its solubility in an acidic environment is due to the protonation of amino groups, while in a basic environment it is due to the formation of carboxylate ions (-COO-). However, it is insoluble within the pH range from 4.5 to 6.8 due the isoelectric point of equimolar co-existing –NH+
3 and –COO- (Yan et al. 2006a,b). Chitosan is soluble in acidic media, and it pre-cipitates above pH 6.5. The advantages of NSC are that it is biocompatible, biodegradable, bioadhesive, and pos-sesses long-term retention properties both in vitro and in vivo (Kato et al. 2000a,b). Due to the abovementioned properties, it has considerable applications in the medical field as a drug carrier to prepare conjugates with antican-cer drugs, and has exhibited excellent antitumor activity. Other applications include wound dressings, tissue engi-neering, growth factor delivery, gene delivery, and cos-metic applications. It has been applied in wound healing for arthritis treatment in Japan (patent 06107551).
3.2 O-succinyl chitosan
O-succinyl chitosan is a succinyl derivative of chitosan. It has properties similar to NSC. There are two reactive func-tional groups (-NH2, -OH) present in chitosan. The amino groups are more responsive than the hydroxyl groups; therefore, the formation of NSC derivatives is more likely to occur compared to O-succinyl chitosan. Zhang et al. established a procedure for the preparation of water- soluble O-succinyl chitosan, shown in Figure 5. The syn-thesis of O-succinyl chitosan requires a more complex
procedure compared to the methodology for NSC prepa-ration. It involves the protection of strongly hydrophilic amino groups by phthaloyl groups in DMF as the solvent. Then, succinyl groups are introduced, followed by removal of the phthaloyl groups using hydrazine hydrate (Naruphontjirakul and Viravaidya-Pasuwat 2011).
3.3 Characterization of NSC
Characterization is a very important step when discuss-ing NSC due to the effects of the degree of substitution, the degree of deacetylation, and the number of –COOH groups. Table 2 describes the characterization in detail and numerous characterization techniques.
4 Characteristics of NSCNSC is an amphiprotic derivative containing amine, hydroxyl, and carboxyl groups. It was not focused on by researchers previously but has attained considerable attention in the last decade. Research on NSC has shown researchers that it is a versatile biomaterial for biomedi-cal applications. It is formed by the introduction of acyl groups into chitosan. The introduction of this group bestows it with excellent physical, chemical, and biologi-cal properties, as required for biomedical applications.
4.1 Physicochemical characteristics of NSC
4.1.1 Solubility and moisture retention properties
NSC is the preferred modification due to the enhanced water solubility of NSC in a large pH range; this is a very important factor for researchers. Due to its amphiprotic property, it is soluble in acidic as well as basic media.
Figure 4: Synthesis of NSC (Yan et al. 2006a,b).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 7
However, its water solubility strongly depends on the degree of acylation and the preparation conditions. Water solubility can be explained based on the increase in car-boxyl groups as the degree of substitution (DS) increases. First, with an increase in the number of carboxyl groups, hydrogen bonds between water molecules and carboxyl groups on the polymer increase. Second, intermolecular hydrogen bonds increase between molecular chains. The presence of carboxylate ions in the NSC chain acts as a driving force to enhance solubility in water. Apart from establishing a relationship between the water solubility of NSC, temperature, pH, and molecular structure with respect to the degree of deacetylation, the DS has a sub-stantial effect on moisture absorption (Ra) and retention (Rh). Ying et al. showed that NSC was prepared with vari-able degrees of deacetylation (DD 6–94%) and degrees of substitution (DS 0.14–0.79) by varying the reaction time. The study revealed that Ra and Rh were high when DD was 50%, i.e. less than the relative humidity. Ra and Rh decreased when the DD value varied from 50%. Rh was low under dry conditions when DD was 50%. Ra and Rh increased with a rise in DS (Ying et al. 2007). Fan et al. (2010) assessed the water retention properties of pure alg-inate and alginate/NSC blend fibers and found that pure alginate blend fibers had poorer water retention proper-ties than alginate/NSC blend fibers.
Electrostatic repulsion and intermolecular H-bonding between carboxylate ions are responsible for the aggrega-tion of NSC in water (Aiping et al. 2006). The modification of hydrophilic and hydrophobic groups has a consider-able effect on the rheological properties of NSC, while the ionic strength has no significant effect on the aggregation behavior of NSC and parent chain of chitosan. NSC is poly-ampholytic in nature and forms a clear solution at acid and alkaline pH (Sui et al. 2008). In addition, DS has a poten-tial effect on the solubility of chitosan. NSC has a signifi-cant ability to synthesize hyaluronan-like substances with an excellent ability to retain moisture. Hyaluronan is an important cosmetic ingredient due to its moisture reten-tion property. This property has also led to the production of NSC for moisturizing agents on an industrial scale.
4.1.2 Adsorption and chelating properties
The presence of a large number of amino and hydroxyl groups in the structure of chitosan allows for the appro-priate configuration to form metal complexes. The adsorp-tion characteristics of chitosan are mainly due to the novel functionality and flexibility of the polymer chain (Wang et al. 2004). The interpretation of the chitosan metal ion complex formation mechanism has been proposed based
Figure 5: Synthesis of O-succinyl chitosan (Naruphontjirakul and Viravaidya-Pasuwat 2011).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
8 S. Bashir et al.: Chitosan derivatives
Table 2: Characterization of NSC.
Technique Comment References
FTIR The stretching band at 1730–1700 cm-1 represents –COOH group and asymmetric stretch near 1600 or symmetric stretch near 1400 cm-1 represents -COO-, the absorption band near 1660 cm-1 (amide I), 1600 cm-1 (amide II) and 1380 cm-1 (amide III). The bands at 1130, 1085, and 1030 cm-1 represent saccharide backbone.
(Aiping et al. 2006, Mukhopadhyay et al. 2014)
1H NMR The peak at δ = 2.3 indicated 1H of methylene in succinyl group and 3.3–3.7 indicating H1, H3, H4, H5, and H6. The peak near 2.95 represented H2.The ratio of integral peak of 1H of methylene and H2 in chitosan determined the degree of substitution.
(Aiping et al. 2006, Yan et al. 2006a,b)
13C NMR The 13C NMR spectra of NSC at 270 MHz are reported in D2O.The peak at 178.9 for –NH (CO)-CH2-CH2- and at 183.7 for –COONa indicated succinylation of chitosan.
(Aiping et al. 2006)
XRD XRD spectrum was reported by many researchers.The XRD spectrum of chitosan shows distinct peak at 11° and 20°. This is due to the strong inter- and intramolecular H-bonding and makes chitosan water insoluble and forms a crystalline region.However, the peak at 11° vanishes, and the weak peak at 20° indicates succinylation of chitosan and decrease in H-bonding capacity.
(Zhou and Wang 2009, Mukhopadhyay et al. 2014)
DSC DSC analysis was performed in sealed aluminium pans under N2 atmosphere in the temperature range of 0°C–325°C and 10°C/min.DSC thermogram of chitosan showed two peaks: one endothermic peak at around 100°C and one exothermic peak at 315°C, but in case of NSC, it is less evident.
(Mura et al. 2011a,b)
TGA TGA thermograms were obtained by heating in the range of 50°C–600°C at the rate of 10°C/min under nitrogen atmosphere.TGA curve showed weight loss at around 50°C–100°C due to moisture in the sample and 293°C–330°C due to degradation of chitosan, while NSC curve showed weight loss at around 40°C–94°C. Polymer degradation was observed at around 221°C–300°C, and a new third peak at 462°C–503°C indicated degradation of succinyl group of NSC.
(Zhou and Wang 2009, Kajjari et al. 2013)
Titrimetry The degree of substitution (DS) was determined by potentiometric titration.
(Mukhopadhyay et al. 2014)
TNBS method The degree of substitution was also determined by TNBS method. It was reported that DS depends on Mw of chitosan and reaction time. After 6 h of reaction time, DS was 64%, 63%, and 62% when chitosan 20, 50, and 100 kDa was used, respectively.
(Na et al. 2013)
Elemental analysis Elemental analysis was used to determine the degree of substitution. It was the ratio of succinyl group to glucosamine unit.
(Sui et al. 2008, Mura et al. 2011a,b)
Ninhydrin assay Ninhydrin reagent used to determine DS. In this assay, aqueous solution of NSC was treated with ninhydrin reagent in sodium acetate buffer (pH 5.5), and DS was found to be 0.61 and 0.38.
(Lü et al. 2010, Tan et al. 2013)
Gel permeation chromatography (GPC)
GPC analysis was performed to determine weight average molecular weight (Mw) of NSC by using the equation:Lg (Mw) = -0.7840 Ve + 10.0903The Mw of NSC was increased with the increase in reaction time and substitution degree. The Mw was 147.6 × 106 for 0.79 DS.
(Ying et al. 2007)
Size exclusion chromatography (SEC)
Mw of NSC was characterized by SEC. SEC was equiped with three detectors on line, and Mw was found to be 231,900±9230.
(Lü et al. 2010, Tan et al. 2013)
Agarose gel electrophoresis
Agarose gel electrophoresis assay showed good binding capability of NSC complexes with DNA. The increase in DS of NSC decreased the binding capability with DNA.
(Lu et al. 2009, Toh et al. 2011)
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 9
on two main hypotheses, i.e. the metal ion bridge model via inter- or intramolecular complex formation with amino groups from the same or a different chain of chitosan. The metal ions are bound to chitosan in a pendant fashion (Nieto et al. 1992). The initial research on the chelation of chitosan derivatives with heavy metals showed that the addition of a modified chitosan solution to modified chitosan metal ion chelates immediately insolubilized the latter (Muzzarelli et al. 1982). NSC also shows adsorption and chelation properties. These properties are due to the succinyl and amino groups on the main chain of NSC. The succinyl groups are helpful in adsorption because these groups are more active than amino groups. The degree of succinylation has a significant effect on adsorption. The adsorption properties of NSC and cross-linked NSC resin with Pb(II) ions as the template reported that NSC as a template had always had higher adsorption proper-ties than NSC. Single metal adsorption and co-adsorption experiments revealed that the template NSC resin selec-tively adsorbed Pb(II) ions from the solution. The degree of succinylation had a significant effect on the adsorption of Pb(II) ions. It was also revealed that the template NSC resin showed excellent reusability and adsorption proper-ties for Pb(II) ions after being reused 10 times (Sun and Wang 2006). The same research group studied the adsorp-tion capacities of NSC for Cu(II) ions and found an effect of the degree of succinylation. The cross-linked NSC tem-plate and NSC could selectively adsorb Cu(II) ions from a mixture of Cu(II), Zn(II), Ni(II), and Co(II) ions. Experi-ments also revealed that the template NSC always had greater adsorption properties than NSC (Sun et al. 2007). The complexation of NSC-g-PEI with DNA as a non-viral gene vector delivery method was investigated by Lu et al. The results revealed that the binding ability of the copoly-mer was strongly dependent on the surface charge. The complexation of NSC-g-PEI with DNA was higher due to the increased surface charge on the copolymer (Lu et al. 2009). Recently, the adsorption of succinylated chitosan
for simultaneous removal of a cationic dye and zinc from binary mixtures was investigated. Table 3 presents brief details on NSC complexation with metal ions and various other ligands.
4.2 Biological characteristics of NSC
This section presents the biological characteristics of NSC. Biological characteristics like apoptosis inhibitory activ-ity, enzyme immobilization, as well as antimicrobial and antioxidant properties are under discussion.
4.2.1 Apoptosis inhibitory activity
Cancer therapy remains a major challenge in spite of many developments in medical science. The importance of chi-tosan has been shown by the analysis of its growth inhibi-tory effect on various tumor cells 5637 by cell counting and calorimetric assays. Prominent caspase-3 activity and DNA fragmentation in cancer cells treated by chitosan also indi-cate the apoptosis-inducing activity of chitosan (Hasegawa et al. 2001). The antitumor effect of NSC nanoparticles was evaluated by apoptosis induction in K562 cells. Many apo-ptotic changes were observed in K562 cells when using NSC, while normal nuclear morphology was exhibited by untreated cells. The results revealed that NSC inhibited the proliferation of K562 cells; ultrastructural morphology indicated an effect of NSC of chromatin, microvilli, and organelles. NSC-treated cells showed ultrastructural fea-tures of apoptosis like condensed, fringed, and fragmented chromatin structure, loss of microvilli, and disruption of cell membranes due to necrosis and apoptosis. Mitochon-dria play a key role in directing and integrating death signals towards the caspase cascade; this leads to the loss of mitochondrial transmembrane potential, changes in electron transport, and the release of caspase activators.
Table 3: Metal ions/compounds adsorption by NSC based on adsorption/chelation.
Matrix Metal ion/compound References
NSC Pb (II) ions (Sun and Wang 2006)NSC Cu (II) ions (Sun et al. 2007)NSC/OCMC hydrogel Mucin adsorption (Lü et al. 2010)NSC-g-PEI DNA (Lu et al. 2009)Glactosyl NSC-g-PEI DNA (Lu et al. 2010)NSC p-nonylphenol, bisphenol A (Aoki et al. 2003)N-succinyl-O-carboxymethyl chitosan Fe3O4 (Zhu et al. 2008)N-acylated chitosan Iron oxide (Bhattarai et al. 2007)NSC Zn and Remacryl Red TGL(dye) (Kyzas et al. 2015)NSC polyelectrolyte film Human plasma protein (Graisuwan et al. 2012)
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
10 S. Bashir et al.: Chitosan derivatives
NSC caused a decrease in negative potential on the surface of K562 cells by inducing positive cell surface potential, indicating the neutralization of surface charge. This was the first interaction observed between the cell membrane and nanoparticles. The positive surface charge and the decrease in membrane potential confirmed the membrane-perturbing activity of NSC. Reactive oxygen species (ROS) act as intracellular messengers, and an ROS burst in the cytosol occurs due to mitochondrial membrane disruption. Increased intracellular ROS and Ca+2 ions were observed, indicating membrane disruption. A DNA fragmentation study revealed the antitumor effect of NSC achieved by apoptosis induction in K562 cells. DNA fragmentation is a biochemical hallmark of apoptosis. Cellular DNA internu-cleosomal fragmentation by endonuclease to 180 bp can be detected by agarose gel electrophoresis. It was found that the degree of DNA fragmentation was directly depend-ent on the NSC concentration (Luo et al. 2010). In another study, the mechanisms of apoptotic pathway differentia-tion, cytosolic cytochrome c levels, Bcl-2, and Bax expres-sion were analyzed. Apoptosis is a common form of cell death that occurs in steps; it acts as a physiological suicide mechanism to preserve tissue homeostasis. Cytochrome c is essential for the initiation of apoptosis. It has been shown that apoptosis is induced due to the accumulation of cytochrome C in the cytosol. The cytochrome c content in the cytoplasm presented a time- and dose-dependent increase when cells were treated with NSC. Bcl-2 belongs to the protein family of apoptosis antagonists. Bcl-2 con-tributes to neoplastic cell expansion by inhibiting normal cells through physiological cell death mechanisms. The expression of Bcl-2/Bax is closely related to cell destiny. Elevated expression of the apoptosis-promoting protein Bax was found along with lower levels of the apoptosis-inhibiting protein Bcl-2; this was time and dose dependent when K562 cells were treated with NSC. Colorimetric and fluorescent analysis indicated caspase-3 and -9 activities, which confirmed mitochondria-dependent apoptosis in K562 cells (Luo et al. 2012). Zhang et al. (Zhang et al. 2014) studied the apoptosis inhibitory activity of NSC nanopar-ticles coupled with low-density lipoprotein (LDL) loaded with osthole (OST/LDL-NSC nanoparticles) on HepG2 cells. Flow cytometry results revealed that Ost/LDL-NSC nano-particles inhibited the proliferation of HepG2 cells by trig-gering apoptosis.
4.2.2 Enzyme immobilization
Enzyme immobilization is very useful in the recovery and reusability of enzymes. It significantly improves enzyme
activity, stability, resistance to temperature, pH selectiv-ity, and specificity. Enzymes act as biocatalysts, and the development of applications generally requires enzyme support (Prashanth and Tharanathan 2007). Chitosan has been shown to be a promising matrix for enzyme immobi-lization because of its biocompatibility. The drawbacks of the use of insoluble chitosan in bioconversion are its slow catalysis/binding due to diffusion-controlled mass trans-fer, steric hindrance in an already biphasic system involv-ing water-insoluble substrates, and its low geometrical congruence with protein surfaces. Chitosan derivatives are excellent supporting materials for enzyme immobi-lization as they exhibit significant thermal stability as compared to the free enzyme. NSC is a prominent mate-rial for enzyme immobilization due to its reversible water-soluble–insoluble property with changing pH and due to its improved mechanical strength. It is an inexpensive and readily available chitosan derivative. Both partially N-succinylated chitosan and N-succinyl glycolchitosan have been used for enzyme immobilization. Enzymes were covalently bonded to partial NSC and N-succinyl glycolchitosan. The activities of β-fructosidase, D-glucose isomerase, and glucoamylase were 64.3%, 65.2%, and 58.8%, respectively. D-glucose isomerase activity was not affected by the succinylation degree, while the activity of the other enzymes was dependent on the degree of succi-nylation (Yamaguchi et al. 1982). Immobilized alliinase in NSC was used to catalyze the conversion of alliin to allicin. Glutaraldehyde (GA) is expected to form a Schiff base upon nucleophilic attack by the amino groups of NSC or amino groups of lysine residues in the protein. Aldehyde groups were cross-linked with the amino groups of alliinase and NSC. Immobilization was performed to protect Lys251 from reaction with the activated carrier through excessive pyridoxal-5-phosphate. The properties of immobilized alliinase were observed by varying the pH, temperature, and crosslinking agent. The maximum pH of the free and immobilized enzyme was 6.0 and 7.0, respectively. The pH shift may have been due to the carboxyl groups sticking out of NSC, which kept the pH of the solution around allii-nase lower than that of the solution distant from alliinase. The maximum temperature for free and immobilized allii-nase was 30°C and 40°C, respectively. The thermostability of free and immobilized alliinase was also investigated. The free enzyme retained at most 5.8% of its original activity following heat treatment at 45°C for 3 h, while the immobilized enzyme retained 40% of its initial activity. The increased stability may have been due to the cova-lent bonding of alliinase to the polymer. The immobilized enzyme retained 85% of its activity after being reused five times. Moreover, the affinity of immobilized alliinase
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 11
towards the substrate was significantly increased as com-pared to the free enzyme. Figure 6 presents a detailed mechanism of the catalytic conversion of alliin to alliicin (Zhou and Wang 2009). In another study, a protease was immobilized on chitosan, carboxymethyl chitosan, and NSC hydrogels for the depolymerization of chitosan. In this study, mixtures of endo and exoenzymes were used. The Michaelis-Menten kinetic parameters revealed a sig-nificant effect of the chemical nature of the support on immobilized enzyme properties. The support provided a microenvironment for the protease and changed the hydrolytic process. The results also revealed that low Mw products obtained by immobilized enzymes on chitosan and carboxymethyl chitosan were collected after 8 h, while products obtained by immobilized enzymes on NSC were collected after 4 h, indicating completion of the reaction. The Mws of the products obtained with the
neutral protease immobilized on chitosan, carboxymethyl chitosan, and NSC hydrogels were 3.4, 3.2, and 1.9 kDa, respectively. The stability of products in relation to the hydrolyzing time and the amount of product (reducing sugars) was observed using Schale’s modified method. The production of reducing sugars during biocatalytic conversion by the enzyme immobilized on chitosan and carboxymethyl chitosan stopped after the hydrolyzing time (8 h) but continued after the hydrolyzing time (4 h) with NSC. This extensive degradation of chitosan was attributed to the non-specific digesting behavior of the immobilized protease on the NSC hydrogel, which allowed the digestion process to be more complete. However, the same amount of protease was immobilized on all hydro-gels, but the protease immobilized on the NSC hydrogel was stronger than chitosan and carboxymethyl chitosan hydrogels. The results indicated that the chitosan chain
Figure 6: Enzyme catalytic conversion of Alliin to Allicin (Zhou and Wang 2009).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
12 S. Bashir et al.: Chitosan derivatives
contained cleavage points that were more susceptible to the enzyme immobilized on the NSC hydrogel. The proper-ties of the immobilized enzymes were strongly dependent on the nature of the support. The supports provided dif-ferent microenvironments to the enzyme and changed the depolymerization process of chitosan (Li et al. 2012a,b).
4.2.3 Antimicrobial properties
The polycationic nature of chitosan and its derivatives is a prerequisite for its antimicrobial activity. The mechanism of the antimicrobial activities of chitosan and its deriva-tives is still under investigation. The antimicrobial activ-ity of chitosan and its derivatives is affected by numerous factors such as Mw, pH, temperature, ionic strength, charge density, and chelating capacity. The pKa value of chitosan and its derivatives plays a vital role when the environmental pH is less than the pKa value. In addition, the charge density, degree of deacetylation, hydropho-bicity, and amino groups have direct effects on antimi-crobial activity (Kong et al. 2010). Antibacterial activity significantly changes due to the modification of chitosan at amino groups due to changes in electrostatic attraction between the amino groups of chitosan and the carboxyl negative charges on the cell walls of bacteria. If chitosan is modified by quaternization at an amino or hydroxy group, its activity increases due to an increase in charge density, which directly depends on the degree of substitu-tion (Sun et al. 2006). N-acyl derivatives have no potential antibacterial activity due to a decrease in charge density as compared to chitosan. Fan et al. (2010) observed anti-bacterial activity against Staphylococcus using untreated and AgNO3-treated NSC/alginate blend fibers. The results revealed that untreated fibers had no antibacterial activ-ity, but AgNO3-treated fibers showed good antibacterial activity. Aziz et al. (2012) studied the antibacterial activ-ity of an NSC/dextran hydrogel against Gram-positive and Gram-negative bacteria and found that none of the bacte-ria were susceptible to the NSC hydrogel. This was due to a reduction in the positive charge on the amino groups, and thus, NSC was not able to interact electrostatically with the negative charge on the cell walls of the bacteria. Inta et al. (2014) compared the antibacterial activity of hydro-phobically modified dodecyl succinyl phthaloyl chitosan and chitosan. The antibacterial activity was assessed against Gram-positive bacteria (S. aureus and B. subtilis) and a Gram-negative bacterium (E. coli) by determining the minimum inhibitory concentration (MIC). Chitosan showed MIC value of 0.312 mg/mL against all bacterial strains. However, dodecyl succinyl phthaloyl chitosan
showed MIC value of 0.078 mg/mL against Gram-positive bacteria and MIC value of 0.312 mg/mL against the Gram-negative bacterium. The results indicated greater growth inhibition against Gram-positive bacteria as compared to Gram-negative bacteria. This might be due to the differ-ent cell structure. Gram-negative bacteria possess more negative charges on the cell surface as compared to Gram-positive bacteria. The greater antibacterial activity against Gram-positive bacteria was possibly due to hydrophobic interactions between the dodecyl succinyl chains and the bacterial cell wall proteins, facilitating the absorption of dodecyl succinyl phthaloyl chitosan to the cell wall. The weak antibacterial activity against Gram-negative bacteria was likely due to the interaction of the remaining proto-nated amino groups on dodecyl succinyl phthaloyl chi-tosan and negatively charged groups on the cell surface. Modified dodecyl succinyl phthaloyl chitosan showed better water vapor barrier properties under neutral pH conditions than the chitosan film.
4.2.4 Antioxidant activity
The antioxidant activity of chitosan and its derivatives depends on the Mw, charge density, reducing power, and electron-withdrawing effect of the substituted groups. The antioxidant activity of chitosan and its derivatives has also been attributed to its hydroxyl groups. The number and activity of hydroxyl groups are important factors associ-ated with antioxidant activity. Furthermore, low Mw, high charge density, and strong reducing power chitosan deriv-atives have better antioxidant activity. With a decrease in the Mw of chitosan derivatives, inter- and intramolecular hydrogen bonding become weaker, resulting in increased antioxidant activity (Vinsova and Vavrikova 2011). The antioxidant activity of chitosan is strongly influenced by the properties of the substituting group and the degree of substitution. The electron-withdrawing capacity also has a significant effect on the antioxidant activity of chitosan derivatives. Sun et al. (Sun et al. 2011) reported on the anti-oxidant activity of N-maleoyl and NSC oligosaccharides with the same degree of substitution. The antioxidant activity was determined by scavenging superoxide anions and hydroxyl free radicals, as well as by the reducing power of the acyl groups. Superoxide anion and hydroxyl free radical scavenging activity was determined by chemi-luminescence assays using a biochemical luminometer (Sun et al. 2004). Superoxide anions were produced by luminol-enhanced autoxidation of pyprogallol, while hydroxyl free radicals were produced in a Fe(II)-H2O2-luminol system. The method of Yen and Chen was used
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 13
to determine the reducing power of the samples (Yen and Chen 1995). The scavenging effects of superoxide anions and hydroxyl free radicals showed that NSC and N-male-oyl chitosan oligosaccharides had antioxidant activity. The reducing properties of both derivatives also revealed the antioxidant activity of NSC and N-maleoyl chitosan. NSC and N-maleoyl chitosan had reducing power due to their succinyl and maleoyl groups. The reducing power of succinyl and maleoyl groups is comparable. Both succinyl and maleoyl groups have electron-withdrawing capacity as well. These groups destroy intermolecular and intramolec-ular hydrogen bonds and enhance the activity of hydroxyl and amino groups. In another study, quaternized chitosan showed better antioxidant activity than chitosan due to its higher positive charge density. Quaternization enhanced the activity of the amino and hydroxy groups, resulting in increased antioxidant activity (Guo et al. 2008).
4.3 Biopharmaceutical characteristics of NSC
The cytotoxicity of a biomaterial is very important in its applications. NSC is a well-known biocompatible mate-rial and, like chitosan, can be used in the preparation of hydrogels, microparticles, nanoparticles, and nano-spheres. Lu et al. prepared a NSC/carboxymethyl cellulose hydrogel, and the MTT assay was applied to observe the influence of the hydrogel on the proliferation of HEK 293T cells. The results demonstrated 93% cell viability after 48 h (Lü et al. 2010). Moreover, Lu et al. prepared NSC-g-PEI copolymers. The MTT assay was applied to observe cell viability in 293T, CHO, and HeLa cells. In this assay, cells were seeded in 96-well plates in 100 μL of DMEM containing 10% FBS at a density of 6000 cells/well. The copolymer solution was added after 48 h, and cell viabil-ity was assessed. The results revealed that cytotoxicity was minimal in the presence of NSC as compared to PEI alone. This was due to the biocompatibility of chitosan, which reduced the cytotoxicity of the copolymer (Lu et al. 2009). The cytotoxicity of a polymer depends on its charge. Cationic polymers cause cytotoxicity due to inter-actions with cells with negatively charged components in the plasma membrane. The same research group prepared an NSC-g-PEI- lactobionic acid (LA) copolymer, and cyto-toxicity was assessed in 293T, HeLa, and HepG2 cells. The results revealed that NSC-g-PEI-LA had lower cytotoxicity compared to PEI. They noted the effect of LA on cytotoxic-ity by using different amounts of LA; a small amount of LA reduced the surface charge, which increased cytotoxicity and cell specificity (Lu et al. 2010). Chitosan is non-toxic
and tissue compatible, but NSC is a synthesized derivative and is known to significantly promote cell proliferation. Zhu et al. assessed in vitro cell toxicity and found that NSC had no negative effects on 3T3 fibroblasts up to 0.25 mg/mL (Aiping et al. 2006).
Apart from water solubility and cytotoxicity, NSC has considerable biodegradability in vitro as well as in vivo. Normally, the degradability of a hydrogel depends upon the pH, temperature, oxygen content, water potential, structure of the polymer network, crystalline nature of the polymer, and type of linkages. Degradability is observed by a change in weight over a specific period of time. The biodegradability of NSC was confirmed using an NSC/OCMC hydrogel in phosphate buffer solution at 37°C. In this hydrogel, Schiff-base linkages were hydrolytically susceptible and not stable, resulting in an enlarged lattice size of the network. This phenomenon led to disintegra-tion of the hydrogel (Ito et al. 2007). Tan et al. also studied biodegradation of an NSC/PEG hybrid hydrogel in phos-phate buffer solution at 37°C. The results revealed that the hydrogel was biodegradable.
Chitosan has better mucoadhesive properties and a nasal absorption-enhancing effect. NSC has inherent permeation-enhancing properties with acceptable safety, which is favorable for intranasal drug delivery. The per-meation-enhancing property and ciliotoxicity of NSC were evaluated by Na et al. (Aiping et al. 2006) using an in situ toad palate model. The safety profile of NSC was compared with chitosan in cell culture. The results revealed that NSC was sufficient to achieve a permeation-enhancing effect in vivo. The ciliotoxicity study demonstrated that NSC had a better safety profile than chitosan. Similarly, NSC has an inherent mucoadhesive property that has been observed by many researchers. Mukhopadhyay et al. (2014) com-pared the mucoadhesive property of chitosan and NSC hydrogels in vitro as well as in vivo. The results revealed that 39% and 77% of mucin was adsorbed by chitosan and NSC hydrogels in vitro. NSC had better mucoadhesive properties than chitosan in the mouse intestine even after continuous washing of the intestine with phosphate buffer solution at pH 7.4. This profile indicates its applicability in the medical field. Figure 7 illustrates the numerous for-mulations, physicochemical properties, and outstanding biological characteristics of NSC.
Mura et al. (2011a,b) also observed the mucoadhesive properties of NSC matrices and microparticles in differ-ent parts of the colon. The small intestine showed weaker mucoadhesion than the large intestine, which was defi-nitely due to the difference in the epithelia of the small and large intestines. It might be due to the presence of villi in the small intestine and the absence in the large
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
14 S. Bashir et al.: Chitosan derivatives
intestine, which resulted in stronger mucoadhesion in the large intestine than the small one. Another factor might be the goblet cell ratio in both intestines, which is greater in the large intestine. Two distinct systems showed stronger mucoadhesion in an inflamed colon among the different studied parts. This might be due to the presence of posi-tively charged proteins in the inflamed region, which has been reported by many researchers. Mucoadhesion is the interaction between the mucosa and polymers due to van der Waals forces and other physical entanglements. These forces depend mainly on the chemical structure of polymers. Polymers having hydroxyl, amine, amide, and carboxyl groups show strong mucoadhesive proper-ties. These properties also depend on the strength of the anionic charge, chain flexibility, Mw, and surface energy. NSC has all the abovementioned properties, so it is a strongly mucoadhesive polymer.
4.3.1 Biodistribution
NSC has prolonged systemic circulation and a longer plasma half-life than other polymer molecules, at about 100 and 43 h in normal tissues and tumor tissues, respec-tively. The reason for the long circulation time of NSC is its negatively charged carboxylate ions, which prolong the plasma half-life. This long-term retention property enhances its anticancer efficiency. The biodistribution of labeled NSC was observed in mice as a potential cancer therapy. The NSC was labeled with fluorescein isothiocy-anate (NSC-FITC), and the biodistribution was monitored after intraperitoneal (i.p.) and intravenous (i.v.) adminis-tration to sarcoma 180 tumor-bearing mice. No significant distribution difference was observed between i.p. and i.v. administration of NSC-FITC (Kato et al. 2001a,b). In another study, NSC, lactosaminated-NSC (Lac-NSC), and
Figure 7: It describes the surprising and outstanding formulations, physiochemical and biological characteristics of NSC that contribute towards the appropriateness in biomedical applications.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 15
galactosaminated-NSC (Gal-NSC) were labeled with FITC. A biodistribution study among NSC-FITC, Lac-NSC-FITC, and Gal-NSC-FITC was performed 8 h after i.v. administra-tion in the mouse liver. The results revealed that Gal-NSC-FITC was eliminated from the plasma 8 h after injection. NSC-FITC was maximally distributed in the systemic circulation and scantily in the liver as compared to Lac-NSC-FITC. Generally, Lac-NSC-FITC was found to be an excellent liver targeting drug carrier (Kato et al. 2001a,b). Jain et al. prepared lactosaminated-NSC nanoparticles and studied the biodistribution of Lac-NSC nanoparticles conjugated with acyclovir in the liver, spleen, kidney, and lungs 24 h after i.v. administration. Figure 8 comprehen-sively describes the biodistribution (Jain et al. 2014).
5 Interaction between N-succcinyl chitosan and drugs
The production of protein is feasible on a large scale for pharmaceutical applications, but the administration of a protein to the body is a major challenge because proteins are hydrophilic in nature and are unstable. Synthetic and natural polymers are suitable carriers for protein administration. Polylactide and poly ε-caprolactone are polymers that can be used as protein carriers, but these synthetic polymers have certain limitations due to their hydrophobic moieties. In addition, the use of harmful organic solvents during polymer production is
also discouraged (Kajihara et al. 2001); these harmful solvents degrade proteins. In order to avoid these limita-tions, the use of natural polymers, especially chitosan, is suggested. Chitosan and its derivatives have mucoad-hesive properties and lead to transient opening of epi-thelial tight junctions, as described earlier (Artursson et al. 1994). Among the chitosan derivatives, NSC is a suitable protein carrier because it can self-assemble into nanospheres in distilled water without the use of organic solvents, surfactants, or special experimen-tal techniques (Aiping et al. 2006). Zhu et al. (2007) studied the interaction between NSC and bovine serum albumin (BSA). UV and fluorescence results indicated interactions between the hydrophobic moieties of NSC and BSA. Circular dichroism (CD) results demonstrated that a complex formed between NSC and BSA, and the conformation of BSA did not change during chain entanglement. Isothermal calorimetric results showed that binding of BSA with NSC occurred in a molar ratio of 30:1. Hence, a protein drug can be entrapped in the polymer network. NSC is an excellent carrier of hydro-philic biomacromolecules. In another study, the inter-action between an injectable NSC/OCMC hydrogel and a protein was observed by synchronous spectra at Δλ = 60 nm. During analysis, tryptophan residues were observed after 12 days. The protein did not denature during the formulation, storage, and release from the injectable hydrogel. The reason for this preservation of protein was its non-toxicity and the maintenance of plasma concentrations in the bloodstream.
30
25
20
15
10
Dos
e re
cove
ry (
%)
5
1 h 8 h 24 h
Liver
Lung
Spleen
Kidney
Time (h)
Plain
drug
N-Suc
-CS-N
Ps
Lac-
N-Suc
-CS-N
Ps
Plain
drug
N-Suc
-CS-N
Ps
Lac-
N-Suc
-CS-N
Ps
Plain
drug
N-Suc
-CS-N
Ps
Lac-
N-Suc
-CS-N
Ps
0
Figure 8: Biodistribution after i.v. administration of plain drug, drug-conjugated N-Suc-CS Nps and Lac-N-Suc-CS Nps (Jain et al. 2014). Reprinted with permission from Journal of Nanoparticles Research 16, 2136 (2014); copyright 2014 Springer.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
16 S. Bashir et al.: Chitosan derivatives
6 Formulations of NSC-based carriers
Different formulations of NSC have been developed for biomedical applications using distinct methods. Hydro-gels and nanoparticle-based formulations are described in this review. Different researchers have prepared hydro-gels using various methods based on the requirements of the host organ/tissue. Additionally, preparation methods also depend on the nature of the drug. NSC molecules are either physically associated or chemically cross-linked with other materials in the hydrogel. The major mecha-nisms of physical association leading to gel formation with NSC are ionic interactions, hydrophobic associa-tions, hydrogen bonding, van der Waals forces, and inter-polymer complexes. Physically associated hydrogels are advantageous due to the absence of toxic cross-linking agents. However, there are also certain limitations as well. These limitations include difficulties in maintain-ing pore size, dissolution, and degradation of the gel. Physically associated hydrogels show unpredictable per-formance in vivo. On the other hand, chemically cross-linked hydrogels are formed through covalent bonding with the assistance of certain cross-linking agents. These hydrogels possess excellent mechanical properties and controlled biodegradation. These properties of hydrogels play a vital role in tissue engineering. Chemically cross-linked hydrogels have one major limitation, i.e. the toxic-ity of the cross-linking agent. Lu et al. (2010) synthesized an NSC/OCMC hydrogel using Schiff’s base mechanism, which is chemical cross-linking. In this method, NSC and OCMC solutions were prepared separately in phosphate-buffered saline. The reaction mechanism is shown in Figure 9.
Dai et al. (2008) prepared NSC/sodium alginate hydrogel beads through chemical cross-linking. NSC and alginate solutions were dropped into a CaCl2 solution. NSC and alginate cross-linked with Ca+2 ions to form hydro-gel beads. Tan et al. (2013) synthesized physically cross-linked NSC/PEG hydrogels. First of all, methacrylic acid was grafted onto NSC and dimethacrylate onto PEG. Then, methacrylated NSC was irradiated with UV to sterilize it, and dissolved in phosphate saline buffer. Dimethacrylate was sterilized by filtration through a syringe and dissolved in a buffer solution. In order to prepare the gel, these were mixed with the photoinitiator Irgacure at 37°C. The mixture was irradiated by UV light at 365 nm for 15 min, and the gel was formed.
The latest progress and promising developments in nanotechnology will have a considerable impact on drug delivery. Mostly, drugs act non-specifically as untargeted, toxic entities with devastating side effects. Nanoscale drug delivery will confer the ability to increase the ther-apeutic effect by enhancing drug half-life, and provide significant improvements in the water solubility of drugs, decrease immunogenicity, and allow for controlled release of the drug. In addition, nanoparticles can accu-mulate in tissues, specifically tumors, due to increased permeability. NSC nanoparticles have become known as significant nanovehicles in drug delivery systems. There are various methods used in nanoparticle prepa-ration such as self-aggregation, sonication, solvent dif-fusion, micro-emulsion, solvent evaporation, spraying, co-precipitation, and ionic gelation. Lu et al. (2009), 2010) prepared NSC-g-PEI and galactosyl NSC-g-PEI nanoparti-cles by ionic cross-linking. The size of the nanoparticles was found to be 150 nm. Hou et al. (2010) prepared NSC nanoparticles by a sonication method. NSC was dispersed in deionized water by shaking and then sonicated for 24 h.
Figure 9: Synthetic scheme of NSC/OCMC (Lü et al. 2010).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 17
The suspension was filtered and dried under vacuum. Transmission electron microscopy observed 30–200 nm sized nanoparticles. Other formulations included micro-particles, matrices, beads, blends, and nanospheres. The preparation of microparticles, matrices, beads, and nano-spheres involves different methods such as spray drying, freeze drying, ionic gelation, and emulsion cross-linking. Numerous NSC formulations are given in Table 4.
7 Applications of NSCNSC is a biocompatible polymer, and it has potential appli-cations in drug delivery, tissue engineering, and wound dressings. This section provides a detailed overview of these applications.
7.1 Drug delivery
7.1.1 Protein delivery
Effective delivery of protein is a challenge for research-ers due to several drawbacks, such as instability, speedy proteolysis, and short plasma half-life (Chen et al. 1995). Protein drugs undergo rapid degradation through enzyme proteolytic activity in blood. Additionally, protein drugs are small and rapidly pass through the kidney (Kontos and Hubbell 2012). Frequent administration of a protein in the body extends the therapeutic effect of the protein. However, it is not a good idea because it will stimulate the immune response and cause dangerous side effects, leading to patient discomfort and increased cost of treat-ment. The abovementioned limitations can be overcome
Table 4: NSC-based formulations and preparation techniques.
NSC based Formulation
Composition Method of preparation References
Hydrogel NSC/oxidized carboxymethly cellulose Schiff’s base mechanism (Lü et al. 2010)Hydrogel NSC/polyethylene glycol Radiation cross-linking (Tan et al. 2013)Hydrogel NSC/sodium alginate Ionic gelation (Rogalsky et al. 2011)Hydrogel NSC/RGD grafted sodium alginate Schiff’s base mechanism (Liu et al. 2013)Hydrogel NSC/oxidized hyaluronic acid Schiff’s base mechanism (Tan et al. 2009)Hydrogel Dex-grafted NSC/AHA Schiff’s base mechanism (Sun et al. 2013)X NSC/RGD grafted sodium alginate/LIPUS X Hydrogel NSC/dextran aldehyde Schiff’s base mechanism (Wang et al. 2014)Hydrogel NSC/polyacrylamide Hemiacetal formation (Aziz et al. 2012)Hydrogel NSC Chemical cross-linking (Mukhopadhyay et al. 2014)Hydrogel NSC/alginate Covalent cross-linking Hydrogel Ost/NSC Enzymatic cross-linking Nanoparticles NSC(hydroxycamptothecin) Emulsion solvent diffusion (Zhang et al. 2014)Nanoparticles 5-FU-NSC Sonication (Hou et al. 2010)Nanoparticles Lac-NSC(acyclovir) Emulsion solvent diffusion (Yan et al. 2006a,b)Nanoparticles Folate modified NSC Ionotropic gelation (Rogalsky et al. 2011)Nanoparticles NSC-g-PEI-DNA Self-assembly (Yan et al. 2015)Nanoparticles Glactosyl conjugated NSC-g-PEI-DNA Chemical cross-linking (Lu et al. 2009)Nanoparticles NSC Chemical cross-linking (Lu et al. 2010)Nanosphere Lauryl succinyl chitosan Self-assembly (Aiping et al. 2006)Microparticles NSC TPP cross-linking (Rekha and Sharma 2009)Microparticles NSC Spray drying (Mura et al. 2012)Microparticles NSC-MMC Ionic cross-linking (Rekha and Sharma 2008)Microparticles NSC/PVA Emulsion technique (Onishi et al. 2001)Microsphere NSC/sodium alginate w/o emulsion (Kajjari et al. 2013)Beads NSC(enzyme) Ionic gelation (Dai et al. 2008)Beads NSC/oxidized alginate Ionic gelation (Li et al. 2012a,b)Blend NSC/alginate Ca+2 or Zn+2 ion cross-linking (Straccia et al. 2014)Blend fibers NSC/β-cyclodextrine Electrospinning method (Fan et al. 2010)Matrices NSC Freeze drying (Mura et al. 2011a,b)Polyelectrolyte film NSC Multilayer assembly (Graisuwan et al. 2012)Thin film NSC-MMC Gamma irradiation (Vanichvattanadecha et al. 2010)Conjugate Cholestrol succinyl chitosan Covalent bonding (Song et al. 1992)Liposomes Incubation method (Wang et al. 2010)
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
18 S. Bashir et al.: Chitosan derivatives
by using hydrogels as a protein drug delivery vehicle. The large amount of water in the hydrogel may provide an aqueous environment for the protein, which protects and preserves the characteristics and bioactivity of the protein drug. It has already been observed that proteins entrapped in polymeric networks are more resistant to denaturation (Kim et al. 2009). The delivery of insulin as a protein drug is important because of poor patient compliance. Numer-ous routes have been considered, but oral delivery is the most common method of insulin delivery. However, there are multiple barriers in the gastrointestinal (GI) tract that limit the applicability of oral delivery. Researchers have suggested many natural or synthetic polymer carriers that may enhance oral route administration ( Mukhopadhyay et al. 2013). Chitosan is a suitable polymer carrier for oral insulin delivery. However, due to the poor water solubil-ity of chitosan, NSC has been selected due to its greater solubility. Mukhopadhyay et al. (2014) prepared a copoly-mer hydrogel of NSC grafted to polyacrylamide and chi-tosan grafted to polyacrylamide for insulin delivery. They studied the synthesis of copolymer hydrogels and the behavior of insulin release. Insulin loading and in vitro release were observed in both hydrogels, and it was found that NSC/polyacrylamide (AAm) hydrogels showed better insulin loading efficiency of 46% and encapsulation efficiency (EE) of 76%. The concentration of acrylamide had a significant impact on insulin loading. In vitro, it was found that 98% of the insulin was released from the NSC/AAm hydrogel and 20–26% was released from the chitosan/AAm hydrogel at pH 7.4. The in vivo pharmaco-logical responses to the insulin-loaded NSC/AAm and chi-tosan/AAm hydrogels were investigated. The NSC/AAm hydrogel showed a more intense hypoglycemic effect than the chitosan/AAm hydrogel in diabetic mice after delivery and led to 4.43% relative bioavailability.
There are several disadvantages of conventional hydrogels in applications requiring placement in the body for medical purposes. After release of the drug, surgery is required to remove the implanted polymeric material. Recently, there has been an increasing interest in hydro-gels, which can be formed in situ after being injected subcutaneously, intraperitoneally, or intravenously in the body (Li et al. 2012a,b). Exploitation of injectable hydrogels will overcome the drawback of conventional hydrogels and eliminate the need for further surgery. These hydrogels can be administered safely by an inva-sive procedure, providing maximum molecule release in the body at a specific site of action without any physical or chemical transformation. Hydrogels reduce patient discomfort, treatment cost, recovery time, and the risk of infection. With these distinctions, hydrogels have
been explored as suitable formulations for the controlled release of protein drugs. In situ gel formation serves as a controlled release depot and maintains drug plasma con-centration within the therapeutic range, which prolongs the medicinal effect over an extended period of time (He et al. 2008, Yeom et al. 2010, Overstreet et al. 2012, Parisi-Amon et al. 2013).
Lu et al. (2010) introduced oxidized carboxymethyl-cellulose (CMC)/NSC injectable hydrogels for the con-trolled release of a protein. Hydrogel formation occurred at physiological temperature and pH. The gelation rate was directly dependent on the degree of oxidation of CMC. BSA release was rapid in the first 90 min, and the release rate was fast as compared to the degradation rate of the hydrogel. In the second phase (90 min to 10 h), the release rate was constant. Drug diffusion from the hydrogel occurred in parallel with the degradation of the hydro-gel; this was verified by assessing the weight loss of the polymer in the first 10 h. The release of BSA in the third stage was very slow as compared to the first and second stages. Drug release rate was dependent on both diffu-sion and degradation, not only diffusion of drug from the polymer hydrogel. The release of BSA was also dependent on the characteristics of the hydrogel, in particular cross-linking density and pore size. Protein delivery from inject-able hydrogels was very slow as BSA was not completely released in 12 days.
7.2 Targeted drug delivery
New discoveries in health science have caused dramatic changes in quality of life, longevity, and productivity by improving treatment methods and drugs. The develop-ment of drugs is highly challenging and expensive. The discovery of a new drug takes approximately 15 years, and the estimated cost of drug development is around $802 million US, which increases by 7.4% annually. The high cost of drug discovery and price inflation is an eco-nomic burden on society. Another major issue with drugs is poor outcome because drugs do not have the ability to traffic to a specific site. A large amount of administered drug distributes over normal tissues/organs instead of the specific area to be treated. This issue often causes severe side effects. An efficient advancement to counter-act this serious issue is to develop a drug release system that can target the therapeutic agent to a specific site. This approach is called targeted drug delivery. Targeted drug delivery has considerable advantages such as site-specific delivery, increased patient compliance, therapeutic effi-cacy, and decrease in the therapeutic dose. Over a century
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 19
ago, Paul Ehrlich developed a concept of a site-specific drug delivery that selectively destroyed affected cells without damaging healthy cells. He described this sup-posed drug as a “magic bullet”. With this concept, many researchers have focused on the development of ideal site-specific drug carriers that selectively destroy dis-eased cells. Numerous site-specific drug delivery carriers have been developed using lipids, dendrites, surfactants, as well as natural and synthetic polymers. Among these carriers, natural polymers have attracted considerable attention due to their various advantages. NSC is a deriva-tive of a natural polymer, which protects bioactive agents from the hostile environment of the body and releases drugs at a specific site in a controlled manner. Numerous NSC-based formulations have been used for site-specific drug delivery such as hydrogels, matrices, microparticles, nanoparticles, beads, liposomes, and blends.
7.2.1 Anti-cancer drug delivery
Medical science has made tremendous achievements, but cancer is still a major human killer. Chemotherapy is a therapy that uses chemical compounds to kill cancer cells. The chemical compounds used in chemotherapy are mostly water insoluble and cannot be applied directly to the human body to kill cancer cells. Water-insolu-ble compounds need organic solvents or formulations
for biomedical applications, which incurs certain side effects. Therefore, drug delivery systems have been devel-oped that encapsulate anti-cancer drugs and specifically deliver them to cancer cells. These systems include bio-compatible polymer carriers. Sato et al. prepared water-soluble glutaric mitomycin C (glu-MMC) conjugated with NSC in DMF (Nagai 1996). Kato et al. prepared water-sol-uble mitomycin C conjugated with highly N-succinylated chitosan (Kato et al. 2000a,b). The drug contents in the water-soluble conjugates were 1.3% and 12%, respectively. These conjugates were compared with water-insoluble NSC-MMC conjugates produced by Song et al. A small amount of the drug was found in water-soluble conjugates as compared to water-insoluble conjugates. In vitro, the release characteristics of MMC showed pH dependency for both water-soluble conjugates. In addition, MMC showed a faster release rate from water-soluble conjugates as com-pared to water-insoluble conjugates. It was proposed that the slow drug release from water-insoluble conjugates was due to tight cross-linking. Water-soluble conjugates are shown in Figure 10. Onishi et al. prepared water-sol-uble microparticle conjugates of NSC-MMC and further improved the release rate of MMC.
Tang et al. prepared different Mw NSC samples labeled with FITC. They were used to observe the affinity of NSC toward cancer cells, including lung, breast, colon carci-noma, and pancreatic carcinoma, as well as fibroblasts. Fluorescence microscopy and flow cytometry revealed the
Figure 10: Conjugation of NSC with MMC and glu-MMC (Nagai 1996, Kato et al. 2000a,b).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
20 S. Bashir et al.: Chitosan derivatives
influence of Mw on the affinity toward cancer cells. Moreo-ver, strong affinity of NSC toward lung cancer cells was also reported (Jayakumar et al. 2010).
One study described the synthesis of NSC and hydrox-ycamptothecin-loaded NSC (NSCH) nanoparticles by soni-cation (Hou et al. 2010). The effect of drug concentration on the size of the nanoparticles, as well as the in vitro and in vivo release behavior of drug, was evaluated. TEM revealed that the size of NSC and drug-loaded NSC nano-particles was 30 and 200 nm, respectively. A high drug loading efficiency of 68.5% was observed. Burst release of 20.76% after 24 h occurred due to unencapsulated drug followed by slow release in phosphate buffer solution of 7.4 pH. In vivo studies showed tumor targeting of NSCH and depression of tumor growth after injection into S180 sarcoma tumor-bearing mice. The lethal effect of NSCH was observed by histopathological analysis. Yan et al. (2006a,b) prepared NSC and 5-FU-loaded nanoparticles by an emulsion solvent diffusion method. The effect of the initial concentration of the drug on the characteristics of the nanoparticles and the release rate in phosphate-buff-ered saline was observed. TEM and zeta sizing reported that nanoparticles were in a size range of 202–273 nm. The highest loading capacity (19%) and the highest release (61%) were observed at 1000 μg/mL as the initial con-centration of 5-FU. The 5-FU-loaded NSC nanoparticles showed mild toxicity and good anti-tumor activity against S180 sarcoma tumor cells. In another study, osthole-loaded NSC nanoparticles were prepared and coupled with LDL through amide linkages. Osthole showed a sustained-release effect in vitro as compared to the native material. Subcellular localization in vitro and near-IR fluo-rescence real-time imaging in vivo showed that nanopar-ticles had high liver tumor targeting efficiency. Zhu et al. (2014) prepared LDL loaded with cholesterol-conjugated siRNA and coupled with NSC nanoparticles and then loaded them with doxorubicin (dox). In vivo tumor target-ing showed that Cy7-labeled Dox-LDL-NSC nanoparticles had significantly accumulated in liver tumors. The results revealed that LDL-NSC nanoparticles were effective tumor targeting agents. Some formulations of NSC used in drug delivery are presented in Table 5.
7.2.2 Colon-targeted drug delivery
Colon delivery refers to targeted delivery of drugs into the lower GI tract, which occurs primarily in the large intes-tine (colon). The site-specific delivery of drugs to the lower parts of the GI tract is advantageous for localized treatment of several colonic diseases, mainly inflammatory bowel
disease, irritable bowel syndrome, and colon cancer. Other potential applications of colonic delivery include chronotherapy, prophylaxis of colon cancer, and the treat-ment of nicotine addiction (Reddy et al. 1999, Chourasia and Jain 2003). It has also gained increased importance not just in the delivery of drugs for the treatment of local diseases but as a potential site for the systemic delivery of therapeutic agents (Philip et al. 2009).
The oral route is the most convenient and is pre-ferred over other delivery methods. Rectal administra-tion offers the shortest route for targeting drugs to the colon. However, reaching the proximal part of colon via this route is difficult. Rectal administration can also be uncomfortable for patients, and compliance may be less than optimal (Mastiholimath et al. 2007). The colon is rich in lymphoid tissue, and the binding of antigens to mast cells in the colonic mucosa causes rapid local release of inflammatory mediators. The most critical challenge in drug delivery is to preserve the formulation of the drug during its passage through the stomach. Oral administra-tion of drugs has certain limitations because a very small amount of the drug reaches the target site, as a signifi-cant amount of the drug is rapidly absorbed in the upper part of the GI tract. This results in decreased efficacy of treatment because treatment depends upon the required amount of drug and drug release at a specific site in a con-trolled manner. Drug carriers minimize these limitations. Drugs can be orally delivered to the colon using a syn-thetic or natural polymer like chitosan and its derivatives as carriers. Recently, chitosan derivatives have garnered substantial attention regarding colon-targeted drug deliv-ery. Mura et al. studied colon-targeted delivery of 5-ASA using NSC. They investigated drug release using four dif-ferent matrices, prepared by a freeze drying process. The release rate of 5-ASA from chitosan matrices was fast in acidic medium, while the release rate from NSC was rapid in alkaline medium in vitro. Chitosan/ASA/cyclodextrin matrices released more drug at pH 1.2 because, at this pH, chitosan matrices are in an unswollen state while NSC matrices are in a swollen state, and slow drug release occurs. The reasons for fast release in the unswollen state are related to the inertness of the matrix and the forma-tion of an infusion complex between 5-ASA and cyclodex-trin, which enhanced drug solubility and improved drug delivery. With a pH gradient, the drug was released from NSC matrices and cyclodextrin containing chitosan matri-ces. NSC-based matrices were better because drug release was 15% while other matrices delivered more than 49% of the drug in an acidic medium. Furthermore, at alkaline pH, a maximum of 82% of the drug was released from chitosan matrices, and greater than 92% of the drug was
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 21
released from NSC. Mucoadhesive properties, such as the maximum detachment force F(max) and work of adhesion (Wad), were studied in different parts of the rat intestine. All matrices showed good mucoadhesion in the colon.
Microparticles are particulate systems with a size range of 50 μm to 200 mm. These microparticles are widely used in oral drug delivery due to enhanced drug absorp-tion, targeting of bioactive agents, sustained drug action, improved bioavailability, reduced toxicity, and increased gastric tolerance of agents irritating to the stomach (Desai
et al. 1996). Mura et al. (2012) prepared NSC microparti-cles (MP) by spray drying and matrices by freeze drying. Different properties of the microparticles, i.e. drug entrap-ment and release behavior, were comparatively observed. In vitro, drug release was greater than 92% from micropar-ticles and at most 85% from matrices in 24 h. Rheological studies confirmed that microparticles were less viscous than matrices because of their large surface area and strong interactions with water, which increased the swell-ing ratio and drug release. Moreover, microparticles were
Table 5: Various formulations and anti-cancer, antiviral, and protein drug delivery.
Drug Formulation Method and outcome References
Mitomycin C NSC-MMC conjugates
Water-insoluble conjugates were prepared by condensation of NSC with mitomycin C by using EDC.HCl at 5 pH. Conjugates were attained with 33% MMC contents. In vitro drug release studies showed very slow release behavior and pseudo first-order kinetics. In another study, in vivo chemotherapeutic effect of NSC-MMC conjugates was observed using mice bearing B16 melanoma and L1210 leukemia. Conjugates showed remarkable growth inhibitory effect.
(Song et al. 1992, Song et al. 1993)
Mitomycin C NSC-MMC conjugates
Water-soluble conjugates of NSC-glu-MMC were prepared. Drug-polymer covalent bonding was observed by gel chromatography. In vivo evaluation, intraperitonial, intratumoral, and i.v. administration of NSC-glu-MMC conjugates against P238 leukemia and Sarcoma 180 showed significant suppression of tumor growth.
(Nagai 1996)
5-Fluororacil NSC Nps Emulsification solvent diffusion method was used to prepare 5-Fu-loaded NSC Nps. The size of Nps was 220–260 nm, with a -26 mV zeta potential. 5-FU-loaded Nps were mainly distributed in the liver and tumor. A small amount was distributed in lung and heart. The Nps also reduced the toxic effect of 5-FU in the lung and heart.
(Yan et al. 2010)
Hydroxycamptothecin NSC Nps Hydroxycamptothecin-loaded Nps were prepared by sonication. The size of Nps was found to be 300 nm. The biphasic drug release behavior was observed. The results revealed sustained local delivery of Nps for hydrophobic antitumor drug.
(Hou et al. 2010)
Doxorubicin NSC Nps The Dox-siRNA/LDL-NSC Nps were prepared with an average size of 206 nm. Drug loading amounts of 12.35% and EE of 71% were observed. In vitro study revealed drug-loaded Nps significantly inhibited the cell growth.
(Zhu et al. 2014)
Insulin NSC Mps NSC microparticles with two different degrees of substitution were prepared (SCP-3 and SCP-6). Minimum insulin release was observed from SCP-6 at pH 1.2 and found it promising for in vivo evaluation. SCP-6 reduced glucose level of rat up to 56% at 4th hour and showed excellent mucoadhesive properties.
(Rekha and Sharma 2008)
Insulin Lauryl succinyl chitosan (LSC) particles
LSC nano-/microparticles were prepared by sodium tripolyphosphate cross-linking in the size range from 315 nm to 1.09 μm. The succinyl groups inhibited insulin release at pH 1.2 while hydrophobic moities controlled insulin release at intestinal pH. Results also revealed that hydrophobic and hydrophilic groups improved mucoadhesivity on jejunum of rat intestine and permeability in Caco 2 of insulin.
(Rekha and Sharma 2009)
Acyclovir Lac-NSC Nps Lactose-conjugated NSC Nps were prepared by ionotropic gelation. Lactose is an asialoglycoprotein receptor ligand for targeting of hepatic parenchymal cells.
(Rogalsky et al. 2011)
Nps were spherical in shape with average size of 220 nm. Drug loading efficiency and cumulative release of acyclovir were 62.5% and 27.3%, respectively.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
22 S. Bashir et al.: Chitosan derivatives
excellent drug carriers because of increased drug bioavail-ability, leading to a lower drug dose and a reduction in toxicity and side effects. Another considerable advantage of microparticles was that they were better able to pass through the GI tract, leading to less inter- and intra-subject variability. Microparticles performed to a greater degree in vivo than matrices as they spread throughout the colon due to their large surface area, leading to less irrita-tion and reproducible drug release.
A comparative study of colon-targeted drug delivery in vivo was also performed using matrices and micro-particles. Inflamed colons were treated with a drug and succinyl chitosan suspension, drug-loaded matrices, and microparticles. Drug-loaded matrices and microparticles improved the recovery of the colon. The colon to body weight ratio, clinical activity score, histological evalua-tion, and distribution of CD3+ T cells and CD20+ B cells also confirmed the recovery of the colon in the presence of drug-loaded systems (Mura et al. 2011a,b).
Blends are hydrophilic polymer biomaterials that are composed of two or more components having differ-ent physical and chemical properties. Hydrophilic blends can be exploited in the biomedical field (Islam and Yasin 2012). Praveen et al. (Kajjari et al. 2013) prepared NSC/polyvinyl alcohol (PVA) hydrogel to blend microspheres using GA as a cross-linking agent by the water in oil emul-sion method. Different concentrations of polymers (SCS and PVA) and GA were used to assess the EE and equilib-rium of swelling (ES). EE and ES were strongly depend-ent on the concentration of NSC and GA. An increase in the concentration of NSC increased EE and ES, while a decrease in GA increased EE and ES.
Popular drug encapsulation techniques involve toxic organic solvents. Traces of toxic solvents cause toxicity and inflammation that restrict their use in drug delivery. Thus, researchers have developed encapsulation tech-niques achieved in toxic solvent-free media. Beads can be prepared by different methods by using natural and syn-thetic polymers. Beads serve as a substrate on which the drug is encapsulated or coated. Beads provide sustained release and uniform drug distribution within the GI tract. Furthermore, beads also enhance the bioavailability of formulated drugs (Patil et al. 2010). Dai et al. prepared NSC/alginate beads by an ionic gelation method for the controlled release of nifedipine. In vitro, nifedipine release was very low at pH 1.5 (11%) in the first 16 h, 5.5% at pH 2.5, 75.8% at pH 7.4, and 68% at pH 8.4. According to the drug release mechanism, the significant drug release at pH 7.4 was due to the ionization of carboxyl groups present in succinyl-chitosan (Dai et al. 2008). Zhu et al. (2010) used NSC/alginate hydrogel beads for the controlled delivery
of nifedipine in rabbits. A statistical program was used to calculate the pharmacokinetic parameters. The main parameters of nifedipine were the area under plasma concentration (AUC), the maximum plasma concentra-tion (Cmax), the elimination half-life (t1/2), the time to reach Cmax (Tmax), and the mean residence time (MRT). Nifedipine release was 8% in simulated gastric fluid (SGF), which was far less than the release in simulated intestinal fluid (SIF) (68%). The results of the pharmacokinetic param-eters can be used as a clinical reference.
7.2.3 Nasal drug delivery
Nasal infection, allergy, and rhinal congestion have con-ventionally been treated by the nasal drug delivery route. Nasal drug delivery systems can also be exploited for sys-temic drug delivery. Diverse therapeutic compounds can be delivered through intranasal administration, which is a viable drug delivery route (Illum 2003). This is due to the large surface area of the nasal mucosa and the swift onset of the therapeutic effect with sufficient ability to deliver drugs to the central nervous system directly and non-invasively. All these factors increase patient comfort, compliance, and convenience. Intranasal administra-tion is painless, with no need for sterilization and can be delivered by the patient or the physician. However, two barriers limit rhinal drug delivery or nasal absorption, i.e. low membrane permeability and rapid clearance of drugs administered to the nasal cavity (Costantino et al. 2005, 2007, Johnson and Quay 2005). Different absorp-tion enhancers have been observed to circumvent this problem. Intranasal absorption of salmon calcitonin was assessed by exploiting chitosan and β-cyclodextrin in rats. The results demonstrated a better absorption-enhancing effect of chitosan than β-cyclodextrin (Sinswat and Tengamnuay 2003). Na et al. (2010) compared differ-ent absorption enhancers for the intranasal absorption of isosorbide dinitrate (ISDN) in rats. These enhancers were hydroxypropyl-β-cyclodextrin, poloxamer 188, and chi-tosan. All formulations significantly enhanced intranasal absorption, but poloxamer 188 showed superior perme-ation-enhancing effects of ISDN in rats. Due to certain limitations of chitosan, such as solubility and the absorp-tion-enhancing effect in alkaline media, modification of chitosan is a better choice to increase its biomedical appli-cations. In order to improve the solubility and absorption-enhancing property of chitosan in acidic as well as in basic media, Van der Merwe et al. (2004) synthesized trimethyl-chitosan (TMC) and showed that TMC was more effective in the induction of the immune response to ovalbumin in
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 23
mice. Unfortunately, it showed cytotoxicity in L929 mouse fibroblasts (Mao et al. 2005). Moreover, the effects of TMC on transepithelial resistance were not reported with differ-ent degrees of quaternization in Caco-2 cells at pH 6.2 and 7.4 (Kotzé et al. 1999). The limitations of TMC led research-ers to look for another suitable derivative of chitosan with a good safety profile and enhanced permeation effects. Na et al. studied the permeation-enhancing effect of NSC on the intranasal absorption of ISDN and compared it with chitosan. The results revealed significantly improved intranasal absorption of ISDN. An enhanced permeation effect was found using 0.1% NSC rather than using 0.5% chitosan. Nasal ciliotoxicity studies showed a good safety profile of NSC and excellent pharmacokinetic parameters. All these studies revealed that NSC is a better intranasal absorption enhancer than chitosan (Na et al. 2013).
7.2.4 Ophthalmic drug delivery
Ophthalmic diseases include diabetic retinopathy, cata-racts, age-related macular degeneration, and glaucoma. Ocular diseases like eye infections are treated by apply-ing medicines to the outer surface of the eye or intraocu-lar treatment through the cornea. However, these typical disease treatment methods have several limitations like systemic toxicity and poor ophthalmic bioavailability. Poor ocular bioavailability is due to the impermeability of corneal membrane, nasolacrimal drainage, and the dynamics of tears. These factors are also responsible for poor drug delivery to the posterior eye segment. The hydrophilic/hydrophobic drug/membrane nature causes poor drug absorption across mucosal membranes (Le Bourlais et al. 1998, Clark and Yorio 2003). In order to minimize these problems, significant attempts have been made to improve ocular-controlled drug delivery systems. Recently, several ophthalmic-controlled drug release systems have been developed to treat eye dis-eases. Among these systems, polymer hydrogels represent one such system and have demonstrated prolonged drug residence at the target site and improved bioavailabil-ity. Chitosan-based hydrogels have shown considerable potential in ophthalmic-controlled drug delivery systems. Chitosan-based delivery systems demonstrate delayed drug residence times in precorneal tissue, mucoadhesive properties, and increased viscosity (Gupta et al. 2002, Pijls et al. 2005). Cao et al. investigated chitosan-PNIPAAm hydrogels in situ for the ocular delivery of timolol maleate. They also compared the hydrogel delivery method with the conventional eye drop method. The results revealed that the chitosan-PNIPAAm in situ gel forming material
had significant potential for ocular drug delivery (Cao et al. 2007). Genta et al. synthesized chitosan hydrogel microspheres to improve the bioavailability of chitosan for the ocular delivery of acyclovir in rabbits. The results demonstrated that a significant concentration of the drug was released over a prolonged period of time (Genta et al. 1997). Chitosan has certain limitations in ophthalmic drug delivery such as drug-polymer complex formation, insufficient permeability of anionic drugs, and the well-known solubility problem. Due to these limitations, modi-fied chitosan is a better choice for ocular drug delivery. Braga et al. (2008) exploited three chitosan derivatives in the ophthalmic delivery of timolol maleate and flurbi-profen. NSC, N-carboxymethyl, and N-carboxybutyl chi-tosan were impregnated with drugs using a supercritical solvent impregnation (SSI) method. The results revealed that NSC, N-carboxymethyl, and N-carboxybutyl chitosan showed controlled release of timolol maleate and flur-biprofen following the impregnation process. Out of the three chitosan derivatives, N-carboxymethyl chitosan had been reported as a better drug carrier. Overall, N-chitosan derivative-based ophthalmic drug delivery systems were easily controlled according to the desired drug level.
7.2.5 Gene delivery
In the last few decades, many researchers have investigated the possibility of gene therapy for the purpose of benefi-cial vaccine (DNA) production, defective gene compensa-tion, and silencing genes (shRNA and siRNA). However, nucleic acid delivery is restricted by several hurdles. In order to overcome these hurdles, viral vectors have been exploited for gene encapsulation, but viral vectors have severe side effects like oncogenicity, immunogenicity, and cytotoxicity. Substitution with non-viral vectors protects nucleic acids and allows them to reach specific therapeu-tic sites (Riva et al. 2011). Numerous non-viral gene vectors have been reported. Chitosan is the only non-viral natural cationic vector that has the capability to bind with DNA effectively. It is also less toxic than cationic polymers such as polylysine, polyethylene imine, and polyarginine. The potential of chitosan as a non-viral gene vector was first described by Mumper et al. (1995). However, the low trans-fection efficiency of chitosan is a disadvantage due to the strong interaction between chitosan and DNA. Other drawbacks include insufficient endosomal release, poor solubility, and weak buffering capacity (Chae et al. 2005, Mao et al. 2010). To counteract these drawbacks, chitosan derivatives have been studied as non-viral vectors. NSC has been reported in several studies. Toh et al. (2011)
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
24 S. Bashir et al.: Chitosan derivatives
intensively studied chitosan/DNA and NSC/DNA poly-plexes as non-viral gene delivery vectors. Chitosan formed stable DNA polyplexes, which decreased the intracellular release of DNA and provided poor gene transfection effi-ciency. Succinyl groups reduced the tight binding capa-bility of NSC with DNA and improved DNA dissociation in the endolysosomal compartment. This dissociation of DNA improved gene transfection. The degree of substitu-tion greatly affected the gene transfection efficiency of NSC. NSC with 5% and 10% DS had better gene transfec-tion efficiency as compared to NSC with 20% DS. Highly substituted succinyl chitosan had increased water solu-bility but formed stable complexes with DNA, leading to low gene transfection efficiency. Lu et al. (2009) reported NSC-g-PEI as a serum-resistant gene vector for binding with DNA. Transfection of NSC-g-PEI-DNA complexes was investigated using CHO, HeLa, and 293T cells in vitro. The results showed significant transfection efficiency in the presence of serum. The presence of different concentra-tions of serum did not affect the transfection efficiency. Due to limited cell specificity, Lu et al. (2010) synthesized hepatocyte targeting gene vector galactosyl conjugated NSC-g-PEI copolymers. This copolymer was bound with DNA to form NSC-g-PEI-LA/DNA complexes. The transfec-tion efficiency of the complex was investigated in 293T, HeLa, and HepG2 cells. The complex showed excellent transfection activity and HepG2 cell specificity. The trans-fection efficiency of galactosyl NSC-g-PEI was lower than that of NSC-g-PEI as a result of increased substitution with galactose moieties, which caused a decrease in the charge density of amino nitrogen after the introduction of galac-tose. siRNA is a strongly negatively charged and high Mw nucleic acid. It is unstable in serum due to the presence of nucleases. Therefore, transport and transfection of siRNA are hindered, which create problems with the use of siRNA to silence genes in the cytoplasm. Another obstacle is macrophage phagocytosis in the delivery of siRNA in vivo. In order to overcome this disadvantage, the preparation of an effective siRNA carrier is important. Negatively charged siRNA can easily combine with cationic poly-mers. Zhu et al. (2014) coupled low density protein (LDL) with NSC nanoparticles for effective co-delivery of siRNA and doxorubicin. LDL is used because of its non-toxicity, endogenous degradability, and ability to escape from the reticuloendothelial system (RES). The results of reverse transcription-PCR and confocal microscopy revealed a decrease in mdr1 mRNA expression and highly efficient uptake of siRNA. LDL coupled NSC nanoparticles also pro-tected siRNA from macrophage phagocytosis by dynamic observation using live cell station. Toh et al. (2011) inten-sively studied chitosan/DNA and NSC/DNA polyplexes as
nonviral gene delivery vectors. Chitosan formed a stable DNA polyplex that decreased intracellular release of DNA and poor gene transfection efficiency. Succinyl group reduced the tight binding capability of NSC with DNA and improved the DNA dissociation from the endolysosomal compartment. This dissociation of DNA improved gene transfection. Degree of substitution greatly affected the gene transfection efficiency of NSC. NSC with 5% and 10% DS have better gene transfection efficiency as compared to NSC with 20% DS. Highly substituted succinyl chitosan has increased water solubility but forms stable complex with DNA and poor gene transfection efficiency. Yan et al. (2015) prepared Tat-tagged and folate-modified NSC (Tat-NSC-FA) self-assembly copolymer nanoparticles for tumor gene therapy. The copolymer was complexed with DNA at various weight ratios and characterized by dynamic light scattering, TEM, and zeta potential. The copolymer nanoparticles were less toxic as compared to native chi-tosan. Agarose gel electrophoresis revealed the efficient DNA condensation of Tat-NSC-FA at a weight ratio of 1.5:1, which meant that the copolymer had a greater binding capacity for DNA. These results were inconsistent regard-ing the zeta potentials. Tat-tagged folate modification of NSC is shown in Figure 11.
7.3 Tissue engineering
In the early 1990s, Langer and Vacanti put forward the concept of tissue engineering. Tissue engineering generally involves the integration of cell into a tissue-engineered scaffold. Basically, tissue engineering is the fabrication of living body parts. Numerous strategies have been proposed for the replacement of body parts, which should be biologically and functionally active after replacement. These efforts include the incorporation of cells and growth factors using different types of materials and methods (Griffith and Naughton 2002).
7.3.1 Cartilage tissue engineering
Cartilage tissue engineering is considered a promising approach for cartilage restoration. A biomaterial loaded with chondrocytes, genes, growth factors, and bioactive agents can be injected at the site of the lesion to induce cell differentiation and proliferation in situ. Cartilage structures and functions have been naturally mimicked with scaffolds (Hunziker 2002, Martin et al. 2004, Adkis-son et al. 2010). Polymeric hydrogels are a cross-linked three-dimensional network with the ability to encapsulate
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 25
cells and growth factors, thereby promoting adhesion, dif-ferentiation, cell proliferation, extracellular matrix (ECM) secretion, and enhanced tissue regeneration. Therefore, these materials may be excellent candidates as scaffolds in cartilage tissue engineering (Tibbitt and Anseth 2009, Kim et al. 2011). Among the natural polymers, chitosan is a very imperative biomaterial, primarily due to its properties. With its biocompatibility and biodegradability, it does not induce an inflammatory response pathologically and has intrinsic antibacterial activity. Chitosan has gained consid-erable attention due to its structure analogous to glycosa-minoglycan units (Nettles et al. 2002). Chitosan hydrogels are usually synthesized to mimic a collagen network to enhance the growth of chondrocytes in articular cartilage. Chitosan forms ionic complexes with anionic polyelectro-lytes such as sulfate glycosaminoglycan (sGAGs) due to its
polycationic nature and forms a water-insoluble network. Teixeira et al. (Jin et al. 2009) prepared chitosan hydrogels by conjugating chitosan with glycolic acid. SEM revealed that chondrocytes had a spherical morphology, and chon-drocytes maintained their round morphology inside the hydrogel for up to 14 days. The chondrocyte-preserving nature of chitosan hydrogels is a prerequisite for efficient matrix production. The same research group also reported on dextran-tyramine hydrogels. Chondrocytes managed to sustain their round morphology but not as well as in the chitosan hydrogel (Jin et al. 2007).
Chitosan has several limitations, which have been mentioned already. The chemical modification of chi-tosan into NSC improves all these properties, especially cell adhesion, short-term retention in the body, and enhanced mechanical strength, which are necessary for
Figure 11: Tat-tagged folate modification of NSC (Yan et al. 2015).
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
26 S. Bashir et al.: Chitosan derivatives
cartilage tissue engineering. The inadequate strength of the natural polymer, specifically chitosan, limits its appli-cations. Tan et al. prepared NSC/aldehyde hyaluronic acid (AHA) injectable composite hydrogels through a Schiff base mechanism. It was observed that the compressive modulus, which is an important factor for cartilage tissue engineering, improved with an increasing amount of NSC in the hybrid hydrogel. This composite hydrogel had excel-lent cell EE and preserved the chondrocyte phenotype, which was almost same as in natural cartilage. Chondro-cytes encapsulated in hydrogel had a spherical morphol-ogy as in regular cartilage, which supports the potential application of this hydrogel (Tan et al. 2009). From SEM images, it was clearly seen that the preservation of chon-drocytes was better in the NSC/AHA composite hydrogels after 24 h in culture. Chondrocytes retained a more regular and spherical morphology in the NSC hydrogel than in the chitosan hydrogel.
7.3.2 Adipose tissue engineering
The differentiation process of pre-adipocytes into adipo-cytes is called adipogenesis. It is an extensively studied cell differentiation process. The important characteristics of differentiated adipocytes are growth capture, morphologi-cal change, and high expression of lipogenic genes. The cell differentiation process can be significantly enhanced by using growth factors known as adipogenic factors. These growth factors also support angiogenesis, i.e. insulin, dexamethasone (Dex), IBMX, ciglitazone, and insulin-like growth factor (IGF-1). However, the major challenge is the delivery of adipogenic factors. Insulin delivery requires glucose sensing ability. Slow release of insulin in a normal glucose environment is difficult because it may cause gly-cemia in patients (Ravaine et al. 2008, Zhao et al. 2009). In such cases, glucose-sensitive hydrogels possess special features for insulin delivery because hydrogels possess swelling ability, and the swelling ratio varies with the con-centration of glucose. Tan et al. (2010) synthesized glucose-responsive hydrogels of NSC and AHA. The key adipogenic factor, insulin, has a negative charge, and immobilized enzymes known as glucose oxidase (GOD) and catalase (CTL) were incorporated into the hydrogel network by Schiff bond cross-linking. GOD modified glucose to gluconic acid and hydrogen peroxide in the presence of oxygen. CTL converted hydrogen peroxide into water and oxygen. The conversion of glucose to gluconic acid reduced the pH of the environment, which influenced insulin release. It was reported that 10.8% of insulin was released after 8 h, but insulin release decreased gradually due to a loss of enzyme
160AHA-SCSAHA-SCS-Dex
120
80
40
01 3 5
Time (days)
Rel
ativ
e D
NA
con
tent
(%
)
Figure 12: Relative DNA content that represents proliferation of ASCs as a function of culture time in Dex-grafted hydrogel and control sample (NSC/AHA) (Sun et al. 2013). Reprinted with permis-sion from Journal of Applied Polymer Science 129, 682–688 (2013); copyright 2013 John Wiley & Sons, Ltd.
bioactivity. Immobilized GOD/CTL enhanced the release of insulin from glucose-containing hydrogels due to the pro-duction of gluconic acid from glucose.
Dex is an anti-inflammatory drug and adipogenic factor used to proliferate cell differentiation. In this study (Sun et al. 2013), Dex was grafted on NSC/AHA. Human adipose-derived stem cells (ASCs) were encapsulated into hydrogels in order to evaluate the biological performance of hydrogels as a cell carrier in vitro. The results showed increased adhesion of cells to the Dex-grafted hydrogel as compared to the control sample. The DNA content of cells indicated proliferation, and the DNA content sequentially increased in the Dex-grafted hydrogel than in the control hydrogel after 5 days of culture, as shown in Figure 12.
In another study (Tan et al. 2013), a methacrylate NSC/PEG hydrogel was synthesized by photo cross-linking, and insulin was entrapped in this hydrogel. Maximum insulin entrapment was 88%, and release was 41%. The biological performance and cell carrier ability of the hydrogel were observed by seeding ASCs. PEG had effects on insulin entrapment, the release of insulin, and the adhesion of cells, which could be changed by varying the amount of PEG. Maximum cell adhesion was observed on the insulin-loaded SCS/PEG-10 hydrogel, and there was no significant difference between the SCS and NSC/PEG-10 hydrogels. The DNA content also showed remark-able discrepancy among the hydrogels.
7.3.3 Bone tissue engineering
Most often, defective bone heals without any surgical intervention, but often requires bone regeneration. Due to several disadvantages of conventional bone grafting,
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 27
there is an alternative therapeutic concept of bone regen-eration, which is bone tissue engineering. Bone tissue engineering unites many characteristics of stem cells, bioreactors, growth factors, and scaffolds. The scaffold is the key component as it provides a structural template for the development of tissue. The scaffold also has a remarkable effect on cell behavior (Comisar et al. 2007) and should be made of an osteoconductive material with mechanical properties similar to host bone. Secondly, the bioreactor maintains the temperature and pH, con-trols the transport of nutrients, and provides oxygen to the cells. The cells are isolated from tissue and expanded in vitro prior to bone construct preparation. Mesenchymal stem cells (MSCs) can be obtained from various tissues like bone marrow and have the capability to differentiate into adipose tissue and bone (Zuk et al. 2001). Another source of bone tissue is ASCs, which have the potential to differentiate into adipogenic, osteogenic, and endothelial lineages (Caplan 2007). MSCs have the ability to differen-tiate, which depends upon the culture media, conditions, and presence of growth factors. In the recent study, osteo-genic differentiation of MSCs in a simple bone niche was reported in vivo. The differentiation of MSCs is regulated by osteoblasts and osteocytes, which was observed using conditioned media and co-culture (Birmingham et al. 2012). MSCs can also be differentiated in vitro into multi-ple lineages, including endothelial, chondrocytes, osteo-cytes, adipocytes, and skeletal muscle cells (Pittenger et al. 1999, Siddiqui 2013).
New bone formation and vascularization are critical factors in bone tissue engineering. Actually, newly formed bone faces necrosis due to poor vascularization, which ulti-mately results in the failure of repairing the bone defect. Natural and synthetic scaffolds serve as temporary skel-etons at the site of defected bone and stimulate new bone regeneration. These scaffolds can be formulated in the form of hydrogel scaffolds due to their excellent biocom-patibility, biodegradability, structural integrity, ECM-like structure, ability to deliver proteins to tissues, and desir-able biodegradability (Li et al. 2005). Among the materials used for the preparation of hydrogels, NSC is a biologically renewable, non-toxic, and biofunctional natural polymer. It promotes the adhesion, proliferation, and differentiation of cells and evokes a minimum foreign body reaction upon implantation. It is osteoconductive and has the ability to enhance bone formation in vitro and in vivo. Due to these advantages, NSC hydrogels can be used in bone tissue engineering. Osteogenic and endothelial differentiation of bone marrow-derived MSCs was observed by using an RGD-grafted oxidized sodium alginate (OSA)/NSC hydro-gel as the scaffold. Hydrogels have excellent mechanical
properties, which depend on the ratio of NSC to alginate. Hydrogels with 8:2 volume ratio of VNSC to VOSA showed excel-lent maximum strength, indicating that NSC is responsible for compressive strength. This RGD-grafted hydrogel led to enhanced cell adhesion and proliferation. Alkaline phos-phatase activity, as well as alkaline phosphatase and aliza-rin red staining, confirmed the osteogenic differentiation of MSCs. MSCs showed endothelial differentiation in the presence of VEGF and bFGF (Liu et al. 2013).
Many researchers have studied cell proliferation, dif-ferentiation, and the development of therapeutic appli-cations using low intensity pulsed ultrasound (LIPUS). Ultrasound stimulation is sound energy with a frequency below the detection of human hearing. LIPUS is mul-tifunctional and produces significant effects in bone regeneration. LIPUS is used for mechanical stimulation in wound healing and induces osteogenesis (Lim et al. 2013). An RGD-grafted OSA/NSC composite hydrogel was exploited to achieve a high level of bone formation and vascularization. LIPUS was used along with the hydrogel scaffold as a mechanical stimulator. The results suggested that this hybrid hydrogel had excellent biological proper-ties, and an MTT assay showed that the number of cells was increased significantly in the presence of LIPUS and RGD (Wang et al. 2014).
7.4 Drug delivery in combination with tissue engineering
Previous research has focused on the development of drug delivery systems in which drugs/bioactive molecules are encapsulated in the bulk phase and delivered to a specific site of the body, while recent approaches have opened up new possibilities of constructing tissue engineering scaffolds that provide not only control over confiscation but also controlled release of drugs/bioactive factors at a specific site to enhance the regeneration process (Tabata 2005). The bioactive molecules/drugs embedded in the scaffold heal or regenerate tissue, and the scaffold acts as a substrate for tissue organization and differentiation. In the field of reconstructive surgery and regenerative medicine, considerable efforts have gone into improving drug deliv-ery, wound dressing, and tissue engineering technologies. The regeneration of tissue involves monitoring the micro-environment, which closely reproduces that of the host for a preferred cellular response. This microenvironment is provided by three-dimensional scaffolds, which act as an architectural model. The reconstruction of diseased or injured tissues/organs requires rehabilitation, which not only provides structural and mechanical nobility to the
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
28 S. Bashir et al.: Chitosan derivatives
tissue/organ but also sustains the controlled release of growth factors. Thus, the scaffold can augment the regen-eration and healing process while providing diffusivity and structural support to facilitate cellular infiltration.
Extensive research has been performed on the applica-tions of three-dimensional scaffolds to allow for the steady release of therapeutics/growth factors, specifically adipo-genic, osteogenic, and endothelial drugs for adipose and bone tissue regeneration. The shift that has taken place to utilize drug delivery and tissue engineering advances towards repairing structures by exploiting scaffolds that are capable of sustained drug delivery has been reviewed exhaustively (Moioli et al. 2007). However, in the case of NSC, not enough research has been done on three-dimen-sional scaffold drug delivery systems. Tan et al. (Tan et al. 2013) prepared a biodegradable covalently cross-linked methacrylate water-soluble NSC/PEG hybrid hydrogel to deliver insulin as an adipogenic factor. The results revealed that the hydrogel with 10 wt% PEG showed excellent effi-ciency in terms of insulin delivery compared to the control. ASCs were seeded into the insulin-loaded NSC/PEG hydro-gel to evaluate the biological performance and potential of the hydrogel as a cell carrier. These characteristics provide potential opportunities for NSC/PEG hydrogels as a scaf-fold in adipose tissue engineering. Sun et al. (Sun et al. 2013) prepared an injectable biodegradable NSC/AHA Dex-grafted hydrogel in which ASCs were encapsulated to eval-uate the biological performance and capability as a cell carrier. The results revealed that the Dex-grafted hydrogel gave rise to proliferation and cell adhesion.
7.5 Wound dressings
Insoluble polymer hydrogels have large-scale applica-tions in wound dressings because they contain up to 95% water and hence keep the wound environment moist. Additionally, polymer hydrogels have significant poten-tial to absorb wound exudates, transmit oxygen, and regulate the moisture content. However, fluid and bac-terial permeability strongly depends on the materials involved. Polymer hydrogels rehydrate non-viable tissues and promote the debridement of wounds, which assists in autolysis. Hydrogels have been used as state-of-the-art management for necrotic, painful, and sloughy wounds, but are not indicated for wounds with a high amount of exudate. Hydrogels have significant advantages in wound dressings as they provide moisture to the wound, such that removal of dead tissue, epidermis repair, and granula-tion are facilitated. They also provide a cool sensation to relieve pain and minimize the risk of infection. Moreover,
their transparency allows observation of the status of the wound (Murakami et al. 2010). Alginate hydrogels have sig-nificant applications in wound dressings because of their excellent absorbance of exudates, leading to increased epithelialization and reduced bacterial contamination. Alginate hydrogels blended with other polymers such as chitosan, hyaluronic acid, and collagen have been dis-cussed elsewhere (Muzzarelli 2009, Na et al. 2012). Among these polymers, chitosan is highly recommended due to its intrinsic antibacterial properties. However, chitosan has several drawbacks; therefore, chemical modification pro-vides more satisfactory results. Chitosan has no significant antimicrobial activity but can be involved in a synergistic cooperation between hydrogels and textile fibers. NSC has better water retention properties, so it can be exploited in wound dressings (Abdelrahman and Newton 2011).
Straccia et al. (2014) synthesized NSC/sodium algi-nate hydrogel blends containing microcellulose, cross-linked by calcium or zinc ions. Antimicrobial activity was assessed against two common skin pathogens, E. coli and S. aureus. The development of NSC in alginate hydrogels increased the swelling of the hydrogel due to the presence of many carboxyl groups; this decreased syneresis and increased the stability of the hydrogel in saline solution. The water vapor transmission rate (WVTR) was higher in the presence of NSC. Bulky succinyl groups are present in NSC, which reduced molecular entanglements and increased water permeation through the hydrogel film, which is good for the maintenance of a moist environment in the wound bed. A moist environment helps in regenera-tion and epithelialization. Fan et al. (2010) observed the water retention properties of NSC/alginate blend fibers and found that water retention values increased gradually with an increase in the amount of NSC in the fibers. The high water retention property of NSC is advantageous in wound dressing applications. Miyata et al. (1992) reported on NSC/collagen derivatives as wound dressing materials.
8 Current challenges and future perspectives
NSC-based systems have recently attracted the extensive attention of researchers in drug delivery, wound dressing, and tissue engineering platforms. However, these uses are still limited to the stage of research and development experiments and are not extensively used in industrial for-mulations. NSC has different water solubility as compared to its parent chitosan with enhanced solubility in acidic as well as alkaline media. The hydrophobicity of NSC remains
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 29
a considerable drawback regarding its application in the biomedical field. The assessment of the physicochemical properties of a polymer is necessary for the development of effective drug carrier formulations. Another drawback responsible for the limited use of NSC is its poor degra-dation behavior. Therefore, NSC has been used in com-bination with other polymers such as hydrogels, beads, composites, matrices, blends, nanoparticles, microparti-cles, and copolymers in order to improve its degradation behavior and physicochemical properties. The applica-tions of NSC-based systems in drug delivery, tissue engi-neering, and wound healing are at the trial stage. Various drugs and growth factors have been encapsulated in NSC formulations, and the effects of the formulations on encap-sulation were studied. It has been reported that NSC-based systems have enhanced bioavailability and the ability to stimulate endothelial and osteogenic differentiation. NSC in combination with other polymers such as hyaluronic acid and alginate is being studied in tissue engineering due to its compatible mechanical properties, but its prop-erties are not sufficiently compatible with host tissue, which restrict its use in tissue engineering. The combina-tion of NSC with AHA improves the mechanical properties of NSC formulations, which is vital for this application. NSC-based scaffolds with an ECM-like structure have been extremely useful in formulations for repairing and regenerating damaged tissues/organs in tissue engineer-ing. Despite all these properties, formulations of NSC are still not commercialized clinically for applications such as tissue engineering and drug delivery. However, its formula-tions are commercialized and patented in wound-healing applications. The main aim is to improve its degradation, mechanical strength, and stability in a biological microen-vironment and to enhance its antibacterial properties. The interaction of NSC formulations with the biological envi-ronment, enhanced drug loading efficiency, transport, as well as controlled and targeted release still require special attention. After successful research on these issues, com-mercialization will be a major concern in clinical tissue engineering and drug delivery using NSC-based formula-tions in future research.
9 SummaryChitosan is a semi-synthetic amino polysaccharide with a unique structure. It is a copolymer of β-(1-4)-linked 2-acetamido-2-deoxy-β D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose. It is obtained by the ther-mochemical deacetylation of chitin in the presence of a
base. Numerous limitations such as colloidal stability, degradability, and poor aqueous solubility prevent its utilization as a biomaterial. Therefore, modification of chitosan seems to be promising to enhance its exploita-tion in biomedical applications. Different derivatives are obtained by the modification of chitosan. NSC is a novel acyl chitosan derivative that involves a simple preparation procedure, free from toxic reagents, a low production cost, excellent water solubility, mucoadhesion, bioavailability, and biodegradability. Exploration of NSC-based formu-lations in the biomedical field is still at an early stage as research work only started in the past 5 years. Now, there are convincing studies supporting the use of NSC for applications in the biomedical field. NSC improves solu-bility to poorly soluble drugs and can also be exploited to improve the oral and parenteral bioavailability of drugs. Numerous therapeutic agents such as proteins, as well as anti-inflammatory, anticancer, antihypertensive, antian-ginal, and antibiotic drugs, have been incorporated in various NSC formulations to achieve controlled and site-specific drug delivery. However, NSC-based formulations are the result of research and development laboratories within an academic framework. Results from these labo-ratories can enhance the significance of NSC-based for-mulations that might be applicable in the industry. The NSC formulations described in this review may be helpful in the controlled release and targeted delivery of various drugs. NSC has intrinsic tissue engineering potential as a biomaterial with excellent mucoadhesive ability, a well-defined pore size, and regulated biodegradation for growth factor delivery and as a scaffold in cartilage and adipose tissue engineering. These formulations have been used successfully in endothelial and osteogenic differen-tiation in bone tissue engineering. NSC-based hydrogels possess excellent water permeation properties, minimal syneresis, and good stability in saline solution. NSC-based hydrogels have considerable potential in wound dress-ing applications. In conclusion, NSC-based formulations with derived and intact properties make them suitable for biomedical applications. It can be summarized that NSC is undeniably a versatile biocompatible, mucoadhesive, non-toxic, and biodegradable chitosan derivative with significant potential in biomedical applications to drive comprehensive explorations.
Acknowledgments: This work was supported by the High Impact Research Grant (H-21001-F000046) from the Ministry of Education, Malaysia, and University of Malaya Research Grants (RP025A-14AFR and RP017C-14AFR).Conflict of interest statement: The authors have no conflict of interest statement.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
30 S. Bashir et al.: Chitosan derivatives
ReferencesAbdelrahman T, Newton H. Wound dressings: principles and
practice. Surgery 2011; 29: 491–495.Adkisson HD, Martin JA, Amendola RL, Milliman C, Mauch KA,
Katwal AB, Seyedin M, Amendola A, Streeter PR, Buckwalter JA. The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage. Am J Sports Med 2010; 38: 1324–1333.
Aiping Z, Tian C, Lanhua Y, Hao W, Ping L. Synthesis and characterization of N-succinyl-chitosan and its self-assembly of nanospheres. Carbohydr Polym 2006; 66: 274–279.
Alves N, Mano J. Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications. Int J Biol Macromol 2008; 43: 401–414.
Aoki N, Nishikawa M, Hattori K. Synthesis of chitosan derivatives bearing cyclodextrin and adsorption of p-nonylphenol and bisphenol A. Carbohydr Polym 2003; 52: 219–223.
Artursson P, Lindmark T, Davis SS, Illum L. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res 1994; 11: 1358–1361.
Aziz MA, Cabral JD, Brooks HJ, Moratti SC, Hanton LR. Antimicrobial properties of a chitosan dextran-based hydrogel for surgical use. Antimicrob Agents Chemother 2012; 56: 280–287.
Bhattarai SR, Remant KC, Aryal S, Khil MS, Kim HY. N-Acylated chitosan stabilized iron oxide nanoparticles as a novel nano-matrix and ceramic modification. Carbohydr Polym 2007; 69: 467–477.
Birmingham E, Niebur G, McHugh P. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cell Mater 2012; 23: 13–27.
Braga ME, Pato MTV, Silva HS, Ferreira EI, Gil MH, Duarte CM, de Sousa HC. Supercritical solvent impregnation of ophthalmic drugs on chitosan derivatives. J Supercrit Fluids 2008, 44: 245–257.
Cao Y, Zhang C, Shen W, Cheng Z, Yu LL, Ping Q. Poly (N-isopropylacrylamide)–chitosan as thermosensitive in situ gel-forming system for ocular drug delivery. J Controlled Release 2007; 120: 186–194.
Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 2007; 213: 341–347.
Casettari L, Vllasaliu D, Castagnino E, Stolnik S, Howdle S, Illum L. PEGylated chitosan derivatives: synthesis, characterizations and pharmaceutical applications. Prog Polym Sci 2012; 37: 659–685.
Chae SY, Son S, Lee M, Jang M-K, Nah J-W. Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier. J Controlled Release 2005; 109: 330–344.
Champagne LM. The synthesis of water soluble N-acyl chitosan derivatives for characterization as antibacterial agents, Louisiana, USA: Xavier University of Louisiana, 2008.
Chen J, Jo S, Park K. Polysaccharide hydrogels for protein drug delivery. Carbohydr Polym 1995; 28: 69–76.
Choi CY, Kim SB, Pak PK, Yoo DI, Chung YS. Effect of N-acylation on structure and properties of chitosan fibers. Carbohydr Polym 2007; 68: 122–127.
Chourasia M, Jain S. Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Pharm Sci 2003; 6: 33–66.
Clark AF, Yorio T. Ophthalmic drug discovery. Nat Rev Drug Discovery 2003; 2: 448–459.
Comisar WA, Kazmers NH, Mooney DJ, Linderman JJ. Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach. Biomaterials 2007; 28: 4409–4417.
Costantino H, Sileno A, Johnson P. Pharmacokinetic attributes of intranasal delivery: case studies and new opportunities. Drug Deliv 2005; 3: 8–11.
Costantino HR, Illum L, Brandt G, Johnson PH, Quay SC. Intranasal delivery: physicochemical and therapeutic aspects. Int J Pharm 2007; 337: 1–24.
Dai YN, Li P, Zhang JP, Wang AQ, Wei Q. A novel pH sensitive N-succinyl chitosan/alginate hydrogel bead for nifedipine delivery. Biopharm Drug Dispos 2008; 29: 173–184.
Desai MP, Labhasetwar V, Amidon GL, Levy RJ. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharmaceut Res 1996; 13: 1838–1845.
Domard A, Rinaudo M, Terrassin C. New method for the quaternization of chitosan. Int J Biol Macromol 1986; 8: 105–107.
Eweis M, Elkholy S, Elsabee M. Antifungal efficacy of chitosan and its thiourea derivatives upon the growth of some sugar-beet pathogens. Int J Biol Macromol 2006; 38: 1–8.
Fan L, Yu L, Xu Y, Yi C, Cai J, Li M, Huang J. The novel alginate/ N-succinyl-chitosan antibacterial blend fibers. J Appl Polym Sci 2010; 116: 2151–2156.
Fan M, Hu Q, Shen K. Preparation and structure of chitosan soluble in wide pH range. Carbohyd Polym 2009; 78: 66–71.
Genta I, Conti B, Perugini P, Pavanetto F, Spadaro A, Puglisi G. Bioadhesive microspheres for ophthalmic administration of acyclovir. J Pharm Pharmacol 1997; 49: 737–742.
Graisuwan W, Wiarachai O, Ananthanawat C, Puthong S, Soogarun S, Kiatkamjornwong S, Hoven VP. Multilayer film assembled from charged derivatives of chitosan: Physical characteristics and biological responses. J Colloid Interf Sci 2012; 376: 177–188.
Griffith LG, Naughton G. Tissue engineering–current challenges and expanding opportunities. Science 2002; 295: 1009–1014.
Guo Z, Xing R, Liu S, Zhong Z, Li P. Synthesis and hydroxyl radicals scavenging activity of quaternized carboxymethyl chitosan. Carbohyd Polym 2008; 73: 173–177.
Gupta P, Vermani K, Garg S. Hydrogels: from controlled release to pH-responsive drug delivery. Drug Discov Today 2002; 7: 569–579.
Ha C-S, Gardella JA. Surface chemistry of biodegradable polymers for drug delivery systems. Chem Rev 2005; 105: 4205–4232.
Hasegawa M, Yagi K, Iwakawa S, Hirai M. Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells. Cancer Science 2001; 92: 459–466.
Hawary DL, Motaleb MA, Farag H, Guirguis OW, Elsabee MZ. Water-soluble derivatives of chitosan as a target delivery system of 99m Tc to some organs in vivo for nuclear imaging and biodistribution. J Radioanal Nucl Ch 2011; 290: 557–567.
He C, Kim SW, Lee DS. In situ gelling stimuli-sensitive block copolymer hydrogels for drug delivery. J Control Release 2008;127: 189–207.
Hirano S, Moriyasu T. Some novel N-(carboxyacyl) chitosan filaments. Carbohyd Polym 2004; 55: 245–248.
Hirano S, Yamaguchi Y, Kamiya M. Novel N-saturated-fatty-acyl derivatives of chitosan soluble in water and in aqueous acid and alkaline solutions. Carbohyd Polym 2002; 48: 203–207.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 31
Hou Z, Han J, Zhan C, Zhou C, Hu Q, Zhang Q. Synthesis and evaluation of N-succinyl-chitosan nanoparticles toward local hydroxycamptothecin delivery. Carbohyd Polym 2010; 81: 765–768.
Hunziker E. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cartilage 2002; 10: 432–463.
Illum L. Nasal drug delivery – possibilities, problems and solutions. J Control Release 2003; 87: 187–198.
Inta O, Yoksan R, Limtrakul J. Hydrophobically modified chitosan: A bio-based material for antimicrobial active film. Materials Science and Engineering: C 2014; 42: 569–577.
Islam A, Yasin T. Controlled delivery of drug from pH sensitive chitosan/poly (vinyl alcohol) blend. Carbohyd Polym 2012; 88: 1055–1060.
Ito T, Yeo Y, Highley CB, Bellas E, Benitez CA, Kohane DS. The prevention of peritoneal adhesions by in situ cross-linking hydrogels of hyaluronic acid and cellulose derivatives. Biomaterials 2007; 28: 975–983.
Jain N, Rajoriya V, Jain PK, Jain AK. Lactosaminated-N-succinyl chitosan nanoparticles for hepatocyte-targeted delivery of acyclovir. J Nanopart Res 2014; 16: 1–14.
Jayakumar R, Nwe N, Tokura S, Tamura H. Sulfated chitin and chitosan as novel biomaterials. Int J Biol Macromol 2007; 40: 175–181.
Jayakumar R, Prabaharan M, Nair S, Tokura S, Tamura H, Selvamurugan N. Novel carboxymethyl derivatives of chitin and chitosan materials and their biomedical applications. Prog Mater Sci 2010; 55: 675–709.
Jin R, Hiemstra C, Zhong Z, Feijen J. Enzyme-mediated fast in situ formation of hydrogels from dextran–tyramine conjugates. Biomaterials 2007; 28: 2791–2800.
Jin R, Moreira Teixeira L, Dijkstra PJ, Karperien M, Van Blitterswijk C, Zhong Z, Feijen J. Injectable chitosan-based hydrogels for cartilage tissue engineering. Biomaterials 2009; 30: 2544–2551.
Johnson PH, Quay SC. Advances in nasal drug delivery through tight junction technology. Expert Opin Drug Del 2005; 2: 281–298.
Kajihara M, Sugie T, Hojo T, Maeda H, Sano A, Fujioka K, Sugawara S, Urabe Y. Development of a new drug delivery system for protein drugs using silicone (II). J Control Release 2001; 73: 279–291.
Kajjari PB, Manjeshwar LS, Aminabhavi TM. Novel blend microspheres of poly (vinyl alcohol) and succinyl chitosan for controlled release of nifedipine. Polymer bulletin 2013; 70: 3387–3406.
Karadeniz F, Karagozlu MZ, Pyun S-Y, Kim S-K. Sulfation of chitosan oligomers enhances their anti-adipogenic effect in 3T3-L1 adipocytes. Carbohyd Polym 2011; 86: 666–671.
Kato Y, Onishi H, Machida Y. Biological fate of highly-succinylated N-succinyl-chitosan and antitumor characteristics of its water-soluble conjugate with mitomycin C at iv and ip administration into tumor-bearing mice. Biol Pharm Bull 2000a; 23: 1497–1503.
Kato Y, Onishi H, Machida Y. Evaluation of N-succinyl-chitosan as a systemic long-circulating polymer. Biomaterials 2000b; 21: 1579–1585.
Kato Y, Onishi H, Machida Y. Biological characteristics of lactosaminated N-succinyl-chitosan as a liver-specific drug carrier in mice. J Control Release 2001a; 70: 295–307.
Kato Y, Onishi H, Machida Y. Lactosaminated and intact N-succinyl-chitosans as drug carriers in liver metastasis. Int J Pharm 2001b; 226: 93–106.
Khor E. Chitin: a biomaterial in waiting. Curr Opin Solid St M 2002; 6: 313–317.
Kim S, Kim J-H, Jeon O, Kwon IC, Park K. Engineered polymers for advanced drug delivery. Eur J Pharm Biopharm 2009; 71: 420–430.
Kim IL, Mauck RL, Burdick JA. Hydrogel design for cartilage tissue engineering: a case study with hyaluronic acid. Biomaterials 2011; 32: 8771–8782.
Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol 2010; 144: 51–63.
Kontos S, Hubbell JA. Drug development: longer-lived proteins. Chem Soc Rev 2012; 41: 2686–2695.
Kotzé AF, Thanou MM, Lueben HL, A. De Boer, Verhoef J, Junginger HE. Enhancement of paracellular drug transport with highly quaternized N-trimethyl chitosan chloride in neutral environments: In vitro evaluation in intestinal epithelial cells (Caco-2). J Pharm Sci 1999; 88: 253–257.
Kumar MNR. A review of chitin and chitosan applications. React Funct Polym 2000; 46: 1–27.
Kurita K. Chemical modifications of chitin and chitosan. Springer: Chitin in Nature and Technology, 1986: 287–293.
Kurita K. Controlled functionalization of the polysaccharide chitin. Prog Polym Sci 2001; 26: 1921–1971.
Kurita K. Chitin and chitosan: functional biopolymers from marine crustaceans. Mar Biotechnol 2006; 8: 203–226.
Kyzas GZ, Siafaka PI, Pavlidou EG, Chrissafis KJ, Bikiaris DN. Synthesis and adsorption application of succinyl-grafted chitosan for the simultaneous removal of zinc and cationic dye from binary hazardous mixtures. Chem Eng J 2015; 259: 438–448.
Le Bourlais C, Acar L, Zia H, Sado PA, Needham T, Leverge R. Ophthalmic drug delivery systems – recent advances. Prog Retin Eye Res 1998; 17: 33–58.
Li Z, Ramay HR, Hauch KD, Xiao D, Zhang M. Chitosan–alginate hybrid scaffolds for bone tissue engineering. Biomaterials 2005; 26: 3919–3928.
Li Y, Rodrigues J, Tomas H. Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications. Chem Soc Rev 2012a; 41: 2193–2221.
Li J, Cai J, Zhong L, Du Y. Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan. Carbohyd Polym 2012b; 87: 2697–2705.
Lim K, Kim J, Seonwoo H, Park SH, Choung P-H, Chung JH. In vitro effects of low-intensity pulsed ultrasound stimulation on the osteogenic differentiation of human alveolar bone-derived mesenchymal stem cells for tooth tissue engineering. Bio Med Res Int 2013; 2013: 1–15.
Liu X, Peng W, Wang Y, Zhu M, Sun T, Peng Q, Zeng Y, Feng B, Lu X, Weng J. Synthesis of an RGD-grafted oxidized sodium alginate–N-succinyl chitosan hydrogel and an in vitro study of endothelial and osteogenic differentiation. J Mater Chem B 2013; 1: 4484–4492.
Lu B, Sun Y-X, Li Y-Q, Zhang X-Z, Zhuo R-X. N-Succinyl-chitosan grafted with low molecular weight polyethylenimine as a serum-resistant gene vector. Mol BioSystems 2009; 5: 629–637.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
32 S. Bashir et al.: Chitosan derivatives
Lu B, Wu D-Q, Zheng H, Quan C-Y, Zhang X-Z, Zhuo R-X Galactosyl conjugated N-succinyl-chitosan-graft-polyethylenimine for targeting gene transfer. Mol Biosystems 2010; 6: 2529–2538.
Lü S, Liu M, Ni B. An injectable oxidized carboxymethylcellulose/ N-succinyl-chitosan hydrogel system for protein delivery. Chem Eng J 2010; 160: 779–787.
Luo H, Li J, Chen X. Antitumor effect of N-succinyl-chitosan nanoparticles on K562 cells. Biomed Pharmacother 2010; 64: 521–526.
Luo H, Su H, Wang X, Wang L, Li J. N-Succinyl-chitosan nanoparticles induced mitochondria-dependent apoptosis in K562. Mol Cell Probe 2012; 26: 164–169.
Mao S, Shuai X, Unger F, Wittmar M, Xie X, Kissel T. Synthesis, characterization and cytotoxicity of poly (ethylene glycol)-graft-trimethyl chitosan block copolymers. Biomaterials 2005; 26: 6343–6356.
Mao S, Sun W, Kissel T. Chitosan-based formulations for delivery of DNA and siRNA. Adv Drug Deliver Rev 2010; 62: 12–27.
Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends Biotechnol 2004; 22: 80–86.
Mastiholimath V, Dandagi P, Jain SS, Gadad A, Kulkarni A. Time and pH dependent colon specific, pulsatile delivery of theophylline for nocturnal asthma. Int J Pharm 2007; 328: 49–56.
Matevosyan G, Yukha YS, Zavlin P. Phosphorylation of chitosan. Russ J Gen Chem 2003; 73: 1725–1728.
Mima S, Miya M, Iwamoto R, Yoshikawa S. Highly deacetylated chitosan and its properties. J Appl Polym Sci 1983; 28: 1909–1917.
Miyata T, Kodaira K, Higashijima H, Kimura T, Noishiki Y. Wound dressing, succinylchitosan, atelocollagen, Google Patents. 1992.
Moioli EK, Clark PA, Xin X, Lal S, Mao JJ. Matrices and scaffolds for drug delivery in dental, oral and craniofacial tissue engineering. Adv Drug Deliver Rev 2007; 59: 308–324.
Mourya V, Inamdar NN. Chitosan-modifications and applications: opportunities galore. React Funct Polym 2008; 68: 1013–1051.
Mukhopadhyay P, Sarkar K, Chakraborty M, Bhattacharya S, Mishra R, Kundu P. Oral insulin delivery by self-assembled chitosan nanoparticles: In vitro and in vivo studies in diabetic animal model. Mater Sci Eng C 2013; 33: 376–382.
Mukhopadhyay P, Sarkar K, Bhattacharya S, Bhattacharyya A, Mishra R, Kundu P. pH sensitive N-succinyl chitosan grafted polyacrylamide hydrogel for oral insulin delivery. Carbohyd Polym 2014; 112: 627–637.
Mumper R, Wang J, Claspell J, Rolland A. Novel polymeric condensing carriers for gene delivery. In: Proceedings of the international symposium on controlled release bioactive materials. 1995; 22: 178–179.
Mura C, Manconi M, Valenti D, Manca ML, Díez-Sales O, Loy G, Fadda AM. In vitro study of N-succinyl chitosan for targeted delivery of 5-aminosalicylic acid to colon. Carbohyd Polym 2011a; 85: 578–583.
Mura C, Nácher A, Merino V, Merino-Sanjuan M, Carda C, Ruiz A, Manconi M, Loy G, Fadda A, Diez-Sales O. N-Succinyl-chitosan systems for 5-aminosalicylic acid colon delivery: In vivo study with TNBS-induced colitis model in rats. Int J Pharm 2011b; 416: 145–154.
Mura C, Nácher A, Merino V, Merino-Sanjuan M, Manconi M, Loy G, Fadda A, Díez-Sales O. Design, characterization and in vitro evaluation of 5-aminosalicylic acid loaded N-succinyl-chitosan microparticles for colon specific delivery. Colloid Surface B 2012; 94: 199–205.
Murakami K, Aoki H, Nakamura S, Nakamura S-i, Takikawa M, Hanzawa M, Kishimoto S, Hattori H, Tanaka Y, Kiyosawa T. Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings. Biomaterials 2010; 31: 83–90.
Muzzarelli RA. Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohyd Polym 2009; 76: 167–182.
Muzzarelli RA, Tanfani F, Emanuelli M, Mariotti S. N-(carboxymethylidene) chitosans and N-(carboxymethyl) chitosans: novel chelating polyampholytes obtained from chitosan glyoxylate. Carbohyd Res 1982; 107: 199–214.
Na L, Mao S, Wang J, Sun W. Comparison of different absorption enhancers on the intranasal absorption of isosorbide dinitrate in rats. Int J Pharm 2010; 397: 59–66.
Na SY, Oh SH, Song KS, Lee JH. Hyaluronic acid/mildly crosslinked alginate hydrogel as an injectable tissue adhesion barrier. J Mat Sci Mat M 2012; 23: 2303–2313.
Na L, Wang J, Wang L, Mao S. A novel permeation enhancer: N-succinyl chitosan on the intranasal absorption of isosorbide dinitrate in rats. Eur J Pharm Sci 2013; 48: 301–306.
Nagai T. In vivo drug release and antitumor characteristics of water-soluble conjugates of mitomycin C with glycol-chitosan and N-succinyl-chitosan. Biol Pharm Bull 1996; 19: 1170–1177.
Naruphontjirakul P, Viravaidya-Pasuwat K. Development of doxorubicin – Core shell o-succinyl chitosan graft pluronic® 127 copolymer nanoparticles to treat human cancer. Int. J. Biosci. Biochem. Bioinforma 2011; 1: 131–136.
Nettles DL, Elder SH, Gilbert JA. Potential use of chitosan as a cell scaffold material for cartilage tissue engineering. Tissue Eng 2002; 8: 1009–1016.
Nieto J, Peniche-Covas C, Del Bosque J. Preparation and characterization of a chitosan-Fe (III) complex. Carbohyd Polym 1992; 18: 221–224.
Onishi H, Takahashi H, Yoshiyasu M, Machida Y. Preparation and in vitro properties of N-succinylchitosan-or carboxymethylchitin-mitomycin C conjugate microparticles with specified size. Drug Dev Ind Pharm 2001; 27: 659–667.
Overstreet DJ, Dutta D, Stabenfeldt SE, Vernon BL. Injectable hydrogels. J Polym Sci Pol Phys 2012; 50: 881–903.
Parisi-Amon A, Mulyasasmita W, Chung C, Heilshorn SC. Protein-engineered injectable hydrogel to improve retention of transplanted adipose-derived stem cells. Adv Healthc Mater 2013; 2: 428–432.
Patil J, Kamalapur M, Marapur S, Kadam D. Ionotropic gelation and polyelectrolyte complexation: the novel techniques to design hydrogel particulate sustained, modulated drug delivery system: a review. Dig J Nanomater Bios 2010; 5: 241–248.
Philip AK, Dabas S, Pathak K. Optimized prodrug approach: A means for achieving enhanced anti-inflammatory potential in experimentally induced colitis. J Drug Target 2009; 17: 235–241.
Pijls RT, Sonderkamp T, Daube GW, Krebber R, Hanssen HH, Nuijts RM, Koole LH. Studies on a new device for drug delivery to the eye. Eur J Pharm Biopharm 2005; 59: 283–288.
Pillai O, Panchagnula R. Polymers in drug delivery. Curr Opin Chem Biol 2001; 5: 447–451.
Pillai C, Paul W, Sharma CP. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog Polym Sci 2009; 34: 641–678.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 33
potential of adult human mesenchymal stem cells. Science 1999; 284: 143–147.
Polnok, A, Verhoef J, Borchard G, Sarisuta N, Junginger HE. In vitro evaluation of intestinal absorption of desmopressin using drug-delivery systems based on superporous hydrogels. Int J Pharm 2004; 269: 303–310.
Prashanth KH, Tharanathan R. Chitin/chitosan: modifications and their unlimited application potential – an overview. Trends Food Sci Tech 2007; 18: 117–131.
Ramesh HP, Tharanathan RN. Carbohydrates-the renewable raw materials of high biotechnological value. Cr Rev Biotechn 2003; 23: 149–173.
Ravaine V, Ancla C, Catargi B. Chemically controlled closed-loop insulin delivery. J Control Release 2008; 132: 2–11.
Reddy SM, Sinha VR, Reddy DS. Novel oral colon-specific drug delivery systems for pharmacotherapy of peptide and nonpeptide drugs. Drugs Today 1999; 35: 537–580.
Rekha M, Sharma CP. pH sensitive succinyl chitosan microparticles: a preliminary investigation towards oral insulin delivery. Trends Biomater Artif Organs 2008; 21: 107–115.
Rekha M, Sharma CP. Synthesis and evaluation of lauryl succinyl chitosan particles towards oral insulin delivery and absorption. J Control Release 2009; 135: 144–151.
Richardson S, Gorton L. Characterisation of the substituent distribution in starch and cellulose derivatives. Anal Chim Acta 2003; 497: 27–65.
Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006; 31: 603–632.
Rinaudo M. Main properties and current applications of some polysaccharides as biomaterials. Polym Int 2008; 57: 397–430.
Riva R, Ragelle H, des Rieux A, Duhem N, Jérôme C, Préat V. Chitosan and chitosan derivatives in drug delivery and tissue engineering. Chitosan for Biomaterials II, Springer: 2011; 19–44.
Rogalsky AD, Kwon H, Lee-Sullivan P. Compressive stress–strain response of covalently crosslinked oxidized-alginate/ N-succinyl-chitosan hydrogels. J Biomed Mater Res A 2011; 99: 367–375.
Sashiwa H, Yamamori N, Ichinose Y, Sunamoto J, Aiba Si. Chemical Modification of Chitosan, 17. Macromol Biosci 2003; 3: 231–233.
Siddiqui I. A. In vitro differentiation of mesenchymal stem cells into endothelial cells. Der Pharmacia Sinica 2013; 4: 106–112.
Sinswat P, Tengamnuay P. Enhancing effect of chitosan on nasal absorption of salmon calcitonin in rats: comparison with hydroxypropyl-and dimethyl-β-cyclodextrins. Int J Pharm 2003; 257: 15–22.
Song Y, Onishi H, Nagai T. Synthesis and drug-release characteristics of the conjugates of mitomycin C with N-succinyl-chitosan and carboxymethyl-chitin. Chem Pharm Bull 1992; 40: 2822–2825.
Song Y, Onishi H, Nagai T. Pharmacokinetic characteristics and antitumor activity of the N-succinyl-chitosan-mitomycin C conjugate and the carboxymethyl-chitin-mitomycin C conjugate. Biol Pharm Bull 1993; 16: 48–54.
Straccia MC, Romano I, Oliva A, Santagata G, Laurienzo P. Crosslinker effects on functional properties of alginate/ N-succinylchitosan based hydrogels. Carbohyd Polym 2014; 108: 321–330.
Sui W, Wang Y, Dong S, Chen Y. Preparation and properties of an amphiphilic derivative of succinyl-chitosan. Colloids Surf A Physicochem Eng Asp 2008; 316: 171–175.
Sun S, Wang A. Adsorption properties of N-succinyl-chitosan and cross-linked N-succinyl-chitosan resin with Pb (II) as template ions. Sep Purif Technol 2006; 51: 409–415.
Sun T, Xie W, Xu P. Superoxide anion scavenging activity of graft chitosan derivatives. Carbohyd Polym 2004; 58: 379–382.
Sun L, Du Y, Fan L, Chen X, Yang J. Preparation, characterization and antimicrobial activity of quaternized carboxymethyl chitosan and application as pulp-cap. Polymer 2006; 47: 1796–1804.
Sun S, Wang Q, Wang A. Adsorption properties of Cu (II) ions onto N-succinyl-chitosan and crosslinked N-succinyl-chitosan template resin. Biochem Eng J 2007; 36: 131–138.
Sun T, Zhu Y, Xie J, Yin X. Antioxidant activity of N-acyl chitosan oligosaccharide with same substituting degree. Bioorg Med Chem Lett 2011; 21: 798–800.
Sun J, Xiao C, Tan H, Hu X. Covalently crosslinked hyaluronic acid-chitosan hydrogel containing dexamethasone as an injectable scaffold for soft tissue engineering. J Appl Polym Sci 2013; 129: 682–688.
Tabata Y. Significance of release technology in tissue engineering. Drug Discov Today 2005; 10: 1639–1646.
Tan H, Chu CR, Payne KA, Marra KG. Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials 2009; 30: 2499–2506.
Tan H, Rubin JP, Marra KG. Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration. Organogenesis 2010; 6: 173–180.
Tan H, Luan H, Hu Y, Hu X. Covalently crosslinked chitosan-poly (ethylene glycol) hybrid hydrogels to deliver insulin for adipose-derived stem cells encapsulation. Macromol Res 2013; 21: 392–399.
Thanou M, Verhoef J, Marbach P, Junginger H. Intestinal absorption of octreotide: N-trimethyl chitosan chloride (TMC) ameliorates the permeability and absorption properties of the somatostatin analogue in vitro and in vivo. J Pharm Sci 2000; 89: 951–957.
Tharanathan R. Biodegradable films and composite coatings: past, present and future. Trends Food Sci Tech 2003; 14: 71–78.
Tharanathan RN, Kittur FS. Chitin – the undisputed biomolecule of great potential. Crit Rev Food Sci Nutr 2003; 43: 61–87.
Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng 2009; 103: 655–663.
Tiwari P, Panthari P, Katare DP, Kharkwal H. Natural polymers in drug delivery. Int J Pharm Pharm Sci 2014, 3: 1395–1409.
Toh EK-W, Chen H-Y, Lo Y-L, Huang S-J, Wang L-F. Succinated chitosan as a gene carrier for improved chitosan solubility and gene transfection. Nanomed Nanotechnol 2011; 7: 174–183.
Van der Merwe S, Verhoef J, Verheijden J, Kotze A, Junginger H. Trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs. Eur J Pharm Biopharm 2004; 58: 225–235.
Vanichvattanadecha C, Supaphol P, Nagasawa N, Tamada M, Tokura S, Furuike T, Tamura H, R. Rujiravanit Effect of gamma radiation on dilute aqueous solutions and thin films of N-succinyl chitosan. Polym Degrad Stabil 2010; 95: 234–244.
Vinsova J, Vavrikova E. Chitosan derivatives with antimicrobial, antitumour and antioxidant activities-a review. Curr Pharm Design 2011; 17: 3596–3607.
Wang X, Du Y, Liu H. Preparation, characterization and antimicrobial activity of chitosan–Zn complex. Carbohyd Polym 2004; 56: 21–26.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
34 S. Bashir et al.: Chitosan derivatives
Wang Y, Tu S, Li R, Yang X, Liu L, Zhang Q. Cholesterol succinyl chitosan anchored liposomes: preparation, characterization, physical stability, and drug release behavior. Nanomed-Nanotechnol 2010; 6: 471–477.
Wang Y, Peng W, Liu X, Zhu M, Sun T, Peng Q, Zeng Y, Feng B, Zhi W, Weng J. Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound. Acta biomaterialia 2014; 10: 2518–2528.
Wongpanit P, Sanchavanakit N, Pavasant P, Supaphol P, Tokura S, Rujiravanit R. Preparation and characterization of microwave-treated carboxymethyl chitin and carboxymethyl chitosan films for potential use in wound care application. Macromol Biosci 2005; 5: 1001–1012.
Wu Y, Seo T, Sasaki T, Irie S, Sakurai K. Layered structures of hydrophobically modified chitosan derivatives. Carbohyd Polym 2006; 63: 493–499.
Yamaguchi R, Arai Y, Kaneko T, Itoh T. Utilization of partially N-succinylated derivatives of chitosan and glycolchitosan as supports for the immobilization of enzymes. Biotechnol Bioeng 1982; 24: 1081–1091.
Yan C, Chen D, Gu J, Qin J. Nanoparticles of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization – in-vitro drug release and anti-tumour activity. J Pharm Pharmacol 2006a; 58: 1177–1181.
Yan C, Chen D, Gu J, Hu H, Zhao X, Qiao M. Preparation of N-succinyl-chitosan and their physical-chemical properties as a novel excipient. Yakuga Zasshi 2006b; 126: 789–793.
Yan C, Gu J, Guo Y, Chen D. In vivo biodistribution for tumor targeting of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) nanoparticles. Yakuga Zasshi 2010; 130: 801–804.
Yan C-y, Gu J-w, Hou D-p, Jing H-y, Wang J, Guo Y-z, Katsumi H, Sakane T, Yamamoto A. Synthesis of Tat tagged and folate modified N-succinyl-chitosan self-assembly nanoparticles as a novel gene vector. Int J Biol Macromol 2015; 72: 751–756.
Yen G-C, Chen H-Y. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agr Food Chem 1995; 43: 27–32.
Yeom J, Bhang SH, Kim B-S, Seo MS, Hwang EJ, Cho IH, Park JK, Hahn SK. Effect of cross-linking reagents for hyaluronic acid hydrogel dermal fillers on tissue augmentation and regeneration. Bioconjugate Chem 2010; 21: 240–247.
Ying GQ, Yang H, Yi Y, Xu F. Relationships between the molecular structure and moisture-absorption and moisture-retention abilities of succinyl chitosan. Poly Bull 2007; 59: 509–516.
Zhang C-g, Zhu Q-l, Zhou Y, Liu Y, Chen W-l, Yuan Z-Q, Yang S-d, Zhou X-f, Zhu A-j, Zhang X-n. N-Succinyl-chitosan nanoparticles coupled with low-density lipoprotein for targeted osthole-loaded delivery to low-density lipoprotein receptor-rich tumors. Int J Nanomed 2014; 9: 2919–2932.
Zhao Y, Trewyn BG, Slowing II, Lin VS-Y. Mesoporous silica nanoparticle-based double drug delivery system for glucose-responsive controlled release of insulin and cyclic AMP. J Am Chem Soci 2009; 131: 8398–8400.
Zhong Z, Xing R, Liu S, Wang L, Cai S, Li P. Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro. Carbohyd Res 2008; 343: 566–570.
Zhou JQ, Wang JW. Immobilization of alliinase with a water soluble–insoluble reversible N-succinyl-chitosan for allicin production. Enzyme Microb Tech 2009; 45: 299–304.
Zhu A-p, Yuan L-h, Chen T, Wu H, Zhao F. Interactions between N-succinyl-chitosan and bovine serum albumin. Carbohyd Polym 2007; 69: 363–370.
Zhu A, Yuan L, Dai S. Preparation of well-dispersed super paramagnetic iron oxide nanoparticles in aqueous solution with biocompatible N-succinyl-O-carboxymethylchitosan. J Phys. Chem. C 2008; 112: 5432–5438.
Zhu X-J, Yuan W, Li P, Liu X, He J-Q. Pharmacokinetics of a novel nifedipine and pH-sensitive N-succinyl chitosan/alginate hydrogel bead in rabbits. Drug Dev Ind Pharm 2010; 36: 1463–1468.
Zhu Q-l, Zhou Y, Guan M, Zhou X-f, Liu Y, Chen W-l, Zhang C-g, Yuan Z-q, Liu C, Zhu A-j. Low-density lipoprotein-coupled N-succinyl chitosan nanoparticles co-delivering siRNA and doxorubicin for hepatocyte-targeted therapy. Biomaterials 2014; 35: 5965–5976.
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001; 7: 211–228.
BionotesShahid Bashir Faculty of Science, Department of Chemistry, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia
Shahid Bashir earned his Master’s degree in Analytical Chemistry from Institute of Chemistry, University of the Punjab (PUIC), Pakistan, in 2012. He joined University of Malaya as a PhD Scholar in February 2014. His research interests include biopolymers based stimuli sensitive hydrogels: synthesis, characterization, properties and applications in drug delivery.
Yin Yin TeoFaculty of Science, Department of Chemistry, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia
Yin Yin Teo obtained her PhD degree in Chemistry from University of Malaya in 2013. She is currently a senior lecturer at Department of Chemistry, University of Malaya. Her current research interest includes preparation and characterization of nanocarriers and controlled drug delivery.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
S. Bashir et al.: Chitosan derivatives 35
S. RameshCentre for Ionics, Faculty of Science, Department of Physics, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia,[email protected]
S. Ramesh received his BSc and MTech (Materials Science) from University of Malaya. He received his PhD from University of Malaya, Malaysia in the field of Advanced Materials in year 2004. He cur-rently serves as a Professor in University of Malaya. His research interests center on improving the understanding, design, and performance of polymer electrolytes, mainly through the application of electrochemical devices such as secondary batteries, solar cells, fuel cells, supercapacitors etc. He has obtained many awards and recognitions. More recently in 2014 he was selected as one of the Top Research Scientists Malaysia.
K. RameshCentre for Ionics, Faculty of Science, Department of Physics, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia
K. Ramesh is currently working as a Senior Lecturer in Department of Physics. He has obtained his PhD degree from University of Malaya. His research interests focus on organic coatings, corro-sion protection, antifouling coatings and polymer electrolytes. He has been awarded as the Outstanding Reviewer for Pigment & Resin Technology in the Emerald Literati Network 2015 Awards for Excellence.
Amir Azam KhanFaculty of Engineering, Department of Mechanical and Manufacturing Engineering, Universiti Sarawak (UNIMAS), 94300 Kota Samarahan, Sarawak, Malaysia
Amir Azam Khan is presently working as Professor at the Faculty of Engineering, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Malaysia. His present research interests include renew-able energy Materials, surface engineering of ceramics, polymer and natural composites, synthesis of nanopowders through sol-gel, water repellant polymeric films and ceramic electrolytes for solid oxide fuel cells. He was awarded Best Young Scientist Award in 2001 (Chemistry) by the Third World Academy of Sciences (TWAS) and Palmes Académiques by the French Government for his invaluable services in the education and research. He was also invited by the Max Planck Institute, Stuttgart, Germany, as an invited researcher and speaker in 2001.
Brought to you by | provisional accountAuthenticated | [email protected]
Download Date | 9/17/15 11:02 AM
