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Review
The implications of recent advances in carboxymethyl chitosan basedtargeted drug delivery and tissue engineering applications
OFLaxmi Upadhyaya a,⁎, Jay Singh b,⁎, Vishnu Agarwal c, Ravi Prakash Tewari a
a Department of Applied Mechanics, MotiLal Nehru National Institute of Technology, Allahabad 211004, Indiab Department of Applied Chemistry & Polymer Technology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, Delhi 110042, Indiac Department of Applied Mechanics (Biotechnology), MotiLal Nehru National Institute of Technology, Allahabad 211004, India
Please cite this article as: L. Upadhyaya, et altissue engineering applications, J. Control. Re
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Article history:Received 10 February 2014Accepted 23 April 2014Available online xxxx
Keywords:Carboxymethyl chitosanDrug deliveryTissue engineeringFormulation of drug
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ROver the last decade carboxymethyl chitosan (CMCS) has emerged as a promising biopolymer for the develop-ment of newdrug delivery systems and improved scaffolds alongwith other tissue engineering devices for regen-erative medicine that is currently one of the most rapidly growing fields in the life sciences. CMCS is amphiproticether, derived from chitosan, exhibiting enhanced aqueous solubility, excellent biocompatibility, controllablebiodegradability, osteogenesis ability and numerous other outstanding physicochemical and biological proper-ties. More strikingly, it can load hydrophobic drugs and displays strong bioactivity which highlight its suitabilityand extensive usage for preparing different drug delivery and tissue engineering formulations respectively. Thisreview provides a comprehensive introduction to various types of CMCS based formulations for delivery of ther-apeutic agents and tissue regeneration and further describes their preparation procedures and applications in dif-ferent tissues/organs. Detailed information of CMCS based nano/micro systems for targeted delivery of drugswith emphasis on cancer specific and organ specific drug delivery have been described. Further, we havediscussed various CMCS based tissue engineering biomaterials along with their preparation procedures and ap-plications in different tissues/organs. The article then, gives a brief account of therapy combining drug deliveryand tissue engineering. Finally, identification of major challenges and opportunities for current and ongoing ap-plication of CMCS based systems in the field are summarised.
., The implications of recent advances in carboxymethyl chitosan based targeted drug delivery andlease (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.043
Natural polysaccharides, due to their non-toxicity, biocompatibilityand biodegradability, are widely being studied as biomaterial for drugdelivery and tissue engineering applications. Among them, chitosan,which is known to be biocompatible, biodegradable, non-toxic,mucoadhesive and antimicrobial, has been exhaustively exploited for de-veloping different formulations for controlled delivery of biotherapeuticsand in regenerative medicine. But limited solubility of chitosan in waterand other organic solvents has prevented its full exploitation in drug de-livery and tissue repair and reconstruction [1,2]. In addition to this, thelimited colloidal stability of chitosan based particulate drug delivery sys-tems are known to exhibit immunogenicity [3] and degradability of chi-tosan based formulations in tissue regeneration and drug delivery isuncontrollable [4]. Therefore derivatisation of chitosan seems a promis-ing way to get rid of these limitations of chitosan and widening rangeof drug delivery and tissue engineering applications. In fact, life sciencesand bio-technologies is the realm where chitosan and chitosan deriva-tives have raised greater scientific interest because of their remarkablestructural and functional properties. Among them, carboxymethyl chito-san (CMCS), a water soluble derivative of chitosan, has attracted boom-ing interests in several fields such as in vitro diagnostics [5–7],theranostics [8] bioimaging [9], biosensors [10,11], wound healing [12],gene therapy [13–16] and food technology [17,18] but its greatest impacthas been in the area of drug delivery and tissue engineering. CMCS is po-tentially biologically compatible material that is chemically versatile (–NH2 and –COOH) groups and various molecular weight, (MW). The pos-itive facets of increased water solubility, excellent biocompatibility [19],admirable biodegradability, high moisture retention ability [20], im-proved antioxidant property [21], enhanced antibacterial [22] and anti-fungal [23] activity and non-toxicity as compared to chitosan hasprovided ample opportunities to the drug delivery and tissue engineer-ing scientists to create a plethora of formulations and scaffolds. In addi-tion, it is also known to be more bioactive [24], promotes osteogenesis
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
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[25] and its safety evaluation on compounds [26], in vitro models,blood systems [27] and tumor application [28] has beenwell established.All these favorable physical, chemical and biological properties of CMCSmake it a promising biomedical material for drug delivery and tissue en-gineering applications in several formulations. Recently numerous ex-perimental results have been reported on the potential therapeuticapplications of CMCS in reduction in post surgical adhesion formation[29], antibacterial biomaterial [30] and accelerated wound healing [31].The most exhaustively investigated CMCS based drug delivery formula-tions include hydrogels [32], microspheres [26], beads [33], micelles[34], aggregates [35], nanoparticles [36,37], films [38] and membranes[39]. Similarly, repairing and reconstruction of tissues like bone, cartilage,and nerve by CMCS based tissue engineering devices like scaffolds [40],injectable gels [41], and biocomposites has been reported by various re-searchers in recent years.
2. Scope of the present review
In consideration of the above, the scope of the present review is toidentify the lines of applied research that are now consolidating majoradvances made with the CMCS during the last decade in the field ofdrug delivery and tissue engineering. The novelty of these facts isunderlined by the fact that a previous attempt to review the literatureon CMCS has focused primarily on the general biomedical applicationsof CMCS with only a minor part dealing with drug delivery and tissueengineering applications [42]. While another review article authoredby Mourya et al. [43] mainly covers literature regarding the synthesisand characterisation of CMCS along with its pharmaceutical applica-tions. CMCS being inherently bestowed with astonishing physical,chemical and biological features, emerging trends show that it is highlysuitable for the delivery of numerous bioactive and therapeutic com-pounds and for the repair and reconstruction of damaged and/or dis-eases tissues. Excellent biocompatibility, improved biodegradability,enhanced antimicrobial activity, better chelating ability, moisture
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absorption and retention capacity, increased antioxidant property, andhigher bioactivity of CMCS compared to its parent polymer chitosanhas contributed to the exhaustive usage of this biopolymer in drug de-livery and tissue engineering applications both in vitro and in vivo.This review therefore intends to convey to the reader the detailed infor-mation on various CMCS based formulations for drug delivery and tissueengineering and their preparative techniques alongwith applications intissues like bone, cartilage and nerves in last few years. The review givesan overview of CMCS based formulations for targeted delivery focusingcancer specific and organ specific drug delivery. The article also gives abrief discussion about the fact that tissue engineering applications areoften combined with drug delivery strategy. In this regard, it is very im-portant to understand the basic anatomy of a drug delivery vehiclewhich is elucidated in the section discussed later.
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3. Limitations with the use of chitosan in drug delivery
The drug absorption enhancing property of chitosan due to its bio/mucoadhesive nature (i.e. delayed clearance of the formulation fromthe absorption site) and the transient opening of the tight junctions be-tween the epithelial cells of the mucosal membrane are well experi-mentally demonstrated and reported in literature. But the majorlimitation associated with the use of chitosan in efficaciously increasingmucosal drug absorption is its poor solubility at pH higher than its pKa(∼pH 5.5–6.5). Chitosan is soluble in aqueous dilute acid such as hydro-chloric acid and aqueous organic acid such as formic, acetic, oxalic andlactic acids when the degree of deacetylation (DD) of chitin reachesabout 50%.While it remains insoluble at neutral and alkaline pH values.The main mechanism involved in the solubilisation of chitosan is pro-tonation of the –NH2 function on the C-2 position of the D-glucosamine monomeric unit of chitosan, whereby the polysaccharideis converted to a polyelectrolyte in acidic media. The main factorsgoverning the solubility of chitosan are DD, the ionic concentration,the pH, the MW, the nature of the acid used for protonation, the distri-bution of acetyl groups along the chain and the conditions of the meth-od of isolation and drying of the polysaccharide. Highly deacetylatedchitosans (N85%) are soluble only up to a pH of 6.5. The dependenceof the degree of ionisation on the pH and the pKa of the acid havebeen experimentally displayed by examination of the role of proton-ation in the presence of acetic acid and hydrochloric acid on solubilityof chitosan [44]. The decrement of intermolecular interactions and thelower crystallinity causing change in the microstructure of chitosan,which facilitate the permeation of water, LiOH hydrates and urea hy-drates, thereby enhancing chitosan dissolution in an aqueous solutionof LiOH/urea [45].
It is a well known fact that the pH of the small intestines increasesfrom the duodenum to the terminal ileum from pH about 4.5 to 7.4.Therefore, in the lower part of intestine, chitosan will not be soluble.And in order to achieve higher oral bioavailability of drugs throughthe use of chitosan as an absorption enhancing delivery system, dissolu-tion of chitosan at the pH values present in the lumen of small andlarge intestines is must. In consideration of the above facts, several at-tempts have been made and a large body of research exists onchemical modification of chitosan through derivatisation of the amineand/or hydroxyl groups to enhance the water solubility of chitosan.These derivatives mainly include sulfonation [46], quaternarisation[47], carboxymethylation [48], N- andO-hydroxyalkylation [49] anddif-ferent grafted copolymers of chitosan [50–52]. Among these, chitosanderivatives, CMCS has drawn significant attention of the researchersfor drug delivery applications due to its outstandingly enhanced watersolubility, superior bio/mucoadhesive property, ease of preparationand numerous other admirable physicochemical and biological charac-teristics that are elaborated in Fig. 1 which make it highly suitable forfabricating different drug delivery formulations and tissue engineeringbiomaterials.
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4. Biopharmaceutical and toxicological profile of CMCS
As a result of the research undertaken over the last decade there isan acceptable understanding of suitability of CMCS as a versatile func-tional biomaterial for delivering different therapeutic moieties and tis-sue engineering applications. Among the water soluble chitosanderivative, CMCS is an amphiprotic ether derivative containing carboxyland amine groups in the molecule. Chemically, chitosan is poly-1,4-linked β-D-glucosamine, a cationic polysaccharide prepared by alkalineN-deacetylation of chitin. The inherent properties of chitosan itself likebiocompatibility, non-toxicity, biodegradability and antimicrobial char-acteristic makes it suitable for various drug delivery [53,54] and tissueengineering applications [55,56]. But the limited solubility of chitosanin common solvents including water hinders its full potential exploita-tion in biomedical and pharmaceutical industry. Therefore introducingcarboxymethyl group into the chitosan polymer chain endows it withsome outstanding physical, chemical and biological properties suitablefor delivery of therapeutic compounds and tissue repair and reconstruc-tion purpose. The water solubility property of CMCS at various pH anddifferent preparation conditions has been experimentally demonstrat-ed. The study showed that degree of carboxymethylation has critical ef-fect on aqueous solubility of CMCS. The study demonstrated that thewater insolubility of CMCS at different pHs varied with the degree ofcarboxymethylation. The experiment also showed that the increase inthe reaction temperature increased the fraction of carboxymethylationand thereby increased the insolubility at lower pHs. Also the decreasein the fraction of carboxymehylation increased the insolubility at higherpHs [57]. Similarly, DD and degree of substitution (DS) are known tohave critical effect on the moisture-absorption and moisture-retentionabilities of CMCS. The study revealed that under conditions of high rel-ative humidity, themaximummoisture absorption andmoisture reten-tion were obtained at DD values of about 50%. Also when the DD valuesdeviated from 50% moisture absorption and moisture retention de-creased. While both moisture absorption and moisture retention in-creased with the increase in DS value [58]. Currently, a quantitativestudy of the acid base equilibrium of CMCS has been carried out by agroup of researchers which can be useful for many biomedical applica-tions. The study investigated the effect of metal ion properties on thestability of the complexes. The results showed that the study of com-plexes can be ordered as Mn(N,O-CMCS) b Co(N,O-CMCS),Ni(N,O-CMCS) b Cu(N,O-CMCS) b Zn(N,O-CMCS) [59]. Apart from these, theantibacterial characteristics of CMCS [60], its fungistatic activity [61], an-timicrobial properties of modified forms of CMCS [62–64] and CMCSbased composites [65,66] have been well reported in literature.
CMCS has been known to be highly biocompatible and is also knownto promote the proliferation of the normal skin fibroblast significantlybut inhibited the proliferation of keloid fibroblast [19]. Tao et al. pre-pared CMCS characterisedwith different sulfate content and concentra-tion. MTT method was applied to evaluate effect of CMCS on fibroblastproliferation. The results of the study demonstrated that CMCSwith sul-fate content 26.26% at the concentration of 100 μg/mL shows best po-tential for skin fibroblast proliferation [67]. Currently, it is also shownthat oligo-chitosan, N,O-CMCS and N-CMCS in sheets and pastes arecytocompatible with potential of wound healing when cytotoxicitywas evaluated using primary normal human dermal fibroblast culturesand hypertrophic scars [68]. The biological safety of CMCS in blood sys-tems of rats has been experimentally established [27]. Moreover, CMCSis known to be safe in vivo and slightly inhibited growth of sarcoma 180and enhanced body immunity via elevation of serum IL-2 and TNF-αlevel in treatedmice [28]. Apart from this, the excellent biodegradabilityof CMCS by in vitro and in vivo evaluation has been experimentally dem-onstrated in rats. This study revealed that liver played central role inbiodegradation of CMCS [69]. The better biodegradability of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) crosslinked CMCS films in lysozyme solution (pH 7.4, 37 °C) has been report-ed. Also, thesefilms enhanced the spread of Neuro-2a cells and provided
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Fig. 1. Represent desirable property of CMCS and which makes it highly suitable for fabricating different drug delivery formulations and tissue engineering biomaterials nanosystem.
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a good proliferation substratum for Neuro-2a cells, as compared to chi-tosan films [70]. The biodegradation study on CMCS-g-medium chainlength polyhydoxyalkanoates by Bhatt and coworkers showed that de-pends on several factors such as temperature, pH, water potential, oxy-gen content, stereo regularity and crystallinity of the polymer, and itsmaterial processing [71]. The spontaneous degradation of CMCS in soiland by enzymeswith satisfactory results has been earlier reported in lit-erature [72]. In this view, controlling the degradation of covalentlycross-linked CMCS utilizing bimodal MW distribution as reported byGuangyuan and coworkers is worth mentioning [73]. Recently, a studydemonstrating the significant effect of MW of CMCS on its uptakefrom the lumen of abdomen and blood vessels to peripheral tissues,the distribution of this chemical and urinary excretion after intraperito-neal administration has been carried out [74].
The earliest study establishing the low toxicity of CMCS dates back toearly 1990swhen Tokura et al. [75] studied the biological activities of dif-ferent biodegradable polysaccharides.With regard to the in vivo toxicity,no acute toxicity was detected in blood systems of rats after CMCS wasabsorbed in the abdominal cavity and degraded gradually in the blood[76]. Finally a recent study exploring the in vitro cytotoxicity profile ofchitosan, O-CMCS and N,O-CMCS nanoparticles to breast cancer cells-MCF-7 showed less toxicity (almost 98% viability was found) [77]. Inthis context, the cytocompatibility of some of themodified CMCS formu-lations like CMCS-2, 2′ ethylenedioxy bisethylamine-folate [78], CMCS-Polyamidoamine dendrimer nanoparticles [79] and N-octyl-N,O-CMCS[80] demonstrated currently is worthmentioning. In addition to the tox-icological profile of CMCS in different forms, the ability of CMCS in pre-vention and reduction of postsurgical adhesion formation [81–83] andin vivowoundhealing ability [84] have been experimentally demonstrat-ed. Finally, the inherent excellentmucoadhesive and absorption enhanc-ing property of chitosan are retained in CMCSwhich is favorable for drugdelivery applications. Therefore, this interesting biopharmaceutical andtoxicological profile of CMCS has encouraged its application as drug de-livery and tissue engineering biomaterial. Fig. 2 illustrates different out-standing and astonishing physicochemical and biological properties ofCMCS that contribute for its suitability in numerous drug delivery andtissue engineering applications.
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ED5. Synthesis of various carboxymethyl chitosans (CMCS)
Synthesis of different types of CMCS by different chemical routes andeffects of DD and DS along with relationship between molecular struc-ture and moisture absorption and moisture retention abilities of CMCShas been well described earlier by several researchers [85–89]. The ef-fect of various parameters like acid, pH, ionic concentration on the ag-gregation behavior of the polymer in aqueous systems has also beenreported [90,91]. In fact, recently, Kong and coworkers developed anovel method for simultaneously determining DD, DS, and the distribu-tion factor of –COONa or –COOH in CMCS by potentiometric titration[92]. Fig. 3 shows the preparative methods of different types of CMCS
5.1. Synthesis of O-carboxymethyl chitosans (O-CMCS)
Among the water soluble derivatives of CMCS, O-CMCS is known tobe an amphiprotic ether derivativewhich contains –COOHgroups and –
NH2 groups in the molecule. The reaction medium used for preparationof O-CMCS is strongly alkaline. O-CMCS is prepared by suspending chi-tosan, sodium hydroxide and solvent isopropanol into flask and stirringthe alkaline slurry at room temperature for 1 h. Subsequently,monochloroacetic acid dissolved in isopropanol is added to reactionmixture dropwisewithin 30min. Thewhole reactionmixture is reactedfor 4 h at 55 °C. Finally, the solid is filtered and washed with ethyl alco-hol and dried in vacuum. The preparation conditions and degree ofcarboxymethylation govern water solubility of O-CMCS as experimen-tally demonstrated by Chen at al. [58].
5.2. Synthesis of N-carboxymethyl chitosans (N-CMCS)
Synthesis of N-CMCS takes place in slightly acidic medium. To pre-pare N-CMCS, free amino group of chitosan is reacted with glyoxylicacid to give soluble aldimine and then aldimine is reduced with reduc-ing agent sodium cyanoborohydride. The reaction neither requireswarming, nor cooling. The proportions of acetyl, carboxymethyl andfree amino groups are determined by DA and MW of the chitosanused and the quantity of glyoxylic acid used [42].
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Fig. 2. Illustrates the different outstanding and astonishing physicochemical and biological properties of CMCS that contribute for its suitability in numerous drug delivery and tissue en-gineering applications.
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5.3. Synthesis of N,O-carboxymethyl chitosans (N,O-CMCS)
N,O-CMCS is hydrophilic and amphoteric polyelectrolyte which bearscarboxymethyl substituents at some of the amino and primary hydroxyl
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Fig. 3. Preparative methods of
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
EDsites of the glucosamine units of the chitosan structure. The main attrac-
tive features of N,O-CMCS are moisture retention, gel formation andgood biocompatibility along with increased water solubility and en-hanced antibacterial property that makes this polymer derivative highly
different types of CMCS.
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suitable forwide variety of biomedical applications.N,O-CMCS canbepre-pared by using chitosan, sodium hydroxide, isopropanol and chloroaceticacid [93,94].
5.4. Synthesis of N,N-dicarboxymethyl chitosans (N,N-di-CMCS)
The most crucial parameters for preparing N,N-di-CMCS are the con-centration of chitosan, water, glacial acetic acid, glyoxylic acid, and sodi-um borohydride. In order to synthesise N,N-di-CMCS, to a fixedconcentration of chitosan (30 g) suspended in demineralised water(3 l), 27 g of glacial acetic acid is added and stirred for 20 min. Afterthis glyoxylic acid is added (178 mL 50% v/v corresponding to 119 g ofglyoxylic acid) and the molar ratio of amine/glyoxylic acid is set to1:9 at pH 2–3. Finally, with the help of peristaltic pump (1.2 ml min−1),sodium borohydride (90 g) in water (2.5 l) is delivered as a 3.6% solutionto the reaction vessel. TheN,N-di-CMCS prepared by thismethod exhibitsgood film forming ability, good capacity to chelate metal ions and alsopossesses excellent osteoinductive properties with calcium phosphate[89].
6. Preparation techniques of CMCS based formulations for drugdelivery
CMCS based formulations for drug delivery applications have beenprepared by different techniques and methods by various researchersas per the requirement of the host tissue/organ and the type of drugbeing delivered. CMCS based hydrogels are one of the major formula-tions for drug delivery applications and can be prepared by differentmethods. In each process, CMCS is either physically associated or chem-ically cross-linked. Themajor schemes of physical interactions that leadto gelation of CMCS solution in hydrogels are ionic interaction, polyelec-trolyte complex; inter polymer complex, and hydrophobic associations.Moreover the physically cross linked hydrogels have the advantage ofgel formation without the use of cross-linking moieties. But they havecertain limitations like difficulty in accurately controlling the physicalgel pore size, chemical functionalisation, dissolution and degradation,thereby providing inconsistent in vivo performance. Cross linkedhydrogels often possess better mechanical properties where crosslinks can be incorporated either chemically or through irradiation. Butchemical cross linking method for preparation of hydrogels suffersfrom disadvantage of having toxic residues of cross linking chemicals.On the other hand preparation of microspheres, nanoparticles and mi-celles/aggregates can be carried out by different methods like emulsioncross linking, sonication, coacervation/precipitation, spray drying, ionicgelation, sieving, emulsion droplet-coalescence and reverse micellarmethods. Each method has its own advantage and certain limitationsand therefore the choice of preparative technique depends on the typeof particulate being synthesised, its size range, composition, the drugbeing loaded or encapsulated and the target tissue/organ where drugis to be delivered. Similarly, layer-by-layer assembly and cross linkingare the main preparative methods for the synthesis of composites andblending, casting and drying methods are themost popular preparativetechniques for films and fibers for the purpose of delivery of differentdrugs. Table 1 shows preparation techniques of CMCS based formula-tions for drug delivery.
7. CMCS formulations for drug delivery applications
7.1. CMCS hydrogels
In the recent few decades, the pharmaceutical industries havewitnessed the emergence of drug delivery technologies as a powerfuland efficient tool in order to effectively use the existing drugs and devel-op newdrug candidates successfully [131]. A hydrogel can be defined ascross linked network formed from a macromolecular hydrophilic poly-mer capable of absorbing large amount of water. The volume of water
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
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absorbed by hydrogels may range from 10% to thousands of times ofits own volume. Thehighwater contentmakes hydrogels biocompatiblewhile coherence of their mechanical properties with the soft tissues as-sists and enhances healing phenomenon. The compatibility of mechan-ical properties of hydrogels with the soft tissues also mimicsmorphological and functional characteristics of organ tissue. The visco-elastic property of hydrogels causes minimised damaged to the sur-rounding tissues in the host. Natural polymer based hydrogels havealso attracted the attention of scientists and researchers because oftheir improved biocompatibility, biodegradability and notable capabili-ty of controlled delivery of bioactive molecules [132–134].
As far as covalent cross linking is concerned, it leads to the formationof hydrogels with permanent network structure because irreversiblechemical links are formed. Therefore, this type of linkage not only al-lows absorption of water and/or bioactive compounds without dissolu-tion but also permits drug release by diffusion. Broadly covalentlycrosslinked chitosan hydrogels can be divided into three categories: chi-tosan cross linked with itself, hybrid polymer network (HPN) and semior full interpenetrating networks (IPN). In chitosan cross linked with it-self, crosslinking involves two structural units that may or may not be-long to the same chitosan polymeric chain [135]. The overall finalstructure of this type of hydrogel can be considered as a crosslinkedgel network dissolved in a second entangled network formed by chito-san chains with restricted mobility [136]. In hybrid polymer networks(HPN) based hydrogels the crosslinking reaction occurs between astructural unit of chitosan chain and a structural unit of polymericchain of another type. Although crosslinking of two structural units ofthe same type and/or belonging to the same polymeric chain cannotbe excluded. Finally semi or full IPNs contain a non reacting polymerwhich is added to the chitosan solution before crosslinking. Thus crosslinked polymer network in which the non reacting polymer isentrapped (semi-IPN) are thus formed. This additional polymer can fur-ther be crosslinked in order to have two entangled crosslinked net-works forming a full IPN whose microstructure and properties can bequite different from its corresponding semi-IPN [137]. Ionically crosslinked hydrogels leads to the formation of non permanent networkstructure since reversible links are formed [138]. Ionically cross linkedhydrogels show higher swelling sensitivity to pH changes as comparedto hydrogels having covalent cross linking. The entities reacting withchitosan are ions or ionic molecules with well defined MW in ionicallycrosslinking. It is different from polyelectrolyte complexation as the en-tities reacting with chitosan are polymer with a broadMW distribution[139]. In contrast to covalent crosslining ionic cross linking is a simpleand mild procedure as no auxillary molecules such as catalyst are re-quired [140]. Ionic cross linking can be assured by the classical methodof preparing a cross linked network namely by the addition ofcrosslinker either solubilised [141] or dispersed [142,143] to the chito-san solution.
Superporous hydrogels containing poly (acrylic acid-co-acrylamide)/O-CMCS IPNs were prepared by cross-linking O-CMCS with glutaralde-hyde (GA) after superporous hydrogel was synthesised by Yin et al. Anenhanced capacity of loading insulin was reported for the superporoushydrogels as compared to the non-porous hydrogels. Due to improvedmechanical properties, in vitromuco-adhesive force and loading capaci-ties, these IPNs showed potential of muco-adhesive system for peroraldelivery of peptide and protein drugs [97]. Similarly, Chen et al. prepareda novel type of IPNhydrogelmembrane of poly (N isopropylacrylamide)/CMCS and systematically studied the effects of the feed ratio of compo-nents, swelling medium and irradiation dose on the swelling anddeswelling properties of the hydrogel. The results showed that a combi-nation of pH and temperature can be coupled to control the responsivebehavior of poly(N-isopropylmethacrylamide (PNIPAAM)/CMCShydrogels [95]. There are several CMCS based semi-IPNs reported in lit-erature prepared by crosslinking with genipin [20], GA or cross-linked/grafted with ethylene glycol diglycidyl ether [102], and N,N′-methylenebisacrylamide [99]. Min Wang and coworkers synthesised
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Film Poly-vinyl alcohol/CMCS Blending/casting [129]t1:40
Fibers Ag nanoparticles/poly vinyl alcohol/CMCS Electrospinning technique [130]t1:41
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kinetics study showed fast adsorption of Fe (III) ions onto CMCSgels. Thiswas primarily due to the coordination of Fe (III) ions with amino, hy-droxyl and carboxyl groups of CMCS molecules [105]. Also an ESRstudy on CMCS radicals in a highly concentrated CMCS aqueous solutionwhich forms hydrogels by ionizing radiation was performed by SeiichiSaiki et al. [103]. Physically cross linked alginate-N,O-CMCS hydrogelswith calcium for oral delivery of protein drugs was prepared by Lin andcoworkers [96]. Substantial research has been done in chemicallycross-linked hydrogels also. Currently, pH sensitive hydrogels composedof CMCS, chemically cross linked by GA was prepared and evaluatedin vitro as a potential carrier for colon targeted drug delivery ofornidazole [100]. In this regard, the synthesis of novel polyampholytehydrogels based on CMCS of varying DD and DS cross linked with GAby Chen et al. [97] is worth mentioning. With increasing DD or DSvalue, the hydrogel changed from polyampholyte into polycations orpolyanions, respectively. The release of bovine serum albumin (BSA)was much quicker at pH 7.4 buffer than pH 1.2 solutions. In recentyears radiation cross linking has proved to be safer, clean and effectivemethod of hydrogel synthesis. The products formed are free of toxicityadditives as neither initiator nor cross linker is required as in convention-al chemical routines. The synthesis of CMCS using γ-rays radiation [104,105] and ionizing radiation [103] has been reported in the literature.Yang et al. [144] have in situ synthesised dark bluish coloured 5-fluorouracil (5-FU) loaded CMCS hydrogels using genepin as the crosslinker and it was speculated that the dark bluish colour of the hydrogelsprepared resulted from the cross linking reaction between genipin andthe amino groups of CMCS (Fig. 4A). The possible cross linking
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
mechanism for the reaction of CMCS with genepin was speculated as il-lustrated in Fig. 4B [144]. Similarly, Yang et al. [145] prepared hydrogelbeads based on methoxy poly (ethylene glycol) grafted carboxymethylchitosan (mPEG-g-CMC) and alginate in order to construct interpen-etrating polymeric matrix. Fig. 5 [145] illustrates the synthesis ofmPEG-g-CMC copolymer using Schiff's base method. Some of the CMCSbased hydrogels for delivery of different therapeutics are summarisedin Table 2.
7.2. CMCS microspheres
Microspheres are spherical free flowing particles having size rangebetween 50 μm and 2 mm with drugs entrapped inside them. Whilein matrix type microspheres drug is mainly released by erosion mecha-nism, release of drug takes place by diffusion and erosion in matrix andreservoir typemicrospheres [152]. In general drug release rate dependsupon solubility, diffusion, biodegradability of the matrix, drug loadingefficiency, and size of the microspheres. Therefore the drug releasemechanism can be altered by varyingpolymer employed and its proper-ties. Microspheres are mainly exploited in controlled drug delivery butby derivatisation and surface modification they can also be used fordrug targeting purpose. The partition coefficient determines the drugdistribution within the microspheres. CMCS based microspheres haveemerged as efficient drug carriers due to their ability to encapsulate avariety of drugs, biocompatibility, protection of fragile drugs, high bio-availability and sustained drug release characteristics. Currently,carboxymethyl cellulose and CMCS based biodegradable and highly po-rous microspheres were prepared with an inverse emulsion-cross-
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Fig. 4. Possible representations for (A) in situ synthesis of the drug loaded CMCS hydrogel and its photo and (B) cross linking mechanism between CMCS and genipin (Reproduced withpermission from [144]).
8 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
linking method without incorporating any extraneous cross-linkingagents capable of loading anticancer drug doxorubicin (DOX)whose re-lease profile was adjustable. The in vitro and in vivo studies indicatedthat these microspheres can be used as biocompatible and biodegrad-able embolic agents for transarterial embolisation [153]. Recently,quaternised carboxymethyl chitosan (QCMCS) was intercalated intothe interlayer of organic montmorillonite (OMMT) to obtain theQCMCS/OMMT nanocomposites. The cross linked alginate-Q-CMCS-or-ganic montmorillonite (AQCOM) microspheres were prepared by
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Fig. 5. Synthesis of the methoxy poly (ethylene glycol) grafted carboxymethyl ch
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ED Pcrosslinking with CaCl2 and the drug-controlled release was evaluated
by using BSA as model drug. The AQCOM microsphere displayedmore excellent encapsulation and controlled release capacities thanthe microsphere without OMMT and in vitro active cutaneous anaphy-laxis test on Guinea pigs did not cause anaphylaxis [121]. Fig. 6 showsthe pictures of Guinea pigs after ACA test of AQCOM-3 microspheres.Significant anaphylaxis such as swelling and even ulcerated inducedby 2,4-dinitrophenol was observed (as shown in Fig. 6f). Again the in-duce contact and even challenge exposure on Guinea pig treated by
itosan (mPEG-g-CMC) copolymer (reproduced with permission from [145]).
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t2:1 Table. 2t2:2 Summarizes different CMCS based hydrogels for delivery of different therapeutics in drug delivery applications.
Composition of hydrogel Remark Refs.t2:3
Photocrosslinked methacrylated O-CMCS & PEG diacrylate OCMCS-based hydrogels with varying concentrations of carboxy and methacrylate groups are envisaged asattractive and versatile synthetic extracellular matrices that can effectively sequester, protect and controllablyrelease basic proteins. Varying the carboxyl and methacrylate groups respectively allows variation of charge andwater content/pore size which ultimately allows control of the absorption and dynamic release, via substratebiodegradation, of basic proteins.
[146]t2:4
Covalently crosslinked N,O-CMCS and oxidized alginatehydrogel
Good cytocompatibility against NH3T3 cells by in vitro test and no obvious cytotoxicity for major organs duringthe period of 21-day intraperitoneal administrationwas observed by acute toxicity test. The hydrogels developedalso showed good hemocompatibility.
[147]t2:5
Nanocomposite hydrogel composed of Curcumin,N,O-CMCS and oxidized alginate
Nano curcumin with improved stability by using methoxy poly(ethylene glycol)-b-poly( -caprolactone)copolymer as carrierwas slowly released from thehydrogelwith the diffusion controllablemanner at initial phasefollowed by the corrosion manner of hydrogel at terminal phase. In vivo wound healing study showed that thehydrogel could significantly enhance there-epithelialisation of epidermis and collagen deposition in the woundtissue.
[32]t2:6
CMCS and carbopol 934 hydrogel for delivery oftheophylline
In vitro swelling studies have shown little swelling in acidic pH 432%at the end of 2 h and 1631% in basic pH at theend of 12 h. The pH-sensitive hydrogel of CMCS can be used for extended release of theophylline in intestine andcan be highly useful in treating symptoms of nocturnal asthma.
[148]t2:7
CMCS hydrogel A simple and economical method with no toxic chemicals involved was introduced. The steam-inducedcrosslinking of CMCS sodium salt was observed to be quite efficacious. Depending on the harshness of steamingconditions used, the DS of the hydrogels was found to be up to 36. The increasing temperature and duration ofsteam exposure changed the coloration of the samples from light beige to brown.
[149]t2:8
In situ gelable hydrogel composed of N-CMCS and oxidizeddextran (Odex)
The rate of gelationwas directly related to the degree of oxidation of Odex and the hydrogels underwent fastmassloss in the first 2 weeks, followed by a more moderate degradation. The in vivo studies in mice full-thicknesstranscutaneous wound models showed its ability of enhanced wound healing ability.
[150]t2:9
Physically crosslinked hydrogels of CMCS with celluloseethers including hydroxyethylcelluloseand methylcellulose
With the increase in the interaction of component polymers, the swelling and drug release rate of hydrogelsdecrease. Component polymer ratio controlled the swelling and drug release from hydrogels.
[151]t2:10
5-FU or bevacizumab loaded N,O-CMCS hydrogelscrosslinked with genepin
The in vitro drug release experiments showed that nearly 100% of −5-FU was released from the drug-loadedhydrogels within 8 h, but less than 20% bevacizumab was released after 53 h. The hydrogels provided greatopportunity to increase the therapeutic efficacy of glaucoma filtration surgery.
[144]t2:11
Fig. 6.Photos of Guineapigs by ACA test: (a) experimental procedure, (b) thefirst day after treating byAQCOM-3microsphere, (c) the seventh day after treating byAQCOM-3microsphere,(d) the eighth day after treating byAQCOM-3microsphere, (e) the eighth day after negative control, and (f) the eighth day after positive control (reproducedwith permission from [121]).
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Please cite this article as: L. Upadhyaya, et al., The implications of recent advances in carboxymethyl chitosan based targeted drug delivery andtissue engineering applications, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.043
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AQCOM-3microspheres did not induce the anaphylaxis (b–d) which il-lustrated that AQCOM-3 microspheres did not induce the anaphylaxis[121]. Similarly, clozapine loaded spherical microspheres of chitosanand CMCS within size range of 100 ± 0.25 μm to 140 ± 0.25 μm wereprepared by spray drying technique. The in vitro drug release studyshowed that CMCS microspheres released most part of the drug in theintestine compared to chitosan microspheres hence controlled drug re-lease was achieved [154]. Ma et al. [119] developed a simple method tofabricate BSA loaded self-assembled CMCS and CMCS-graft-poly (N,N-diethylacrylamide) microcapsules and microparticles whose in vitrodrug release rate and encapsulation efficiency depended on pH value.The microspheres were found to be non-cytotoxic against L02 humanhepatic natural cell and release of BSA could be sustained. Also sustainedrelease of BSA from microspheres based on mixtures of ionotropicallycross linked sodium alginate and chemicallymodified CMCS and coatedthrough polyelectrolyte complexation with chitosan grafted with poly(ethylene glycol) has been reported in literature. Fig. 7 [26] illustratesthe carboxymethylation of chitosan and synthesis of CMCS grafted sodi-um acrylate copolymer. Again, high encapsulation efficiency andsustained release of model anticancer drug DOX has been achieved bypreparing CMCS-CaCO3 microspheres by Wang and coworkers [118].There is also information regarding preparation of two kinds of O-CMCS bounded with iron oxide particles by in situ co-precipitationand incorporationmethods reported in literature. Themagnetic proper-ties of thesemicrospheres were studied to evaluate its potential in drugdelivery applications [120].
7.3. CMCS micelles/aggregates
When polar or non-polar ends of amphiphilic polymer self assembleby hydrophobic or ion-pair interaction in a monophasic or biphasic liq-uid, nanosised (200 nm to 0.5 μm) structures are formed which are re-ferred to as micelles. In an aqueous environment, the amphiphilicpolymers form hydrophobic core by assembly of lipophilic parts andvice versa. The repositioning of hydrophobic and hydrophilic drug with-in the hydrophobic or hydrophilic core of micelles improves the solubil-ity of drugs in monophasic non-solvent medium often referred to asmicellar solubilisation. In biphasic medium, polymeric segments distri-bution takes place according to their physicochemical characteristics.
In general, the hydrophobicity of the drug and the type of polymerdetermines the preparative method of micelle. In fact the experimentalevidences have led to the conclusion that polymericmicelles exhibit nu-merous promising properties for oral delivery of lipophilic drugs [155].Nano precipitation technique, dialysis method and sonication are some
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Fig. 7. Scheme of carboxymethylation of chitosan and synthesis of CMCS graf
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of the preparativemethods of micellar structures. Formation of micellesby self-assembly is themost popular preparativemethodology in case ofCMCS based or modified CMCS micelles designed and developed fordrug delivery purpose. The nanoscopic dimension, segregated core/shell structure, sheltering effect of hydrophobic core on drugsentrapped and stealthy characteristics by their hydrophilic shellsmakes polymeric micelles suitable for tumor targeting [156,157].CMCS has been extensively exploited by several researchers for devel-oping micelle based nanoformulations for model anticancer drug DOX[158,30]. Aifeng et al. synthesised and characterised DOX loadedoctreotide-modified N-octyl-O,N-CMCS micelles which proved to bepromising carrier for efficient intracellular targeting of antitumordrugs [159]. Similarly self-aggregated nanoparticles from linoleic acid(LA) modified CMCS were prepared by Yu-long Tan and coworkers fordelivery of hydrophobic anticancer drug adriamycin (ADR). The criticalaggregation concentration determined by measuring the fluorescenceintensity of the pyrene as a fluorescent probe, was in the range of0.061–0.081 mg/mL [123]. Gong and coworkers grafted Cis-3-(9H-purin-6-ylthio)-acrylic acid (PTA) on primary amino of CMCS, underthe catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-drochloride EDC·HCl and N-Hydroxysuccinimide (NHS) i.e. the reactiontook following two steps: synthesis of PTA (Fig. 8A) and synthesis of PTAgrafted CMCS (PTA-g-CMCS) Fig. 8B [160]. Amphiphilic polymeric pro-drug PTA-g-CMCS designed and synthesised could self-assemble intospherical micelles with a size ranging from 104 to 285 nm with 6-mercaptopurine loaded into it. The result demonstrated thatnanocarrier was found suitable for controlled drug delivery. Fig. 8Cshows 6-methoxy propyl release from PTA-g-CMCS in the presence ofglutathione, which follows a Michael addition–elimination reac-tion [160]. Jeong et al. [124] synthesised methoxy poly(ethylene gly-col)-grafted carboxymethyl chitosan (CMCPEG) copolymer using awater-soluble carbodiimide. In addition, DOX-incorporated nanoparti-cles using CMCPEG were prepared by ion complex formation betweenthe amine groups of DOX and the carboxyl group of CMCPEG. The selfassembled N-phthaloyl-CMCS based micelles for drug delivery oflevofloxacine hydrochloride and BSA have also been earlier preparedby Peng et al. [161]. Another technique of nano-precipitation wasemployed for the preparation of copolymer polylactide-polyethyleneglycol succinate, 1,3-beta-glucan (Glu), O-CMCS and folate-conjugatedO-CMCS micelles for efficient encapsulation of herbal anticancer drugcurcumin. The nanoformulation displayed better aqueous solubilityand biodegradation along with significantly enhanced the cellular up-take by cancer cell HT29 and HeLa. The anti-tumor-promoting effect ofthe curcumin encapsulated by copolymer in Hep-G2 cell lines after
ted sodium acrylate copolymer (reproduced with permission from [26]).
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Fig. 8. (A) Synthesis of PTA, (B) synthesis of PTA-g-CMCS, (C) 6-MP release from PTA-g CMCS in the presence of glutathione (GSH), which follows aMichael addition–elimination reaction:GSH attacks the β-C of the unsaturated bonds, leading to the cleavage of the unsaturated bonds (reproduced with permission from [160]).
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two weeks of cell growth is shown in (Fig. 9). The sudy showed that inthe cytotoxicity assay there were no distinct differences in the cell sur-vival. Also in the anti-tumor promoting assay, the ration of tumor pro-motion with the Cur, Glu, PLA-TPGS alone was comparable to thecontrol. But contrary to this clear changes in the size and morphologyof tumor between the control and all the tested samples particularlycurcumin encapsulated with glucan copolymer. It was observed thatin comparison to the tumor on the tested wells, in control the tumorsize was much larger and their surface was very rough (Fig. 9). [162].Wang and coworkers prepared Ca–P/CMCS/KALA nanoparticles by selfassembly via electrostatic interaction between positively charged pep-tide KALA and negatively charged Ca–P/CMC nanoparticles in an aque-ous solution after loading anticancer drug DOX·HCl onto the Ca–P/CMCS hybrid nanoparticles. The investigation results showed thatDOX·HCl could be encapsulated with high efficiency and the presenceof KALA peptide significantly enhanced the cell inhibition effect [163].Dialysis method was employed for the preparation of methotrexateconjugated O-CMCS micelles where methotrexate exhibited significantsustained release behavior in PBS solutions (pH 4.0, 7.2 and 9.0). Thecritical micelle concentration (CMC) of O-CMCS–methotrexate conju-gates determined in aqueous media was in the range of 0.0084–0.0424 mg/mL [164]. In addition, sonication method has also beenutilised for the designing and development of CMCS based nanocarriersfor delivery of anticancer drugs like DOX [165] and paclitaxel (PTX)[166]. Table 3 shows different CMCS based self aggregates and thedrugs delivered by these fabrications.
7.4. CMCS nanoparticles
The emerging trends and recent advances in nanotechnology hasmade a significant impact on the development of drug delivery systems,after the liposomes were first described in the 1960s as carriers of pro-teins and drugs for disease treatment [171]. Most of the front-linedrugs are toxic entities that act in unspecific fashion being untargeted,often eliciting unwanted, dose limiting anddebilitating side effects. Com-pared to conventional drug delivery systems, nanoscale drug delivery ve-hicles are capable of enhancing therapeutic activity by prolonging drughalf-life, improving solubility of hydrophobic drugs, reducing potentialimmunogenicity, and/or releasing drugs in a sustained or stimuli-
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
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triggered fashion. Thus, the toxic side effects and administration fre-quency of the drugs can be reduced [172]. In addition, nanoscale particlescan passively accumulate in specific tissues (e.g., tumors) through theenhanced permeability and retention (EPR) effect [173]. Polymers likeCMCS have emerged as a promising drug delivery nanovehicle due to in-herent increased water solubility, biological functionality, compatibility,safety, biodegradability, and antimicrobial nature. In the context ofdrug encapsulated CMCS based nanoparticles, nanoprecipitation, dialy-sis, micro emulsion, emulsion-solvent diffusion technique, solvent evap-oration and ionic gelation methods have been employed so far. Thepreparation of self-aggregated cholesterol modified O-CMCS nanoparti-cles by sonication method were prepared to be used as a novel carrierfor PTX has been reported by Wang et al. [174]. These results suggestthat cholesterol modified O-CMCS self-assembled nanoparticles can ef-fectively solubilise PTX and modify its tissue bio-distribution, whichmay be advantageous in enhancing the therapeutic index and reducingthe toxicity of PTX. Similarly Zhang et al. [113] reported synthesis ofmagnetic Fe3O4 nanoparticles functionalised with CMCS by sprayingco-precipitation method and these magnetic nanoparticles are suitablefor use as nanomagnetic carriers of drugs formulation. In this context,the preparation of vincristine loaded polymeric ethosomes; formedfrom amphiphilic octadecyl Q-CMCS with different DS by micro emul-sion method is worth mentioning [175]. Biocompatible ciprofloxacin-loaded CMCS nanoparticles by ionic cross-linkingmethod and optimisedby using Box–Behnken response surface method by Zhao et al., demon-strated, stronger antibacterial activity against Escherichia coli than thefree ciprofloxacin because they can easily be uptaken by cells. Still fabri-cation of CMCS based nanostructure delivery vehicles by ionic gelation isthe most popular method employed till date [108,109].
A number of CMCS based nanoscale carriers have been constructedin recent years for the efficient delivery of different anticancer, anti-inflammatory drugs, antibiotics, proteins, peptides and vaccines.Model anticancer drug DOX has been encapsulated into a number ofmodified CMCS nanosystems like FA modified CMCS nanoparticles[176] amphiphatic carboxymethyl–hexanoyl chitosan nanocapsules[158], and acylated CMCS nanoaggregates [35]. Also, liposomes modi-fied with CMCS [177] and CMCS capped magnetic nanoparticles [168]have also been exploited for DOX delivery. The drug release profile ofDOX loaded-CMCS-capped-MNP/MMT was tested at pH 7.4 and
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Fig. 9. Anti-tumor-promoting effects of the curcumin encapsulated by copolymer in Hep-G2 cell lines after twoweeks of cell growth on agar: control (a), Cur (b) and Cur-PLA-TPGS(c) under inverted microscope × 100 (reproduced with permission from [162]).
12 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
UNpH 5.0 respectively and the released DOX concentration was deter-
mined by UV–vis measurement. From the drug release curve (Fig. 10)it is clear that the release of DOX from DOX loaded-CMCS-capped-MNP/MMT is pH triggered and the release rate of DOX was higher atpH 5.0 than pH 7.4. It is well known fact that the pH of tumor tissue islower than normal cells. Thus this delivery system will exhibit smallertoxicity towards normal cells as compared to tumor cells thereby illus-trating its advantage of safety [178]. Recently, researchers have also de-veloped O-CMCS [179] and folate conjugated O-CMCS nanosystems[162] for efficient delivery of another anticancer compound: curcumin.The cellular uptake study of curcumin-O,CMCS NPs by normal L929cells and cancer cell line MCF-7 cells using flow cytometry showed(Fig. 11) that thenanoformulation uptakewas concentration dependentand non specific. This meant that both the cell line showed increasinguptake of the nanoformulation with the increase in the concentrationof the curcumin-O-CMCS nanoformulation. Also there is not much
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significant difference in the cellular uptake of curcumin-O,CMCS NPscomparing normal and cancer cell lines [179] Another multifunctionalnanodrug system containing Fe3O4, O-CMCS and curcumin has been re-cently fabricated by Thu et al. [180]. Similarly, Shen et al. [126] preparedCMCS-ferroferric oxide doped cadmium telluride quantum dot nano-particles with folate receptors affinity, magnetic responsiveness and lu-minescent property via layer-by-layer assembly technique in situsurface as shown in Fig. 12. The results indicated that the novel multi-functional folate conjugated carboxymethyl chitosan-ferroferric oxidedoped cadmium telluride quantum dot nanoparticles synthesised ex-hibit a high drug loading efficiency, minimised cytotoxicity and desir-able cell compatibility, and are potential candidates for CMCS-basedtargeted drug delivery and cellular imaging. In addition to this,nanoformulations of other anticancer drugs like vincristine,camptothecin, PTX, and ADR have been developed by different modifi-cations of CMCS such as amphiphilic carboxymethyl–hexanoyl chitosan[181], PEGylated O-CMCS nanoparticles graftedwith cyclic Arg-Gly-Asp(RGD) peptide [182] and LAmodified CMCS nanoparticles [166] respec-tively. Apart from these drugs, CMCS based nanoparticles mediated ve-hicles have been prepared for delivery of anti-inflammatory compounds[183], proteins/peptides [184] and vaccines [185] along with poorlywater soluble drugs like triamcinolone [186] and vitamin D3 [187].Table 4 describes various nanoformulations for delivery of differentdrugs along with brief method and their outcome.
7.5. CMCS films and fibers
In the recent years, drugs and polymers are manufactured into vari-ous film configurations, like coating of some precarious drugs or pro-teins by thin polymer films or generating drug–polymer matrix films.These different matrices are suitable for facilitating diffusion mediateddrug delivery or adaptable for accomplishing transdermal drug delivery.The parameters that determine the efficacy of polymeric films in deliv-ery of therapeutic compounds are thickness, surface morphology, de-gree of swelling, degradation behavior, drug release and therapeuticeffect. The drug release pattern and rate of drug release from the filmcoating systems depend on the coating material, thickness of the filmand circumstance of the applied site. Polymeric films have also gainedimportance as promising drug delivery formulation as its thin layerresists change in crystallinity and segmental motions. As CMCS showsinherent excellent biocompatibility, improved biodegradability, antimi-crobial strength and easily film forming ability, it has emerged as a suit-able biopolymer for films fabrication for drug delivery. The ornidazole(OD) loaded poly-vinyl alcohol PVA/CMCS films prepared by blending/casting method demonstrated excellent antibacterial and biocompati-bility characteristics which were enhanced with increasing CMCS con-tent in the films. The process of preparing the placebo films and theircorresponding drug OD loaded films (loaded OD) via blending/castingis shown in Fig. 13 [38]. The results from in vitro and in vivo study dem-onstrated the blend films as an excellent candidate for local drug deliv-ery system. A similar study was carried out where PVA/CMCS blendfilms prepared by casting and dryingmethod were evaluated as coatingmaterial for site specific drug delivery. The drug release kinetics studyby salicylic acid, theophyline, and OD also revealed that a desired rateof drug release could be obtained by controlling the content of CMCSin the blend film, type and MW of drugs, pH of the medium and thick-ness of the film. As pH of the buffer increased the permeability of ODwith a maximum at pH 7 as shown in Fig. 14. The reason for this phe-nomenon can be attributed to the fact that as pH increased, deproton-ation of the charge groups of CMCS takes place. CMCS moleculesbecame uncoiled and assumed to be elongated thereby causing the ex-pansion of the bulk of films. It caused the enhanced water absorptionability and lead to the increase of permeability [129]. A study on theBSA and bovine fibrinogen equilibrium adsorption amount on thePVA/CMCS films prepared by mechanical blending showed the influ-ence of CMCS content and pH and ionic strength of protein solutions
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t3:1 Table. 3t3:2 Different CMCS based self aggregates.
CMCS based formulation Size range ofself-aggregates
Brief method and outcome Refs.t3:3
O-CMCS aggregates – Not only the aggregates but also the unimers of O-CMCS enhanced the solubilityof camptothecin. The aggregates showed good drug loading capacity and sustaineddrug release demonstrating potential for localised drug delivery.
[167]t3:4
Cholesterol modified O-CMCS aggregates 234.9–100.1 nm A series of cholesterol modified O-CMCS conjugates with different DS of cholesterolwere synthesised and self-aggregates were prepared by probe sonication in water.The relationships between the chemical structure, the amphiphilic property and themorphological characteristics of O-CMCS self-aggregated nanoparticles wereinvestigated in this study.
[112]t3:5
LA-modified CMCS self-aggregates 222.8–1028 nm Covalently conjugated LA to CMCS via EDC mediated reaction to generate self-aggregatednanoparticles by sonication method. Physically entrapped ADR showed slow release whichcan be adjusted by change in medium pH.
[168]t3:6
LA-modified CMCS aggregates 417.8 ± 17.8 nm The LA modified CMCS self aggregates prepared by sonication method exhibited increasedloading capacity and loading efficiency and decreased sustained release for ADR withincreasing DS of LA in CMCS with critical aggregation concentration values in the rangeof 0.061–0.081 mg/mL.
[123]t3:7
Deoxycholic acid-O-CMCS-folic acid conjugates 179–212 nm Deoxycholic acid and FA modified O-CMCS self-aggregates were prepared by sonicationmethod and the mean diameter and critical aggregation concentration value changed withchange in DS and pH values.
[169]t3:8
CMCS nanoclusters 301 nm The results showed that demonstrated that Box–Behnken design methodology was aneffective way to obtain the optimal formulation of tea polyphenols-loaded chitosan nanoclusters,and the nanoclusters complexation synthesizing through ionic gelationbetween CMCS and chitosan hydrochloride was good biomaterials, which couldbe successfully used to encapsulate tea polyphenols
[170]t3:9
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on the adsorption amount of these plasma proteins [199]. Drug loadedbiodegradable polymer based nanofibers intended to deliver drugs atthe targeted site in a sustained fashion avoiding burst release have re-ceived attention by the researchers in the past decade. As far as CMCSbased nanofibers are concerned, electrospinning technique has beenmost widely accepted method. It is relatively a simple approach to con-trol the morphology of ultrafine fibers with outstanding advantages ofvery large surface-to-volume ration and high porosity with a smallpore size [200]. The silver nanoparticles/PVA/CMCS nanofibers of uni-form diameter of 295 to 343 nmwith 4 to 14 nm sized Ag nanoparticleswere synthesised via electrospinning technique which proved suitableas antibacterial biomaterial [201].
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7.6. CMCS composites
Composites are biomaterials that are composedofmore than one con-stituent of different physical and chemical properties that are blended toform macroscopic, microscopic or nano structure. In case of compositesfor delivery of drugs, usually the polymers are modified either to
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Fig. 10. Release profiles of DOX from CMCS-capped-MNP/MMT at different pH. The linesare based on the fitting with the empirical Peppas's model (reproduced with permissionfrom [178]).
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or to enable targeteddelivery bymagnetic or surfacemodification. The al-ginate/chitosan/CMCS composite microcapsules prepared by extrusionmethod encapsulating Lactobacillus casei ATCC 393 proved to be usefulfor the delivery of probiotic cultures to the human gastro-intestinaltract [202]. Geisberger et al. [125] prepared polyoxometalates-trimethylchitosan nanoparticles which are obtained from the direct electrostaticinteraction of positively charged trimethyl chitosan with negativelycharged polyoxometalates as shown in Fig. 15. Table 5 shows differentCMCS based composites synthesised for delivery of different drugs.
8. CMCS based targeted drug delivery
Discovery and development of a new drug is a highly challenging,labor intensive and expensive process. On an average development pro-cess of each new drug takes approximately 15 years with an estimatedcost of about US$802 million. And, this estimated cost has been report-ed tomarkedly increase at an annual rate of 7.4% above general price in-flation [203]. Due to their inability to reach the target site of action,mostof the drugs in the clinical phase fail to achieve desired clinical out-comes. In fact a considerable amount of drug administered gets distrib-uted over normal tissues or organs, which are not involved in thepathological processes causing severe side effects. An effective approachto overcome this critical problem is the development of targeted drugdelivery system that can release bioactive compound at the desiredsite of action. The main advantages of targeted drug delivery includethe accumulation of drug in the action site, increase in therapeutic effi-cacy, reduction of therapeutic dose and toxicity, etc. [204].
The concept of developing a drug that could selectively destroy dis-eased cells without harming healthy cells was proposed by Paul Ehrlichalmost a century ago. He gave this hypothetical drugnameof the “magicbullet” [205]. Since then, out of several polymers that have beenexploited in targeted drug delivery till date, CMCS has received signifi-cant attention due to its active functional groupswhich can easily attachtargeting ligands like FA. It can protect therapeutic agents from hostileconditions in body and can release the entrapped agents selectively atdesired site in a controlled fashion. In addition to it, the inherent phar-macological properties and excellent biological properties of CMCSlike biocompatibility, biodegradability, non-toxicity, increased watersolubility and unique mucoadhesivity also facilitate site specific drugdelivery. Fig. 16 illustrates the route followed by different CMCS based
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Fig. 11. (A) Cellular uptake of curcumin-O-CMCNps by FACS (a) control L929 cells alone (b) and (c) L929 exposed to 1 and5 mg/ml curcumin-O-CMCNps (d) controlMCF-7 cells alone (e)and (f) exposed to 1 and 5 mg/ml curcumin-O-CMCNps (B)Apoptosis assay by FACS (a) control L929 cells exposed to O-CMCNps (b) and (c) L929 exposed to 1 and5 mg/ml curcumin-O-CMC Nps (d) control MCF-7 cells exposed to O-CMC Nps (e) and (f) exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (reproduced with permission from [179]).
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Uformulations during their delivery to the targeted site of action inhuman body. Sahu et al. investigated the use of hydrophobically modi-fied CMCS nanoparticles for the delivery of anticancer drug PTX. The re-sults showed that the nanoparticles exhibit a significant inhibitoryeffect on the folate receptor over expressing tumor cells like HeLa cells[166]. Similarly evaluation of FA modified CMCS nanoparticles loadedwith another anticancer drug DOX has been also reported [206]. Theconfocal and flow cytometry study revealed that the nanoparticlescould target the cancerous cells more effectively than the normal cells.Recently, Wang et al. prepared novel biodegradable deoxycholic acid(DA)-O, CMCS-FA micelles for the delivery of PTX which showed en-hanced level of uptake compared to plain micelles in MCF-7 cells[207]. N-succinyl-O-carboxymethyl chitosan (NSO, CMCS), another
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modified derivative of CMCS has been proven to be an excellent disper-sant to prepare a well-dispersed suspension of superparamagneticFe3O4 nanoparticles due to its amphiphilic polyelectrolyte property. Itsgood cytocompatibility and functional carboxyl groups showed its po-tential for targeted dug delivery [208]. Similarly,magnetic nanoparticlescomplexed with CMCS were prepared through spraying co-precipitation method and their core-shell structure, stability and mag-netic properties were also investigated. The studies revealed that thespraying process is a more practical method due to an increased quan-tity of adsorbed CMCS and a simplified automated preparation proce-dure for targeted drug delivery [113]. In this context, the preparationofwell-dispersed suspension of superparamagnetic Fe3O4 nanoparticlesstabilised by chitosan and O-CMCS respectively by Zhu et al. is worth
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Fig. 12. Schematic diagrams of the fabrication procedure for CMCH-based folate/luminescent/magnetic nanoparticles (reproduced with permission from [126]).
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the surface of Fe3O4 nanoparticles is believed to be the electrostaticand coordination interactions, respectively andwas suitable for site spe-cific drug delivery [209]. Novel multifunctional FA-CMCS-ZnS:Mn (FA-CMCS-Zinc Sulphide: Manganese) nanoparticles developed throughsimple aqueous route byMathew et al. [128], have also shown potentialfor effective targeted drug delivery and imaging of cancer cells.
8.1. Cancer-specific drug delivery based on CMCS
Despite of tremendous developments in the field of medical science,the cure for cancer is still a major challenge. Indiscriminate distributionof most of the anticancer drugs towards disease and healthy cells fol-lowing systemic administration ultimately leading to high toxicity re-mains the critical bottleneck of conventional chemotherapeutics. Inaddition to this, the poor solubility of most of the anticancer drugs inwater creates the need to use organic solvents or detergents for clinicalapplications, resulting in undesirable side effects such as venous irrita-tion and respiratory distress [210]. Therefore, in order to develop a suc-cessful anticancer therapy, it is important to design a distinct carriersystem that can encapsulate large amount of anticancer drug and deliv-er it specifically to the cancerous cells. Till date a large number of CMCSbased matrixes that include nanoparticles, micelles, microspheres,nanofibers, composites, hydrogels, films and fibers have been designedby different researchers for delivery of different anticancer drugs likeDOX, 5-FU, methotrexate, PTX, ADR, curcumin, camptothecin, 6-Mer-captopurine, Vincristine etc. A study of glycol chitosan-carboxymethylcyclodextrins drug carrier for efficient targeted delivery of three hydro-phobic anticancer drugs namely 5-FU, DOX, and vinblastine have beencarried out by Tan et al. [211]. Moreover, the development of multifunc-tional novel folate conjugated CMCS–Fe3O4–CdTe nanoparticlesexhibiting high drug loading efficiency, low cytotoxicity and favorable
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
cell compatibility for the delivery of another hydrophobic anticancerdrug ADR has been reported by Shen et al. [126]. CMCS based deliverysystem of another model anticancer drug has been experimented byJin et al. [165], where the group demonstrated the preparation andin vitro evaluation of amphiphilic DAmodified CMCS based pH sensitiveself aggregated nanoparticles. The results of the experiment showed thegreater cellular uptake and enhanced retention of drug loaded nanopar-ticles in drug-resistant cells thereby confirming its superior efficacyover free DOX. Maya et al. [212] have prepared O-CMC nanoparticlesby ionic gelation technique where negatively charged carboxyl groupsof O-CMC were cross-linked using CaCl2 and PTX was loaded withinthe nanoparticles before the cross-linking step, after allowing the drugto interact with O-CMC. In order to facilitate the targeted delivery ofPTX, antibody Cetuximab (Cet) was conjugated onto the PTX O-CMCnanoparticles by EDC coupling chemistry. The complete reactionscheme for the preparation of Cet-PTX-O-CMC nanoparticles is shownin Fig. 17 [212]. Zou et al. [213] used octreotide–polyethylene glycol–stearic acid (OCT–Phe–PEG–SA) as a targeting molecule for N-octyl-O,N-carboxymethyl chitosan (OCC) micelles loaded with DOX to preparenovel active tumor targeting carrier. Fig. 18 [213] illustrates the proce-dure of self assembly and receptor-mediated cellular internalisation ofOCC-OCT micelles. When OCT conjugated to stearic acid (SA) via poly-ethylene glycol (PEG) spacer (OCT–Phe–PEG–A) was used to modifyOCC micelles. SA could insert into the inner hydrophobic core of OCCwhen it self-assembled in the aqueous environment, thereby increasingthe soundness of modification of OCC. On the other hand, PEG, coveringthe surface of micelles, could increase the blood circulation time of mi-celles whereas OCT increased the targeting efficiency of the micelles totumor cells. The results of the in vitro and in vivo studies indicated thatOCC–OCT micelles might be a promising tumor-targeting carrier forcancer therapy [159]. Table 6 shows different CMCS based matrices forthe delivery of various anticancer drugs.
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t4:1 Table. 4t4:2 Different nanoformulation for delivery of various anti-cancers, anti-inflammatory, anti-microbial drugs, proteins/peptides and vaccines along with their brief outcome.
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Brief method and outcome Refs.t4:3
Anti-cancer Adriamycin Novel CMCS based folate/Fe3O4/CdTe nanoparticles within size range of 170–190 nm possessing intensesuperparamagnetic effect and photoluminescence property at room temperature exhibiting high drugloading efficiency, low cytotoxicity and favorable cell compatibility were prepared.
[126]t4:4
Camptothecin A novel organic–inorganic hybrid molecule consisting of carboxymethyl-hexanoyl chitosan modified with(3-aminopropyl) triethoxysilanewas synthesisedwhich showed self-assembly behavior into nanoparticleswith a stable polygonal geometry consisting of ordered silane layers of 6 nm in thickness. This formulationdemonstrated excellent cytocompatibility and cellular internalisation capability in ARPE-19 cell line alongwith well controlled encapsulation and release profiles.
[181]t4:5
Curcumin Curcumin loaded N,O-CMCS formulation was prepared by simple ionotropic gelation method with sizerange of 150 ± 30 nm having high encapsulation efficiency of 80% and indicated slow, controlled andsustained drug release. The nanoformulation was specifically toxic to cancer cells and non-toxic to normalcells
[188]t4:6
Doxorubicin DOX·HCl loaded nanospheres and microspheres were prepared by precipitation method with highencapsulation efficiency which displayed sustained drug release.
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Doxorubicin DOX loaded methoxy poly(ethylene glycol)-grafted-CMCS nanoparticles were synthesised by ioncomplex formationwith size b300 nm. Drug releasewas faster at acidic pH than neutral or basic pH andshowed promising antitumor activity.
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Doxorubicin Amphiphilic deoxycholic acid modified CMCS based pH-sensitive self-aggregated nanoparticles were pre-pared within size range of 87 to 174 nm
[165]t4:9
5-Fluorouracil 5-FU encapsulated FA–CMC–ZnS:Mn nanoparticles with size range of 130–150 nmwere preparedwhich were found to be suitable for targeting, controlled drug delivery and cancer cell imaging usingBreast cancer cell line MCF-7 and were non-toxic to L929 cells.
[128]t4:10
5-Fluorouracil 80 ± 20 nm sized 5-FU loaded N,O-CMCS nanoparticles were prepared with drug entrapment effi-ciency of 65% and were toxic to breast cancer cells showing good blood compatibility.
[189]t4:11
Methotrexate O-CMCS-methotrexate nanoparticles were prepared by dialysis method with size range of187.2–363.5 nm and zeta potentials ranged from −8.19 mV to −3.08 mV. Drug release behavior wassustained in PBS solution of (pH 4.0, 7.2 and 9.0).
[164]t4:12
6-Mercaptopurine Six 6-Mercaptopurine-CMCS were prepared and structurally characterised. 6-Mercaptopurine-CMCS inpH 7.4 PBS could self-assemble into nanoparticles with mean diameter of 155.8 ± 6.0 nm by DynamicLight Scattering and 100 nm by TEM. 6-Mercaptopurine showed release in media containing 2 mM and10 mM GSH and maximum cumulative release rates were 65.1% and 74.4%, respectively.
[119]t4:13
Paclitaxel Target oriented nanoparticles based on O-CMCSmodified with stearic acid with FA covalently attachedby carbodiimide reaction by sonication method without using surfactants/emulsifiers. The nanoparti-cles exhibited significant inhibitory effect on Folate Receptor over expressing tumor cells like HeLa cells.
[166]t4:14
Vincristine Novel multifunctional O-QCMCS/Cholesterol liposomes were constructed with good physical andthermal stability, excellent solubility in water, and high drug encapsulation efficiency (90.1%) anddisplayed steady release action over 2 weeks.
[190]t4:15
Anti-inflammatory Indomethacin Magnetic N-benzyl-O-CMCS nanoparticles were synthesised by incorporation and in situmethods and in-domethacinwas incorporatedby solvent evaporationmethodwith loading efficiency of 60.8% to74.8%. Thein vitro drug release profile in Simulated Body Fluid (pH 7.4, 37°) displayed an initial fast release, whichbecame slower as time progressed.
[191]t4:16
Ibuprofen Novel O-CMCS/β-cyclodextrin nanoparticles of spherical shape and size with 166 nmwere prepared andIbuprofen was loaded with entrapment efficiency of 93.25 ± 2.89%. The release rate was slower from O-CMCS/β-cyclodextrin than from chitosan/β-CD in Simulated Gastric Medium (pH 1.2) while conversewastrue for Simulated Intestinal Fluid (pH 6.8).
[127]t4:17
Ketoprofen Ampiphillic matrices of CMCS-graft-phosphatidylethanolamine were prepared by a EDC-mediatedcoupling reaction.
[192]t4:18
Dexamethasone Novel surface engineered highly branched CMCS/polyamidoamine dendrimer nanoparticles weresynthesised which did not exhibited significant cytotoxicity in the range of concentration below 1 mg/mLand displayed high internalisation efficiency by both human osteoblast-like cells and rat bone marrowstromal cells. Interesting physicochemical and biological properties were reported for these macromolec-ular systems.
[193]t4:19
Anti-microbial Tetracycline Tetracycline encapsulated O-CMCS nanoparticles were prepared by ionic gelation method which werebiocompatible and 200 nm in size. This nanomedicine was 6-fold more effective in killing intracellularS. aureus compared to Tetracycline alone.
[194]t4:20
Gatifloxacin(GFLX)
Spherical 30–70 nm sized GFLX entrapped O-CMCS nanoparticles were developed which displayed afour-fold lowerMIC value against Gram negative bacteria compared to GFLX solution and a similar MICvalue against Gram-positive bacteria as compared to GFLX solution.
[195]t4:21
Proteins/Peptides/Vaccines Bovine Serum Albumin (BSA) N,O-CMCS modified pristine nanodiamond particles were developed which were biocompatible andshowed no cytotoxicity to cells.
[196]t4:22
Basic Fibroblast Growth Factor (bFGF) Out of a series of chitosan derivatives synthesised, 2-iminothiolane modified 2-N sulfated 6-O-carboxymethylchitosan and chitosan complex was found suitable for preparing nanoparticles by poly-electrolyte self-assembly method which could successfully protect bFGF from inactivation over a 120 hperiod as determined by L929 fibroblast culture tests. The release of bFGF could be successfully controlled.
[197]t4:23
Tetanus Toxoid(TT)
40–400 nm sized N-trimethyl chitosan, chitosan and M,N-CMCS nanoparticles with negative surfacecharge for M,N-CMCS and positive surface charge for chitosan and M,N-CMCS nanoparticles were de-velopedwith TT loadedwith efficiencyN90%m/m. Effective uptake of the Fluorescein isothiocyante-BSAloaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells andenhanced immune response was reported after intranasal application of nanomedicine.
[198]t4:24
Protein drugs Water soluble oleoyl-CMCS was synthesised through covalent modification of chitosan with oleic acidandmonochloroacetic acid prepared by self-assembledmethod. TheOCMCS nanoparticles showedhighloading efficiency and sustained release of extracellular products. Themucoadhesion and internalisationcapability make oleoyl-CMCS nanosystems interesting candidates that could be effective to improveprotein drugs adsorption after oral administration.
[116]t4:25
16 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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Fig. 13. Preparation of the PVA/CMCS blank films and drug films (reproduced with permission from [38]).
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8.2. Organ-specific drug delivery based on CMCS
Organ, or site-specific drug delivery, has several distinct advantagesover other means of delivering drugs. For example, direct infusion intothe target organ or the vasculature surrounding and supplying bloodto the organ ensures that the majority of the drug goes to the site it isintended to act on. This allows for the use of themore toxic drug agents(e.g., chemotherapeutics) in high concentrations, because the exposureof other organs to the compound is limited. Targeting organs or tissuesin vivo can greatly reduce the risk of toxic side effects and significantlyincrease the efficacy of a variety of drugs, including toxic chemothera-peutic agents, pain medications, and gene therapies. The advantagesof site-specific employing CMCS vehicles include pH-sensitivity,bioadhesive ability, solubility and absorbability, controllable biodegrad-ability, nontoxicity of the degradation end products, sustained releasepotential and ease of administration [217]. Many researchers have pre-pared different CMCS based formulations that exclusively deliver vari-ous pharmacological agents at specific organs for their activity. Theseinclude different organs of body like intestine, liver, pancreas, colon,eyes, and others which are elaborated below.
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Fig. 14. Release of ornidazole through 30% C blend film at different pH. Results aremeans 6standard deviation for n ¼ 3 (reproduced with permission from [129]).
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ED P8.2.1. Colon-specific drug delivery using CMCS
Colon-specific delivery of drugs has gainedmuch attention in recentyears for the treatment of various diseases such as Crohn's disease, ul-cerative colitis, and irritable bowel syndrome [218]. The relatively lowproteolytic activities of protein/peptide drugs in the colon and evenfor other nonpeptide drugs such as cardiovascular and antiasthmaticagents has prompted several researchers to focus on colon targeteddrug delivery. The absorption and degradation pathways in the uppergastrointestinal tract are the major hindrances in delivering drugs tothe colon. To overcome these obstacles, several strategies of colon-specific drug delivery have been attempted till date by various scien-tists, that include use of prodrugs which become active at the colon,drug-eluting system responding to the pH, and microflora-activatabledrug delivery systems have gained increasing attention. These strate-gies basically emphasise on preventing loss of the drug at the stomachand the small intestine, thereby facilitating quantitative drug deliveryto the colon.
CMCS based systems have been widely studied for colon targeteddrug delivery as colonic microflora can degrade its glycosidic linkagethus facilitating the release of drugs entrapped specifically in colon.Tavakol et al. [219] investigated the release of sulfasalazine drug fromthe alginate-N,O-CMCS gel beads prepared by ionic gelation method.The in vitro studies revealed that the chitosan coated alginate-N,O-CMCS hydrogel may be used as potential polymeric carrier for colon-specific delivery of sulfasalazine. Tu et al. [220] studied the sigmoidalswelling kinetics of a series of CMCS-g-poly (acrylic acid) hydrogelswhich were pretreated under acidic buffer media. The swelling kineticsof 5-aminosalicylic acid at different pH showed its potential for colon-specific drug delivery. Recently, Vaghani et al. [100] prepared andcharacterised pH sensitive hydrogel composed of CMCS cross linkedwith GA. The group evaluated in vitro as a promising carrier for the ad-ministration of colon targeted drug delivery of OD.
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8.2.2. Liver targeted drug delivery using CMCSLiver targeted delivery of drugs has gained increasing attention for
the treatments of a number of chronic hepatic diseases such as hepatitis,hepatocirrhosis, hepatoma and hepatic carcinoma in the recent years.Considerable effort has been made to exploit CMCS as liver-specificdrug carrier. This is because nanoparticles prepared from such amphi-philic derivative via self-assembly have been recognised as a promising
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Fig. 15. Electrostatic interaction of CMCS (a) and trimethyl chitosan (b) biopolymer matrices with encapsulated polyoxometalates (POMs) (reproduced with permission from [125]).
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drug carrier, since their hydrophobic domain can serve as a depot forsparingly soluble drugs [221]. Zheng et al. [222] synthesised glycyrrhizicacid encapsulated novel thiolated lactosaminated (TLAC)/CMCS nano-particles through ionic gelification method and characterised thesenanoparticles in vitro studies. The in vivo studies of pharmacokinetic pa-rameters were evaluated in rabbits and tissue distribution in mice. Theresults showed that the TLAC-CMCS may be used as a promising drugcarrier for hepatic targeting and controlled release. Similarly, prepara-tion, characterisation and tissue distribution studies of lactosaminatedCMCS nanoparticles have been reported by Zheng and coworkers. Theexperiment demonstrated that the in vitro release of glycyrrhizic acidfrom the nanoparticles exhibited a biphasic pattern, initial burst releaseand consequently sustained release. Also, these nanoparticles modifythe tissue distribution profile of the glycyrrhizic acid solution, the kid-ney excretion rate is reduced and drug accumulation in the liver is in-creased [108]. Recently, Shi et al. [216] prepared O-CMC-methylmethacrylate (MMA) copolymers through the graft copolymerizion of
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Table. 5Different CMCS based composites synthesised for delivery of different drugs.
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
EDhydrophobic MMA and hydrophilic O-CMC, which spontaneously self-
assembled into coreshell O-CMC nanoparticle in aqueous solution. Theadjacent hydroxyls of glycyrrhizin (GL) were turned into aldehydegroups through periodate oxidation in order to obtain O-CMC-GL nano-particle. The preparation procedure of O-CMCnanoparticle, GLmodifiedCMC-GL, and PTXL loaded CMC-GL nanoparticle (PTXL/CMC-GL) nano-particles were illustrated in Fig. 19 [216] In addition, in vitro andin vivo studies of PTX loaded GL-modified O-CMCS nanoparticles werealso carried out by for hepatocellular carcinoma targeted drug deliveryapplication. The results established these nanoformulation as promisingdrug carrier for hepatic cancer.
8.2.3. Ocular drug delivery using CMCSThe field of ocular drug delivery is one of the most interesting and
challenging endeavors facing the researchers and scientist community.Fromdrug point of view, it is very difficult to study the eye as an isolatedorgan. This is due to the presence of highly sensitive ocular tissues like
Refs.
l FA–CMCS–ZnS:Mn nanocomposites prepared by simple aqueous route showedn of 5-FU (92.08%) with controlled release and were found nontoxic to moused toxic to MCF-7 cell line.
[128]
O-CMCS/β cyclodextrin nanocomposites by simple ionic cross-linking methodpsulation efficiency of 93.25 ± 2.89%. The release rate was slower from O-CMCS/βin simulated gastric medium while converse was true for simulated intestinal
[127]
omposites were prepared by direct cross-linking approach. POM–CMCS com-e zeta potentials and larger particle sizes than the positively charged POM–
posites
[125]
nm thick silane layer formations upon self-assembly of the hybrid moleculease of CPT. The hybridmolecule showed excellent cytocompatibility and efficientwith tp ARPE-19 cell line.
[181]
el folate conjugated CMCS/Fe3O4/CdTe NCs were prepared by layer-by-layeritial CMCS concentration, medium pH and reaction time strongly influencedinding mode of CMCS. The multifunctional nanocomposites exhibited high drugcytotoxicity and favorable cell
[126]
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Fig. 16. Illustrates the route followed by different CMCS based formulations during their delivery to the targeted site of action in human body.
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the uveal tract and retina. In addition to this, the presence of tissue bar-riers to drug penetration which include the lipophilic corneal epitheli-um, the hydrophilic corneal and scleral stroma, the conjunctivallymphatics, choroidal vasculature, and the blood-ocular barriersmakes ocular drug delivery even more challenging [223]. The choiceof chitosan for ocular drug delivery has been justified due to its out-standing mucoadhesive and penetration enhancing properties, as wellas by its good biocompatibilitywith the ocular structures [224]. Applica-tion of chitosan based nanostructures in delivery of ocular therapeuticshas beenwell reviewed by Fuente et al. [225]. CMCS has attractedmuchattention in ophthalmic drug delivery due to its inherent low toxicity,
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Fig. 17. Schematic illustration depicting preparation of PTXL-O-CMC nanoparticles and bioconcarboxyl functionality and subsequently linked to Cet through covalent linkage (reproduced w
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
EDbiocompatibility, biodegradability and ability to prolong ophthalmic
drug retention time [226]. In fact the ability of CMCS to prolong theprecorneal drug retention, by virtue of its viscosity-increasing affect, ofits ability to bind ofloxacin and, probably, of its mucoadhesive proper-ties in comparison to chitosan has been experimentally demonstratedby Colo et al. [226]. An in vitro study of gatifloxacin (GFLX) from novelO-CMCS formulation showed that the release was slower than thatfrom GFLX solution and the MIC of OCMCS formulation against Gram-negative bacteria is fourfold lower than the system without OCMCS.The GFLX is a fourth-generation fluoroguinolone and in vitro studieson isolates from bacterial infections of the eye have shown an
jugation of Cet on PTXL-O-CMC Nps through EDC activation chemistry. EDC activated theith permission from [212]).
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Fig. 18. Schematic illustration of the self-assembly and receptor-meditated cellular internalisation of OCC–OCT micelles (reproduced with permission from [213]).
t6:1 Table 6t6:2 Different CMCS based matrices for the delivery of various anticancer drugs.
Anticancer drug CMCS based matrixstructure
Matrix composition Remarks Refs.t6:3
Adriamycin Self aggregatednanoparticles
LA-modified CMCS Self-aggregated nanoparticles exhibitedan increased Loading Capacity and Loading Efficiency, decreased sustained release with an increasingratio of the hydrophobic LAto hydrophilic CMCS
[123]t6:4
Camptothecin Aggregates O-CMCS Not only the aggregates but also the unimers of OCMCS can help to enhance the solubility of CPT. In vitrocancer antiproliferative activity test further confirms theslow release of CPT from OCMCS-drug system.
[167]t6:5
Curcumin Nanoparticles O-CMCS Spherical 150 ± 30 nm sized curcumin loaded O-CMCS nanoparticles were prepared with entrapmentefficiency of 87%. The nanoparticles were toxic to cancer cells and non-toxic to normal cells.
[179]t6:6
Doxorubicin Nanospheres andmicrospheres
CaCO3–CMCS The water soluble DOX·HCl could be effectively loaded in the hybridmicroparticles and nanospheres with a high encapsulationefficiency, and the drug release could be effectively sustained,indicating the hybrid microspheres and nanospheres weresuitable for delivery of water-soluble drugs
[118]t6:7
Self-assembliednanoparticles
Folic acid (FA) modifiedCMCS
267.8 nm sized nanoparticles were prepared by sonication method. The cellular uptake of Folatemodified CMCS nanoparticleswas found to be higher than that of nanoparticlesbased on LA modified CMCS.
[166]t6:8
Self assembledhollownanocapsule
Carboxymethyl-hexanoylchitosan
The model anticancer drug DOX was entrapped with an efficiency of 46.8%, and a corresponding drugrelease from the nanocapsules for a time period exceeded 7 days can be achieved in vitro.
130–150 nm sized novel nanoparticles loaded with 5-FU exhibited non-toxicity to L929 cells. Thenanoparticles could be used for controlled drug delivery and imaging of cancer cells.
[128]t6:12
Methotrexate Nanoparticles FA conjugated CMCS The encapsulation efficiency and loading capacity of Methotrexate in the FA-O-CMCS nanoparticleswere higher, and the particle size of the FA-O-CMCS nanoparticleswas also smaller than those in the FA-CS nanoparticles.
[214]t6:13
Self assemblednanoparticles
Methotrexate conjugatedO-CMCS
Spherical shaped nanoparticles within size range of 187.2–363.5 nmwere prepared by dialysis method.In vitro drug release study by the dynamic dialysis method showed that Methotrexate exhibitedsignificant sustained-release behaviorsin PBS buffer solutions (pH 4.0, 7.2 and 9.0), indicating that these nanoparticles had good in vitro sta-bilityand the potential to be used as a novel drug carrier system
[164]t6:14
6-Mercaptopu-rine
Nanoparticles 6-Mercaptopurine-CMCS The 6-Mercaptopurine release from 6-MP-CMC showed dependence on glutathione concentration. Inaqueous solution 6-MP-CMC could self-assemble into the nanoparticles through the intra- and inter-molecular hydrophobic interactions between 6-MP groups
100–205 nm sized PTX loaded O-CMCS nanoparticles were prepared with encapsulation efficiency of83.7% and performed a biphasic release.
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Nanoparticles FA conjugated CMCS modi-fied with stearic acid
The PTX loaded nanoparticles exhibited many desirable properties like pH-sensitive dissolution, lowcytotoxicity, and high amount drug encapsulation and significant inhibitory effect on the FR over ex-pressing tumor cells like HeLa cells
[166]t6:17
Vincristine Liposomes O-Q-CMCS-Cholesterol Vincristine was encapsulated in polymeric liposomes with high entrapment efficiency (90.1%). Theformulation was stable in aqueous solution and exhibited slow, steady release action over 2 weeksunder physiologic pH (7.4).
[190]t6:18
20 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
Please cite this article as: L. Upadhyaya, et al., The implications of recent advances in carboxymethyl chitosan based targeted drug delivery andtissue engineering applications, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.043
Fig. 19. Synthetic route of O-CMC-methyl methacrylate (MMA) copolymers and glycyrrhizin (GL) and schematic representation depicting the formation of O-CMC nanoparticles, O-CMC-GL nanoparticles, and PTXL loaded O-CMC-GL nanoparticle (PTXL/CMC-GL) (reproduced with permission from [216]).
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RRECencouraging response to it [195]. Currently, Yang et al. investigated both
in vitro and in vivo, the ability of 5-FU or bevacizumab loaded N,O-CMCShydrogels tomodulatewound healing after glaucoma filtration surgery.On one hand the in vitro study showed that the nearly 100% of 5-FU wasreleased from the drug-loaded hydrogels within 8 h, but less than 20%bevacizumab was released after 53 h. On the other hand, the in vivostudy carried out in rabbits showed that the CMCS hydrogels were non-toxic to the cornea andwere gradually biodegraded in the eyes [144]. Re-cently, to investigate the availability of induced pluripotent stem cellsiPSCs as bioengineered substitutes in corneal repair Chien et al. [227] de-veloped a thermo-gelling injectable amphiphatic carboxymethylhexanoylchitosan (CMHC) nanoscale hydrogel and found that such gel increasedthe viability and CD44 þ proportion of iPSCs, and maintained theirstem-cell like gene expression, in the presence of culture media. Thestudy demonstrated that human keratocyte-reprogrammed iPSCs, whencombined with CMHC hydrogel, can be used as a rapid delivery systemto efficiently enhance corneal wound healing.
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U8.2.4. OthersApart from these, there are a number of other organs for which
CMCS based drug delivery systems have been designed by different sci-entists in the recent times. This include synthesis and in vitro study ofmicroencapsulated beads composed of alginate-N,O-CMCS for thedeliv-ery of model protein BSA to different regions of the intestinal tract. Theresults of the study demonstrated excellent pH sensitivity and proved tobe suitable polymeric carrier for site specific delivery of bioactive pro-tein in the intestine. Also the main advantage was that the bioactivityof the protein drugwas preserved due to the preparation of drug loadedbeads in the aqueous medium at neutral environment [96]. Anothersimilar work for the in vitro and in vivo evaluation of pH sensitiveCMCS based hydrogels for intestinal delivery of drug theophylline has
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
also been reported recently by [148]. While the in vitro results showedthat formulation I containing CMC and carbopol in 1:1 ratio showedsustained release, better prolonged action from the prepared hydrogelformulation when compared to (standard) marketed sustained releaseformulation was reported from the in vivo study. Currently, synthesis,characterisation and in vitro evaluation of anticancer drug 5-FU loadedCMCS nanoparticles have been reported by Anitha et al. [188] The toxic-ity of the drug loaded CMCS nanoparticles was showed byMTT, apopto-sis and caspase 3 assays thereby confirming the potential of 5-FU loadedN,O-CMC nanoparticles in breast cancer chemotherapy in which theside effects of conventional chemo treatment could be reduced. Recent-ly, preparation of metformin loaded O-CMCS nanoparticles prepared byionic gelationmethod for delivery to pancreatic cancer cells has showedpH sensitive release of the drug in vitro. While the cytotoxicity studyshowed the preferential toxicity of the drug loaded nanoparticles onpancreatic cancer cells (MiaPaCa-2) compared to normal cells (L929),the nanoparticles exhibited nonspecific internalisation by normal andpancreatic cancer cells [109].
9. Tissue engineering: origin and strategies
Tissue engineering, by definition, is a highly interdisciplinary fieldthat combines the principles andmethods of life sciences and engineer-ing to utilise structural and functional relationships in normal and path-ological tissue to develop biological substitutes to restore, maintain, orimprove biofunction [228]. The term “tissue engineering” was formallycoined in 1987 [229] and since then, it has emerged as a scientific fielddistinct from medical field by providing numerous promising tech-niques with practical clinical applications. The popular theory of themedical field that the human body possesses an inherent capacity toheal itself has been the fundamental principal that has been exploited
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in the field of tissue engineering. In fact, the specific tissue or the organdetermines the level of ability to self-repair and regenerate from dis-ease, damage or an injury. This capacity is limited by the degree of dam-age, loss of function and involvement of multiple tissues [230]. Therecent developments in tissue engineering like xeno-transplantationof tissues, new prosthetics and localised manipulation of lesion sites atthe cellular and molecular level have made significant advancementsin reconstructive surgery in comparison to the conventional approacheslike tissue auto- and allo-grafting [230,231].
The main objective of tissue engineering is to overcome the lack oftissue donors and the immune repulsion between receptors and donors.In fact, by laying emphasis on tissue and cell-based therapy, tissue engi-neering and clinical practices strive to achieve same goals. The tissue en-gineering strategy involves the in vitro seeding and proliferation ofrelevant cells and/or signaling molecules in an appropriately designedtissue engineering biomaterial like scaffold to form a natural tissue.This tissue is implanted into the defect in the patients. While in somecases, the scaffold or scaffold with cells is directly implanted in vivowhere the host body functions as a bioreactor to construct new tissues.Thus, these transplantable constructs enable regeneration of functionaltissue in the host providing an alternative to conventional organ
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Table. 7Preparation techniques of CMCS based biomaterials for tissue engineering.
n-HAP/CMCS composite Particle filtration and lyophilisationfollowed by genipin crosslinking
CMCS/gelatin/n-HAP i-gel Enzymatic crosslinking
CMCS film Covalently crosslinked
CMCS-graft-D-glucuronic acidmembranes
Grafting D-GA onto CMCS inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
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transplantation and tissue reconstruction methods. Over the past40 years, tissue engineering has shown significantmost tissue types, es-pecially those damaged or lost following debilitating health problemssuch as cancer and degenerative disorders [232].
10. Preparation techniques of CMCS based biomaterials for tissueengineering
CMCS is biocompatible, biodegradable, andmore bioactive than chi-tosan with enhanced osteogenesis property and it can be easily formu-lated in a variety of forms like hydrogels, scaffolds, composites, i-gels,films and membranes which have wide range of potential applicationsin tissue engineering. The techniques that are suitable for preparationof different CMCS based hydrogels for tissue regeneration purposehave already been discussed in Section 8. As in hydrogels, the crosslinks can be incorporated either by chemical cross linking method orby radiation cross linking in the scaffolds also as per the requirementof the host tissue and the tissue engineering biomaterial being fabricat-ed. Nowadays, enzymatic cross linking has also gained attention due toits own benefits. Selecting the scaffolding approach for tissue engineer-ing is tissue and application specific. The conventional scaffold
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Series of biodegradable scaffolds were prepared where ultrasonictreatment on the polymer solutions affected the β-TCP particlesdistribution. Excellent biocompatibility and ability of boneregeneration was revealed by in vivo implantation in mandibleof beagle dog.
[130]
Coating of scaffolds with HAP substantially enhanced the viability,attachment, proliferation, and differentiation of the osteoblast anddirected stem cell differentiation to osteoblast
[40]
The scaffolds exhibited 20–500 μm sized pores with regular interconnectionwith appx. 58.9% of porosity determined from microcomputed tomographyanalysis. Average scaffolds consisted of 24% HA and 76% CMCS determinedfrom 2D morphometric analysis.
[233]
The SEM showed that nano/micro particles formed on the surface of thenano-nonwoven CMCS fibrous scaffold. FTIR and XRD confirmed that thenano/micro particles were hydroxyapatite crystalline. HAP particlesappeared to have a great effect on the late stages of osteoblast behavior(alkaline phosphatase).
[234]
The FTIR and XRD results of genipin cross linked n-HAP/CMCS scaffoldsrevealed that CMCS's hydroxyl, amine and amide groups determinednano homogenous distribution of n-HAP and provided nano topographicalfeatures for nanohybrid scaffolds. The scaffolds had pore size of 150 μm sizedpores with less toxicity and more facility for adhesion and proliferation of cells.
[235]
Thermo responsive & core-shell microgels were prepared having phasetransition temperature nearer to that of body compared to pure PNIPAM
[236]
Due to the high water absorption capacity, a similarcompressive modulus with soft tissue, controllable biodegradation,and excellent biocompatibility, the hydrogels have potential as skinscaffolds and wound healing materials.
[106]
The porosity ratio of CMCS/n-HA is about 75% and the compressivestrength can exceed 21 MPa with circular pores of diameter rangingfrom several μm to six hundred μm. The in vivo experiments showedno inflammatory reaction and bone putrescence and toxicity of liver and kidney.
[237]
The composite scaffold with (VEGF)-transfected bone marrow stromalcells (BMSCs) was studied in a rabbit radial defect model. The scaffold isbiocompatible, nontoxic, promotes the infiltration and formation of themicrocirculation, and stimulates bone defect repair with degradationrate matching the growing rate of bone.
[238]
The i-gels prepared susceptible to tyrosinase/p-cresol mediated in situgelling at physiological temperature that may be used in treating irregularsmall bonedefectswithminimal clinical invasion aswell as for bone cell delivery
[41]
Controlling the molecular weight distribution of properly tailored chitosanallows one to regulate the mechanical properties and degradation ofchitosan in a sophisticated manner, while maintaining favorable cellinteractions.
[73]
The membranes showed bioactivity which demonstrated its potential fortissue engineering applications.
[239]
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fabrication techniques include solvent-casting particulate-leachingmethod, gas foaming, fibre meshes/fibre bonding, phase separation,melt moulding, emulsion freeze drying, solution casting, andlyophilisation (freeze drying)method. The particle leachingmethod ex-hibits advantages of creation of scaffolds with big pores, well‐controlledhigh interconnected porosity and pore morphology. The benefits of gasfoaming technique include lack of solvent, eliminating the risk of re-maining residues, and the lowprocessing temperatures preventingdeg-radation of the polymer during processing. Emulsion freeze‐drying isattractive for creation of scaffolds that are relatively thick having largepores. Despite of several positive facets of these traditional methods,the incapability of precisely controlling pore size, pore geometry, spatialdistribution of pores and construction of internal channels within thescaffold, presence of residual organic solvent and poor mechanical in-tegrity are some of the most significant problem facing these conven-tional techniques due to the risks of toxicity and carcinogenicity itposes to cells. Therefore, new techniques like self-assembly systems,solid free-form fabrication and electrospinning technique haveattracted the attention of the researchers as these methods can over-comemany of the demerits of the conventional scaffolding approaches.Sintering method for scaffold design has also received attention and isusually applied in case of ceramics powders, metals, glasses and certainpolymers as well as composites. But most of the composites for tissuerepair are prepared by coprecipitation, porogen leaching and freezedry-ing (lyophilisation) method. In addition to this, injectable-gels, andmembranes can be prepared by enzymatic and covalent cross linkingand graftingmethod respectively. CMCS based films aremost common-ly prepared byblending/castingmethod thatmay include covalent crosslinking or other linkages. Table 7 shows preparation techniques of CMCSbased biomaterials for tissue engineering.
11. CMCS based biomaterials for tissue engineering and regeneration
An ideal biomaterial for tissue engineering is expected tomeet someimportant criteria. It must be biocompatible, easily biodegradable in ap-propriate time window, its degradation products should be non-toxic,and it must support cell adhesion and growth and should exhibit me-chanical strength comparable with the host tissue [240]. The applica-tions of chitosan and its derivatives in the field of tissue engineeringhave been earlier reviewed [241]. CMCS has attracted considerable at-tention for tissue engineering application due to its inherent increased
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Fig. 20. SEM images of gelatin/CM-chitosan/β-TCP composite scaffolds with β-TCP fraction o(reproduced with permission from [130]).
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bioactivity as compared to chitosan and its ability to promote osteogen-esis [25]. Also the capacity of CMCS to chelate calcium frommineralizingsolution containing calcium and phosphate to induce calcium phos-phate or hydroxyapatite (HAP) formation has made it suitable biopoly-mer for tissue engineering [242]. Apart from this, it is well known toexhibit excellent biocompatibility, better biodegradability, non-toxicity, and ability to promote cell adhesion which is desirable in thefield of tissue engineering and regenerative medicine. In the past fewyears, several researchers have utilised CMCS in fabrication of differenttissue engineering biomaterials which include scaffolds, hydrogels,composites, nanofibers, nanoparticles, dendrimer, and membranesand for implant functionalisation [243–245] due to easy processibilityof CMCS into these constructs.
11.1. CMCS scaffolds
Scaffolds are unique tissue engineering biomaterial as they are able toestablish three-dimensional environments for propagated cells and spe-cific signalling molecules that can mimic native tissues environments.Tissue engineering scaffolds can be of natural, synthetic or a hybrid ofboth. CMCS has emerged as a promising scaffolding polymer due to itsinherent excellent biocompatibility, ability to promote cell adhesionand increased bioactivity as compared to chitosan. Also in comparisonto chitosan that has relatively slow and uncontrollable degradability[246]. CMCS shows accelerated degradation rate which can be regulatedthrough different cross-linking extents by EDC, while retaining excellentmechanical properties. However, EDC cross-linked CMCS porous tubularscaffolds were fabricated for nerve regeneration which showed de-creased hydrophilicity and elastic modulus which is desirable for nerverepair [247]. Nanofibrous collagen-coated porous CMCS microcarrierswere successfully fabricated by a simple modified phase separationmethod and thereafter collagen anchoring-assembling. In vitro chondro-cyte culture revealed better cell attachment, proliferation, and differenti-ation on the CMC-MCs immobilised with self-assembled collagennanofibers. Cells were observed to grow into a tissue-like structureafter 7 days of culture. Thus the scaffolds prepared showed potentialfor application as injectable scaffolds for cell delivery in cartilage tissueengineering [73]. Gelatin/CM-chitosan/β-tricalcium phosphate compos-ite scaffolds were prepared using a green fabrication method, i.e.radiation-induced cross linking. Considering their excellent and adjust-able water retention capacity, highly interconnected porous network
f (A) 0%, (B) 5%, (C) 10%, (D) 20%, (E) 30%, and (F) 40%. The ultrasonic time was 20 min
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Fig. 21. Schiff-base formation between amino groups of CMCS and aldehyde groups of oxidized gellan gum (A). In gellan chains, cis-dihydroxyl of rhamnose was oxidized to dialdehyde,the addition of Ca2+ introduced ionic bonds between the carboxyl groups of gellan via electrostatic interaction, subsequently aldehyde groups and amino groups of CM-chitosan formedthe second network via the Schiff-base reaction. The cross linking mechanism of complex hydrogel (B). Gellan gum chains formed double helix conformations with Ca2+, and then CMCSchains link the aldehyde zones to the formation of a three dimensional network, that created the gel (reproduced with permission from [254]).
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structure, proper compressive strength and high porosity, the scaffoldsmet the criteria for bone tissue regeneration. Fig. 20 shows the SEMmor-phology of scaffoldswith different fractions ofβ-TCPwhen the ultrasonictime was set to 20 min. From the Fig it is clear that similar pore size(about 350 μm) were observed in the composite scaffold. Althoughsome agglomeration appeared in thewalls of scaffolds containing higherfractions of β-TCP which is in agreement with the literature [130]. Thebioactivity of a novel CMCS scaffolds with and without incorporatingmineral trioxide aggregate (MTA) in a tooth model was characterised.The deposition of HAP was significantly higher (P b 0.05) on MTA-coated CaC (CaMT) scaffold than that on Cross-linked CMCS scaffold(CaC). Therefore, it can be concluded that the bioactivity of the CMCSscaffold can be enhanced by incorporating MTA [242].
11.2. CMCS hydrogels
In the past decade, hydrogels have made significant progress in thedevelopment of tissue engineering scaffold [248]. A number of syntheticand natural polymers have been exploited in the last few years as hy-drogel biomaterials that include alginate [249], chondroitin sulfate[250], hyaluronic acid [251] and collagen [252]. The insufficient me-chanical performance and relative harsh gelation conditions for cell en-capsulation are the major limitations of hydrogels in tissue engineeringapplication [253]. A double-network complex hydrogelwith significant-ly improved gelation temperature andmechanical properties composedof oxidised gellan gum and CMCS by Ca2+ cross linking and Schiff reac-tion was prepared. Firstly, polymer chains were cleaved into smallersegments by oxidation reaction, which lead to the decrease of polymermolecular weight and the increase of cross linking aldehyde groups.This allowed for the formation of two entangled networks of differentcross linked polymers as described in Fig. 21A [254]. In the secondstep, the chemical cross linking reaction (Schiff-base formation) be-tween the pendant amino groups of CMCS and the aldehyde grouplead to the formation of a complex gel in the oxidised gellan as shownin Fig. 21B [254]. The results showed that the gelation temperaturewas lowered from 42 °C to below physiological temperature by
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
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oxidation, and further reduced by complexingwithCMCS. Also the com-plex hydrogel showed an increased compressive modulus of 278 kPa,and an ability to return to the original shape after release of the com-pressive load. Thus the hydrogels were promising biomaterials for carti-lage tissue engineering. A study on the synthesis and characterisation ofUV cross-linked hydrogels derived from novel water solublemethacrylated O-CMCS and polyethylene glycol diacrylate. The hydro-gel substrates similarly supported attachment and proliferation ofSmooth Muscle Cells (SMCs). The results from the study demonstratedthese hydrogels to be promising biomaterials for tissue regeneration[146]. A novel microgel class consisting of biocompatible CMCS andtemperature-sensitive PNIPAM was designed and synthesised by seed-ed emulsion polymerisation. The presence of PNIPAM inmicrogels con-tributed to the thermoresponsive property to CMCS while CMCS addedto the improved biocompatibility to the microgels which made thesemicrogels suitable for tissue regeneration purpose [236]. Yang et al.[106] fabricated CMCS/gelatin hydrogels by green method i.e. radiationcross linking method with excellent and adjustable water retention ca-pacity (10–700 g/g dry gel) and a similar compressive modulus withthat of soft tissue (10–200 kPa). These hybrid hydrogels have improvedflexibility, antimicrobial and water absorption capacity than gelatinhydrogels and superior handle ability and mechanical properties thanCM-chitosan hydrogels. Apart from these, they exhibited excellent andcontrollable degradability and good cytocompatibility which suggestedtheir potential application in tissue engineering and wound healing.
11.3. CMCS composites
In the recent years, composites have drawn attention of researcherstowards development of biocompatible and biodegradable compositesfor tissue regeneration. In this context, preparation of porousbiocomposites of nano-hydroxyapatite (n-HAP) and CMCS by porogenleaching method and their characterisation by IR, XRD, SEM and com-pressive strength has shown potential application for bone tissue engi-neering. The composites displayed porosity where the pores wereinterconnected and the compressive strength can exceed 21 MPa
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Fig. 22. Synthesis of CMCS-graft-D-glucuronic acid (reproduced with permission from [239]).
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which should be desirable for bone tissue engineering [237]. Similarly,Shi fabricated N-carboxyethyl chitosan/nanohydroxyapatite N-CECS/n-HAP composites for tissue-engineered trachea and investigate itsbiomechanical and biocompatible properties. The N-CECS/n-HAP com-posites exhibited satisfactory tensile strength and Young’s modulusvalues thereby confirming their potential for tissue-engineered trachea[255]. Also, recently, the effect of the n-HAP/CMCS composite with vas-cular endothelial growth factor (VEGF)-transfected bone marrow stro-mal cells (BMSCs) in a rabbit radial defect model was studied [238].This composite was biocompatible, nontoxic, promotes the infiltrationand formation of the microcirculation, stimulates bone defect repairand the degradation rate of the composite matched that of growingbone thus demonstrating its potential for bone defect repair.
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11.4. CMCS injectable gels, films and membranes
Apart from popular tissue engineering devices like scaffolds,hydrogels and composites, injectable gels, films and membranes havealso beendeveloped bydifferent researchers for tissue repair and regen-eration purpose. Development of gelatin and CMCS gels in situ in thepresence of tyrosinase and p-cresol where presence of n-HAP does nothamper in situ gelation of the polymers in physiological pH and temper-ature has been reported by Mishra and coworkers [41]. The resultsclearly indicate the potential of tyrosinase/p-cresol crosslinked CMCS–gelatin gel as injectable gel matrix for cell based bone tissue engineer-ing. Guangyuan et al. [73] preparedCMCS films and carboxymethylationand bimodal MW distribution were successfully combined to regulaterate of degradation of the films formed. The results displayed thatthese CMCS films with tunable degradation rates provide a powerfulmaterial system for tissue engineering. In this view, the developmentof CMCS-graft-D-glucuronic acid (CMCS-g-D-GA) membranes preparedby grafting CMCS with D-glucuronic acid by using EDC catalyst inwater by Jayakumar and coworkers is worth mentioning. The synthesismethod of the CMCS-g-D-GA membranes is shown in Fig. 22 [239]. Theresults of this investigation indicated that as themembraneswere capa-ble of having bioactivity, theywere expected to be suitable for tissue en-gineering purposes.
Fig. 23. In vivo gel stability study. Representativemacroscopic image of post-mortalmouseshowing the location and texture of iGel 24 h post-implantation (reproducedwith permis-sion from [41]).
12. CMCS applications in different tissues/organs
As the inherent ability of body to self-repair from disease or injuryand to regenerate depends on the specific tissue or organ system, itseems obvious that the choice of tissue engineering device or formula-tion largely depends on the tissue or organ being repaired, reconstruct-ed and/or regenerated [229]. In context of CMCS, its fabrication dependsgreatly on the target tissue for which the restorative device is beingmade which may include different organs like bone [256], nerve, carti-lage [73], vascular tissue [243], tooth [242] and even trachea [255].
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12.1. CMCS in bone tissue engineering
In recent years, CMCS based scaffolds, hydrogels and biocompositeshave gained importance in orthopedic research because of their poten-tial to minimise surgical invasiveness. When engineering bone tissue,the tissue engineering device must meet a number of requirementslike being biocompatible, biodegradable in suitable time window, itsdegradation products should be non toxic and capable of being easilyeliminated by the metabolic pathways, must support cell adhesionand growth and exhibit adequate mechanical stability [257,258]. In ad-dition to this, the biomaterial designed should allow new bone in-growths (osteoconductive) [259] and angiogenesis to supply thenewly formed tissue with nutrients while inducing bone formation(osteoinductive) [260]. In this view, the synthesis, characterisationand in vivo study of an enzymatically cross linked CMCS/gelatin/n-HAP injectable in mice reported by Mishra and coworkers have beendemonstrated as promising biomaterials thatmay be used in treating ir-regular small bone defects with minimal clinical invasion as well as forbone cell delivery. The in vivo injectability study of the i-Gels in murinemodels (Fig. 23) showed that the injected i-Gels were successfully re-trieved from the exact position of euthanisedmice. At the site of implan-tation no apparent sign tof inflammation (redness or edema) wasobservedwhich illustrated that the i-Gels were nonimmunogenic in na-ture. Also the yellowish colour instead of purple colour of the retrievedi-Gels can be attributed to the limitation of ample molecular oxygen in-side the human body. The in vivo study also demonstrated that the i-Gelswill have lesser gel strength in vivo as compared to in vitro situation[41]. Oliveira et al. reported high efficiency of DOX-loaded CMCS/
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Fig. 24.Optical microscopy images of Alizarin Red stained osteoblasts after culturing for 14 days on (a) Ti, (b) Ti-CMCS, (c) Ti-CMCS-VEGF, (d) Ti-HAC and (e) Ti-HAC-VEGF. Initial seedingwas carried outwith 3104 cells/cm2. (f) shows the Ti-CMCS substratewhich had been placed in cell culturemedium for 14 dayswithout cell seeding after Alizarin Red staining. Scale bar¼200 mm (reproduced with permission from [244]).
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RPAMAMdendrimer nanoparticles on being internalised by different celltypes, in vitro, promoting the osteogenic differentiation of rat bonemar-row stromal cells in tissue culture polystyrene dishes [261]. Similarly,the low generation poly(amidoamine) (PAMAM) dendrimerswith CMCS, CMCS/PAMAM dendrimer nanoparticles were surfaceengineered and loaded with DOX with the expectation of exhibitinghigh drug loading efficiency and non cytotoxicity compared to amine-terminated PAMAM dendrimers of high generation [262]. Xuefeng Hucarried out a study on an in vitro assessment of titanium functionalisedwith dopamine followed by CMCS or hyaluronic acid catechol (HAC)conjugated with vascular endothelial growth factor (VEGF). It provedto be promising alternative for enhanced osteointegration and inhibi-tion of bacterial inhibition. Fig. 24 displays the results of mineralisationof cells after two weeks culture on different substrates which wasassessed by staining by Alizarin Red. The degree of staining on the Ti-CMCS substrates (Fig. 24b) and Ti-HAC substrates (Fig. 24d) is notsubstantially higher than pristine Ti (a). Figure shows an absence ofpurplish red stains which was control experiment carried out with Ti-CMCS in cell culturemediumwithout cells. As shown by the dense cov-erage of calciumdeposits on the Ti-CMCS-VEGF (Fig) and Ti-CMCS-HAC(Fig) substrates mineralisation is greatly enhanced which can be attrib-uted to the presence of immobilised VEGF [244]. Also the developmentof hydroxyapatite scaffolds with macroporous structure and non-cytotoxicity which could efficiently support the adhesion, proliferation
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and osteogenic differentiation of rat bone marrow stromal cells in thepresence of 0.01mgml−1 DOX-loaded CMCS/PAMAMdendrimer nano-particles, in vitro has been reported by several research groups [262,263].
12.2. CMCS in cartilage tissue engineering
The choice of biomaterial is very critical for the success of tissue en-gineering approaches, particularly in cartilage repair [264]. The struc-tural similarity of chitosan with various glycosaminoglycans found inarticular cartilage hasmade it a suitable scaffolding biomaterial in artic-ular cartilage engineering [265,266]. Therefore, CMCS has been nowexperimented for different cartilage tissue engineering applications.Earlier, N,N-di-CMCS as delivery agent for bone morphogenetic proteinin the repair of articular cartilage has already been experimented [267].Guangyuan et al. [73] successfully fabricated nanofibrous collagen-coated porous CMCS microcarriers for cultivating cells and for applica-tion in cartilage tissue engineering as injectable scaffolds for cell deliv-ery. Recently, double-network complex hydrogel with significantlyimproved gelation temperature and mechanical properties have beenprepared by mixing oxidised gellan gum with CMCS which was foundto be promising material for cartilage tissue engineering [254]. Apartfrom these research studies, the ability of chitosan and CMCS to protectchondrocytes from apoptosis, significantly suppress the degeneration of
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cartilage in osteoarthritis and to protect chondrocytes from the IL-1betainduced catabolism have also been reported in literature [268,269]
12.3. CMCS in nerve tissue engineering
Researchers have investigated different biodegradable biomaterials,including collagen, polyglycolic acid, polylactic acid and co-polymers ofL-lactide and -caprolactone, for nerve repair [270,271]. A research studyon the degradation of covalently cross-linked CMCS in vitro for the firsttime and its potential application for peripheral nerve regeneration hasbeen carried out by Lu et al. [70]. The results revealed that the fasterdegradation of EDC-crosslinked CMCS than chitosan and decrease inthe hydrophilicity and elastic modulus of CMCS films are beneficial forapplication of CMCS in nerve repair. The study also suggested that theEDC cross-linked CMCS films enhanced the spread and provided agood proliferation substratum of Neuro-2a cells. Similarly the tunabledegradation rates of CMCS have been experimentally demonstrated re-cently by Guangyuan et al. [73]. He described the ability of Neuro-2acells to adhere and proliferate when cultured on binary and unaryCMCS films was found to be comparable to those on non-modifiedunary chitosan films which established CMCS as a promising materialfor neural tissue engineering. Recently, ability of CMCS to stimulate pro-liferation of Schwann cells in vitro by activating the intracellular signal-ing cascades of extracellular signal-regulated kinase (ERK1/2) andphosphatidylinositil-3 kinase (PI3K/Akt) has been reported by Bin Heand coworkers [272].
12.4. CMCS in wound healing
CMCS-basedmaterials, produced in varying formulations, have beenused in a number of wound healing applications. The inherent acceler-ativewoundhealing effects of CMCS on seconddegree burnmodels per-formed in rats was demonstrated in vitro and in vivo by Peng et al. [273].In another experimental study, N,CMCS was used as biomaterial to healdeep second-degree burnwounds. The results demonstrated that the N,CMCS was efficient in accelerating wound healing via activatingtransforming growth factor-β1/Smad3 signaling pathway [274]. The im-proved wound healing ability of CMCS of different MW has earlier beenexperimentally investigated by Chen et al. [20] by using a cell culturewhich showed positive results. The wound dressings of CMCS devel-oped by Qin and coworkers evaluated for wound healing abilityin vitro also displayed promising results [275]. CMCS, not only in its in-herent form, but also in many modified forms or in combination with
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Fig. 25. In vitro Ag release curves of AgNPs/PVA/CM-chitosan nanofibers in PBS(reproduced with permission from [201]).
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other biomaterials has demonstrated potential of enhanced woundhealing property. In fact, oligo-chitosan, N,O-CMCS and N,CMCS insheet and paste forms were evaluated in vitro for possible utilisationin wound dressing applications for wound healing [68]. Similarly, N,O-CMCS/Collagen matrixes containing chondroitin sulfate or an acellulardermal matrix developed by Chen et al. [84] demonstrated potentialas wound dressings for clinical applications. An in vivo experiment toevaluate the wound healing effect of water soluble chitosan/heparincomplex on the full thickness skin excision performed on the backs ofthe rat displayed that the complex is most effective in wound healing[276]. Also, in an in vivo animal experiment using a burn woundmodel water-soluble O-CMCS derivatives modified with furfuryl glyc-idyl ether (O-CMCS/FGE) displayed wound healing effects. The resultsof the study showed that O-CMCS/FGE would be a promising candidateas an anti-adhesionmaterial for biomedical applications [31]. A study onsubcutaneous implantation of the ornidazole loaded (PVA)/CMCS filmsprepared by blending/casting method in the surgical wound did notpromote any adverse effect. Over a long period of time, it is expectedthat these drug film would be absorbed and the wound would becicatrised by new forming tissues eventually [34]. Angiogenesis of full-thickness burnwounds repairedwith collagen-sulfonated CMCS porousscaffold encoding VEGF, DNA plasmids has been reported by Teng et al.[277]. Recently, Ag nanoparticles/(PVA)/CMCS nanofibers prepared byelectrospinning technique by Zhao et al. showed potential for wounddressing biomaterial. Fig. 25 shows the in vitro Ag released amount ofcrosslinks AgNPs/PVA/CMCS nanofibers as a function of time. At thethird hour the release of Ag in both the samples was fast and afterthat became relatively slow. Finally in both the samples after 24 h theconcentration of Ag+ reached equilibrium [201]. Apart from enhancedwound healing capacity, CMCS has also been experimentally proven ef-ficacious in preventing aswell as reducing post-operative surgical adhe-sions by several researchers [278–280].
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13. Tissue engineering application is often combined with drugdelivery strategy
In the area of regenerativemedicine and reconstructive surgery sub-stantial input has been made from developments in tissue engineeringand drug delivery technologies. The regeneration of functional tissue re-quires an appropriate microenvironment that closely mimics the hostsite for desired cellular responses which is typically provided by 3-D tis-sue engineering scaffold that acts as an architectural template [281]. Butrepair and reconstruction of diseased and/or damaged tissues/organsdemands therapy that cannot only provide mechanical and structuralintegrity to the tissue but also maintains sustained/controlled deliveryof therapeutics and/or growth factors in order to enhance the healingand regeneration process. While scaffold provides structural support,diffusivity to enable cellular infiltration and acts as substrate for tissuedifferentiation and organisation, the drugs/bioactive molecules embed-ded in it, cues for the surrounding tissues to heal and regenerate. Recentadvances in the field of tissue engineering and drug delivery have en-abled the design and fabrication of scaffolds that can deliver growth fac-tors/therapeutic agents in a more controlled fashion over a definedperiod of time. In fact the control over the regenerative and repairingpotential of tissue engineering scaffolds has dramatically improved inrecent years, mainly by using drug releasing scaffolds or by incorpora-tion of drug delivery devices in the tissue engineering scaffolds[282–284]. While previous approaches of developing drug delivery for-mulations mainly focused on the encapsulation or embedment of drugswithin the bulk phase and targeted delivery, recent strategies of tissueengineering open up the new possibility of constructing scaffolds thatcan provide the control over the sequestration and delivery of specificbioactive factors to enhance and guide the regeneration process [285,286]. Fig. 26 shows themore efficient and effective approach of combin-ing tissue engineering applications with drug delivery strategy for
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Fig. 26. Shows the more efficient and effective schematic approach of combining tissue engineering applications with drug delivery strategy for enhanced repairing and regeneration ofdamaged and/or diseased tissues/organs.
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enhanced repairing and regeneration of damaged and/or diseased tis-sues/organs.
Extensive overviews of recent research studies on development andapplications of three dimensional scaffolds with potential capabilitiesfor the controlled delivery of therapeutic drugs particularly osteogenicdrugs for bone regeneration are reported in literature [287,288]. Infact, this paradigm shift that has taken place to utilise the tissue engi-neering and drug delivery approaches towards the regeneration of den-tal, oral and craniofacial structures usingmatrices and scaffolds capableof controlled drug release has earlier been exhaustively reviewed [289].In this context, the researchwork for development of scaffolds with po-tential of drug delivery for neural [290] and bone [291,292] tissue engi-neering is worth mentioning. As far as CMCS is considered, not muchresearch work has been done in 3D scaffolding delivery system. ButCMCS/gelatin/n-HAP i-gels susceptible to tyrosinase/p-cresol mediatedin situ gelling at physiological temperature capable of treating irregularsmall bone defects have been investigated to exhibit potential of bonecell delivery [41]. Reves et al. [293] prepared microspheres cross linkedby twodifferentmethodswhichwere incorporated successfully into thecomposite scaffolds. The X-CMCS beads (obtained by carbodiimidechemistry cross linking) displayed good potential for use in bone tissueengineering applications in which degradation and local drug deliveryare desired as compared to Gen-X CMCS (cross linked by genepin)which showed poor degradation and drug release profiles.
14. CMCS based systems: current challenges and opportunities
CMCS based nanocarriers have become one of the most extensivelystudied nanometric drugdelivery platforms. But these trials are still lim-ited to experimental purposes and are not implicated widely asmarketed formulation. Despite of its increased aqueous solubility ascompared to nativemolecule chitosan, its significant hydrophobicity re-mains the major drawback responsible for its limited use in biomedicalfield. A drug requires compatible physicochemical properties of thema-trix polymer for developing a formulation successfully. CMCS alone or incombination with other polymers, metals, and metal oxides has been
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used either as blends, co-polymers or composites to alter the physico-chemical characteristics and degradation behavior. CMCS has beentrialled in almost all novel drug delivery systems of current drug deliv-ery approach due to its compatibility with wide range of polymers andits suitability with drugs of different properties. A number of researchworks have been reported where same drug being encapsulated in dif-ferent CMCS formulations. Therefore, during the process of formulationdevelopment for a particular drug, such information can be used forcomparative efficacy improvement studies between two or more for-mulations. Also, owing to its enhanced bioactivity and its ability to pro-mote osteogenesis, CMCS is being studied at preclinical and clinical levelensuring its potentiality in tissue engineering. CMCS can chelate calciumfrom mineralizing solution containing calcium and phosphate and in-duce calcium phosphate or HAP formation thereby making it suitablefor bone and cartilage regeneration. But being polymer, the mechanicalproperties of CMCS are not compatible enough with the host tissuesparticularly with bone which restricts its usage as such in tissue engi-neering applications. Thus, efforts to improve themechanical propertiesof CMCS based formulations are essential for this type of application.Therefore, CMCS has often been exploited in the form of either compos-ites with ceramics or HAP or in combination with some other polymerswith better mechanical strength in order to make it suitable for tissueengineering. CMCS based 3D scaffolds mimic the extracellular matrixand have proved to be highly useful formulations for repair and recon-struction of damaged organs in general and tissues in particular. But de-spite of its versatility, CMCS based formulations are not commercialisedwidely in clinical drug delivery and tissue engineering practice. Also,there is a paucity of studies regarding the development of technologicalstrategies to integrate and position drug delivery devices with asubmicrometric spatial resolution within the scaffolds. Nevertheless,numerous studies involving study of CMCS as drug delivery carriersand tissue engineering devices are being patented and some are underpreclinical or clinical investigation. The main goals are to improvetheir stability in the biological environment, tomediate the bio distribu-tion of active compounds, enhanced drug loading, targeting, transport,release, and interaction with biological barriers. The cytotoxicity of
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nanoparticles or their degradation products remains a major problem,and improvements in biocompatibility obviously are a major concernof future research in CMCS based drug delivery.
15. Summary and future remarks
CMCS based drug delivery and tissue engineering formulations re-search has reached significant maturity in the last few years. There arenumerous compelling evidences for the potential of CMCS as biomateri-al for many novel challenging drug delivery and tissue engineering ap-plications. CMCS has been shown to improve the dissolution rate ofmany otherwise poorly soluble drugs, and thus, can be exploited for bio-availability improvement of drugs. Various therapeutic agents, such asanticancer, anti-inflammatory, antibiotics, antithrombotic, proteins,and amino acids have been effectively incorporated in CMCS-based sys-tems to increase bioavailability and to achieve targeted and/or con-trolled release. While this concept is still mostly within the academicframe, results from several laboratories studies confirm enhancementsin the bioavailability of these macromolecules to a level that might suf-fice for industrial development. Various CMCS based formulations pre-sented herein this review like hydrogels, microspheres, nanoparticles,films, fibers, composites and scaffolds can be helpful in deciding thecontext of using CMCS in selectively capturing a therapeutic payloadand control release in a target site as well as for tissue engineering pur-poses. CMCS's innate tissue engineering potential as a biopolymer togenerate structures with predictable pore sizes and controllable degra-dation rates makes it particularly suitable for bone and cartilage regen-eration. In concluding remarks, CMCS, the polymer with intact orderived properties makes it suitable to use and prepare all kinds ofnovel drug delivery and tissue engineering formulations. From theabove investigations itmay be concluded that CMCS is indeed a versatilebiodegradable polymer derivative having tremendous potential inpharmaceutical drug delivery and tissue engineering application innear future.
Acknowledgement
The authors would like to thankMinistry of Human Resource Devel-opment, Govt. of India for providing fellowship for research. The authorsalsowant to acknowledge the Director, Motilal Nehru National Instituteof Technology, Allahabad, India for providing other necessary facilitiesfor research work.
References
[1] L. Casettari, D. Vllasaliu, E. Castagnino, S. Stolnik, S. Howdle, L. Illum, PEGylated chi-tosan derivatives: synthesis, characterizations and pharmaceutical applications,Prog. Polym. Sci. 37 (2012) 659–685.
[2] D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K. Maiti, Enzymaticallycrosslinked carboxymethyl-chitosan/gelatin/nano-hydroxyapatite injectable gelsfor in situ bone tissue engineering application, Mater. Sci. Eng. C 31 (2011)1295–1304.
[3] L.A. Caetano, R. Amaral, L. Figueiredo, A.J. Almeida, L.M.D. Goncalves, Chitosan-alginate microparticulate system for an alternative route of administration ofBCG vaccine, Biomater. Nanobiotechnol. Tissue Eng. (2013).
[4] K. Tomihata, Y. Ikada, Preparation of cross-linked hyaluronic acid films of lowwater content, Biomaterials 18 (1997) 189–195.
[5] D. Hawary, M.Motaleb, H. Farag, O. Guirguis, M. Elsabee,Water-soluble derivativesof chitosan as a target delivery system of 99mTc to some organs in vivo for nuclearimaging and biodistribution, J. Radioanal. Nucl. Chem. 290 (2011) 557–567.
[6] Z. Shi, K.G. Neoh, E.T. Kang, B. Shuter, S.C. Wang, C. Poh, (Carboxymethyl)chitosan-modified superparamagnetic iron oxide nanoparticles for magnetic resonance im-aging of stem cells, ACS Appl. Mater. Interfaces 1 (2008) 328–335.
[7] Y. Liu, X. Fu, Y. Bai, M. Zhai, Y. Liao, J. Liao, Improvement of reproducibility and sen-sitivity of CE analysis by using the capillary coated dynamically withcarboxymethyl chitosan, Anal. Bioanal. Chem. 399 (2011) 2821–2829.
[8] A. Bava, F. Cappellini, E. Pedretti, F. Rossi, E. Caruso, E. Vismara, Heparin andcarboxymethylchitosan metal nanoparticles: an evaluation of their cytotoxicity,Biomed. Res. Int. (2013) 10.
[9] M. Xie, H.-H. Liu, P. Chen, Z.-L. Zhang, X.-H. Wang, Z.-X. Xie, CdSe/ZnS-labeledcarboxymethyl chitosan as a bioprobe for live cell imaging, Chem. Commun. 44(2005) 5518–5520.
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
ED P
RO
OF
[10] L.-X. Zhang, X.-H. Cao, Y.-B. Zheng, Y.-Q. Li, Covalent modification of single glassconical nanopore channel with 6-carboxymethyl-chitosan for pH modulated ioncurrent rectification, Electrochem. Commun. 12 (2010) 1249–1252.
[11] Q. Xu, C. Mao, N.-N. Liu, J.-J. Zhu, J. Sheng, Direct electrochemistry of horseradishperoxidase based on biocompatible carboxymethyl chitosan-gold nanoparticlenanocomposite, Biosens. Bioelectron. 22 (2006) 768–773.
[12] G.Wang, G. Lu, Q. Ao, Y. Gong, X. Zhang, Preparation of cross-linked carboxymethylchitosan for repairing sciatic nerve injury in rats, Biotechnol. Lett. 32 (2010) 59–66.
[13] A.P. Zhu, N. Fang, M.B. Chan-Park, V. Chan, Interaction between O-carboxymethylchitosan and dipalmitoyl-sn-glycero-3-phosphocholine bilayer,Biomaterials 26 (2005) 6873–6879.
[14] Y.-f. Zhang, P. Yin, X.-q. Zhao, J. Wang, J. Wang, C.-d. Wang, O-Carboxymethyl-chi-tosan/organosilica hybrid nanoparticles as non-viral vectors for gene delivery,Mater. Sci. Eng. C 29 (2009) 2045–2049.
[15] P. Li, D.H. Liu, L. Miao, C.X. Liu, X.L. Sun, Y.J. Liu, N. Zhang, A pH-sensitive multifunc-tional gene carrier assembled via layer-by-layer technique for efficient gene deliv-ery, Int. J. Nanomedicine 7 (2012) 925–939.
[16] B. Shi, Z. Shen, H. Zhang, J. Bi, S. Dai, Exploring N-imidazolyl-O-carboxymethyl chi-tosan for high performance gene delivery, Biomacromolecules 13 (2011) 146–153.
[17] A. Khanjari, I.K. Karabagias, M.G. Kontominas, Combined effect of N,O-carboxymethylchitosan and oregano essential oil to extend shelf life and control Listeriamonocytogenes in raw chickenmeat fillets, LWT—Food Sci. Technol. 53 (2013) 94–99.
[18] Y. Luo, Z. Teng, X.Wang, Q. Wang, Development of carboxymethyl chitosan hydro-gel beads in alcohol-aqueous binary solvent for nutrient delivery applications,Food Hydrocoll. 31 (2013) 332–339.
[19] X.-G. Chen, Z. Wang, W.-S. Liu, H.-J. Park, The effect of carboxymethyl-chitosan onproliferation and collagen secretion of normal and keloid skin fibroblasts, Biomate-rials 23 (2002) 4609–4614.
[20] S.-C. Chen, Y.-C. Wu, F.-L. Mi, Y.-H. Lin, L.-C. Yu, H.-W. Sung, A novel pH-sensitivehydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked bygenipin for protein drug delivery, J. Control. Release 96 (2004) 285–300.
[21] D. Zhao, J. Huang, S. Hu, J. Mao, L. Mei, Biochemical activities of N, O-carboxymethylchitosan from squid cartilage, Carbohydr. Polym. 85 (2011) 832–837.
[22] L.X. Fei, G.Y. Lin, Y.D. Zhi, Z. Li, Y.K. De, Antibacterial action of chitosan andcarboxymethylated chitosan, J. Appl. Polym. Sci. 79 (2001) 1324–1335.
[23] F. Seyfarth, S. Schliemann, P. Elsner, U.C. Hipler, Antifungal effect of high- and low-molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligo-saccharide and N-acetyl-D-glucosamine against Candida albicans, Candida kruseiand Candida glabrata, Int. J. Pharm. 352 (2008) 139–148.
[24] I.B. Leonor, E.T. Baran, M. Kawashita, R.L. Reis, T. Kokubo, T. Nakamura, Growth of abonelike apatite on chitosan microparticles after a calcium silicate treatment, ActaBiomater. 4 (2008) 1349–1359.
[25] R.A.A. Muzzarelli, Chitins and chitosans for the repair of wounded skin, nerve, car-tilage and bone, Carbohydr. Polym. 76 (2009) 167–182.
[26] I.M. El-Sherbiny, Enhanced pH-responsive carrier system based on alginate andchemically modified carboxymethyl chitosan for oral delivery of protein drugs:preparation and in-vitro assessment, Carbohydr. Polym. 80 (2010) 1125–1136.
[27] D. Fu, B. Han, W. Dong, Z. Yang, Y. Lv, W. Liu, Effects of carboxymethyl chitosan onthe blood system of rats, Biochem. Biophys. Res. Commun. 408 (2011) 110–114.
[28] M. Zheng, B. Han, Y. Yang, W. Liu, Synthesis, characterization and biological safetyof O-carboxymethyl chitosan used to treat Sarcoma 180 tumor, Carbohydr. Polym.86 (2011) 231–238.
[29] J. Zhou, J.M. Lee, P. Jiang, S. Henderson, T.D.G. Lee, Reduction in postsurgical adhe-sion formation after cardiac surgery by application of N, O-carboxymethyl chito-san, J. Thorac. Cardiovasc. Surg. 140 (2010) 801–806.
[30] X. Yin, J. Chen, W. Yuan, Q. Lin, L. Ji, F. Liu, Preparation and antibacterial activity ofSchiff bases from O-carboxymethyl chitosan and para-substituted benzaldehydes,Polym. Bull. 68 (2012) 1215–1226.
[31] H.-N. Na, S.-H. Park, K.-I. Kim, M. Kim, T.-I. Son, Photocurable O-carboxymethyl chi-tosan derivatives for biomedical applications: synthesis, in vitro biocompatibility,and their wound healing effects, Macromol. Res. 20 (2012) 209–1209.
[32] X. Li, S. Chen, B. Zhang, M. Li, K. Diao, Z. Zhang, In situ injectable nano-compositehydrogel composed of curcumin, N, O-carboxymethyl chitosan and oxidized algi-nate for wound healing application, Int. J. Pharm. 437 (2012) 110–119.
[33] N.A. Gujarathi, B.R. Rane, J.K. Patel, pH sensitive polyelectrolyte complex of O-carboxymethyl chitosan and poly (acrylic acid) cross-linked with calcium forsustained delivery of acid susceptible drugs, Int. J. Pharm. 436 (2012) 418–425.
[34] X.-Y. Gong, Y.-H. Yin, Z.-J. Huang, B. Lu, P.-H. Xu, H. Zheng, Preparation, character-ization and in vitro release study of a glutathione-dependent polymeric prodrugCis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan, Int. J.Pharm. 436 (2012) 240–247.
[35] K.-H. Liu, B.-R. Chen, S.-Y. Chen, D.-M. Liu, Self-assembly behavior and doxorubicin-loading capacity of acylated carboxymethyl chitosans, J. Phys. Chem. B 113 (2009)11800–11807.
[36] Z. Teng, Y. Luo, Q. Wang, Carboxymethyl chitosan–soy protein complex nanoparti-cles for the encapsulation and controlled release of Vitamin D3, Food Chem. 141(2013) 524–532.
[37] Z. Wang, T. Yue, Y. Yuan, R. Cai, C. Niu, C. Guo, Kinetics of adsorption of bovineserum albumin on magnetic carboxymethyl chitosan nanoparticles, Int. J. Biol.Macromol. (2013) 57–65.
[38] L.-C. Wang, X.-G. Chen, D.-Y. Zhong, Q.-C. Xu, Study on poly(vinyl alcohol)/carboxymethyl-chitosan blend film as local drug delivery system, J. Mater. Sci.Mater. Med. 18 (2007) 1125–1133.
[39] S.-H. Yu, F.-L. Mi, S.-S. Shyu, C.-H. Tsai, C.-K. Peng, J.-Y. Lai, Miscibility, mechanicalcharacteristic and platelet adhesion of 6-O-carboxymethylchitosan/polyurethanesemi-IPN membranes, J. Membr. Sci. 276 (2006) 68–80.
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
30 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
UNCO
RREC
[40] R. Budiraharjo, K.G. Neoh, E.T. Kang, Hydroxyapatite-coated carboxymethyl chito-san scaffolds for promoting osteoblast and stem cell differentiation, J. Colloid Inter-face Sci. 366 (2012) 224–232.
[41] D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K. Maiti, Enzymaticallycrosslinked carboxymethyl-chitosan/gelatin/nano-hydroxyapatite injectable gelsfor in situ bone tissue engineering application, Mater. Sci. Eng. C 31 (2011)1295–1304.
[42] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Biomedical applications ofcarboxymethyl chitosans, Carbohydr. Polym. 91 (2013) 452–466.
[43] V.K. Mourya, N.N. Inamdar, A. Tiwari, Carboxymethyl chitosan and its applications,Adv. Mater. Lett. 1 (2010) 11–33.
[44] M. Rinaudo, G. Pavlov, J. Desbrières, Solubilization of chitosan in strong acid medi-um, Int. J. Polym. Anal. Charact. 5 (1999) 267–276.
[45] M. Fan, Q. Hu, K. Shen, Preparation and structure of chitosan soluble in wide pHrange, Carbohydr. Polym. 78 (2009) 66–71.
[46] G.E. Bannikova, P.P. Sukhanova, G.A. Vikhoreva, V.P. Varlamov, L.S. Gal'braikh, Hy-drolysis of chitosan sulfate with an enzyme complex from Streptomyceskurssanovii, Appl. Biochem. Microbiol. 38 (2002) 413–415.
[47] A. Polnok, J.C. Verhoef, G. Borchard, N. Sarisuta, H.E. Junginger, In vitro evaluationof intestinal absorption of desmopressin using drug-delivery systems based onsuperporous Hydrogels, Int. J. Pharm. 269 (2004) 303–310.
[48] P. Wongpanit, N. Sanchavanakit, P. Pavasant, P. Supaphol, S. Tokura, R. Rujiravanit,Preparation and characterization of microwave-treated carboxymethyl chitin andcarboxymethyl chitosan films for potential use in wound care application,Macromol. Biosci. 5 (2005) 1001–1012.
[49] S. Richardson, L. Gorton, Characterisation of the substituent distribution in starchand cellulose derivatives, Anal. Chim. Acta. 497 (2003) 27–65.
[50] J.-W. Shim, Y.-C. Nho, Preparation of poly(acrylic acid)-chitosan hydrogels bygamma irradiation and in vitro drug release, J. Appl. Polym. Sci. 90 (2003)3660–3667.
[51] F. Yao, C. Liu, W. Chen, Y. Bai, Z. Tang, K. Yao, Synthesis and characterization of chi-tosan grafted oligo(L-lactic acid), Macromol. Biosci. 3 (2003) 653–656.
[52] M. Yazdani-Pedram, J. Retuert, Homogeneous grafting reaction of vinyl pyrrolidoneonto chitosan, J. Appl. Polym. Sci. 63 (1997) 1321–1326.
[53] A.M. de Campos, Y. Diebold, E.L. Carvalho, A. Sánchez, M.J. Alonso, Chitosan nano-particles as new ocular drug delivery systems: in vitro stability, in vivo fate, andcellular toxicity, Pharm. Res. 21 (2004) 803–810.
[54] C. Prego, P. Paolicelli, B. Díaz, S. Vicente, A. Sánchez, Á. González-Fernández,Chitosan-based nanoparticles for improving immunization against hepatitis B in-fection, Vaccine 28 (2010) 2607–2614.
[55] L. Zhao, E.F. Burguera, H.H.K. Xu, N. Amin, H. Ryou, D.D. Arola, Fatigue and humanumbilical cord stem cell seeding characteristics of calcium phosphate-chitosan-biodegradable fiber scaffolds, Biomaterials 31 (2010) 840–847.
[56] Q. Lian, D. Li, Z. Jin, J. Wang, A. Li, Z. Wang, Fabrication and in vitro evaluation ofcalcium phosphate combined with chitosan fibers for scaffold structures, J. Bioact.Compat. Polym. 24 (2009) 113–124.
[57] X.-G. Chen, H.-J. Park, Chemical characteristics of O-carboxymethyl chitosans relat-ed to the preparation conditions, Carbohydr. Polym. 53 (2003) 355–359.
[58] L. Chen, Y. Du, X. Zeng, Relationships between the molecular structure andmoisture-absorption and moisture-retention abilities of carboxymethyl chitosan:II. Effect of degree of deacetylation and carboxymethylation, Carbohydr. Res. 338(2003) 333–340.
[59] A. Shoukry, W. Hosny, Coordination properties of N, O-carboxymethyl chitosan(NOCC). Synthesis and equilibrium studies of some metal ion complexes. Ternarycomplexes involving Cu(II) with (NOCC) and some biorelevant ligand, Cent. Eur. J.Chem. 10 (2012) 59–70.
[60] Z. Zhong, P. Li, R. Xing, S. Liu, Antimicrobial activity of hydroxylbenzenesulfonailidesderivatives of chitosan, chitosan sulfates and carboxymethyl chitosan, Int. J. Biol.Macromol. 45 (2009) 163–168.
[61] R.A.A. Muzzarelli, C. Muzzarelli, R. Tarsi, M. Miliani, F. Gabbanelli, M. Cartolari, Fungi-static activity of modified chitosans against Saprolegnia parasitica, Biomacromolecules2 (2000) 165–169.
[62] Y. Wen, Z. Tan, F. Sun, L. Sheng, X. Zhang, F. Yao, Synthesis and characterization ofquaternized carboxymethyl chitosan/poly(amidoamine) dendrimer core-shellnanoparticles, Mater. Sci. Eng. C 32 (2012) 2026–2036.
[63] M.W. Sabaa, N.A. Mohamed, R.R. Mohamed, N.M. Khalil, S.M. Abd El Latif, Synthe-sis, characterization and antimicrobial activity of poly (N-vinyl imidazole) graftedcarboxymethyl chitosan, Carbohydr. Polym. 79 (2010) 998–1005.
[64] L. Sun, Y. Du, L. Fan, X. Chen, J. Yang, Preparation, characterization and antimicro-bial activity of quaternized carboxymethyl chitosan and application as pulp-cap,Polymer 47 (2006) 1796–1804.
[65] N.T. An, N.T. Dong, P.T.B. Hanh, T.T.Y. Nhi, D.A. Vu, D.T.N. Que, Silver-N-carboxymethyl chitosan nanocomposites: synthesis and its antibacterial activities,J. Bioterror. Biodef. 1 (2010) 102.
[66] A. El.Shafei, A. Abou-Okeil, ZnO/carboxymethyl chitosan bionano-composite to im-part antibacterial and UV protection for cotton fabric, Carbohydr. Polym. 83 (2011)920–925.
[67] S. Tao, S. Gao, Y. Zhou, M. Cao, W. Xie, H. Zheng, Preparation of carboxymethyl chi-tosan sulfate for improved cell proliferation of skin fibroblasts, Int. J. Biol.Macromol. 54 (2013) 160–165.
[68] M.S.B. Abdull Rasad, A.S. Halim, K. Hashim, A.H.A. Rashid, N. Yusof, S. Shamsuddin,In vitro evaluation of novel chitosan derivatives sheet and paste cytocompatibilityon human dermal fibroblasts, Carbohydr. Polym. 79 (2010) 1094–1100.
[69] W. Dong, B. Han, Y. Feng, F. Song, J. Chang, H. Jiang, Pharmacokinetics and biodeg-radation mechanisms of a versatile carboxymethyl derivative of chitosan in rats:in vivo and in vitro evaluation, Biomacromolecules 11 (2010) 1527–1533.
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
ED P
RO
OF
[70] G. Lu, L. Kong, B. Sheng, G. Wang, Y. Gong, X. Zhang, Degradation of covalentlycross-linked carboxymethyl chitosan and its potential application for peripheralnerve regeneration, Eur. Polym. J. 43 (2007) 3807–3818.
[71] R. Bhatt, B. Panchal, K. Patel, V.K. Shina, U. Trivedi, Synthesis, characterization,and biodegradation of carboxymethylchitosan-g-medium chain lengthpolyhydroxyalkanoates, J. Appl. Polym. Sci. 110 (2008) 975–982.
[72] J.M. Wasikiewicz, H. Mitomo, N. Nagasawa, T. Yagi, M. Tamada, F. Yoshii, Radiationcrosslinking of biodegradable carboxymethylchitin and carboxymethylchitosan, J.Appl. Polym. Sci. 102 (2006) 758–767.
[73] L. Guangyuan, S. Baiyang, W. Gan, W. Yujun, G. Yandao, Z. Xiufang, Controlling thedegradation of covalently cross-linked carboxymethyl chitosan utilizing bimodalmolecular weight distribution, J. Biomater. Appl. 23 (2009) 435–451.
[74] W. Dong, B. Han, K. Shao, Z. Yang, Y. Peng, Y. Yang, Effects of molecular weightson the absorption, distribution and urinary excretion of intraperitoneally adminis-trated carboxymethyl chitosan in rats, J. Mater. Sci. Mater. Med. 23 (2012)2945–2952.
[75] S. Tokura, S.-I. Nishimura, N. Sakairi, N. Nishi, Biological activities of biodegradablepolysaccharide, Macromol. Symp. 101 (1996) 389–396.
[76] Z. Yang, B. Han, D. Fu, W. Liu, Acute toxicity of high dosage carboxymethyl chitosanand its effect on the blood parameters in rats, J. Mater. Sci. Mater. Med. 23 (2012)457–462.
[77] A. Anitha, V.V. Divya Rani, R. Krishna, V. Sreeja, N. Selvamurugan, S.V. Nair, Synthe-sis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N, O-carboxymethyl chitosan nanoparticles, Carbohydr.Polym. 78 (2009) 672–677.
[78] S.P. Chakraborty, S.K. Sahu, P. Pramanik, S. Roy, Biocompatibility of folate-modifiedchitosan nanoparticles, Asian Pac. J. Trop. Biomed. 2 (2012) 215–219.
[80] M. Huo, Y. Zhang, J. Zhou, A. Zou, J. Li, Formation, microstructure, biodistributionand absence of toxicity of polymeric micelles formed by N-octyl-N, O-carboxymethyl chitosan, Carbohydr. Polym. 83 (2011) 1959–1969.
[81] R. Kennedy, D.J. Costain, V.C. McAlister, T.D. Lee, Prevention of experimental post-operative peritoneal adhesions by N, O-carboxymethyl chitosan, Surgery 120(1996) 866–870.
[82] D.J. Costain, R. Kennedy, C. Ciona, V.C. McAlister, T.D.G. Lee, Prevention of postsur-gical adhesions with N, O-carboxymethyl chitosan: examination of the most effica-cious preparation and the effect of N, O-carboxymethyl chitosan on postsurgicalhealing, Surgery 121 (1997) 314–319.
[83] J. Zhou, J.M. Lee, P. Jiang, S. Henderson, T.D.G. Lee, Reduction in postsurgical adhe-sion formation after cardiac surgery by application of N, O-carboxymethyl chito-san, J. Thorac. Cardiovasc. Surg. 140 (2010) 801–806.
[84] R.-N. Chen, G.-M. Wang, C.-H. Chen, H.-O. Ho, M.-T. Sheu, Development of N, O-(Carboxymethyl)chitosan/collagen matrixes as a wound dressing,Biomacromolecules 7 (2006) 1058–1064.
[85] A.-P. Zhu, N. Fang, Adhesion dynamics, morphology, and organization of 3 T3 fibro-blast on chitosan and its derivative: the effect of O-carboxymethylation,Biomacromolecules 6 (2005) 2607–2614.
[87] R.A.A. Muzzarelli, M. Weckx, O. Filippini, C. Lough, Characteristic properties of N-carboxybutyl chitosan, Carbohydr. Polym. 11 (1989) 307–320.
[88] C. Yu, L. Yun-fei, T. Hui-min, J. Jian-xin, Synthesis and characterization of a novelsuperabsorbent polymer of N, O-carboxymethyl chitosan graft copolymerizedwith vinyl monomers, Carbohydr. Polym. 75 (2009) 287–292.
[89] R.A.A. Muzzarelli, V. Ramos, V. Stanic, B. Dubini, M.M. Belmonte, G. Tosi, Osteogen-esis promoted by calcium phosphate N, N-dicarboxymethyl chitosan, Carbohydr.Polym. 36 (1998) 267–276.
[90] H.C. Ge, D.K. Luo, Preparation of carboxymethyl chitosan in aqueous solution undermicrowave irradiation, Carbohydr. Res. 340 (2005) 1351–1356.
[91] H.T. Pang, X.G. Chen, H.J. Park, D.S. Cha, J.F. Kennedy, Preparation and rheologicalproperties of deoxycholate-chitosan and carboxymethyl-chitosan in aqueous sys-tems, Carbohydr. Polym. 69 (2007) 419–425.
[92] X. Kong, Simultaneous determination of degree of deacetylation, degree of substi-tution and distribution fraction of –COONa in carboxymethyl chitosan by potenti-ometric titration, Carbohydr. Polym. 88 (2012) 336–341.
[93] Y. Chen, Y.F. Liu, H.M. Tan, J.X. Jiang, Synthesis and characterization of a novel su-perabsorbent polymer of N, O-carboxymethyl chitosan graft copolymerized withvinyl monomers, Carbohydr. Polym. 75 (2009) 287–292.
[94] L. Wang, A. Wang, Adsorption behaviors of Congo red on the N, O-carboxymethyl-chitosan/montmorillonite nanocomposite, Chem. Eng. J. 143 (2008) 43–50.
[95] J. Chen, J. Sun, L. Yang, Q. Zhang, H. Zhu, H. Wu, Preparation and characteriza-tion of a novel IPN hydrogel memberane of poly(N-isopropylacrylamide)/carboxymethyl chitosan (PNIPAAM/CMCS), Radiat. Phys. Chem. 76 (2007)1425–1429.
[97] L. Chen, Z. Tian, Y. Du, Synthesis and pH sensitivity of carboxymethyl chitosan-based polyampholyte hydrogels for protein carrier matrices, Biomaterials 25(2004) 3725–3732.
[98] L. Yin, L. Fei, F. Cui, C. Tang, C. Yin, Superporous hydrogels containing poly(acrylicacid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer net-works, Biomaterials 28 (2007) 1258–1266.
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
31L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
UNCO
RREC
[99] B.-L. Guo, Q.-Y. Gao, Preparation and properties of a pH/temperature-responsivecarboxymethyl chitosan/poly(N-isopropylacrylamide)semi-IPN hydrogel for oraldelivery of drugs, Carbohydr. Res. 342 (2007) 2416–2422.
[100] S.S. Vaghani, M.M. Patel, C.S. Satish, Synthesis and characterization of pH-sensitivehydrogel composed of carboxymethyl chitosan for colon targeted delivery ofornidazole, Carbohydr. Res. 347 (2012) 76–82.
[101] S.-H. Yu, F.-L. Mi, S.-S. Shyu, C.-H. Tsai, C.-K. Peng, J.-Y. Lai, Miscibility, mechanicalcharacteristic and platelet adhesion of 6-O-carboxymethylchitosan/polyurethanesemi-IPN membranes, J. Membr. Sci. 276 (2006) 68–80.
[102] R. Mohamed, R. Seoudi, M.W. Sabaa, Synthesis and characterization of antibacterialsemi-interpenetrating carboxymethyl chitosan/poly (acrylonitrile) Hydrogels, Cel-lulose 19 (2012) 947–958.
[103] S. Saiki, N. Nagasawa, A. Hiroki, N. Morishita, M. Tamada, Y. Muroya, ESR study oncarboxymethyl chitosan radicals in an aqueous solution, Radiat. Phys. Chem. 79(2010) 276–278.
[104] A. Hiroki, H.T. Tran, N. Nagasawa, T. Yagi, M. Tamada, Metal adsorption ofcarboxymethyl cellulose/carboxymethyl chitosan blend hydrogels prepared byGamma irradiation, Radiat. Phys. Chem. 78 (2009) 1076–1080.
[105] M.Wang, L. Xu, M. Zhai, J. Peng, J. Li, G. Wei, γ-ray radiation-induced synthesis andFe(III) ion adsorption of carboxymethylated chitosan hydrogels, Carbohydr. Polym.74 (2008) 498–503.
[106] C. Yang, L. Xu, Y. Zhou, X. Zhang, X. Huang, M. Wang, A green fabrication approachof gelatin/CM-chitosan hybrid hydrogel for wound healing, Carbohydr. Polym. 82(2010) 1297–1305.
[107] Y. Zhou, Y. Zhao, L. Wang, L. Xu, M. Zhai, S. Wei, Radiation synthesis and character-ization of nanosilver/gelatin/carboxymethyl chitosan hydrogel, Radiat. Phys. Chem.81 (2012) 553–560.
[108] L.-M. Yang, J. Chen, S. Wang, Preparation and characterization of N-isopropylacrylamide/carboxymethylated chitosan hydrogel, J. Shanghai Univ.(Eng. Ed.) 14 (2010) 106–110.
[109] H. Zheng, X. Zhang, F. Xiong, Z. Zhu, B. Lu, Y. Yin, Preparation, characterization, andtissue distribution in mice of lactosaminated carboxymethyl chitosan nanoparti-cles, Carbohydr. Polym. 83 (2011) 1139–1145.
[111] X.-F. Liang, J.-Y. Hu, F.-H. Chen, Z.-H. Li, J. Chang, Characterization of chitosanpolymeric ethosomes capable of encapsulating hydrophobic and hydrophilicdrugs prepared by a microemulsion method, Acta Phys.-Chim. Sin. 28 (2012)897–902.
[112] W. Yinsong, L. Lingrong, W. Jian, Q. Zhang, Preparation and characterization of self-aggregated nanoparticles of cholesterol-modifed O-carboxymethyl chitosan conju-gates, Carbohydr. Polym. 69 (2007) 597–606.
[113] W. Zhang, H. Shen, M.Q. Xie, L. Zhuang, Y.Y. Deng, S.L. Hu, Synthesis ofcarboxymethyl-chitosan-bound magnetic nanoparticles by the spraying co-precipitation method, Scri. Mater. 59 (2008) 211–214.
[114] V.H. Pereira, A.J. Salgado, J.M. Oliveira, S.R. Cerqueira, A.M. Frias, J.S. Fraga, In vivobiodistribution of carboxymethylchitosan/poly(amidoamine) dendrimer nanopar-ticles in rats, J. Bioact. Compat. Polym. 26 (2011) 619–627.
[115] J. Ji, S. Hao, J. Dong, D. Wu, B. Yang, Y. Xu, Preparation, evaluation, and in vitro re-lease study of o-carboxymethyl chitosan nanoparticles loaded with gentamicinand salicylic acid, J. Appl. Polym. Sci. 123 (2012) 1684–1689.
[116] Y. Liu, X. Cheng, Q. Dang, F. Ma, X. Chen, H. Park, Preparation and evaluation ofoleoyl-carboxymethy-chitosan (OCMCS) nanoparticles as oral protein carriers, J.Mater. Sci. Mater. Med. 23 (2012) 375–384.
[117] Y. Li, S. Zhang, X. Meng, X. Chen, G. Ren, The preparation and characterization of anovel amphiphilic oleoyl-carboxymethyl chitosan self-assembled nanoparticles,Carbohydr. Polym. 83 (2011) 130–136.
[118] J. Wang, J.-S. Chen, J.-Y. Zong, D. Zhao, F. Li, R.-X. Zhuo, Calcium carbonate/carboxymethyl chitosan hybrid microspheres and nanospheres for drug delivery,J. Phys. Chem. C 114 (2010) 18940–18945.
[119] L. Ma, M. Liu, X. Shi, pH- and temperature-sensitive self-assembly microcapsules/microparticles: synthesis, characterization, in vitro cytotoxicity, and drug releaseproperties, J. Biomed. Mater. Res. B 100B (2012) 305–313.
[120] A. Dusza, M. Wojtyniak, N. Nedelko, A. Slawska-Waniewska, J.M. Greneche, C.A.Rodrigues, Magnetic behavior of O-carboxymethylchitosan bounded with ironoxide particles, IEEE Trans. Magn. 46 (2010) 459–462.
[121] B. Liu, J. Luo, X. Wang, J. Lu, H. Deng, R. Sun, Alginate/quaternized carboxymethylchitosan/clay nanocomposite microspheres: preparation and drug-controlled re-lease behaviour, J. Biomater. Sci. Polym. Ed. (2012) 1–17.
[122] H. Liu, Y. He, Ke P. Zhaoying, Preparation of ambroxol hydrochloridecarboxymethyl chitosan micropheres without burst release, Afr. J. Pharm.Pharmacol. 5 (2011) 1063–1069.
[123] Y.-l. Tan, C.-G. Liu, Self-aggregated nanoparticles from linoleic acid modifiedcarboxymethyl chitosan: synthesis, characterization and application in vitro, Col-loids Surf. B: Biointerfaces 69 (2009) 178–182.
[124] Y.I. Jeong, S.G. Jin, I.Y. Kim, J. Pei, M. Wen, T.Y. Jung, Doxorubicin-incorporatednanoparticles composed of poly(ethylene glycol)-grafted carboxymethyl chitosanand antitumor activity against glioma cells in vitro, Colloids Surf. B: Biointerfaces79 (2010) 149–155.
[125] G. Geisberger, E.B. Gyenge, C. Maake, G.R. Patzke, Trimethyl and carboxymethylchitosan carriers for bio-active polymer–inorganic nanocomposites, Carbohydr.Polym. 91 (2013) 58–67.
[126] J.-M. Shen, W.-J. Tang, X.-L. Zhang, T. Chen, H.-X. Zhang, A novel carboxymethylchitosan-based folate/Fe3O4/CdTe nanoparticle for targeted drug delivery andcell imaging, Carbohydr. Polym. 88 (2012) 239–249.
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
ED P
RO
OF
[127] J. Ji, S. Hao, W. Liu, J. Zhang, D. Wu, Y. Xu, Preparation and evaluation of O-carboxymethyl chitosan/cyclodextrin nanoparticles as hydrophobic drug deliverycarriers, Polym. Bull. 67 (2011) 1201–1213.
[128] M.E. Mathew, J.C. Mohan, K. Manzoor, S.V. Nair, H. Tamura, R. Jayakumar, Folateconjugated carboxymethyl chitosan-manganese doped zinc sulphide nanoparti-cles for targeted drug delivery and imaging of cancer cells, Carbohydr. Polym. 80(2010) 442–448.
[130] Y. Zhou, L. Xu, X. Zhang, Y. Zhao, S.Wei, M. Zhai, Radiation synthesis of gelatin/CM-chitosan/β-tricalcium phosphate composite scaffold for bone tissue engineering,Mater. Sci. Eng. C 32 (2012) 994–1000.
[131] K. Park, Superporous hydrogels for pharmaceutical and other applications, DrugDeliv. Technol. 2 (2002) 38–44.
[132] X. Han, H.C. Liu, D. Wang, F. Su, L. El, X. Wu, Effects of injectable chitosanthermosensitive hydrogel on dog bone marrow stromal cells in vitro, Shanghai J.Stomatol. 20 (2011) 113–118.
[133] L. Zhao, L. Zhu, F. Liu, C. Liu, D. Shan, Q. Wang, pH triggered injectable amphiphilichydrogel containing doxorubicin and paclitaxel, Int. J. Pharmacol. 410 (2011)83–91.
[135] O.A.C. Monteiro, C. Aeroldi, Some studies of crosslinking chitosan-glutaraldehydeinteraction in a homogenous system, Int. J. Biol. Macromol. 26 (1999) 119–128.
[136] W. Arguelles-Monal, F.M. Goycoolea, C. Peniche, I. Higuera-Ciapra, Rheologicalstudy of the chitosan/glutaraldehyde chemical gel system, Polym. Gels. Netw. 6(1998) 429–440.
[137] M.Z. Wang, Y. Fang, D.D. Hu, Preparation and properties of chitosan poly-(N-Isopropylacrylamide) full IPN-hydrogel, React. Funct. Polym. 48 (2001) 215–221.
[138] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and interac-tions in covalently and ionically crosslinked chitosan hydrogels for biomedical ap-plications, Eur. J. Pharm. Biopharm. 57 (2004) 19–34.
[139] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurney, Structure and inter-actions in chitosan hydrogels formed by complexation or aggregation for biomed-ical applications, Eur. J. Pharm. Biopharm. 57 (2004) 35–52.
[140] X.Z. Shu, K.J. Zhu, W. Song, Novel pH-sensitive citrate-crosslinked chitosan film fordrug controlled release, Int. J. Pharm. 212 (2001) 1928.
[141] E. Ruel-Gariepy, A. Chenite, C. Chaput, S. Guirguise, J.C. Leroux, Characterizations ofthermosensitive chitosan gels for the sustained delivery of drugs, Int. J. Pharm. 203(2000) 89–98.
[142] H.P. Brack, S.A. Tirmizi, W.M. Risen, A spectroscopic and viscosimetric study of themetal ion-induced gelation of the biopolymer chitosan, Polymer 38 (1997)2351–2362.
[143] K.I. Draget, K.M. Varum, E. Moen, H. Gynnild, O. Smidsrod, Chitosan crosslinked withMo(VI) polyoxyanions: a new gelling systems, Biomaterials 13 (1992) 635–638.
[144] L.-Q. Yang, Y.-Q. Lan, H. Guo, L.-Z. Cheng, J.-Z. Fan, X. Cai, L.-M. Zhang, R.-F. Chen, H.-S. Zhou, Ophthalmic drug-loaded N, O-carboxymethyl chitosan hydrogels: synthe-sis, in vitro and in vivo evaluation, Acta Pharmacol. Sin. 31 (2010) 1625–1634.
[145] J. Yang, J. Chen, D. Pan, Y. Wan, Z. Wang, pH-sensitive interpenetrating networkhydrogels based on chitosan derivatives and alginate for oral drug delivery,Carbohydr. Polym. 92 (2013) 719–725.
[146] Y.F. Poon, Y.B. Zhu, J.Y. Shen, M.B. Chan-Park, S.C. Ng, Cytocompatible hydrogelsbased on photocrosslinkable methacrylated O-carboxymethylchitosan with tunablecharge: synthesis and characterization, Adv. Funct. Mater. 17 (2007) 2139–2150.
[147] X. Li, X. Kong, Z. Zhang, K. Nan, L. Li, X.Wang, H. Chen, Cytotoxicity and biocompat-ibility evaluation of N, O-carboxymethyl chitosan/oxidized alginate hydrogel fordrug delivery application, Int. J. Biol. Macromol. 50 (2012) 1299–1305.
[148] H. Kumar Singh Yadav, H.G. Shivakumar, In vitro and in vivo evaluation of ph-sensitive hydrogels of carboxymethyl chitosan for intestinal delivery of theophyl-line, ISRN Pharm. (2012) 763127.
[149] W. Janvikul, B. Thavornyutikarn, New route to the preparation ofcarboxymethylchitosan hydrogels, J. Appl. Polym. Sci. 90 (2003) 4016–4020.
[150] L. Weng, A. Romanov, J. Rooney, W. Chen, Non-cytotoxic, in situ gelable hydrogelscomposed of N-carboxyethyl chitosan and oxidized dextran, Biomaterials 29(2008) 3905–3913.
[151] S. Yan, J. Yin, L. Tang, X. Chen, Novel physically crosslinked hydrogels ofcarboxymethyl chitosan and cellulose ethers: structure and controlled drug re-lease behaviour, J. Appl. Polym. Sci. 119 (2011) 2350–2358.
[152] N.K. Varde, D.W. Pack, Microspheres for controlled release drug delivery, Expert.Opin. Biol. Ther. 4 (2004) 35–51.
[153] L. Weng, P. Rostamzadeh, N. Nooryshokry, H.C. Le, J. Golzarian, In vitro and in vivoevaluation of biodegradable embolic microspheres with tunable anticancer drugrelease, Acta Biomater. 13 (2013) (S1742–7061,00074–3).
[154] M.S. Khan, G.D. Vishakante, A. Bathool, R. Kumar, Preparation and evaluation ofspray dried microparticles using chitosan and novel chitosan derivative for con-trolled release of an antipsychotic drug, Int. J. Biol. Pharm. Res. 3 (2012) 113–121.
[155] G. Gaucher, P. Satturwar, M.-C. Jones, A. Furtos, J.-C. Leroux, Polymeric micelles fororal drug delivery, Eur. J. Pharm. Biopharm. 76 (2010) 147–158.
[156] J. Wu, X. Pan, Y. Zhao, Time-dependent shrinkage of polymeric micelles of amphi-philic block copolymers containing semirigid oligocholate hydrophobes, J. ColloidInterface Sci. 1353 (2011) 420–425.
[157] J. Yin, Z. Li, T. Yang, J. Wang, X. Zhang, Q. Zhang, Cyclic RGDyK conjugation facili-tates intracellular drug delivery of polymeric micelles to integrin-overexpressingtumor cells and neovasculature, J. Drug Target. 19 (2011) 25–36.
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
[159] A. Zou, M. Huo, Y. Zhang, J. Zhou, X. Yin, C. Yao, Octreotide-modified N-octyl-O, N-carboxymethyl chitosan micelles as potential carriers for targeted antitumor drugdelivery, J. Pharm. Sci. 101 (2012) 627–640.
[160] X.-Y. Gong, Y.-H. Yin, Z.-J. Huang, B. Lu, P.-H. Xu, H. Zheng, Preparation, character-ization and in vitro release study of a glutathione-dependent polymeric prodrugCis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan, Int. J.Pharm. 436 (2012) 240–247.
[161] X. Peng, L. Zhang, Formation and morphologies of novel self-assembled micellesfrom chitosan derivatives, Langmuir 23 (2007) 10493–10498.
[162] P.T. Ha, M.H. Le, T.M.N. Hoang, T.T.H. Le, T.Q. Duong, T.H.H. Tran, Preparation andanti-cancer activity of polymer-encapsulated curcumin nanoparticles, Adv. Nat.Sci. Nanosci. Nanotechnol. 3 (2012) 035002.
[163] J. Wang, B. Chen, D. Zhao, Y. Peng, R.-X. Zhuo, S.-X. Cheng, Peptide decorated calci-um phosphate/carboxymethyl chitosan hybrid nanoparticles with improved drugdelivery efficiency, Int. J. Pharm. 446 (2013) 205–210.
[164] Y. Wang, X. Yang, J. Yang, Y. Wang, R. Chen, J. Wu, Self-assembled nanoparticles ofmethotrexate conjugated O-carboxymethyl chitosan: preparation, characterizationand drug release behavior in vitro, Carbohydr. Polym. 86 (2011) 1665–1670.
[165] Y.-H. Jin, H.-Y. Hu, M.-X. Qiao, J. Zhu, J.-W. Qi, C.-J. Hu, pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity:preparation and in vitro evaluation, Colloids Surf. B: Biointerfaces 94 (2012)184–191.
[166] S.K. Sahu, S. Maiti, T.K. Maiti, S.K. Ghosh, P. Pramanik, Hydrophobically modifiedcarboxymethyl chitosan nanoparticles targeted delivery of paclitaxel, J. Drug Tar-get. 19 (2011) 104–113.
[167] Z. Aiping, L. Jianhong, Y. Wenhui, Effective loading and controlled release ofcamptothecin by O-carboxymethylchitosan aggregates, Carbohydr. Polym. 63(2006) 89–96.
[168] C. Liu, W. Fan, X. Chen, C. Liu, X. Meng, H.J. Park, Self-assembled nanoparticlesbased on linoleic-acid modified carboxymethyl-chitosan as carrier of adriamycin(ADR), Curr. Appl. Phys. 7 (2007) e125–e129.
[169] F. Wang, D. Zhang, C. Duan, L. Jia, F. Feng, Y. Liu, Preparation and characterizationsof a novel deoxycholic acid-O-carboxymethylated chitosan–folic acid conjugatesand self-aggregates, Carbohydr. Polym. 84 (2011) 1192–1200.
[170] J. Liang, F. Li, Y. Fang, W. Yang, X. An, L. Zhao, Response surface methodology in theoptimization of tea polyphenols-loaded chitosan nanoclusters formulations, Eur.Food Res. Technol. 231 (2010) 917–924.
[171] A.D. Bangham, R.W. Horne, Negative staining of phospholipids and their structuralmodification by surface-active agents as observed in the electron microscope, J.Mol. Biol. 8 (1964) 660-IN10.
[172] J. Shi, A.R. Votruba, O.C. Farokhzad, R. Langer, Nanotechnology in drug delivery andtissue engineering: from discovery to applications, Nano Lett. 10 (2010)3223–3230.
[173] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in can-cer chemotherapy: mechanism of tumoritropic accumulation of proteins and theantitumor agent Smancs, Cancer Res. 46 (1986) 6387–6392.
[174] Y.S. Wang, Q. Jiang, R.S. Li, L.L. Liu, Q.Q. Zhang, Y.M. Wang, J. Zhao, Self-assemblednanoparticles of cholesterol-modified O-carboxymethyl chitosan as a novel carrierfor paclitaxel, 19 (2008) 145101.
[175] X.F. Liang, J.Y. Hu, F.H. Chen, Z.H. Li, J. Chang, Characterization of chitosan polymericethosomes capable of encapsulating hydrophobic and hydrophilic drugs preparedby a microemulsion method, Wuli Huaxue Xuebao/Acta. Phys. -Chim. Sin. 28(2012) 897–902.
[176] Y.-l. Tan, C.-G. Liu, Preparation and characterization of self-assemblied nanoparti-cles based on folic acid modified carboxymethyl chitosan, J. Mater. Sci. Mater.Med. 22 (2011) 1213–1220.
[177] S.X. Yang, X.Z. Qian, L.D. Wang, Y.L. Xu, Preparation of pH-sensitive doxorubicin li-posomes modified with carboxymethyl chitosan and in vitro cell research,Huadong Ligong Daxue Xuebao/J. East China Univ. Sci. Technol. 38 (2012)183–185.
[178] T.S. Anirudhan, S. Sandeep, Synthesis, characterization, cellular uptake and cyto-toxicity of a multi-functional magnetic nanocomposite for the targeted deliveryand controlled release of doxorubicin to cancer cells, J. Mater. Chem. 22 (2012)12888–12899.
[179] A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, Efficient watersoluble O-carboxymethyl chitosan nanocarrier for the delivery of curcumin to can-cer cells, Carbohydr. Polym. 83 (2011) 452–461.
[180] H.P. Thu, L.T.T. Huong, H.T.M. Nhung, N.T. Tham, N.D. Tu, H.T.M. Thi, Fe3O4/o-carboxymethyl chitosan/curcumin-based nanodrug system for chemotherapyand fluorescence imaging in HT29 cancer cell line, Chem. Lett. 40 (2011)1264–1266.
[182] P.-P. Lv, Y.-F. Ma, R. Yu, H. Yue, D.-Z. Ni, W. Wei, Targeted delivery of insolublecargo (paclitaxel) by PEGylated chitosan nanoparticles grafted with Arg-Gly-Asp(RGD), Mol. Pharmacol. 9 (2012) 1736–1747.
[183] O. D'Agostini-Junior, C.L. Petkowicz, A.G. Couto, S.F. de Andrade, R.A. Freitas, Simul-taneous in situ monitoring of acrylic acid polymerization reaction on N-carboxymethyl chitosan using multidetectors: formation of a new bioadhesiveand gastroprotective hybrid particle, Mater. Sci. Eng. C 31 (2011) 677–682.
[184] H. Wu, A. Zhu, L. Yuan, Interactions between O-carboxymethylchitosan and bovineserum albumin, Mater. Chem. Phys. 112 (2008) 41–46.
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
ED P
RO
OF
[185] B. Sayın, S. Somavarapu, X.W. Li, D. Sesardic, S. Şenel, O.H. Alpar, TMC-MCC (N-trimethyl chitosan-mono-N-carboxymethyl chitosan) nanocomplexes for mucosaldelivery of vaccines, Eur. J. Pharm. Sci. 38 (2009) 362–369.
[186] T.R. de Oliveira Rosa, A. Debrassi, R.M.L. da Silva, C. Bressan, R.A. de Freitas, C.A.Rodrigues, Synthesis of N-benzyl-O-carboxymethylchitosan and application inthe solubilization enhancement of a poorly water-soluble drug (triamcinolone),J. Appl. Polym. Sci. 124 (2012) 4206–4212.
[187] Y. Luo, Z. Teng, Q. Wang, Development of Zein nanoparticles coated withcarboxymethyl chitosan for encapsulation and controlled release of vitamin D3,J. Agric. Food Chem. 60 (2012) 836–843.
[188] A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Curcumin-loaded N, O-carboxymethyl chitosan nanoparticles for cancer drug delivery, J.Biomater. Sci. Polym. Ed. 23 (2012).
[189] A. Anitha, K.P. Chennazhi, S.V. Nair, R. Jayakumar, 5-flourouracil loaded N, O-carboxymethyl chitosan nanoparticles as an anticancer nanomedicine for breastcancer, J. Biomed. Nanotechnol. 8 (2012) 29–42.
[190] X.F. Liang,H.J.Wang, H. Luo,H. Tian, B.B. Zhang, L.J. Hao, Characterizationof novelmul-tifunctional cationic polymeric liposomes formed from octadecyl quaternizedcarboxymethyl chitosan/cholesterol and drug encapsulation, Langmuir 24 (2008)7147–7153.
[191] A. Debrassi, C. Bürger, C.A. Rodrigues, N. Nedelko, A. Ślawska-Waniewska, P.Dłużewski, Synthesis, characterization and in vitro drug release of magnetic N-benzyl-O-carboxymethylchitosan nanoparticles loaded with indomethacin, ActaBiomater. 7 (2011) 3078–3085.
[192] M. Prabaharan, R.L. Reis, J.F. Mano, Carboxymethyl chitosan-graft-phosphatidylethanolamine: amphiphilic matrices for controlled drug delivery,React. Funct. Polym. 67 (2007) 43–52.
[193] J.M. Oliveira, N. Kotobuki, A.P. Marques, R.P. Pirraco, J. Benesch, M. Hirose, Surfaceengineered carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticlesfor intracellular targeting, Adv. Funct. Mater. 18 (2008) 1840–1853.
[194] S. Maya, S. Indulekha, V. Sukhithasri, K.T. Smitha, S.V. Nair, R. Jayakumar, et al., Ef-ficacy of tetracycline encapsulated O-carboxymethyl chitosan nanoparticlesagainst intracellular infections of Staphylococcus aureus, Int. J. Biol. Macromol. 51(2012) 392–399.
[195] A. Zhu, W. Jin, L. Yuan, G. Yang, H. Yu, H. Wu, O-Carboxymethylchitosan-basednovel gatifloxacin delivery system, Carbohydr. Polym. 68 (2007) 693–700.
[196] H.-D. Wang, Q. Yang, C.H. Niu, Functionalization of nanodiamond particles with N,O-carboxymethyl chitosan, Diam. Relat. Mater. 19 (2010) 441–444.
[197] Y.-C. Ho, S.-J. Wu, F.-L. Mi, Y.-L. Chiu, S.-H. Yu, N. Panda, H.-W. Sung, Thiol-modifiedchitosan sulfate nanoparticles for protection and release of basic fibroblast growthfactor, Bioconjug. Chem. 21 (2010) 28–38.
[198] B. Sayın, S. Somavarapu, X.W. Li, M. Thanou, D. Sesardic, H.O. Alpar, Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan (TMC) nanoparticlesfor non-invasive vaccine delivery, Int. J. Pharm. 363 (2008) 139–148.
[199] L.C. Wang, X.G. Chen, Q.C. Xu, C.S. Liu, L.J. Yu, Y.M. Zhou, Plasma protein adsorptionpattern and tissue-implant reaction of poly(vinyl alcohol)/carboxymethyl-chitosanblend films, J. Biomater. Sci. Polym. Ed. 19 (2008) 113–129.
[200] T.J. Sill, H.A. von Recum, Electrospinning: applications in drug delivery and tissueengineering, Biomaterials 29 (2008) 1989–2006.
[201] Y. Zhao, Y. Zhou, X. Wu, L. Wang, L. Xu, S. Wei, A facile method for electrospinningof Ag nanoparticles/poly (vinyl alcohol)/carboxymethyl-chitosan nanofibers, Appl.Surf. Sci. 258 (2012) 8867–8873.
[202] X.Y. Li, X.G. Chen, Z.W. Sun, H.J. Park, D.-S. Cha, Preparation of alginate/chitosan/carboxymethyl chitosan complex microcapsules and application in Lactobacilluscasei ATCC 393, Carbohydr. Polym. 83 (2011) 1479–1485.
[203] J.A. DiMasi, R.W. Hansen, H.G. Grabowski, The price of innovation: new estimatesof drug development costs, J. Health Econ. 22 (2003) 151–185.
[204] W.J. Lin, T.D. Chen, C.W. Liu, Synthesis and characterization of lactobionic acidgrafted pegylated chitosan and nanoparticles complex application, Polymer 50(2009) 4166–4174.
[205] K. Strebhardt, A. Ullrich, Paul Ehrlich's magic bullet concept: 100 years of progress,Nat. Rev. Cancer 8 (2008) 473–480.
[206] S. Sahu, S. Mallick, S. Santra, T. Maiti, S. Ghosh, P. Pramanik, In vitro evaluation offolic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubicinfor targeted delivery, J. Mater. Sci. Mater. Med. 21 (2010) 1587–1597.
[207] F. Wang, Y. Chen, D. Zhang, Q. Zhang, D. Zheng, L. Hao, Folate-mediated targetedand intracellular delivery of paclitaxel using a novel deoxycholic acid-O-carboxymethylated chitosan-folic acid micelles, Int. J. Nanomedicine 7 (2012)325–337.
[208] A. Zhu, L. Yuan, S. Dai, Preparation of well-dispersed superparamagnetic iron oxidenanoparticles in aqueous solution with biocompatible N-succinyl-O-carboxymethylchitosan, J. Phys. Chem. C 112 (2008) 5432–5438.
[209] A. Zhu, L. Yuan, T. Liao, Suspension of Fe3O4 nanoparticles stabilized by chitosanand o-carboxymethylchitosan, Int. J. Pharm. 350 (2008) 361–368.
[210] V.P. Torchilin, Targeted polymeric micelles for delivery of poorly soluble drugs,Cell. Mol. Life Sci. 61 (2004) 2549–2559.
[211] H. Tan, F. Qin, D. Chen, S. Han, W. Lu, X. Yao, Study of glycol chitosan-carboxymethyl β-cyclodextrins as anticancer drugs carrier, Carbohydr. Polym. 93(2013) 679–685.
[212] S. Maya, L.G. Kumar, B. Sarmento, N. Sanoj Rejinold, D. Menon, S.V. Nair, Cetuximabconjugated O-carboxymethyl chitosan nanoparticles for targeting EGFR overex-pressing cancer cells, Carbohydr. Polym. 93 (2013) 661–669.
[213] A. Zou, Y. Chen, M. Huo, J. Wang, Y. Zhang, J. Zhou, Q. Zhang, In vivo studies ofoctreotide-modified N-octyl-O, N-carboxymethyl chitosan micelles loadedwith doxorubicin for tumor-targeted delivery, J. Pharm. Sci. 102 (2013)126–135.
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
33L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
UNCO
RREC
[214] J. Ji, D. Wu, L. Liu, J. Chen, Y. Xu, Preparation, evaluation, and in vitro release of folicacid conjugated O-carboxymethyl chitosan nanoparticles loaded with methotrex-ate, J. Appl. Polym. Sci. 125 (2012) E208–E215.
[215] H. Zheng, Y. Rao, Y. Yin, X. Xiong, P. Xu, B. Lu, Preparation, characterization, andin vitro drug release behavior of 6-mercaptopurine-carboxymethyl chitosan,Carbohydr. Polym. 83 (2011) 1952–1958.
[216] L. Shi, C. Tang, C. Yin, Glycyrrhizin-modified O-carboxymethyl chitosan nanoparti-cles as drug vehicles targeting hepatocellular carcinoma, Biomaterials 33 (2012)7594–7604.
[217] Y.Wang, Q. Jiang, L.R. Liu, Q. Zhang, The interaction between bovine serumalbuminand the self-aggregated nanoparticles of cholesterol-modified O-carboxymethylchitosan, Polymer 48 (2007) 4135–4142.
[218] S.K. Jain, A. Jain, Target-specific drug release to the colon, Exp. Opin. Drug Deliv. 5(2008) 483–498.
[219] M. Tavakol, E. Vasheghani-Farahani, T. Dolatabadi-Farahani, S. Hashemi-Najafabadi, Sulfasalazine release from alginate-N, O-carboxymethyl chitosan gelbeads coated by chitosan, Carbohydr. Polym. 77 (2009) 326–330.
[220] H. Tu, Y. Qu, X. Hu, Y. Yin, H. Zheng, P. Xu, Study of the sigmoidal swelling kineticsof carboxymethylchitosan-g-poly(acrylic acid) hydrogels intended for colon-specific drug delivery, Carbohydr. Polym. 82 (2010) 440–445.
[221] J.H. Park, G. Saravanakumar, K. Kim, I.C. Kwon, Targeted delivery of low molec-ular drugs using chitosan and its derivatives, Adv. Drug Deliv. Rev. 62 (2010)28–41.
[222] H. Zheng, X. Zhang, Y. Yin, F. Xiong, X. Gong, Z. Zhu, In vitro characterization, andin vivo studies of crosslinked lactosaminated carboxymethyl chitosan nanoparti-cles, Carbohydr. Polym. 84 (2011) 1048–1053.
[223] A.L. Weiner, B.C. Gilger, Advancements in ocular drug delivery, Vet. Ophthalmol. 13(2010) 395–406.
[224] M.J. Alonso, A. Sanchez, The potential of chitosan in ocular drug delivery, J. Pharm.Pharmacol. 55 (2003) 1451–1463.
[225] M. de la Fuente, M. Raviña, P. Paolicelli, A. Sanchez, B. Seijo, M.J. Alonso, Chitosan-based nanostructures: a delivery platform for ocular therapeutics, Adv. Drug Deliv.Rev. 62 (2010) 100–117.
[226] G. Di Colo, Y. Zambito, S. Burgalassi, I. Nardini, M.F. Saettone, Effect of chitosan andof N-carboxymethylchitosan on intraocular penetration of topically appliedofloxacin, Int. J. Pharmacol. 273 (2004) 37–44.
[227] Y. Chien, Y.-W. Liao, D.-M. Liu, H.-L. Lin, S.-J. Chen, H.-L. Chen, Corneal repair by humancorneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethyl-hexanoylchitosan hydrogel, Biomaterials 33 (2012) 8003–8016.
[228] R. Shalak, C.F. Fox, Preface, in: R. Shalak, C.F. Fox (Eds.), Tissue Engineering, Alan R.Liss, New York, 1988, pp. 26–29.
[229] C.A. Vacanti, Foreword, in: R.P. Lanza, R. Langer, J.P. Vacanti (Eds.), Principles of Tis-sue Engineering, 2nd ed., Academic Press, New York, 2000, p. xxix.
[230] M. Artico, L. Ferrante, F.S. Pastore, E.O. Ramundo, D. Cantarelli, D. Scopelliti, Boneautografting of the calvaria and craniofacial skeleton: historical background, surgicalresults in a series of 15 patients, and review of the literature, Surg. Neurol. 60(2003) 71–79.
[231] J.R. Bain, Peripheral nerve allografting: review of the literature with relevance tocomposite tissue transplantation, Transplant. Proc. 30 (1998) 2762–2767.
droxyapatite/carboxymethylchitosan composite scaffolds prepared through an in-novative “autocatalytic” electroless coprecipitation route, J. Biomed. Mater. Res. A88A (2009) 470–480.
[234] X. Bao, Y. Li, A. Teramoto, K. Abe, Carboxymethyl chitosan/hydroxyapatite compos-ite scaffold for bone regeneration, 2010. 299–302.
[235] Z.H. Lu, D.M. Zhao, K.N. Sun, Preparation and characterization of a bio-compositesscaffold containing nano-hydroxyapatite/carboxymethyl chitosan, Adv. Mater. Res.(2012) 2055–2058.
[236] S.-b. Chen, H. Zhong, L.-l. Zhang, Y.-f. Wang, Z.-p. Cheng, Y.-l. Zhu, Synthesis andcharacterization of thermoresponsive and biocompatible core-shell microgelsbased on N-isopropylacrylamide and carboxymethyl chitosan, Carbohydr. Polym.82 (2010) 747–752.
[237] A.H. Liu, K.N. Sun, D.M. Zhao, A.M. Li, Study of preparation and properties aboutnano-hydroxyapatite/carboxymethyl chitosan porous biocomposites, J. Synth.Cryst. 36 (2007) 276–280.
[238] H.P. Si, Z.H. Lu, Y.L. Lin, J.J. Li, Q.F. Yin, D.M. Zhao, Transfect bone marrow stromalcells with pcDNA3.1-VEGF to construct tissue engineered bone in defect repair,Chin. Med. J. 125 (2012) 906–911.
[239] R. Jayakumar, M. Rajkumar, H. Freitas, P.T. Sudheesh Kumar, S.V. Nair, T. Furuike,Bioactive and metal uptake studies of carboxymethyl chitosan-graft-D-glucuronicacid membranes for tissue engineering and environmental applications, Int. J.Biol. Macromol. 45 (2009) 135–139.
[240] J.P. Vacanti, R. Langer, Tissue engineering: the design and fabrication of living re-placement devices for surgical reconstruction and transplantation, Lancet 354(1999) SI32–SI34.
[241] I.-Y. Kim, S.-J. Seo, H.-S. Moon, M.-K. Yoo, I.-Y. Park, B.-C. Kim, Chitosan and its de-rivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008) 1–21.
[242] R. Budiraharjo, K.G. Neoh, E.T. Kang, A. Kishen, Bioactivity of novel carboxymethylchitosan scaffold incorporating MTA in a tooth model, Int. Endod. J. 43 (2010)930–939.
[243] A.P. Zhu, F. Zhao, N. Fang, Regulation of vascular smooth muscle cells on poly(eth-ylene terephthalate) film by O-carboxymethylchitosan surface immobilization, J.Biomed. Mater. Res. A 86 (2008) 467–476.
[244] X. Hu, K.-G. Neoh, Z. Shi, E.-T. Kang, C. Poh, W. Wang, An in vitro assessment of ti-tanium functionalized with polysaccharides conjugated with vascular endothelial
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
ED P
RO
OF
growth factor for enhanced osseointegration and inhibition of bacterial adhesion,Biomaterials 31 (2010) 8854–8863.
[245] Q. Lin, X. Lan, Y. Li, Y. Yu, Y. Ni, C. Lu, Anti-washout carboxymethyl chitosan mod-ified tricalcium silicate bone cement: preparation, mechanical properties andin vitro bioactivity, J. Mater. Sci. Mater. Med. 21 (2010) 3065–3076.
[246] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and itsdeacetylated derivatives, Biomaterials 18 (1997) 567–575.
[247] G. Lu, L. Kong, B. Sheng, G. Wang, Y. Gong, X. Zhang, Degradation of covalentlycross-linked carboxymethyl chitosan and its potential application for peripheralnerve regeneration, Eur. Polym. J. 43 (2007) 3807–3818.
[249] C.K. Kuo, P.X. Ma, Ionically crosslinked alginate hydrogels as scaffolds for tissue en-gineering: Part 1, Structure, gelation rate and mechanical properties, Biomaterials22 (2001) 511–521.
[250] P.S. Chan, J.P. Caron, M.W. Orth, Effects of glucosamine and chondroitin sulfate onbovine cartilage explants under long-term culture conditions, Am. J. Vet. Res. 68(2007) 709–715.
[251] D.A. Wang, S. Varghese, B. Sharma, I. Strehin, S. Fermanian, J. Gorham, Multifunc-tional chondroitin sulphate for cartilage tissue-biomaterial integration, Nat.Mater. 6 (5) (2007) 385–392.
[252] C. Liu, Z. Xia, J.T. Czernuszka, Design and development of three-dimensional scaf-folds for tissue engineering, Chem. Eng. Res. Des. 85 (2007) 1051–1064.
[253] J.T. Oliveira, R.L. Reis, Polysaccharide-based materials for cartilage tissue engineer-ing applications, J. Tissue Eng. Regen. Med. 5 (2011) 421–436.
[254] Y. Tang, J. Sun, H. Fan, X. Zhang, An improved complex gel of modified gellan gumand carboxymethyl chitosan for chondrocytes encapsulation, Carbohydr. Polym. 88(2012) 46–53.
[255] H. Shi, W. Wang, D. Lu, H. Li, L. Chen, Y. Lu, Y. Zeng, Cellular biocompatibility andbiomechanical properties of N-carboxyethylchitosan/nanohydroxyapatite com-posites for tissue-engineered trachea, Artif. Cells Blood Substit. Immobil.Biotechnol. 40 (2012) 120–124.
[256] X.X. Bao, A. Teramoto, K. Abe, Carboxymethyl chitosan nonwoven scaffold for boneregeneration, Key Eng. Mater. 464 (2011) 712–716.
[257] P.X. Ma, Scaffolds for tissue engineering, Mater. Today 7 (2004) 30–40.[258] X. Liu, P.X. Ma, Polymeric scaffolds for bone tissue engineering, Ann. Biomed. Eng.
32 (2004) 477–486.[259] T. Kawakami, M. Antoh, H. Hasegawa, T. Yamagishi, M. Ito, S. Eda, Experimental
study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste, Biomaterials 13 (1992) 759–763.
[260] M. Honda, Cartilage formation by cultured chondrocytes in a new scaffold made ofpoly(L-lactide-epsilon-caprolactone) sponge, J. Maxillofac. Surg. 58 (2000) 767–775.
[261] J.M. Oliveira, N. Kotobuki, M. Hirose, J.F. Mano, R.L. Reis, H. Ohgushi, Intracellularcarboxymethylchitosan/poly(amidoamine) nanocarriers loaded with dexametha-sone enhances osteogenic differentiation of RBMSCs, In Vitro—Tissue Eng. (2007)1719.
[262] J.M. Oliveira, R.A. Sousa, N. Kotobuki, M. Tadokoro, M. Hirose, The osteogenic differ-entiation of rat bone marrow stromal cells cultured with dexamethasoneloadedcarboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles, Biomaterials30 (2008) 804–813.
[263] J.M. Oliveira, S.S. Silva, P.B. Malafaya, M.T. Rodrigues, M.E. Gomes, N. Kotobuki,Macroporous hydroxyapatite scaffolds for bone tissue engineering applications:physicochemical characterization and assessment of rat bone marrow stromalcells viability, J. Biomed. Mater. Res. A 91 (2009) 175–186.
[264] D.A. Grande, C. Halberstadt, G. Naughton, R. Schwartz, R. Manji, Evaluation of ma-trix scaffolds for tissue engineering of articular cartilage grafts, J. Biomed. Mater.Res. 34 (1997) 211–220.
[265] J.K. Francis Suh,H.W.T.Matthew,Applicationof chitosan-basedpolysaccharidebioma-terials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589–2598.
[266] A. Lahiji, A. Sohrabi, D.S. Hungerford, C.G. Frondoza, Chitosan supports the expres-sion of extracellular matrix proteins in human osteoblasts and chondrocytes, J.Biomed. Mater. Res. 51 (2000) 586–595.
[267] M. Mattioli-Belmonte, A. Gigante, R.A.A. Muzzarelli, R. Politano, A. Benedittis, N.Specchia, N, N-dicarboxymethyl chitosan as delivery agent for bone morphogeneticprotein in the repair of articular cartilage,Med. Biol. Eng. Comput. 37 (1999) 130–134.
[268] M. Lei, S.-q. Liu, Y.-l. Liu, Resveratrol protects bonemarrowmesenchymal stem cellderived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory ef-fect of interleukin-1, Acta Pharmacol. Sin. 29 (2008) 1350–1356.
[269] S.Q. Liu, B. Qiu, L.Y. Chen, H. Peng, Y.M. Du, The effects of carboxymethylated chitosanon metalloproteinase-1, -3 and tissue inhibitor of metalloproteinase-1 gene expres-sion in cartilage of experimental osteoarthritis, Rheumatol. Int. 26 (2005) 52–57.
[270] B.L. Seal, T.C. Otero, A. Panitch, Polymeric biomaterials for tissue and organ regen-eration, Mater. Sci. Eng. R 34 (2001) 147–230.
[271] B. Bini, S. Gao, S. Wang, S. Ramakrishna, Development of fibrous biodegradable poly-mer conduits for guided nerve regeneration, J. Mater. Sci. Mater. Med. 16 (2005)367–375.
[272] B. He, S.Q. Liu, Q. Chen, H.H. Li, W.J. Ding, M. Deng, Carboxymethylated chitosanstimulates proliferation of Schwann cells in vitro via the activation of the ERKand Akt signaling pathways, Eur. J. Pharmacol. 667 (2011) 195–201.
[273] S. Peng, W. Liu, B. Han, J. Chang, M. Li, X. Zhi, Effects of carboxymethyl-chitosan onwound healing in vivo and in vitro, J. Ocean Univ. China 10 (2011) 369–378.
[274] J. Chang, W. Liu, B. Han, S. Peng, He, Z. Gu, Investigation of the skin repair andhealing mechanism of N-carboxymethyl chitosan in second-degree burn wounds,Wound Repair Regen. 21 (2012) 113–121.
[275] Y.M. Qin, L.Q. Huang, The preparation and characterization of carboxymethyl chito-san wound dressings, Adv. Mater. Res. (2011) 465–468.
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
34 L. Upadhyaya et al. / Journal of Controlled Release xxx (2014) xxx–xxx
[276] D.K. Kweon, S.B. Song, Y.Y. Park, Preparation of water-soluble chitosan/heparincomplex and its application as wound healing accelerator, Biomaterials 24(2003) 1595–1601.
[277] J.Y. Teng, R. Guo, J. Xie, D.J. Sun, J.J. Wu, X.N. Pang, Angiogenesis of full-thickness burnwounds repaired with collagen-sulfonated carboxymethyl chitosan porous scaffoldencoding vascular endothelial growth factor DNA plasmids, 91 (2011) 2568–2572.
[278] R. Kennedy, D.J. Costain, V.C. McAlister, T.D. Lee, Prevention of experimental post-operative peritoneal adhesions by N, O-carboxymethyl chitosan, Surgery 120(1996) 866–870.
[279] D.J. Costain, R. Kennedy, C. Ciona, V.C. McAlister, T.D. Lee, Prevention of postsurgi-cal adhesions with N, O-carboxymethyl chitosan: examination of the most effica-cious preparation and the effect of N, O-carboxymethyl chitosan on postsurgicalhealing, Surgery 121 (1997) 314–319.
[280] J. Zhou, C. Elson, T.D. Lee, Reduction in postoperative adhesion formation and re-formation after an abdominal operation with the use of N, O-carboxymethyl chito-san, Surgery 135 (2004) 307–312.
[281] B. Kundu, S.C. Kundu, Osteogenesis of human stem cells in silk biomaterial for re-generative therapy, Prog. Polym. Sci. 35 (2010) 1116–1127.
[282] R. Chen, D. Mooney, Polymeric growth factor delivery strategies for tissue engi-neering, Pharm. Res. 20 (2003) 1103–1112.
[283] J.K. Tessmar, A.M. Göpferich, Matrices and scaffolds for protein delivery in tissueengineering, Adv. Drug Deliv. Rev. 59 (2007) 274–291.
[284] T. Holland, A. Mikos, Review: biodegradable polymeric scaffolds. improvements inbone tissue engineering through controlled drug delivery, in: K. Lee, D. Kaplan(Eds.), Tissue Engineering I, Springer, Berlin Heidelberg, 2006, pp. 161–185.
UNCO
RRECT
Please cite this article as: L. Upadhyaya, et al., The implications of recent atissue engineering applications, J. Control. Release (2014), http://dx.doi.or
OF
[285] D.W. Hutmacher, Scaffold design and fabrication technologies for engineering tis-sues state of the art and future perspectives, J. Biomater. Sci. Polym. Ed. 12(2001) 107–124.
[286] Y. Tabata, Significance of release technology in tissue engineering, Drug Discov.Today 10 (2005) 1639–1646.
[287] S. Cartmell, Controlled release scaffolds for bone tissue engineering, J. Pharm. Sci.98 (2009) 430–441.
[288] V. Mouriño, A.R. Boccaccini, Bone tissue engineering therapeutics: controlled drugdelivery in three-dimensional scaffolds, J. R. Soc. Interface 7 (2010) 209–227.
[289] E.K. Moioli, P.A. Clark, X. Xin, S. Lal, J.J. Mao, Matrices and scaffolds for drug deliveryin dental, oral and craniofacial tissue engineering, Adv. Drug Deliv. Rev. 59 (2007)308–324.
[290] S.M. Willerth, S.E. Sakiyama-Elbert, Approaches to neural tissue engineering usingscaffolds for drug delivery, Adv. Drug Deliv. Rev. 59 (2007) 325–338.
[291] H. Liu, L. Zhang, P. Shi, Q. Zou, Y. Zuo, Y. Li, Hydroxyapatite/polyurethane scaffoldincorporatedwith drug-loaded ethyl cellulosemicrospheres for bone regeneration,J. Biomed. Mater. Res. B Appl. Biomater. 95B (2010) 36–46.
[292] M. Chen, D.Q. Le, S. Hein, P. Li, J.V. Nygaard, M. Kassem, J. Kjems, F. Besenbacher, C.Bünger, Fabrication and characterization of a rapid prototyped tissue engineeringscaffold with embedded multicomponent matrix for controlled drug release, Int.J. Nanomedicine 7 (2012) 4285–4297.
[293] B.T. Reves, J.D. Bumgardner, W.O. Haggard, Fabrication of crosslinkedcarboxymethylchitosan microspheres and their incorporation into composite scaf-folds for enhanced bone regeneration, J. Biomed. Mater. Res. B Appl. Biomater.101B (2013) 630–639
ED P
RO
dvances in carboxymethyl chitosan based targeted drug delivery andg/10.1016/j.jconrel.2014.04.043
