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Journal of Controlled Release 186 (2014) 5487

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Journal of Controlled Release


journal homepage: www.elsevier.com/locate/jconrel

Review

The implications of recent advances in carboxymethyl chitosan based


targeted drug delivery and tissue engineering applications
Laxmi 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, India
b
Department of Applied Chemistry & Polymer Technology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, New Delhi 110042, India
c
Department of Applied Mechanics (Biotechnology), MotiLal Nehru National Institute of Technology, Allahabad 211004, India

a r t i c l e i n f o a b s t r a c t

Article history: Over the last decade carboxymethyl chitosan (CMCS) has emerged as a promising biopolymer for the develop-
Received 10 February 2014 ment of new drug delivery systems and improved scaffolds along with other tissue engineering devices for regen-
Accepted 23 April 2014 erative medicine that is currently one of the most rapidly growing elds in the life sciences. CMCS is amphiprotic
Available online 5 May 2014
ether, derived from chitosan, exhibiting enhanced aqueous solubility, excellent biocompatibility, controllable
biodegradability, osteogenesis ability and numerous other outstanding physicochemical and biological proper-
Keywords:
Carboxymethyl chitosan
ties. More strikingly, it can load hydrophobic drugs and displays strong bioactivity which highlight its suitability
Drug delivery and extensive usage for preparing different drug delivery and tissue engineering formulations respectively. This
Tissue engineering review provides a comprehensive introduction to various types of CMCS based formulations for delivery of ther-
Formulation of drug 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 drugs
with emphasis on cancer specic and organ specic drug delivery have been described. Further, we have
discussed 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 delivery
and tissue engineering. Finally, identication of major challenges and opportunities for current and ongoing ap-
plication of CMCS based systems in the eld are summarised.
2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2. Scope of the present review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3. Limitations with the use of chitosan in drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4. Biopharmaceutical and toxicological prole of CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5. Synthesis of various carboxymethyl chitosans (CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.1. Synthesis of O-carboxymethyl chitosans (O-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2. Synthesis of N-carboxymethyl chitosans (N-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.3. Synthesis of N,O-carboxymethyl chitosans (N,O-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.4. Synthesis of N,N-dicarboxymethyl chitosans (N,N-di-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
6. Preparation techniques of CMCS based formulations for drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Abbreviations: ADR, adriamycin; AQCOM, alginate-Q-CMCS-organic montmorillonite; bFGF, basic broblast growth gactor; BSA, bovine serum albumin; BMSCs, bone marrow stromal
cells; CPT, camptothecin; CMC, critical micelle concentration; CMCS, carboxymethyl chitosan; O-CMCS, O-carboxymethyl chitosans; N-CMCS, N-carboxymethyl chitosans; N,O-CMCS, N,O-
carboxymethyl chitosans; N,N-di-CMCS, N,N-di-carboxymethyl chitosans; CMHC, carboxymethylhexanoyl chitosan; CMCPEG, methoxy poly(ethylene glycol)-grafted carboxymethyl chi-
tosan; CMCS-g-D-GA, CMCS-graft-D-glucuronic acid; CD, cyclodextrin; DD, degree of deacetylation; DS, degree of substitution; DA, deoxycholic acid; DOX, doxorubicin; EPR, enhanced per-
meability and retention; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride; FA, folic acid; 5-FU, 5-uorouracil; GA, glutaraldehyde; GL, glycyrrhizin; GFLX, gatioxacin;
HAP, hydroxyapatite; IPN, interpenetrating; iPSCs, induced pluripotent stem cells; LA, linoleic acid; mPEG-g-CMC, methoxy poly (ethylene glycol) grafted carboxymethyl chitosan; MW,
molecular weight; MTA, mineral trioxide aggregate; MIC, minimum inhibitory concentration; MMA, methyl methacrylate; N-CECS/nano-HAP, N-carboxyethyl chitosan/
nanohydroxyapatite; OCT, octreotide; OCC, N-octyl-O,N-carboxymethyl chitosan; OD, ornidazole; OMMT, organic montmorillonite; PTA, Cis-3-(9H-purin-6-ylthio)-acrylic acid; PTX, pac-
litaxel; PNIPAM, poly(N-isopropylmethacrylamide); PBS, phosphate buffer saline; PEG, polyethylene glycol; PAMAM, poly(amidoamine); PVA, poly-vinyl alcohol; QCMCS, quaternised
carboxymethyl chitosan; SA, stearic acid; SMCs, smooth muscle cells; TLAC, thiolated lactosaminated; VEGF, vascular endothelial growth factor.
Corresponding authors. Tel.: +91 11127871045 (ofce), +91 9871765453 (mobile).
E-mail addresses: laxmi4.phd@gmail.com (L. Upadhyaya), jay_singh143@yahoo.co.in (J. Singh).

http://dx.doi.org/10.1016/j.jconrel.2014.04.043
0168-3659/ 2014 Elsevier B.V. All rights reserved.
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 55

7. CMCS formulations for drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59


7.1. CMCS hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
7.2. CMCS microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
7.3. CMCS micelles/aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
7.4. CMCS nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
7.5. CMCS lms and bers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
7.6. CMCS composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8. CMCS based targeted drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8.1. Cancer-specic drug delivery based on CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
8.2. Organ-specic drug delivery based on CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.1. Colon-specic drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.2. Liver targeted drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.3. Ocular drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
8.2.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
9. Tissue engineering: origin and strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
10. Preparation techniques of CMCS based biomaterials for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
11. CMCS based biomaterials for tissue engineering and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
11.1. CMCS scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
11.2. CMCS hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
11.3. CMCS composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
11.4. CMCS injectable gels, lms and membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12. CMCS applications in different tissues/organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12.1. CMCS in bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12.2. CMCS in cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
12.3. CMCS in nerve tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
12.4. CMCS in wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13. Tissue engineering application is often combined with drug delivery strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
14. CMCS based systems: current challenges and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
15. Summary and future remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

1. Introduction [25] and its safety evaluation on compounds [26], in vitro models,
blood systems [27] and tumor application [28] has been well established.
Natural polysaccharides, due to their non-toxicity, biocompatibility All these favorable physical, chemical and biological properties of CMCS
and biodegradability, are widely being studied as biomaterial for drug make it a promising biomedical material for drug delivery and tissue en-
delivery and tissue engineering applications. Among them, chitosan, gineering applications in several formulations. Recently numerous ex-
which is known to be biocompatible, biodegradable, non-toxic, perimental results have been reported on the potential therapeutic
mucoadhesive and antimicrobial, has been exhaustively exploited for de- applications of CMCS in reduction in post surgical adhesion formation
veloping different formulations for controlled delivery of biotherapeutics [29], antibacterial biomaterial [30] and accelerated wound healing [31].
and in regenerative medicine. But limited solubility of chitosan in water The most exhaustively investigated CMCS based drug delivery formula-
and other organic solvents has prevented its full exploitation in drug de- tions include hydrogels [32], microspheres [26], beads [33], micelles
livery and tissue repair and reconstruction [1,2]. In addition to this, the [34], aggregates [35], nanoparticles [36,37], lms [38] and membranes
limited colloidal stability of chitosan based particulate drug delivery sys- [39]. Similarly, repairing and reconstruction of tissues like bone, cartilage,
tems are known to exhibit immunogenicity [3] and degradability of chi- and nerve by CMCS based tissue engineering devices like scaffolds [40],
tosan based formulations in tissue regeneration and drug delivery is injectable gels [2], and biocomposites has been reported by various
uncontrollable [4]. Therefore derivatisation of chitosan seems a promis- researchers in recent years.
ing way to get rid of these limitations of chitosan and widening range
of drug delivery and tissue engineering applications. In fact, life sciences 2. Scope of the present review
and bio-technologies is the realm where chitosan and chitosan deriva-
tives have raised greater scientic interest because of their remarkable In consideration of the above, the scope of the present review is to
structural and functional properties. Among them, carboxymethyl chito- identify the lines of applied research that are now consolidating major
san (CMCS), a water soluble derivative of chitosan, has attracted boom- advances made with the CMCS during the last decade in the eld of
ing interests in several elds such as in vitro diagnostics [57], drug delivery and tissue engineering. The novelty of these facts is
theranostics [8] bioimaging [9], biosensors [10,11], wound healing [12], underlined by the fact that a previous attempt to review the literature
gene therapy [1316] and food technology [17,18] but its greatest impact on CMCS has focused primarily on the general biomedical applications
has been in the area of drug delivery and tissue engineering. CMCS is of CMCS with only a minor part dealing with drug delivery and tissue
potentially biologically compatible material that is chemically versatile engineering applications [41]. While another review article authored
(NH2 and COOH) groups and various molecular weight, (MW). The by Mourya et al. [42] mainly covers literature regarding the synthesis
positive facets of increased water solubility, excellent biocompatibility and characterisation of CMCS along with its pharmaceutical applica-
[19], admirable biodegradability, high moisture retention ability [20], tions. CMCS being inherently bestowed with astonishing physical,
improved antioxidant property [21], enhanced antibacterial [22] and chemical and biological features, emerging trends show that it is highly
antifungal [23] activity and non-toxicity as compared to chitosan has suitable for the delivery of numerous bioactive and therapeutic com-
provided ample opportunities to the drug delivery and tissue engineer- pounds and for the repair and reconstruction of damaged and/or dis-
ing scientists to create a plethora of formulations and scaffolds. In addi- eases tissues. Excellent biocompatibility, improved biodegradability,
tion, it is also known to be more bioactive [24], promotes osteogenesis enhanced antimicrobial activity, better chelating ability, moisture
56 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

absorption and retention capacity, increased antioxidant property, and 4. Biopharmaceutical and toxicological prole of CMCS
higher bioactivity of CMCS compared to its parent polymer chitosan
has contributed to the exhaustive usage of this biopolymer in drug de- As a result of the research undertaken over the last decade there is
livery and tissue engineering applications both in vitro and in vivo. an acceptable understanding of suitability of CMCS as a versatile func-
This review therefore intends to convey to the reader the detailed infor- tional biomaterial for delivering different therapeutic moieties and tis-
mation on various CMCS based formulations for drug delivery and tissue sue engineering applications. Among the water soluble chitosan
engineering and their preparative techniques along with applications in derivative, CMCS is an amphiprotic ether derivative containing carboxyl
tissues like bone, cartilage and nerves in last few years. The review gives and amine groups in the molecule. Chemically, chitosan is poly-1,4-
an overview of CMCS based formulations for targeted delivery focusing linked -D-glucosamine, a cationic polysaccharide prepared by alkaline
cancer specic and organ specic drug delivery. The article also gives a N-deacetylation of chitin. The inherent properties of chitosan itself like
brief discussion about the fact that tissue engineering applications are biocompatibility, non-toxicity, biodegradability and antimicrobial char-
often combined with drug delivery strategy. In this regard, it is very im- acteristic makes it suitable for various drug delivery [52,53] and tissue
portant to understand the basic anatomy of a drug delivery vehicle engineering applications [54,55]. But the limited solubility of chitosan
which is elucidated in the section discussed later. in common solvents including water hinders its full potential exploita-
tion in biomedical and pharmaceutical industry. Therefore introducing
carboxymethyl group into the chitosan polymer chain endows it with
3. Limitations with the use of chitosan in drug delivery some outstanding physical, chemical and biological properties suitable
for delivery of therapeutic compounds and tissue repair and reconstruc-
The drug absorption enhancing property of chitosan due to its bio/ tion purpose. The water solubility property of CMCS at various pH and
mucoadhesive nature (i.e. delayed clearance of the formulation from different preparation conditions has been experimentally demonstrat-
the absorption site) and the transient opening of the tight junctions be- ed. The study showed that degree of carboxymethylation has critical ef-
tween the epithelial cells of the mucosal membrane are well experi- fect on aqueous solubility of CMCS. The study demonstrated that the
mentally demonstrated and reported in literature. But the major water insolubility of CMCS at different pHs varied with the degree of
limitation associated with the use of chitosan in efcaciously increasing carboxymethylation. The experiment also showed that the increase in
mucosal drug absorption is its poor solubility at pH higher than its pKa the reaction temperature increased the fraction of carboxymethylation
(pH 5.56.5). Chitosan is soluble in aqueous dilute acid such as hydro- and thereby increased the insolubility at lower pHs. Also the decrease
chloric acid and aqueous organic acid such as formic, acetic, oxalic and in the fraction of carboxymehylation increased the insolubility at higher
lactic acids when the degree of deacetylation (DD) of chitin reaches pHs [56]. Similarly, DD and degree of substitution (DS) are known to
about 50%. While it remains insoluble at neutral and alkaline pH values. have critical effect on the moisture-absorption and moisture-retention
The main mechanism involved in the solubilisation of chitosan is pro- abilities of CMCS. The study revealed that under conditions of high rel-
tonation of the NH2 function on the C-2 position of the D- ative humidity, the maximum moisture absorption and moisture reten-
glucosamine monomeric unit of chitosan, whereby the polysaccharide tion were obtained at DD values of about 50%. Also when the DD values
is converted to a polyelectrolyte in acidic media. The main factors deviated from 50% moisture absorption and moisture retention de-
governing the solubility of chitosan are DD, the ionic concentration, creased. While both moisture absorption and moisture retention in-
the pH, the MW, the nature of the acid used for protonation, the distri- creased with the increase in DS value [57]. Currently, a quantitative
bution of acetyl groups along the chain and the conditions of the meth- study of the acid base equilibrium of CMCS has been carried out by a
od of isolation and drying of the polysaccharide. Highly deacetylated group of researchers which can be useful for many biomedical applica-
chitosans (N85%) are soluble only up to a pH of 6.5. The dependence tions. The study investigated the effect of metal ion properties on the
of the degree of ionisation on the pH and the pKa of the acid have stability of the complexes. The results showed that the study of com-
been experimentally displayed by examination of the role of proton- plexes can be ordered as Mn(N,O-CMCS) b Co(N,O-CMCS),Ni(N,O-
ation in the presence of acetic acid and hydrochloric acid on solubility CMCS) b Cu(N,O-CMCS) b Zn(N,O-CMCS) [58]. Apart from these, the
of chitosan [43]. The decrement of intermolecular interactions and the antibacterial characteristics of CMCS [59], its fungistatic activity [60], an-
lower crystallinity causing change in the microstructure of chitosan, timicrobial properties of modied forms of CMCS [6163] and CMCS
which facilitate the permeation of water, LiOH hydrates and urea hy- based composites [64,65] have been well reported in literature.
drates, thereby enhancing chitosan dissolution in an aqueous solution CMCS has been known to be highly biocompatible and is also known
of LiOH/urea [44]. to promote the proliferation of the normal skin broblast signicantly
It is a well known fact that the pH of the small intestines increases but inhibited the proliferation of keloid broblast [19]. Tao et al. pre-
from the duodenum to the terminal ileum from pH about 4.5 to 7.4. pared CMCS characterised with different sulfate content and concentra-
Therefore, in the lower part of intestine, chitosan will not be soluble. tion. MTT method was applied to evaluate effect of CMCS on broblast
And in order to achieve higher oral bioavailability of drugs through proliferation. The results of the study demonstrated that CMCS with sul-
the use of chitosan as an absorption enhancing delivery system, dissolu- fate content 26.26% at the concentration of 100 g/mL shows best po-
tion of chitosan at the pH values present in the lumen of small and tential for skin broblast proliferation [66]. Currently, it is also shown
large intestines is must. In consideration of the above facts, several at- that oligo-chitosan, N,O-CMCS and N-CMCS in sheets and pastes are
tempts have been made and a large body of research exists on cytocompatible with potential of wound healing when cytotoxicity
chemical modication of chitosan through derivatisation of the amine was evaluated using primary normal human dermal broblast cultures
and/or hydroxyl groups to enhance the water solubility of chitosan. and hypertrophic scars [67]. The biological safety of CMCS in blood sys-
These derivatives mainly include sulfonation [45], quaternarisation tems of rats has been experimentally established [27]. Moreover, CMCS
[46], carboxymethylation [47], N- and O-hydroxyalkylation [48] and dif- is known to be safe in vivo and slightly inhibited growth of sarcoma 180
ferent grafted copolymers of chitosan [4951]. Among these, chitosan and enhanced body immunity via elevation of serum IL-2 and TNF-
derivatives, CMCS has drawn signicant attention of the researchers level in treated mice [28]. Apart from this, the excellent biodegradability
for drug delivery applications due to its outstandingly enhanced water of CMCS by in vitro and in vivo evaluation has been experimentally dem-
solubility, superior bio/mucoadhesive property, ease of preparation onstrated in rats. This study revealed that liver played central role in
and numerous other admirable physicochemical and biological charac- biodegradation of CMCS [68]. The better biodegradability of 1-ethyl-
teristics that are elaborated in Fig. 1 which make it highly suitable for 3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross
fabricating different drug delivery formulations and tissue engineering linked CMCS lms in lysozyme solution (pH 7.4, 37 C) has been report-
biomaterials. ed. Also, these lms enhanced the spread of Neuro-2a cells and provided
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 57

Fig. 1. Represent desirable property of CMCS and which makes it highly suitable for fabricating different drug delivery formulations and tissue engineering biomaterials nanosystem.

a good proliferation substratum for Neuro-2a cells, as compared to chi- 5. Synthesis of various carboxymethyl chitosans (CMCS)
tosan lms [69]. The biodegradation study on CMCS-g-medium chain
length polyhydoxyalkanoates by Bhatt and coworkers showed that de- Synthesis of different types of CMCS by different chemical routes and
pends on several factors such as temperature, pH, water potential, oxy- effects of DD and DS along with relationship between molecular struc-
gen content, stereo regularity and crystallinity of the polymer, and its ture and moisture absorption and moisture retention abilities of CMCS
material processing [70]. The spontaneous degradation of CMCS in soil has been well described earlier by several researchers [8387]. The ef-
and by enzymes with satisfactory results has been earlier reported in lit- fect of various parameters like acid, pH, ionic concentration on the ag-
erature [71]. In this view, controlling the degradation of covalently gregation behavior of the polymer in aqueous systems has also been
cross-linked CMCS utilizing bimodal MW distribution as reported by reported [88,89]. In fact, recently, Kong and coworkers developed a
Guangyuan and coworkers is worth mentioning [72]. Recently, a study novel method for simultaneously determining DD, DS, and the distribu-
demonstrating the signicant effect of MW of CMCS on its uptake tion factor of COONa or COOH in CMCS by potentiometric titration
from the lumen of abdomen and blood vessels to peripheral tissues, [90]. Fig. 3 shows the preparative methods of different types of CMCS
the distribution of this chemical and urinary excretion after intraperito-
neal administration has been carried out [73]. 5.1. Synthesis of O-carboxymethyl chitosans (O-CMCS)
The earliest study establishing the low toxicity of CMCS dates back to
early 1990s when Tokura et al. [74] studied the biological activities of dif- Among the water soluble derivatives of CMCS, O-CMCS is known to
ferent biodegradable polysaccharides. With regard to the in vivo toxicity, be an amphiprotic ether derivative which contains COOH groups and
no acute toxicity was detected in blood systems of rats after CMCS was NH2 groups in the molecule. The reaction medium used for preparation
absorbed in the abdominal cavity and degraded gradually in the blood of O-CMCS is strongly alkaline. O-CMCS is prepared by suspending chi-
[75]. Finally a recent study exploring the in vitro cytotoxicity prole of tosan, sodium hydroxide and solvent isopropanol into ask and stirring
chitosan, O-CMCS and N,O-CMCS nanoparticles to breast cancer cells- the alkaline slurry at room temperature for 1 h. Subsequently,
MCF-7 showed less toxicity (almost 98% viability was found) [76]. In monochloroacetic acid dissolved in isopropanol is added to reaction
this context, the cytocompatibility of some of the modied CMCS formu- mixture drop wise within 30 min. The whole reaction mixture is reacted
lations like CMCS-2, 2 ethylenedioxy bisethylamine-folate [77], CMCS- for 4 h at 55 C. Finally, the solid is ltered and washed with ethyl alco-
Polyamidoamine dendrimer nanoparticles [78] and N-octyl-N,O-CMCS hol and dried in vacuum. The preparation conditions and degree of
[79] demonstrated currently is worth mentioning. In addition to the tox- carboxymethylation govern water solubility of O-CMCS as experimen-
icological prole of CMCS in different forms, the ability of CMCS in pre- tally demonstrated by Chen at al. [57].
vention and reduction of postsurgical adhesion formation [80,81,29]
and in vivo wound healing ability [82] have been experimentally demon- 5.2. Synthesis of N-carboxymethyl chitosans (N-CMCS)
strated. Finally, the inherent excellent mucoadhesive and absorption en-
hancing property of chitosan are retained in CMCS which is favorable for Synthesis of N-CMCS takes place in slightly acidic medium. To pre-
drug delivery applications. Therefore, this interesting biopharmaceutical pare N-CMCS, free amino group of chitosan is reacted with glyoxylic
and toxicological prole of CMCS has encouraged its application as drug acid to give soluble aldimine and then aldimine is reduced with reduc-
delivery and tissue engineering biomaterial. Fig. 2 illustrates different ing agent sodium cyanoborohydride. The reaction neither requires
outstanding and astonishing physicochemical and biological properties warming, nor cooling. The proportions of acetyl, carboxymethyl and
of CMCS that contribute for its suitability in numerous drug delivery free amino groups are determined by DA and MW of the chitosan
and tissue engineering applications. used and the quantity of glyoxylic acid used [41].
58 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

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.

5.3. Synthesis of N,O-carboxymethyl chitosans (N,O-CMCS) sites of the glucosamine units of the chitosan structure. The main attrac-
tive features of N,O-CMCS are moisture retention, gel formation and
N,O-CMCS is hydrophilic and amphoteric polyelectrolyte which bears good biocompatibility along with increased water solubility and en-
carboxymethyl substituents at some of the amino and primary hydroxyl hanced antibacterial property that makes this polymer derivative highly

Fig. 3. Preparative methods of different types of CMCS.


L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 59

suitable for wide variety of biomedical applications. N,O-CMCS can be pre- absorbed by hydrogels may range from 10% to thousands of times of
pared by using chitosan, sodium hydroxide, isopropanol and chloroacetic its own volume. The high water content makes hydrogels biocompatible
acid [91,92]. while coherence of their mechanical properties with the soft tissues as-
sists and enhances healing phenomenon. The compatibility of mechan-
5.4. Synthesis of N,N-dicarboxymethyl chitosans (N,N-di-CMCS) ical properties of hydrogels with the soft tissues also mimics
morphological and functional characteristics of organ tissue. The visco-
The most crucial parameters for preparing N,N-di-CMCS are the con- elastic property of hydrogels causes minimised damaged to the sur-
centration of chitosan, water, glacial acetic acid, glyoxylic acid, and sodi- rounding tissues in the host. Natural polymer based hydrogels have
um borohydride. In order to synthesise N,N-di-CMCS, to a xed also attracted the attention of scientists and researchers because of
concentration of chitosan (30 g) suspended in demineralised water their improved biocompatibility, biodegradability and notable capabili-
(3 l), 27 g of glacial acetic acid is added and stirred for 20 min. After ty of controlled delivery of bioactive molecules [129131].
this glyoxylic acid is added (178 mL 50% v/v corresponding to 119 g of As far as covalent cross linking is concerned, it leads to the formation
glyoxylic acid) and the molar ratio of amine/glyoxylic acid is set to of hydrogels with permanent network structure because irreversible
1:9 at pH 23. Finally, with the help of peristaltic pump (1.2 ml min1), chemical links are formed. Therefore, this type of linkage not only al-
sodium borohydride (90 g) in water (2.5 l) is delivered as a 3.6% solution lows absorption of water and/or bioactive compounds without dissolu-
to the reaction vessel. The N,N-di-CMCS prepared by this method exhibits tion but also permits drug release by diffusion. Broadly covalently
good lm forming ability, good capacity to chelate metal ions and also crosslinked chitosan hydrogels can be divided into three categories: chi-
possesses excellent osteoinductive properties with calcium phosphate tosan cross linked with itself, hybrid polymer network (HPN) and semi
[87]. or full interpenetrating networks (IPN). In chitosan cross linked with it-
self, crosslinking involves two structural units that may or may not be-
6. Preparation techniques of CMCS based formulations for drug long to the same chitosan polymeric chain [132]. The overall nal
delivery structure of this type of hydrogel can be considered as a crosslinked
gel network dissolved in a second entangled network formed by chito-
CMCS based formulations for drug delivery applications have been san chains with restricted mobility [133]. In hybrid polymer networks
prepared by different techniques and methods by various researchers (HPN) based hydrogels the crosslinking reaction occurs between a
as per the requirement of the host tissue/organ and the type of drug structural unit of chitosan chain and a structural unit of polymeric
being delivered. CMCS based hydrogels are one of the major formula- chain of another type. Although crosslinking of two structural units of
tions for drug delivery applications and can be prepared by different the same type and/or belonging to the same polymeric chain cannot
methods. In each process, CMCS is either physically associated or chem- be excluded. Finally semi or full IPNs contain a non reacting polymer
ically cross-linked. The major schemes of physical interactions that lead which is added to the chitosan solution before crosslinking. Thus cross
to gelation of CMCS solution in hydrogels are ionic interaction, polyelec- linked polymer network in which the non reacting polymer is
trolyte complex; inter polymer complex, and hydrophobic associations. entrapped (semi-IPN) are thus formed. This additional polymer can fur-
Moreover the physically cross linked hydrogels have the advantage of ther be crosslinked in order to have two entangled crosslinked net-
gel formation without the use of cross-linking moieties. But they have works forming a full IPN whose microstructure and properties can be
certain limitations like difculty in accurately controlling the physical quite different from its corresponding semi-IPN [134]. Ionically cross
gel pore size, chemical functionalisation, dissolution and degradation, linked hydrogels leads to the formation of non permanent network
thereby providing inconsistent in vivo performance. Cross linked structure since reversible links are formed [135]. Ionically cross linked
hydrogels often possess better mechanical properties where cross hydrogels show higher swelling sensitivity to pH changes as compared
links can be incorporated either chemically or through irradiation. But to hydrogels having covalent cross linking. The entities reacting with
chemical cross linking method for preparation of hydrogels suffers chitosan are ions or ionic molecules with well dened MW in ionically
from disadvantage of having toxic residues of cross linking chemicals. crosslinking. It is different from polyelectrolyte complexation as the en-
On the other hand preparation of microspheres, nanoparticles and mi- tities reacting with chitosan are polymer with a broad MW distribution
celles/aggregates can be carried out by different methods like emulsion [136]. In contrast to covalent crosslining ionic cross linking is a simple
cross linking, sonication, coacervation/precipitation, spray drying, ionic and mild procedure as no auxillary molecules such as catalyst are re-
gelation, sieving, emulsion droplet-coalescence and reverse micellar quired [137]. Ionic cross linking can be assured by the classical method
methods. Each method has its own advantage and certain limitations of preparing a cross linked network namely by the addition of
and therefore the choice of preparative technique depends on the type crosslinker either solubilised [138] or dispersed [139,140] to the chito-
of particulate being synthesised, its size range, composition, the drug san solution.
being loaded or encapsulated and the target tissue/organ where drug Superporous hydrogels containing poly (acrylic acid-co-acrylamide)/
is to be delivered. Similarly, layer-by-layer assembly and cross linking O-CMCS IPNs were prepared by cross-linking O-CMCS with glutaralde-
are the main preparative methods for the synthesis of composites and hyde (GA) after superporous hydrogel was synthesised by Yin et al. An
blending, casting and drying methods are the most popular preparative enhanced capacity of loading insulin was reported for the superporous
techniques for lms and bers for the purpose of delivery of different hydrogels as compared to the non-porous hydrogels. Due to improved
drugs. Table 1 shows preparation techniques of CMCS based formula- mechanical properties, in vitro muco-adhesive force and loading capaci-
tions for drug delivery. ties, these IPNs showed potential of muco-adhesive system for peroral
delivery of peptide and protein drugs [95]. Similarly, Chen et al. prepared
7. CMCS formulations for drug delivery applications a novel type of IPN hydrogel membrane of poly (N isopropylacrylamide)/
CMCS and systematically studied the effects of the feed ratio of compo-
7.1. CMCS hydrogels nents, swelling medium and irradiation dose on the swelling and
deswelling properties of the hydrogel. The results showed that a combi-
In the recent few decades, the pharmaceutical industries have nation of pH and temperature can be coupled to control the responsive
witnessed the emergence of drug delivery technologies as a powerful behavior of poly(N-isopropylmethacrylamide (PNIPAAM)/CMCS
and efcient tool in order to effectively use the existing drugs and devel- hydrogels [93]. There are several CMCS based semi-IPNs reported in lit-
op new drug candidates successfully [128]. A hydrogel can be dened as erature prepared by crosslinking with genipin [20], GA or cross-linked/
cross linked network formed from a macromolecular hydrophilic poly- grafted with ethylene glycol diglycidyl ether [99], and N,N-
mer capable of absorbing large amount of water. The volume of water methylenebisacrylamide [97]. Min Wang and coworkers synthesised
60 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 1
Preparation techniques of CMCS based formulations for drug delivery.

CMCS based formulation Composition of formulation Method of preparation Refs.

Hydrogel CMCS/poly(N-isopropylacrylamide) (PNIPAAM) Physical cross linking [93]


Hydrogel N,O-CMCS & alginate Chemical cross linking [20]
Hydrogel N,O-CMCS Physical cross linking [94]
Hydrogel CMCS Chemical cross linking [95]
Hydrogel Poly(acrylic acid-co-acrylamide)/O,CMCS Chemical cross linking [96]
Hydrogel CMCS/poly(N-isopropylacrylamide) Chemical cross linking [97]
Hydrogel CMCS Chemical cross linking [98]
Hydrogel 6-O,CMCS/polyurethane Chemical cross linking [39]
Hydrogel CMCS/poly(acrylonitrile) Chemical cross linking [99]
Hydrogel CMCS Radiation cross linking [100]
Hydrogel CM-cellulose/CMCS Radiation cross linking [101]
Hydrogel CMCS Radiation cross linking [102]
Hydrogel CMCS/gelatin Radiation cross linking [103]
Hydrogel Nano-Ag/gelatin/CMCS Radiation cross linking [104]
Hydrogel N-isopropylacrylamide/CMCS Radiation cross linking [105]
Nanoparticles Cross linked lactosaminated CMCS Ionic gelication [106]
Nanoparticles O,CMCS Ionic gelation [107]
Nanoparticles Amphiphilic octadecyl Q-CMCS Microemulsion [108]
Nanoparticles Cholesterol modied O-CMCS Sonication [109]
Nanoparticles CMCS bound Fe3O4 Spraying co-precipitation [110]
Nanoparticles CMCS/poly(amidoamine) Coprecipitation [111]
Nanoparticles O-CMCS Ionic cross-linking [112]
Nanoparticles Oleoyl-CMCS Self-assembly [113]
Nanoparticles Oleoyl-CMCS Oil in water emulsication [114]
Microspheres CaCO3/CMCS Precipitation [115]
Microcapsules and microparticles CMCS & CMCS graft-poly(N,N-diethylacrylamide) Self-assembly [116]
(CMCTS-g-PDEA)
Microspheres O-CMCS bound with iron oxide In situ coprecipitation [117]
and incorporation
Microspheres Alginate/Q-CMCS/clay nanocomposite Ion-crosslinking [118]
Microspheres Alginate & chemically modied CMCS Ionotropically-crosslinked [22]
Microspheres Ambroxol hydrochloride CMCS Emulsion chemical cross-linking [119]
Micelles Linoleic acid modied CMCS Sonication [120]
Micelles PEG-grafted-CMCS Ion complex formation [121]
Composite CMCS/TMCS Direct crosslinking [122]
Composite Folate conjugated CMCS/Fe3O4/CdTe Layer-by-layer assembly technique [123]
Composite O-CMCS/ cyclodextrin Crosslinking method [124]
Composite FACMCSZnS:Mn Ionic crosslinking [125]
Film Poly-vinyl alcohol/CMCS Blending/casting [126]
Fibers Ag nanoparticles/poly vinyl alcohol/CMCS Electrospinning technique [127]

CMCS hydrogels by gamma rays induced cross linking where adsorption mechanism for the reaction of CMCS with genepin was speculated as il-
kinetics study showed fast adsorption of Fe (III) ions onto CMCS gels. This lustrated in Fig. 4B [141]. Similarly, Yang et al. [142] prepared hydrogel
was primarily due to the coordination of Fe (III) ions with amino, hy- beads based on methoxy poly (ethylene glycol) grafted carboxymethyl
droxyl and carboxyl groups of CMCS molecules [102]. Also an ESR chitosan (mPEG-g-CMC) and alginate in order to construct interpen-
study on CMCS radicals in a highly concentrated CMCS aqueous solution etrating polymeric matrix. Fig. 5 [142] illustrates the synthesis of
which forms hydrogels by ionizing radiation was performed by Seiichi mPEG-g-CMC copolymer using Schiff's base method. Some of the CMCS
Saiki et al. [100]. Physically cross linked alginate-N,O-CMCS hydrogels based hydrogels for delivery of different therapeutics are summarised
with calcium for oral delivery of protein drugs was prepared by Lin and in Table 2.
coworkers [94]. Substantial research has been done in chemically
cross-linked hydrogels also. Currently, pH sensitive hydrogels composed 7.2. CMCS microspheres
of CMCS, chemically cross linked by GA was prepared and evaluated
in vitro as a potential carrier for colon targeted drug delivery of Microspheres are spherical free owing particles having size range
ornidazole [98]. In this regard, the synthesis of novel polyampholyte between 50 m and 2 mm with drugs entrapped inside them. While
hydrogels based on CMCS of varying DD and DS cross linked with GA in matrix type microspheres drug is mainly released by erosion mecha-
by Chen et al. [95] is worth mentioning. With increasing DD or DS nism, release of drug takes place by diffusion and erosion in matrix and
value, the hydrogel changed from polyampholyte into polycations or reservoir type microspheres [149]. In general drug release rate depends
polyanions, respectively. The release of bovine serum albumin (BSA) upon solubility, diffusion, biodegradability of the matrix, drug loading
was much quicker at pH 7.4 buffer than pH 1.2 solutions. In recent efciency, and size of the microspheres. Therefore the drug release
years radiation cross linking has proved to be safer, clean and effective mechanism can be altered by varying polymer employed and its proper-
method of hydrogel synthesis. The products formed are free of toxicity ties. Microspheres are mainly exploited in controlled drug delivery but
additives as neither initiator nor cross linker is required as in convention- by derivatisation and surface modication they can also be used for
al chemical routines. The synthesis of CMCS using -rays radiation [101, drug targeting purpose. The partition coefcient determines the drug
102] and ionizing radiation [100] has been reported in the literature. distribution within the microspheres. CMCS based microspheres have
Yang et al. [141] have in situ synthesised dark bluish coloured 5- emerged as efcient drug carriers due to their ability to encapsulate a
uorouracil (5-FU) loaded CMCS hydrogels using genepin as the cross variety of drugs, biocompatibility, protection of fragile drugs, high bio-
linker and it was speculated that the dark bluish colour of the hydrogels availability and sustained drug release characteristics. Currently,
prepared resulted from the cross linking reaction between genipin and carboxymethyl cellulose and CMCS based biodegradable and highly po-
the amino groups of CMCS (Fig. 4A). The possible cross linking rous microspheres were prepared with an inverse emulsion-cross-
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 61

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 with
permission from [141]).

linking method without incorporating any extraneous cross-linking (AQCOM) microspheres were prepared by crosslinking with CaCl2 and
agents capable of loading anticancer drug doxorubicin (DOX) whose re- the drug-controlled release was evaluated by using BSA as model drug.
lease prole was adjustable. The in vitro and in vivo studies indicated The AQCOM microsphere displayed more excellent encapsulation and
that these microspheres can be used as biocompatible and biodegradable controlled release capacities than the microsphere without OMMT and
embolic agents for transarterial embolisation [150]. Recently, quaternised in vitro active cutaneous anaphylaxis test on Guinea pigs did not cause
carboxymethyl chitosan (QCMCS) was intercalated into the interlayer of anaphylaxis [118]. Fig. 6 shows the pictures of Guinea pigs after ACA
organic montmorillonite (OMMT) to obtain the QCMCS/OMMT nano- test of AQCOM-3 microspheres. Signicant anaphylaxis such as swelling
composites. The cross linked alginate-Q-CMCS-organic montmorillonite and even ulcerated induced by 2,4-dinitrophenol was observed (as

Fig. 5. Synthesis of the methoxy poly (ethylene glycol) grafted carboxymethyl chitosan (mPEG-g-CMC) copolymer (reproduced with permission from [142]).
62 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 2
Summarizes different CMCS based hydrogels for delivery of different therapeutics in drug delivery applications.

Composition of hydrogel Remark Refs.

Photocrosslinked methacrylated O-CMCS & PEG diacrylate OCMCS-based hydrogels with varying concentrations of carboxy and methacrylate groups are envisaged as [143]
attractive and versatile synthetic extracellular matrices that can effectively sequester, protect and controllably
release basic proteins. Varying the carboxyl and methacrylate groups respectively allows variation of charge and
water content/pore size which ultimately allows control of the absorption and dynamic release, via substrate
biodegradation, of basic proteins.
Covalently crosslinked N,O-CMCS and oxidized alginate Good cytocompatibility against NH3T3 cells by in vitro test and no obvious cytotoxicity for major organs during [144]
hydrogel the period of 21-day intraperitoneal administration was observed by acute toxicity test. The hydrogels developed
also showed good hemocompatibility.
Nanocomposite hydrogel composed of Curcumin, Nano curcumin with improved stability by using methoxy poly(ethylene glycol)-b-poly( -caprolactone) [32]
N,O-CMCS and oxidized alginate copolymer as carrier was slowly released from the hydrogel with the diffusion controllable manner at initial phase
followed by the corrosion manner of hydrogel at terminal phase. In vivo wound healing study showed that the
hydrogel could signicantly enhance there-epithelialisation of epidermis and collagen deposition in the wound
tissue.
CMCS and carbopol 934 hydrogel for delivery of In vitro swelling studies have shown little swelling in acidic pH 432% at the end of 2 h and 1631% in basic pH at the [145]
theophylline end of 12 h. The pH-sensitive hydrogel of CMCS can be used for extended release of theophylline in intestine and
can be highly useful in treating symptoms of nocturnal asthma.
CMCS hydrogel A simple and economical method with no toxic chemicals involved was introduced. The steam-induced [146]
crosslinking of CMCS sodium salt was observed to be quite efcacious. Depending on the harshness of steaming
conditions used, the DS of the hydrogels was found to be up to 36. The increasing temperature and duration of
steam exposure changed the coloration of the samples from light beige to brown.
In situ gelable hydrogel composed of N-CMCS and oxidized The rate of gelation was directly related to the degree of oxidation of Odex and the hydrogels underwent fast mass [147]
dextran (Odex) loss in the rst 2 weeks, followed by a more moderate degradation. The in vivo studies in mice full-thickness
transcutaneous wound models showed its ability of enhanced wound healing ability.
Physically crosslinked hydrogels of CMCS with cellulose With the increase in the interaction of component polymers, the swelling and drug release rate of hydrogels [148]
ethers including hydroxyethylcellulose decrease. Component polymer ratio controlled the swelling and drug release from hydrogels.
and methylcellulose
5-FU or bevacizumab loaded N,O-CMCS hydrogels The in vitro drug release experiments showed that nearly 100% of 5-FU was released from the drug-loaded [141]
crosslinked with genepin hydrogels within 8 h, but less than 20% bevacizumab was released after 53 h. The hydrogels provided great
opportunity to increase the therapeutic efcacy of glaucoma ltration surgery.

Fig. 6. Photos of Guinea pigs by ACA test: (a) experimental procedure, (b) the rst day after treating by AQCOM-3 microsphere, (c) the seventh day after treating by AQCOM-3 microsphere,
(d) the eighth day after treating by AQCOM-3 microsphere, (e) the eighth day after negative control, and (f) the eighth day after positive control (reproduced with permission from [118]).
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 63

shown in Fig. 6f). Again the induce contact and even challenge exposure Nano precipitation technique, dialysis method and sonication are some
on Guinea pig treated by AQCOM-3 microspheres did not induce the ana- of the preparative methods of micellar structures. Formation of micelles
phylaxis (bd) which illustrated that AQCOM-3 microspheres did not in- by self-assembly is the most popular preparative methodology in case of
duce the anaphylaxis [118]. Similarly, clozapine loaded spherical CMCS based or modied CMCS micelles designed and developed for
microspheres of chitosan and CMCS within size range of 100 0.25 m drug delivery purpose. The nanoscopic dimension, segregated core/
to 140 0.25 m were prepared by spray drying technique. The in vitro shell structure, sheltering effect of hydrophobic core on drugs
drug release study showed that CMCS microspheres released most part entrapped and stealthy characteristics by their hydrophilic shells
of the drug in the intestine compared to chitosan microspheres hence makes polymeric micelles suitable for tumor targeting [153,154].
controlled drug release was achieved [151]. Ma et al. [116] developed a CMCS has been extensively exploited by several researchers for devel-
simple method to fabricate BSA loaded self-assembled CMCS and CMCS- oping micelle based nanoformulations for model anticancer drug DOX
graft-poly (N,N-diethylacrylamide) microcapsules and microparticles [155,30]. Aifeng et al. synthesised and characterised DOX loaded
whose in vitro drug release rate and encapsulation efciency depended octreotide-modied N-octyl-O,N-CMCS micelles which proved to be
on pH value. The microspheres were found to be non-cytotoxic against promising carrier for efcient intracellular targeting of antitumor
L02 human hepatic natural cell and release of BSA could be sustained. drugs [156]. Similarly self-aggregated nanoparticles from linoleic acid
Also sustained release of BSA from microspheres based on mixtures of (LA) modied CMCS were prepared by Yu-long Tan and coworkers for
ionotropically cross linked sodium alginate and chemically modied delivery of hydrophobic anticancer drug adriamycin (ADR). The critical
CMCS and coated through polyelectrolyte complexation with chitosan aggregation concentration determined by measuring the uorescence
grafted with poly (ethylene glycol) has been reported in literature. Fig. 7 intensity of the pyrene as a uorescent probe, was in the range of
[26] illustrates the carboxymethylation of chitosan and synthesis of 0.0610.081 mg/mL [120]. Gong and coworkers grafted Cis-3-(9H-
CMCS grafted sodium acrylate copolymer. Again, high encapsulation ef- purin-6-ylthio)-acrylic acid (PTA) on primary amino of CMCS, under
ciency and sustained release of model anticancer drug DOX has been the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
achieved by preparing CMCS-CaCO3 microspheres by Wang and co- drochloride EDCHCl and N-Hydroxysuccinimide (NHS) i.e. the reaction
workers [115]. There is also information regarding preparation of two took following two steps: synthesis of PTA (Fig. 8A) and synthesis of PTA
kinds of O-CMCS bounded with iron oxide particles by in situ co- grafted CMCS (PTA-g-CMCS) Fig. 8B [34]. Amphiphilic polymeric pro-
precipitation and incorporation methods reported in literature. The mag- drug PTA-g-CMCS designed and synthesised could self-assemble into
netic properties of these microspheres were studied to evaluate its poten- spherical micelles with a size ranging from 104 to 285 nm with 6-
tial in drug delivery applications [117]. mercaptopurine loaded into it. The result demonstrated that
nanocarrier was found suitable for controlled drug delivery. Fig. 8C
7.3. CMCS micelles/aggregates shows 6-methoxy propyl release from PTA-g-CMCS in the presence of
glutathione, which follows a Michael additionelimination reaction [34].
When polar or non-polar ends of amphiphilic polymer self assemble Jeong et al. [121] synthesised methoxy poly(ethylene glycol)-grafted
by hydrophobic or ion-pair interaction in a monophasic or biphasic liq- carboxymethyl chitosan (CMCPEG) copolymer using a water-soluble
uid, nanosised (200 nm to 0.5 m) structures are formed which are re- carbodiimide. In addition, DOX-incorporated nanoparticles using
ferred to as micelles. In an aqueous environment, the amphiphilic CMCPEG were prepared by ion complex formation between the amine
polymers form hydrophobic core by assembly of lipophilic parts and groups of DOX and the carboxyl group of CMCPEG. The self assembled
vice versa. The repositioning of hydrophobic and hydrophilic drug with- N-phthaloyl-CMCS based micelles for drug delivery of levooxacine hy-
in the hydrophobic or hydrophilic core of micelles improves the solubil- drochloride and BSA have also been earlier prepared by Peng et al.
ity of drugs in monophasic non-solvent medium often referred to as [157]. Another technique of nano-precipitation was employed for the
micellar solubilisation. In biphasic medium, polymeric segments distri- preparation of copolymer polylactide-polyethylene glycol succinate,
bution takes place according to their physicochemical characteristics. 1,3-beta-glucan (Glu), O-CMCS and folate-conjugated O-CMCS micelles
In general, the hydrophobicity of the drug and the type of polymer for efcient encapsulation of herbal anticancer drug curcumin. The nano-
determines the preparative method of micelle. In fact the experimental formulation displayed better aqueous solubility and biodegradation
evidences have led to the conclusion that polymeric micelles exhibit nu- along with signicantly enhanced the cellular uptake by cancer cell
merous promising properties for oral delivery of lipophilic drugs [152]. HT29 and HeLa. The anti-tumor-promoting effect of the curcumin

Fig. 7. Scheme of carboxymethylation of chitosan and synthesis of CMCS grafted sodium acrylate copolymer (reproduced with permission from [26]).
64 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

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 a Michael additionelimination reaction:
GSH attacks the -C of the unsaturated bonds, leading to the cleavage of the unsaturated bonds (reproduced with permission from [34]).

encapsulated by copolymer in Hep-G2 cell lines after two weeks of cell triggered fashion. Thus, the toxic side effects and administration fre-
growth is shown in (Fig. 9). The sudy showed that in the cytotoxicity quency of the drugs can be reduced [168]. In addition, nanoscale particles
assay there were no distinct differences in the cell survival. Also in the can passively accumulate in specic tissues (e.g., tumors) through the
anti-tumor promoting assay, the ration of tumor promotion with the enhanced permeability and retention (EPR) effect [169]. Polymers like
Cur, Glu, PLA-TPGS alone was comparable to the control. But contrary CMCS have emerged as a promising drug delivery nanovehicle due to in-
to this clear changes in the size and morphology of tumor between the herent increased water solubility, biological functionality, compatibility,
control and all the tested samples particularly curcumin encapsulated safety, biodegradability, and antimicrobial nature. In the context of
with glucan copolymer. It was observed that in comparison to the drug encapsulated CMCS based nanoparticles, nanoprecipitation, dialy-
tumor on the tested wells, in control the tumor size was much larger sis, micro emulsion, emulsion-solvent diffusion technique, solvent evap-
and their surface was very rough (Fig. 9). [158]. Wang and coworkers oration and ionic gelation methods have been employed so far. The
prepared CaP/CMCS/KALA nanoparticles by self assembly via electro- preparation of self-aggregated cholesterol modied O-CMCS nanoparti-
static interaction between positively charged peptide KALA and nega- cles by sonication method were prepared to be used as a novel carrier
tively charged CaP/CMC nanoparticles in an aqueous solution after for PTX has been reported by Wang et al. [170]. These results suggest
loading anticancer drug DOXHCl onto the CaP/CMCS hybrid nanopar- that cholesterol modied O-CMCS self-assembled nanoparticles can ef-
ticles. The investigation results showed that DOXHCl could be encapsu- fectively solubilise PTX and modify its tissue bio-distribution, which
lated with high efciency and the presence of KALA peptide signicantly may be advantageous in enhancing the therapeutic index and reducing
enhanced the cell inhibition effect [159]. Dialysis method was employed the toxicity of PTX. Similarly Zhang et al. [110] reported synthesis of
for the preparation of methotrexate conjugated O-CMCS micelles where magnetic Fe3O4 nanoparticles functionalised with CMCS by spraying
methotrexate exhibited signicant sustained release behavior in PBS so- co-precipitation method and these magnetic nanoparticles are suitable
lutions (pH 4.0, 7.2 and 9.0). The critical micelle concentration (CMC) of for use as nanomagnetic carriers of drugs formulation. In this context,
O-CMCSmethotrexate conjugates determined in aqueous media was in the preparation of vincristine loaded polymeric ethosomes; formed
the range of 0.00840.0424 mg/mL [160]. In addition, sonication method from amphiphilic octadecyl Q-CMCS with different DS by micro emul-
has also been utilised for the designing and development of CMCS based sion method is worth mentioning [171]. Biocompatible ciprooxacin-
nanocarriers for delivery of anticancer drugs like DOX [161] and paclitax- loaded CMCS nanoparticles by ionic cross-linking method and optimised
el (PTX) [162]. Table 3 shows different CMCS based self aggregates and by using BoxBehnken response surface method by Zhao et al., demon-
the drugs delivered by these fabrications. strated, stronger antibacterial activity against Escherichia coli than the
free ciprooxacin because they can easily be uptaken by cells. Still fabri-
7.4. CMCS nanoparticles cation of CMCS based nanostructure delivery vehicles by ionic gelation is
the most popular method employed till date [105,106].
The emerging trends and recent advances in nanotechnology has A number of CMCS based nanoscale carriers have been constructed
made a signicant impact on the development of drug delivery systems, in recent years for the efcient delivery of different anticancer, anti-
after the liposomes were rst described in the 1960s as carriers of pro- inammatory drugs, antibiotics, proteins, peptides and vaccines.
teins and drugs for disease treatment [167]. Most of the front-line Model anticancer drug DOX has been encapsulated into a number of
drugs are toxic entities that act in unspecic fashion being untargeted, modied CMCS nanosystems like FA modied CMCS nanoparticles
often eliciting unwanted, dose limiting and debilitating side effects. Com- [172] amphiphatic carboxymethylhexanoyl chitosan nanocapsules
pared to conventional drug delivery systems, nanoscale drug delivery ve- [155], and acylated CMCS nanoaggregates [35]. Also, liposomes modi-
hicles are capable of enhancing therapeutic activity by prolonging drug ed with CMCS [173] and CMCS capped magnetic nanoparticles [164]
half-life, improving solubility of hydrophobic drugs, reducing potential have also been exploited for DOX delivery. The drug release prole of
immunogenicity, and/or releasing drugs in a sustained or stimuli- DOX loaded-CMCS-capped-MNP/MMT was tested at pH 7.4 and
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 65

signicant difference in the cellular uptake of curcumin-O,CMCS NPs


comparing normal and cancer cell lines [175] Another multifunctional
nanodrug system containing Fe3O4, O-CMCS and curcumin has been re-
cently fabricated by Thu et al. [176]. Similarly, Shen et al. [123] prepared
CMCS-ferroferric oxide doped cadmium telluride quantum dot nano-
particles with folate receptors afnity, magnetic responsiveness and lu-
minescent property via layer-by-layer assembly technique in situ
surface as shown in Fig. 12. The results indicated that the novel multi-
functional folate conjugated carboxymethyl chitosan-ferroferric oxide
doped cadmium telluride quantum dot nanoparticles synthesised ex-
hibit a high drug loading efciency, minimised cytotoxicity and desir-
able cell compatibility, and are potential candidates for CMCS-based
targeted 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 modi-
cations of CMCS such as amphiphilic carboxymethylhexanoyl chitosan
[177], PEGylated O-CMCS nanoparticles grafted with cyclic Arg-Gly-Asp
(RGD) peptide [178] and LA modied CMCS nanoparticles [162] respec-
tively. Apart from these drugs, CMCS based nanoparticles mediated ve-
hicles have been prepared for delivery of anti-inammatory compounds
[179], proteins/peptides [180] and vaccines [181] along with poorly
water soluble drugs like triamcinolone [182] and vitamin D3 [183].
Table 4 describes various nanoformulations for delivery of different
drugs along with brief method and their outcome.

7.5. CMCS lms and bers

In the recent years, drugs and polymers are manufactured into vari-
ous lm congurations, like coating of some precarious drugs or pro-
teins by thin polymer lms or generating drugpolymer matrix lms.
These different matrices are suitable for facilitating diffusion mediated
drug delivery or adaptable for accomplishing transdermal drug delivery.
The parameters that determine the efcacy of polymeric lms in deliv-
ery of therapeutic compounds are thickness, surface morphology, de-
gree of swelling, degradation behavior, drug release and therapeutic
effect. The drug release pattern and rate of drug release from the lm
coating systems depend on the coating material, thickness of the lm
and circumstance of the applied site. Polymeric lms have also gained
importance as promising drug delivery formulation as its thin layer
resists change in crystallinity and segmental motions. As CMCS shows
inherent excellent biocompatibility, improved biodegradability, antimi-
crobial strength and easily lm forming ability, it has emerged as a suit-
able biopolymer for lms fabrication for drug delivery. The ornidazole
(OD) loaded poly-vinyl alcohol PVA/CMCS lms prepared by blending/
casting method demonstrated excellent antibacterial and biocompati-
bility characteristics which were enhanced with increasing CMCS con-
Fig. 9. Anti-tumor-promoting effects of the curcumin encapsulated by copolymer in Hep-
G2 cell lines after two weeks of cell growth on agar: control (a), Cur (b) and Cur-PLA-TPGS
tent in the lms. The process of preparing the placebo lms and their
(c) under inverted microscope 100 (reproduced with permission from [158]). corresponding drug OD loaded lms (loaded OD) via blending/casting
is shown in Fig. 13 [38]. The results from in vitro and in vivo study dem-
onstrated the blend lms as an excellent candidate for local drug deliv-
pH 5.0 respectively and the released DOX concentration was deter- ery system. A similar study was carried out where PVA/CMCS blend
mined by UVvis measurement. From the drug release curve (Fig. 10) lms prepared by casting and drying method were evaluated as coating
it is clear that the release of DOX from DOX loaded-CMCS-capped- material for site specic drug delivery. The drug release kinetics study
MNP/MMT is pH triggered and the release rate of DOX was higher at by salicylic acid, theophyline, and OD also revealed that a desired rate
pH 5.0 than pH 7.4. It is well known fact that the pH of tumor tissue is of drug release could be obtained by controlling the content of CMCS
lower than normal cells. Thus this delivery system will exhibit smaller in the blend lm, type and MW of drugs, pH of the medium and thick-
toxicity towards normal cells as compared to tumor cells thereby illus- ness of the lm. As pH of the buffer increased the permeability of OD
trating its advantage of safety [174]. Recently, researchers have also de- with a maximum at pH 7 as shown in Fig. 14. The reason for this phe-
veloped O-CMCS [175] and folate conjugated O-CMCS nanosystems nomenon can be attributed to the fact that as pH increased, deproton-
[158] for efcient delivery of another anticancer compound: curcumin. ation of the charge groups of CMCS takes place. CMCS molecules
The cellular uptake study of curcumin-O,CMCS NPs by normal L929 became uncoiled and assumed to be elongated thereby causing the ex-
cells and cancer cell line MCF-7 cells using ow cytometry showed pansion of the bulk of lms. It caused the enhanced water absorption
(Fig. 11) that the nanoformulation uptake was concentration dependent ability and lead to the increase of permeability [126]. A study on the
and non specic. This meant that both the cell line showed increasing BSA and bovine brinogen equilibrium adsorption amount on the
uptake of the nanoformulation with the increase in the concentration PVA/CMCS lms prepared by mechanical blending showed the inu-
of the curcumin-O-CMCS nanoformulation. Also there is not much ence of CMCS content and pH and ionic strength of protein solutions
66 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 3
Different CMCS based self aggregates.

CMCS based formulation Size range of Brief method and outcome Refs.
self-aggregates

O-CMCS aggregates Not only the aggregates but also the unimers of O-CMCS enhanced the solubility [163]
of camptothecin. The aggregates showed good drug loading capacity and sustained
drug release demonstrating potential for localised drug delivery.
Cholesterol modied O-CMCS aggregates 234.9100.1 nm A series of cholesterol modied O-CMCS conjugates with different DS of cholesterol [109]
were synthesised and self-aggregates were prepared by probe sonication in water.
The relationships between the chemical structure, the amphiphilic property and the
morphological characteristics of O-CMCS self-aggregated nanoparticles were
investigated in this study.
LA-modied CMCS self-aggregates 222.81028 nm Covalently conjugated LA to CMCS via EDC mediated reaction to generate self-aggregated [164]
nanoparticles by sonication method. Physically entrapped ADR showed slow release which
can be adjusted by change in medium pH.
LA-modied CMCS aggregates 417.8 17.8 nm The LA modied CMCS self aggregates prepared by sonication method exhibited increased [120]
loading capacity and loading efciency and decreased sustained release for ADR with
increasing DS of LA in CMCS with critical aggregation concentration values in the range
of 0.0610.081 mg/mL.
Deoxycholic acid-O-CMCS-folic acid conjugates 179212 nm Deoxycholic acid and FA modied O-CMCS self-aggregates were prepared by sonication [165]
method and the mean diameter and critical aggregation concentration value changed with
change in DS and pH values.
CMCS nanoclusters 301 nm The results showed that demonstrated that BoxBehnken design methodology was an [166]
effective way to obtain the optimal formulation of tea polyphenols-loaded chitosan nanoclusters,
and the nanoclusters complexation synthesizing through ionic gelation
between CMCS and chitosan hydrochloride was good biomaterials, which could
be successfully used to encapsulate tea polyphenols

on the adsorption amount of these plasma proteins [195]. Drug loaded attenuate the degradation or release behavior of the incorporated drugs
biodegradable polymer based nanobers intended to deliver drugs at or to enable targeted delivery by magnetic or surface modication. The al-
the targeted site in a sustained fashion avoiding burst release have re- ginate/chitosan/CMCS composite microcapsules prepared by extrusion
ceived attention by the researchers in the past decade. As far as CMCS method encapsulating Lactobacillus casei ATCC 393 proved to be useful
based nanobers are concerned, electrospinning technique has been for the delivery of probiotic cultures to the human gastro-intestinal
most widely accepted method. It is relatively a simple approach to con- tract [198]. Geisberger et al. [122] prepared polyoxometalates-trimethyl
trol the morphology of ultrane bers with outstanding advantages of chitosan nanoparticles which are obtained from the direct electrostatic
very large surface-to-volume ration and high porosity with a small interaction of positively charged trimethyl chitosan with negatively
pore size [196]. The silver nanoparticles/PVA/CMCS nanobers of uni- charged polyoxometalates as shown in Fig. 15. Table 5 shows different
form diameter of 295 to 343 nm with 4 to 14 nm sized Ag nanoparticles CMCS based composites synthesised for delivery of different drugs.
were synthesised via electrospinning technique which proved suitable
as antibacterial biomaterial [197]. 8. CMCS based targeted drug delivery

Discovery and development of a new drug is a highly challenging,


7.6. CMCS composites labor intensive and expensive process. On an average development pro-
cess of each new drug takes approximately 15 years with an estimated
Composites are biomaterials that are composed of more than one con- cost of about US$802 million. And, this estimated cost has been report-
stituent of different physical and chemical properties that are blended to ed to markedly increase at an annual rate of 7.4% above general price in-
form macroscopic, microscopic or nano structure. In case of composites ation [199]. Due to their inability to reach the target site of action, most
for delivery of drugs, usually the polymers are modied either to of 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 the
pathological processes causing severe side effects. An effective approach
to overcome this critical problem is the development of targeted drug
delivery system that can release bioactive compound at the desired
site of action. The main advantages of targeted drug delivery include
the accumulation of drug in the action site, increase in therapeutic ef-
cacy, reduction of therapeutic dose and toxicity, etc. [200].
The concept of developing a drug that could selectively destroy dis-
eased cells without harming healthy cells was proposed by Paul Ehrlich
almost a century ago. He gave this hypothetical drug name of the magic
bullet [201]. Since then, out of several polymers that have been
exploited in targeted drug delivery till date, CMCS has received signi-
cant attention due to its active functional groups which can easily attach
targeting ligands like FA. It can protect therapeutic agents from hostile
conditions in body and can release the entrapped agents selectively at
desired site in a controlled fashion. In addition to it, the inherent phar-
macological properties and excellent biological properties of CMCS
Fig. 10. Release proles of DOX from CMCS-capped-MNP/MMT at different pH. The lines
like biocompatibility, biodegradability, non-toxicity, increased water
are based on the tting with the empirical Peppas's model (reproduced with permission solubility and unique mucoadhesivity also facilitate site specic drug
from [174]). delivery. Fig. 16 illustrates the route followed by different CMCS based
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 67

Fig. 11. (A) Cellular uptake of curcumin-O-CMC Nps by FACS (a) control L929 cells alone (b) and (c) L929 exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (d) control MCF-7 cells alone (e)
and (f) exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (B) Apoptosis assay by FACS (a) control L929 cells exposed to O-CMC Nps (b) and (c) L929 exposed to 1 and 5 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 [175]).

formulations during their delivery to the targeted site of action in modied derivative of CMCS has been proven to be an excellent disper-
human body. Sahu et al. investigated the use of hydrophobically modi- sant to prepare a well-dispersed suspension of superparamagnetic
ed CMCS nanoparticles for the delivery of anticancer drug PTX. The re- Fe3O4 nanoparticles due to its amphiphilic polyelectrolyte property. Its
sults showed that the nanoparticles exhibit a signicant inhibitory good cytocompatibility and functional carboxyl groups showed its po-
effect on the folate receptor over expressing tumor cells like HeLa cells tential for targeted dug delivery [204]. Similarly, magnetic nanoparticles
[162]. Similarly evaluation of FA modied CMCS nanoparticles loaded complexed with CMCS were prepared through spraying co-
with another anticancer drug DOX has been also reported [202]. The precipitation method and their core-shell structure, stability and mag-
confocal and ow cytometry study revealed that the nanoparticles netic properties were also investigated. The studies revealed that the
could target the cancerous cells more effectively than the normal cells. spraying process is a more practical method due to an increased quan-
Recently, Wang et al. prepared novel biodegradable deoxycholic acid tity of adsorbed CMCS and a simplied automated preparation proce-
(DA)-O, CMCS-FA micelles for the delivery of PTX which showed en- dure for targeted drug delivery [110]. In this context, the preparation
hanced level of uptake compared to plain micelles in MCF-7 cells of well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles
[203]. N-succinyl-O-carboxymethyl chitosan (NSO, CMCS), another stabilised by chitosan and O-CMCS respectively by Zhu et al. is worth
68 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 12. Schematic diagrams of the fabrication procedure for CMCH-based folate/luminescent/magnetic nanoparticles (reproduced with permission from [123]).

mentioning. The adsorption mechanism of chitosan and O-CMCS onto cell compatibility for the delivery of another hydrophobic anticancer
the surface of Fe3O4 nanoparticles is believed to be the electrostatic drug ADR has been reported by Shen et al. [123]. CMCS based delivery
and coordination interactions, respectively and was suitable for site spe- system of another model anticancer drug has been experimented by
cic drug delivery [205]. Novel multifunctional FA-CMCS-ZnS:Mn (FA- Jin et al. [161], where the group demonstrated the preparation and
CMCS-Zinc Sulphide: Manganese) nanoparticles developed through in vitro evaluation of amphiphilic DA modied CMCS based pH sensitive
simple aqueous route by Mathew et al. [125], have also shown potential self aggregated nanoparticles. The results of the experiment showed the
for effective targeted drug delivery and imaging of cancer cells. greater cellular uptake and enhanced retention of drug loaded nanopar-
ticles in drug-resistant cells thereby conrming its superior efcacy
8.1. Cancer-specic drug delivery based on CMCS over free DOX. Maya et al. [208] have prepared O-CMC nanoparticles
by ionic gelation technique where negatively charged carboxyl groups
Despite of tremendous developments in the eld of medical science, of O-CMC were cross-linked using CaCl2 and PTX was loaded within
the cure for cancer is still a major challenge. Indiscriminate distribution the nanoparticles before the cross-linking step, after allowing the drug
of most of the anticancer drugs towards disease and healthy cells fol- to interact with O-CMC. In order to facilitate the targeted delivery of
lowing systemic administration ultimately leading to high toxicity re- PTX, antibody Cetuximab (Cet) was conjugated onto the PTX O-CMC
mains the critical bottleneck of conventional chemotherapeutics. In nanoparticles by EDC coupling chemistry. The complete reaction
addition to this, the poor solubility of most of the anticancer drugs in scheme for the preparation of Cet-PTX-O-CMC nanoparticles is shown
water creates the need to use organic solvents or detergents for clinical in Fig. 17 [208]. Zou et al. [209] used octreotidepolyethylene glycol
applications, resulting in undesirable side effects such as venous irrita- stearic acid (OCTPhePEGSA) as a targeting molecule for N-octyl-O,
tion and respiratory distress [206]. Therefore, in order to develop a suc- N-carboxymethyl chitosan (OCC) micelles loaded with DOX to prepare
cessful anticancer therapy, it is important to design a distinct carrier novel active tumor targeting carrier. Fig. 18 [209] illustrates the proce-
system that can encapsulate large amount of anticancer drug and deliv- dure of self assembly and receptor-mediated cellular internalisation of
er it specically to the cancerous cells. Till date a large number of CMCS OCC-OCT micelles. When OCT conjugated to stearic acid (SA) via poly-
based matrixes that include nanoparticles, micelles, microspheres, ethylene glycol (PEG) spacer (OCTPhePEGA) was used to modify
nanobers, composites, hydrogels, lms and bers have been designed OCC micelles. SA could insert into the inner hydrophobic core of OCC
by different researchers for delivery of different anticancer drugs like when it self-assembled in the aqueous environment, thereby increasing
DOX, 5-FU, methotrexate, PTX, ADR, curcumin, camptothecin, 6-Mer- the soundness of modication of OCC. On the other hand, PEG, covering
captopurine, Vincristine etc. A study of glycol chitosan-carboxymethyl the surface of micelles, could increase the blood circulation time of mi-
cyclodextrins drug carrier for efcient targeted delivery of three hydro- celles whereas OCT increased the targeting efciency of the micelles to
phobic anticancer drugs namely 5-FU, DOX, and vinblastine have been tumor cells. The results of the in vitro and in vivo studies indicated that
carried out by Tan et al. [207]. Moreover, the development of multifunc- OCCOCT micelles might be a promising tumor-targeting carrier for
tional novel folate conjugated CMCSFe3O4CdTe nanoparticles cancer therapy [156]. Table 6 shows different CMCS based matrices for
exhibiting high drug loading efciency, low cytotoxicity and favorable the delivery of various anticancer drugs.
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 69

Table. 4
Different nanoformulation for delivery of various anti-cancers, anti-inammatory, anti-microbial drugs, proteins/peptides and vaccines along with their brief outcome.

Drug Drug Brief method and outcome Refs.


category used

Anti-cancer Adriamycin Novel CMCS based folate/Fe3O4/CdTe nanoparticles within size range of 170190 nm possessing intense [123]
superparamagnetic effect and photoluminescence property at room temperature exhibiting high drug
loading efciency, low cytotoxicity and favorable cell compatibility were prepared.
Camptothecin A novel organicinorganic hybrid molecule consisting of carboxymethyl-hexanoyl chitosan modied with [177]
(3-aminopropyl) triethoxysilane was synthesised which showed self-assembly behavior into nanoparticles
with a stable polygonal geometry consisting of ordered silane layers of 6 nm in thickness. This formulation
demonstrated excellent cytocompatibility and cellular internalisation capability in ARPE-19 cell line along
with well controlled encapsulation and release proles.
Curcumin Curcumin loaded N,O-CMCS formulation was prepared by simple ionotropic gelation method with size [184]
range of 150 30 nm having high encapsulation efciency of 80% and indicated slow, controlled and
sustained drug release. The nanoformulation was specically toxic to cancer cells and non-toxic to normal
cells
Doxorubicin DOXHCl loaded nanospheres and microspheres were prepared by precipitation method with high [115]
encapsulation efciency which displayed sustained drug release.
Doxorubicin DOX loaded methoxy poly(ethylene glycol)-grafted-CMCS nanoparticles were synthesised by ion [121]
complex formation with size b300 nm. Drug release was faster at acidic pH than neutral or basic pH and
showed promising antitumor activity.
Doxorubicin Amphiphilic deoxycholic acid modied CMCS based pH-sensitive self-aggregated nanoparticles were pre- [161]
pared within size range of 87 to 174 nm
5-Fluorouracil 5-FU encapsulated FACMCZnS:Mn nanoparticles with size range of 130150 nm were prepared [125]
which were found to be suitable for targeting, controlled drug delivery and cancer cell imaging using
Breast cancer cell line MCF-7 and were non-toxic to L929 cells.
5-Fluorouracil 80 20 nm sized 5-FU loaded N,O-CMCS nanoparticles were prepared with drug entrapment ef- [185]
ciency of 65% and were toxic to breast cancer cells showing good blood compatibility.
Methotrexate O-CMCS-methotrexate nanoparticles were prepared by dialysis method with size range of [160]
187.2363.5 nm and zeta potentials ranged from 8.19 mV to 3.08 mV. Drug release behavior was
sustained in PBS solution of (pH 4.0, 7.2 and 9.0).
6-Mercaptopurine Six 6-Mercaptopurine-CMCS were prepared and structurally characterised. 6-Mercaptopurine-CMCS in [116]
pH 7.4 PBS could self-assemble into nanoparticles with mean diameter of 155.8 6.0 nm by Dynamic
Light Scattering and 100 nm by TEM. 6-Mercaptopurine showed release in media containing 2 mM and
10 mM GSH and maximum cumulative release rates were 65.1% and 74.4%, respectively.
Paclitaxel Target oriented nanoparticles based on O-CMCS modied with stearic acid with FA covalently attached [162]
by carbodiimide reaction by sonication method without using surfactants/emulsiers. The nanoparti-
cles exhibited signicant inhibitory effect on Folate Receptor over expressing tumor cells like HeLa cells.
Vincristine Novel multifunctional O-QCMCS/Cholesterol liposomes were constructed with good physical and [186]
thermal stability, excellent solubility in water, and high drug encapsulation efciency (90.1%) and
displayed steady release action over 2 weeks.
Anti-inammatory Indomethacin Magnetic N-benzyl-O-CMCS nanoparticles were synthesised by incorporation and in situ methods and in- [187]
domethacin was incorporated by solvent evaporation method with loading efciency of 60.8% to 74.8%. The
in vitro drug release prole in Simulated Body Fluid (pH 7.4, 37) displayed an initial fast release, which
became slower as time progressed.
Ibuprofen Novel O-CMCS/-cyclodextrin nanoparticles of spherical shape and size with 166 nm were prepared and [124]
Ibuprofen was loaded with entrapment efciency of 93.25 2.89%. The release rate was slower from O-
CMCS/-cyclodextrin than from chitosan/-CD in Simulated Gastric Medium (pH 1.2) while converse was
true for Simulated Intestinal Fluid (pH 6.8).
Ketoprofen Ampiphillic matrices of CMCS-graft-phosphatidylethanolamine were prepared by a EDC-mediated [188]
coupling reaction.
Dexamethasone Novel surface engineered highly branched CMCS/polyamidoamine dendrimer nanoparticles were [189]
synthesised which did not exhibited signicant cytotoxicity in the range of concentration below 1 mg/mL
and displayed high internalisation efciency by both human osteoblast-like cells and rat bone marrow
stromal cells. Interesting physicochemical and biological properties were reported for these macromolec-
ular systems.
Anti-microbial Tetracycline Tetracycline encapsulated O-CMCS nanoparticles were prepared by ionic gelation method which were [190]
biocompatible and 200 nm in size. This nanomedicine was 6-fold more effective in killing intracellular
S. aureus compared to Tetracycline alone.
Gatioxacin Spherical 3070 nm sized GFLX entrapped O-CMCS nanoparticles were developed which displayed a [191]
(GFLX) four-fold lower MIC value against Gram negative bacteria compared to GFLX solution and a similar MIC
value against Gram-positive bacteria as compared to GFLX solution.
Proteins/Peptides/Vaccines Bovine Serum Albumin (BSA) N,O-CMCS modied pristine nanodiamond particles were developed which were biocompatible and [192]
showed no cytotoxicity to cells.
Basic Fibroblast Growth Factor (bFGF) Out of a series of chitosan derivatives synthesised, 2-iminothiolane modied 2-N sulfated 6-O- [193]
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 h
period as determined by L929 broblast culture tests. The release of bFGF could be successfully controlled.
Tetanus Toxoid 40400 nm sized N-trimethyl chitosan, chitosan and M,N-CMCS nanoparticles with negative surface [194]
(TT) charge for M,N-CMCS and positive surface charge for chitosan and M,N-CMCS nanoparticles were de-
veloped with TT loaded with efciency N90% m/m. Effective uptake of the Fluorescein isothiocyante-BSA
loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells and
enhanced immune response was reported after intranasal application of nanomedicine.
Protein drugs Water soluble oleoyl-CMCS was synthesised through covalent modication of chitosan with oleic acid [113]
and monochloroacetic acid prepared by self-assembled method. The OCMCS nanoparticles showed high
loading efciency and sustained release of extracellular products. The mucoadhesion and internalisation
capability make oleoyl-CMCS nanosystems interesting candidates that could be effective to improve
protein drugs adsorption after oral administration.
70 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 13. Preparation of the PVA/CMCS blank lms and drug lms (reproduced with permission from [38]).

8.2. Organ-specic drug delivery based on CMCS 8.2.1. Colon-specic drug delivery using CMCS
Colon-specic delivery of drugs has gained much attention in recent
Organ, or site-specic drug delivery, has several distinct advantages years for the treatment of various diseases such as Crohn's disease, ul-
over other means of delivering drugs. For example, direct infusion into cerative colitis, and irritable bowel syndrome [214]. The relatively low
the target organ or the vasculature surrounding and supplying blood proteolytic activities of protein/peptide drugs in the colon and even
to the organ ensures that the majority of the drug goes to the site it is for other nonpeptide drugs such as cardiovascular and antiasthmatic
intended to act on. This allows for the use of the more toxic drug agents agents has prompted several researchers to focus on colon targeted
(e.g., chemotherapeutics) in high concentrations, because the exposure drug delivery. The absorption and degradation pathways in the upper
of other organs to the compound is limited. Targeting organs or tissues gastrointestinal tract are the major hindrances in delivering drugs to
in vivo can greatly reduce the risk of toxic side effects and signicantly the colon. To overcome these obstacles, several strategies of colon-
increase the efcacy of a variety of drugs, including toxic chemothera- specic drug delivery have been attempted till date by various scien-
peutic agents, pain medications, and gene therapies. The advantages tists, that include use of prodrugs which become active at the colon,
of site-specic employing CMCS vehicles include pH-sensitivity, drug-eluting system responding to the pH, and microora-activatable
bioadhesive ability, solubility and absorbability, controllable biodegrad- drug delivery systems have gained increasing attention. These strate-
ability, nontoxicity of the degradation end products, sustained release gies basically emphasise on preventing loss of the drug at the stomach
potential and ease of administration [213]. Many researchers have pre- and the small intestine, thereby facilitating quantitative drug delivery
pared different CMCS based formulations that exclusively deliver vari- to the colon.
ous pharmacological agents at specic organs for their activity. These CMCS based systems have been widely studied for colon targeted
include different organs of body like intestine, liver, pancreas, colon, drug delivery as colonic microora can degrade its glycosidic linkage
eyes, and others which are elaborated below. thus facilitating the release of drugs entrapped specically in colon.
Tavakol et al. [215] investigated the release of sulfasalazine drug from
the 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-
specic delivery of sulfasalazine. Tu et al. [216] studied the sigmoidal
swelling kinetics of a series of CMCS-g-poly (acrylic acid) hydrogels
which were pretreated under acidic buffer media. The swelling kinetics
of 5-aminosalicylic acid at different pH showed its potential for colon-
specic drug delivery. Recently, Vaghani et al. [98] prepared and
characterised pH sensitive hydrogel composed of CMCS cross linked
with GA. The group evaluated in vitro as a promising carrier for the ad-
ministration of colon targeted drug delivery of OD.

8.2.2. Liver targeted drug delivery using CMCS


Liver 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-specic
Fig. 14. Release of ornidazole through 30% C blend lm at different pH. Results are means 6 drug carrier. This is because nanoparticles prepared from such amphi-
standard deviation for n 3 (reproduced with permission from [126]). philic derivative via self-assembly have been recognised as a promising
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 71

Fig. 15. Electrostatic interaction of CMCS (a) and trimethyl chitosan (b) biopolymer matrices with encapsulated polyoxometalates (POMs) (reproduced with permission from [122]).

drug carrier, since their hydrophobic domain can serve as a depot for hydrophobic MMA and hydrophilic O-CMC, which spontaneously self-
sparingly soluble drugs [217]. Zheng et al. [218] synthesised glycyrrhizic assembled into coreshell O-CMC nanoparticle in aqueous solution. The
acid encapsulated novel thiolated lactosaminated (TLAC)/CMCS nano- adjacent hydroxyls of glycyrrhizin (GL) were turned into aldehyde
particles through ionic gelication method and characterised these groups through periodate oxidation in order to obtain O-CMC-GL nano-
nanoparticles in vitro studies. The in vivo studies of pharmacokinetic pa- particle. The preparation procedure of O-CMC nanoparticle, GL modied
rameters were evaluated in rabbits and tissue distribution in mice. The CMC-GL, and PTXL loaded CMC-GL nanoparticle (PTXL/CMC-GL) nano-
results showed that the TLAC-CMCS may be used as a promising drug particles were illustrated in Fig. 19 [212]. In addition, in vitro and
carrier for hepatic targeting and controlled release. Similarly, prepara- in vivo studies of PTX loaded GL-modied O-CMCS nanoparticles were
tion, characterisation and tissue distribution studies of lactosaminated also carried out by for hepatocellular carcinoma targeted drug delivery
CMCS nanoparticles have been reported by Zheng and coworkers. The application. The results established these nanoformulation as promising
experiment demonstrated that the in vitro release of glycyrrhizic acid drug carrier for hepatic cancer.
from the nanoparticles exhibited a biphasic pattern, initial burst release
and consequently sustained release. Also, these nanoparticles modify 8.2.3. Ocular drug delivery using CMCS
the tissue distribution prole of the glycyrrhizic acid solution, the kid- The eld of ocular drug delivery is one of the most interesting and
ney excretion rate is reduced and drug accumulation in the liver is in- challenging endeavors facing the researchers and scientist community.
creased [105]. Recently, Shi et al. [212] prepared O-CMC-methyl From drug point of view, it is very difcult to study the eye as an isolated
methacrylate (MMA) copolymers through the graft copolymerizion of organ. This is due to the presence of highly sensitive ocular tissues like

Table. 5
Different CMCS based composites synthesised for delivery of different drugs.

Composite Drug used Brief method & output Refs.

Folic acid conjugated CMCS-Mn doped 5-Fluorouracil (5-FU) Novel multi-functional FACMCSZnS:Mn nanocomposites prepared by simple aqueous route showed [125]
ZnS nanocomposite high drug encapsulation of 5-FU (92.08%) with controlled release and were found nontoxic to mouse
broblast L929 cells and toxic to MCF-7 cell line.
O-CMCS/-CD nanocomposite Ibuprofen (IB) 166 nm sized IB loaded O-CMCS/ cyclodextrin nanocomposites by simple ionic cross-linking method [124]
showed high drug encapsulation efciency of 93.25 2.89%. The release rate was slower from O-CMCS/
CD than chitosan/ CD in simulated gastric medium while converse was true for simulated intestinal
medium.
CMCS/TMCS nanocomposite Bioactive 5090 nm sized nanocomposites were prepared by direct cross-linking approach. POMCMCS com- [122]
Polyoxometalates posites display negative zeta potentials and larger particle sizes than the positively charged POM
(POMs) Trimethyl Chitosan composites
Nano-hybrid CMCS-hexanoyl chitosan Camptothecin (CPT) Highly ordered appx. 6 nm thick silane layer formations upon self-assembly of the hybrid molecule [177]
modied with (3-aminopropyl) leads to sustained release of CPT. The hybrid molecule showed excellent cytocompatibility and efcient
triethoxysilane cellular internalisation with tp ARPE-19 cell line.
Folate conjugated CMCS/Fe3O4/CdTe Adriamycin (ADR) 170190 nm sized novel folate conjugated CMCS/Fe3O4/CdTe NCs were prepared by layer-by-layer [123]
nanocomposites assembly technique. Initial CMCS concentration, medium pH and reaction time strongly inuenced
coating amount and binding mode of CMCS. The multifunctional nanocomposites exhibited high drug
loading efciency, low cytotoxicity and favorable cell
Compatibility.
72 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 16. Illustrates the route followed by different CMCS based formulations during their delivery to the targeted site of action in human body.

the uveal tract and retina. In addition to this, the presence of tissue bar- biocompatibility, biodegradability and ability to prolong ophthalmic
riers to drug penetration which include the lipophilic corneal epitheli- drug retention time [222]. In fact the ability of CMCS to prolong the
um, the hydrophilic corneal and scleral stroma, the conjunctival precorneal drug retention, by virtue of its viscosity-increasing affect, of
lymphatics, choroidal vasculature, and the blood-ocular barriers its ability to bind ooxacin and, probably, of its mucoadhesive proper-
makes ocular drug delivery even more challenging [219]. The choice ties in comparison to chitosan has been experimentally demonstrated
of chitosan for ocular drug delivery has been justied due to its out- by Colo et al. [222]. An in vitro study of gatioxacin (GFLX) from novel
standing mucoadhesive and penetration enhancing properties, as well O-CMCS formulation showed that the release was slower than that
as by its good biocompatibility with the ocular structures [220]. Applica- from GFLX solution and the MIC of OCMCS formulation against Gram-
tion of chitosan based nanostructures in delivery of ocular therapeutics negative bacteria is fourfold lower than the system without OCMCS.
has been well reviewed by Fuente et al. [221]. CMCS has attracted much The GFLX is a fourth-generation uoroguinolone and in vitro studies
attention in ophthalmic drug delivery due to its inherent low toxicity, on isolates from bacterial infections of the eye have shown an

Fig. 17. Schematic illustration depicting preparation of PTXL-O-CMC nanoparticles and bioconjugation of Cet on PTXL-O-CMC Nps through EDC activation chemistry. EDC activated the
carboxyl functionality and subsequently linked to Cet through covalent linkage (reproduced with permission from [208]).
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 73

Fig. 18. Schematic illustration of the self-assembly and receptor-meditated cellular internalisation of OCCOCT micelles (reproduced with permission from [209]).

Table 6
Different CMCS based matrices for the delivery of various anticancer drugs.

Anticancer drug CMCS based matrix Matrix composition Remarks Refs.


structure

Adriamycin Self aggregated LA-modied CMCS Self-aggregated nanoparticles exhibited [120]


nanoparticles an increased Loading Capacity and Loading Efciency, decreased sustained release with an increasing
ratio of the hydrophobic LA
to hydrophilic CMCS
Camptothecin Aggregates O-CMCS Not only the aggregates but also the unimers of OCMCS can help to enhance the solubility of CPT. In vitro [163]
cancer antiproliferative activity test further conrms the
slow release of CPT from OCMCS-drug system.
Curcumin Nanoparticles O-CMCS Spherical 150 30 nm sized curcumin loaded O-CMCS nanoparticles were prepared with entrapment [175]
efciency of 87%. The nanoparticles were toxic to cancer cells and non-toxic to normal cells.
Doxorubicin Nanospheres and CaCO3CMCS The water soluble DOXHCl could be effectively loaded in the hybrid [115]
microspheres microparticles and nanospheres with a high encapsulation
efciency, and the drug release could be effectively sustained,
indicating the hybrid microspheres and nanospheres were
suitable for delivery of water-soluble drugs
Self-assemblied Folic acid (FA) modied 267.8 nm sized nanoparticles were prepared by sonication method. The cellular uptake of Folate [162]
nanoparticles CMCS modied CMCS nanoparticles
was found to be higher than that of nanoparticles
based on LA modied CMCS.
Self assembled Carboxymethyl-hexanoyl The model anticancer drug DOX was entrapped with an efciency of 46.8%, and a corresponding drug [155]
hollow chitosan release from the nanocapsules for a time period exceeded 7 days can be achieved in vitro.
nanocapsule
Nanoparticles Methoxy poly(ethylene The nanoparticles showed increased cytotoxicity compared to DOX alone. [121]
glycol)-grafted-CMCS
Self assembled Acylated CMCS The nanoaggregates exhibited an excellent colloidal and structural stability in aqueous medium. [35]
nanoaggregates
5-Fluorouracil Nanoparticles Folate conjugated CMCS- 130150 nm sized novel nanoparticles loaded with 5-FU exhibited non-toxicity to L929 cells. The [125]
Mn doped zinc sulphide nanoparticles could be used for controlled drug delivery and imaging of cancer cells.
Methotrexate Nanoparticles FA conjugated CMCS The encapsulation efciency and loading capacity of Methotrexate in the FA-O-CMCS nanoparticles [210]
were higher, and the particle size of the FA-O-CMCS nanoparticles was also smaller than those in the FA-
CS nanoparticles.
Self assembled Methotrexate conjugated Spherical shaped nanoparticles within size range of 187.2363.5 nm were prepared by dialysis method. [160]
nanoparticles O-CMCS In vitro drug release study by the dynamic dialysis method showed that Methotrexate exhibited
signicant sustained-release behaviors
in PBS buffer solutions (pH 4.0, 7.2 and 9.0), indicating that these nanoparticles had good in vitro sta-
bility
and the potential to be used as a novel drug carrier system
6- Nanoparticles 6-Mercaptopurine-CMCS The 6-Mercaptopurine release from 6-MP-CMC showed dependence on glutathione concentration. In [211]
Mercaptopu- aqueous solution 6-MP-CMC could self-assemble into the nanoparticles through the intra- and inter-
rine molecular hydrophobic interactions between 6-MP groups
Paclitaxel Nanoparticles Glycyrrhizin (GL)-modied 100205 nm sized PTX loaded O-CMCS nanoparticles were prepared with encapsulation efciency of [212]
O-CMCS 83.7% and performed a biphasic release.
Nanoparticles FA conjugated CMCS modi- The PTX loaded nanoparticles exhibited many desirable properties like pH-sensitive dissolution, low [162]
ed with stearic acid cytotoxicity, and high amount drug encapsulation and signicant inhibitory effect on the FR over ex-
pressing tumor cells like HeLa cells
Vincristine Liposomes O-Q-CMCS-Cholesterol Vincristine was encapsulated in polymeric liposomes with high entrapment efciency (90.1%). The [186]
formulation was stable in aqueous solution and exhibited slow, steady release action over 2 weeks
under physiologic pH (7.4).
74 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

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 [212]).

encouraging response to it [191]. Currently, Yang et al. investigated both also been reported recently by [145]. While the in vitro results showed
in vitro and in vivo, the ability of 5-FU or bevacizumab loaded N,O-CMCS that formulation I containing CMC and carbopol in 1:1 ratio showed
hydrogels to modulate wound healing after glaucoma ltration surgery. sustained release, better prolonged action from the prepared hydrogel
On one hand the in vitro study showed that the nearly 100% of 5-FU was formulation when compared to (standard) marketed sustained release
released from the drug-loaded hydrogels within 8 h, but less than 20% formulation was reported from the in vivo study. Currently, synthesis,
bevacizumab was released after 53 h. On the other hand, the in vivo characterisation and in vitro evaluation of anticancer drug 5-FU loaded
study carried out in rabbits showed that the CMCS hydrogels were non- CMCS nanoparticles have been reported by Anitha et al. [184] The toxic-
toxic to the cornea and were gradually biodegraded in the eyes [141]. Re- ity of the drug loaded CMCS nanoparticles was showed by MTT, apopto-
cently, to investigate the availability of induced pluripotent stem cells sis and caspase 3 assays thereby conrming the potential of 5-FU loaded
iPSCs as bioengineered substitutes in corneal repair Chien et al. [223] de- N,O-CMC nanoparticles in breast cancer chemotherapy in which the
veloped a thermo-gelling injectable amphiphatic carboxymethylhexanoyl side effects of conventional chemo treatment could be reduced. Recent-
chitosan (CMHC) nanoscale hydrogel and found that such gel increased ly, preparation of metformin loaded O-CMCS nanoparticles prepared by
the viability and CD44 proportion of iPSCs, and maintained their ionic gelation method for delivery to pancreatic cancer cells has showed
stem-cell like gene expression, in the presence of culture media. The pH sensitive release of the drug in vitro. While the cytotoxicity study
study demonstrated that human keratocyte-reprogrammed iPSCs, when showed the preferential toxicity of the drug loaded nanoparticles on
combined with CMHC hydrogel, can be used as a rapid delivery system pancreatic cancer cells (MiaPaCa-2) compared to normal cells (L929),
to efciently enhance corneal wound healing. the nanoparticles exhibited nonspecic internalisation by normal and
pancreatic cancer cells [106].

8.2.4. Others
Apart from these, there are a number of other organs for which 9. Tissue engineering: origin and strategies
CMCS based drug delivery systems have been designed by different sci-
entists in the recent times. This include synthesis and in vitro study of Tissue engineering, by denition, is a highly interdisciplinary eld
microencapsulated beads composed of alginate-N,O-CMCS for the deliv- that combines the principles and methods of life sciences and engineer-
ery of model protein BSA to different regions of the intestinal tract. The ing to utilise structural and functional relationships in normal and path-
results of the study demonstrated excellent pH sensitivity and proved to ological tissue to develop biological substitutes to restore, maintain, or
be suitable polymeric carrier for site specic delivery of bioactive pro- improve biofunction [224]. The term tissue engineering was formally
tein in the intestine. Also the main advantage was that the bioactivity coined in 1987 [225] and since then, it has emerged as a scientic eld
of the protein drug was preserved due to the preparation of drug loaded distinct from medical eld by providing numerous promising tech-
beads in the aqueous medium at neutral environment [94]. Another niques with practical clinical applications. The popular theory of the
similar work for the in vitro and in vivo evaluation of pH sensitive medical eld that the human body possesses an inherent capacity to
CMCS based hydrogels for intestinal delivery of drug theophylline has heal itself has been the fundamental principal that has been exploited
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 75

in the eld of tissue engineering. In fact, the specic tissue or the organ transplantation and tissue reconstruction methods. Over the past
determines the level of ability to self-repair and regenerate from dis- 40 years, tissue engineering has shown signicant most tissue types, es-
ease, damage or an injury. This capacity is limited by the degree of dam- pecially those damaged or lost following debilitating health problems
age, loss of function and involvement of multiple tissues [226]. The such as cancer and degenerative disorders [228].
recent developments in tissue engineering like xeno-transplantation
of tissues, new prosthetics and localised manipulation of lesion sites at 10. Preparation techniques of CMCS based biomaterials for tissue
the cellular and molecular level have made signicant advancements engineering
in reconstructive surgery in comparison to the conventional approaches
like tissue auto- and allo-grafting [226,227]. CMCS is biocompatible, biodegradable, and more bioactive than chi-
The main objective of tissue engineering is to overcome the lack of tosan with enhanced osteogenesis property and it can be easily formu-
tissue donors and the immune repulsion between receptors and donors. lated in a variety of forms like hydrogels, scaffolds, composites, i-gels,
In fact, by laying emphasis on tissue and cell-based therapy, tissue engi- lms and membranes which have wide range of potential applications
neering and clinical practices strive to achieve same goals. The tissue en- in tissue engineering. The techniques that are suitable for preparation
gineering strategy involves the in vitro seeding and proliferation of of different CMCS based hydrogels for tissue regeneration purpose
relevant cells and/or signaling molecules in an appropriately designed have already been discussed in Section 8. As in hydrogels, the cross
tissue engineering biomaterial like scaffold to form a natural tissue. links can be incorporated either by chemical cross linking method or
This tissue is implanted into the defect in the patients. While in some by radiation cross linking in the scaffolds also as per the requirement
cases, the scaffold or scaffold with cells is directly implanted in vivo of the host tissue and the tissue engineering biomaterial being fabricat-
where the host body functions as a bioreactor to construct new tissues. ed. Nowadays, enzymatic cross linking has also gained attention due to
Thus, these transplantable constructs enable regeneration of functional its own benets. Selecting the scaffolding approach for tissue engineer-
tissue in the host providing an alternative to conventional organ ing is tissue and application specic. The conventional scaffold

Table. 7
Preparation techniques of CMCS based biomaterials for tissue engineering.

Composition of matrix Technique/method Comment Refs.

Gelatin/CMCS/-tricalcium Radiation crosslinking Series of biodegradable scaffolds were prepared where ultrasonic [127]
phosphate composite scaffold and lyophilizing treatment on the polymer solutions affected the -TCP particles
distribution. Excellent biocompatibility and ability of bone
regeneration was revealed by in vivo implantation in mandible
of beagle dog.
Hydroxyapatite coated CMCS scaffolds Freeze drying technique Coating of scaffolds with HAP substantially enhanced the viability, [40]
attachment, proliferation, and differentiation of the osteoblast and
directed stem cell differentiation to osteoblast
HAP/CMCS composite scaffold Coprecipitation method The scaffolds exhibited 20500 m sized pores with regular interconnection [229]
with appx. 58.9% of porosity determined from microcomputed tomography
analysis. Average scaffolds consisted of 24% HA and 76% CMCS determined
from 2D morphometric analysis.
CMCS/HAP Electrospinning The SEM showed that nano/micro particles formed on the surface of the [230]
nano-nonwoven CMCS brous scaffold. FTIR and XRD conrmed that the
nano/micro particles were hydroxyapatite crystalline. HAP particles
appeared to have a great effect on the late stages of osteoblast behavior
(alkaline phosphatase).
n-HAP/CMCS biocomposite Freeze drying technique The FTIR and XRD results of genipin cross linked n-HAP/CMCS scaffolds [231]
scaffold revealed that CMCS's hydroxyl, amine and amide groups determined
nano homogenous distribution of n-HAP and provided nano topographical
features for nanohybrid scaffolds. The scaffolds had pore size of 150 m sized
pores with less toxicity and more facility for adhesion and proliferation of cells.
N-isopropylacrylamide and CMCS Chemical cross linking & Thermo responsive & core-shell microgels were prepared having phase [232]
hydrogels seed emulsion polymerisation transition temperature nearer to that of body compared to pure PNIPAM
Gelatin & CMCS hybrid hydrogels Radiation induced cross linking Due to the high water absorption capacity, a similar [103]
compressive modulus with soft tissue, controllable biodegradation,
and excellent biocompatibility, the hydrogels have potential as skin
scaffolds and wound healing materials.
CMCS/n-HAP composite Porogen leaching method The porosity ratio of CMCS/n-HA is about 75% and the compressive [233]
strength can exceed 21 MPa with circular pores of diameter ranging
from several m to six hundred m. The in vivo experiments showed
no inammatory reaction and bone putrescence and toxicity of liver and kidney.
n-HAP/CMCS composite Particle ltration and lyophilisation The composite scaffold with (VEGF)-transfected bone marrow stromal [234]
followed by genipin crosslinking cells (BMSCs) was studied in a rabbit radial defect model. The scaffold is
biocompatible, nontoxic, promotes the inltration and formation of the
microcirculation, and stimulates bone defect repair with degradation
rate matching the growing rate of bone.
CMCS/gelatin/n-HAP i-gel Enzymatic crosslinking The i-gels prepared susceptible to tyrosinase/p-cresol mediated in situ [2]
gelling at physiological temperature that may be used in treating irregular
small bone defects with minimal clinical invasion as well as for bone cell delivery
CMCS lm Covalently crosslinked Controlling the molecular weight distribution of properly tailored chitosan [72]
allows one to regulate the mechanical properties and degradation of
chitosan in a sophisticated manner, while maintaining favorable cell
interactions.
CMCS-graft-D-glucuronic acid Grafting D-GA onto CMCS in The membranes showed bioactivity which demonstrated its potential for [235]
membranes the presence of 1-ethyl-3-(3- tissue engineering applications.
dimethylaminopropyl)
carbodiimide (EDC)
76 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

fabrication techniques include solvent-casting particulate-leaching bioactivity as compared to chitosan and its ability to promote osteogen-
method, gas foaming, bre meshes/bre bonding, phase separation, esis [25]. Also the capacity of CMCS to chelate calcium from mineralizing
melt moulding, emulsion freeze drying, solution casting, and solution containing calcium and phosphate to induce calcium phos-
lyophilisation (freeze drying) method. The particle leaching method ex- phate or hydroxyapatite (HAP) formation has made it suitable biopoly-
hibits advantages of creation of scaffolds with big pores, wellcontrolled mer for tissue engineering [238]. Apart from this, it is well known to
high interconnected porosity and pore morphology. The benets of gas exhibit excellent biocompatibility, better biodegradability, non-
foaming technique include lack of solvent, eliminating the risk of re- toxicity, and ability to promote cell adhesion which is desirable in the
maining residues, and the low processing temperatures preventing deg- eld of tissue engineering and regenerative medicine. In the past few
radation of the polymer during processing. Emulsion freezedrying is years, several researchers have utilised CMCS in fabrication of different
attractive for creation of scaffolds that are relatively thick having large tissue engineering biomaterials which include scaffolds, hydrogels,
pores. Despite of several positive facets of these traditional methods, composites, nanobers, nanoparticles, dendrimer, and membranes
the incapability of precisely controlling pore size, pore geometry, spatial and for implant functionalisation [239241] due to easy processibility
distribution of pores and construction of internal channels within the of CMCS into these constructs.
scaffold, presence of residual organic solvent and poor mechanical in-
tegrity are some of the most signicant problem facing these conven- 11.1. CMCS scaffolds
tional techniques due to the risks of toxicity and carcinogenicity it
poses to cells. Therefore, new techniques like self-assembly systems, Scaffolds are unique tissue engineering biomaterial as they are able to
solid free-form fabrication and electrospinning technique have establish three-dimensional environments for propagated cells and spe-
attracted the attention of the researchers as these methods can over- cic signalling molecules that can mimic native tissues environments.
come many of the demerits of the conventional scaffolding approaches. Tissue engineering scaffolds can be of natural, synthetic or a hybrid of
Sintering method for scaffold design has also received attention and is both. CMCS has emerged as a promising scaffolding polymer due to its
usually applied in case of ceramics powders, metals, glasses and certain inherent excellent biocompatibility, ability to promote cell adhesion
polymers as well as composites. But most of the composites for tissue and increased bioactivity as compared to chitosan. Also in comparison
repair are prepared by coprecipitation, porogen leaching and freeze dry- to chitosan that has relatively slow and uncontrollable degradability
ing (lyophilisation) method. In addition to this, injectable-gels, and [242]. CMCS shows accelerated degradation rate which can be regulated
membranes can be prepared by enzymatic and covalent cross linking through different cross-linking extents by EDC, while retaining excellent
and grafting method respectively. CMCS based lms are most common- mechanical properties. However, EDC cross-linked CMCS porous tubular
ly prepared by blending/casting method that may include covalent cross scaffolds were fabricated for nerve regeneration which showed de-
linking or other linkages. Table 7 shows preparation techniques of CMCS creased hydrophilicity and elastic modulus which is desirable for nerve
based biomaterials for tissue engineering. repair [60]. Nanobrous collagen-coated porous CMCS microcarriers
were successfully fabricated by a simple modied phase separation
11. CMCS based biomaterials for tissue engineering and regeneration method and thereafter collagen anchoring-assembling. In vitro chondro-
cyte culture revealed better cell attachment, proliferation, and differenti-
An ideal biomaterial for tissue engineering is expected to meet some ation on the CMC-MCs immobilised with self-assembled collagen
important criteria. It must be biocompatible, easily biodegradable in ap- nanobers. Cells were observed to grow into a tissue-like structure
propriate time window, its degradation products should be non-toxic, after 7 days of culture. Thus the scaffolds prepared showed potential
and it must support cell adhesion and growth and should exhibit me- for application as injectable scaffolds for cell delivery in cartilage tissue
chanical strength comparable with the host tissue [236]. The applica- engineering [72]. Gelatin/CM-chitosan/-tricalcium phosphate compos-
tions of chitosan and its derivatives in the eld of tissue engineering ite scaffolds were prepared using a green fabrication method, i.e.
have been earlier reviewed [237]. CMCS has attracted considerable at- radiation-induced cross linking. Considering their excellent and adjust-
tention for tissue engineering application due to its inherent increased able water retention capacity, highly interconnected porous network

Fig. 20. SEM images of gelatin/CM-chitosan/-TCP composite scaffolds with -TCP fraction of (A) 0%, (B) 5%, (C) 10%, (D) 20%, (E) 30%, and (F) 40%. The ultrasonic time was 20 min
(reproduced with permission from [127]).
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 77

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 formed
the 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 CMCS
chains link the aldehyde zones to the formation of a three dimensional network, that created the gel (reproduced with permission from [249]).

structure, proper compressive strength and high porosity, the scaffolds oxidation, and further reduced by complexing with CMCS. Also the com-
met the criteria for bone tissue regeneration. Fig. 20 shows the SEM mor- plex hydrogel showed an increased compressive modulus of 278 kPa,
phology of scaffolds with different fractions of -TCP when the ultrasonic and an ability to return to the original shape after release of the com-
time was set to 20 min. From the Fig it is clear that similar pore size pressive load. Thus the hydrogels were promising biomaterials for carti-
(about 350 m) were observed in the composite scaffold. Although lage tissue engineering. A study on the synthesis and characterisation of
some agglomeration appeared in the walls of scaffolds containing higher UV cross-linked hydrogels derived from novel water soluble
fractions of -TCP which is in agreement with the literature [127]. The methacrylated O-CMCS and polyethylene glycol diacrylate. The hydro-
bioactivity of a novel CMCS scaffolds with and without incorporating gel substrates similarly supported attachment and proliferation of
mineral trioxide aggregate (MTA) in a tooth model was characterised. Smooth Muscle Cells (SMCs). The results from the study demonstrated
The deposition of HAP was signicantly higher (P b 0.05) on MTA- these hydrogels to be promising biomaterials for tissue regeneration
coated CaC (CaMT) scaffold than that on Cross-linked CMCS scaffold [143]. A novel microgel class consisting of biocompatible CMCS and
(CaC). Therefore, it can be concluded that the bioactivity of the CMCS temperature-sensitive PNIPAM was designed and synthesised by seed-
scaffold can be enhanced by incorporating MTA [238]. ed emulsion polymerisation. The presence of PNIPAM in microgels con-
tributed to the thermoresponsive property to CMCS while CMCS added
11.2. CMCS hydrogels to the improved biocompatibility to the microgels which made these
microgels suitable for tissue regeneration purpose [232]. Yang et al.
In the past decade, hydrogels have made signicant progress in the [103] fabricated CMCS/gelatin hydrogels by green method i.e. radiation
development of tissue engineering scaffold [243]. A number of synthetic cross linking method with excellent and adjustable water retention ca-
and natural polymers have been exploited in the last few years as hy- pacity (10700 g/g dry gel) and a similar compressive modulus with
drogel biomaterials that include alginate [244], chondroitin sulfate that of soft tissue (10200 kPa). These hybrid hydrogels have improved
[245], hyaluronic acid [246] and collagen [247]. The insufcient me- exibility, antimicrobial and water absorption capacity than gelatin
chanical performance and relative harsh gelation conditions for cell en- hydrogels and superior handle ability and mechanical properties than
capsulation are the major limitations of hydrogels in tissue engineering CM-chitosan hydrogels. Apart from these, they exhibited excellent and
application [248]. A double-network complex hydrogel with signicant- controllable degradability and good cytocompatibility which suggested
ly improved gelation temperature and mechanical properties composed their potential application in tissue engineering and wound healing.
of oxidised gellan gum and CMCS by Ca2+ cross linking and Schiff reac-
tion was prepared. Firstly, polymer chains were cleaved into smaller 11.3. CMCS composites
segments by oxidation reaction, which lead to the decrease of polymer
molecular weight and the increase of cross linking aldehyde groups. In the recent years, composites have drawn attention of researchers
This allowed for the formation of two entangled networks of different towards development of biocompatible and biodegradable composites
cross linked polymers as described in Fig. 21A [249]. In the second for tissue regeneration. In this context, preparation of porous
step, the chemical cross linking reaction (Schiff-base formation) be- biocomposites of nano-hydroxyapatite (n-HAP) and CMCS by porogen
tween the pendant amino groups of CMCS and the aldehyde group leaching method and their characterisation by IR, XRD, SEM and com-
lead to the formation of a complex gel in the oxidised gellan as shown pressive strength has shown potential application for bone tissue engi-
in Fig. 21B [249]. The results showed that the gelation temperature neering. The composites displayed porosity where the pores were
was lowered from 42 C to below physiological temperature by interconnected and the compressive strength can exceed 21 MPa
78 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 22. Synthesis of CMCS-graft-D-glucuronic acid (reproduced with permission from [235]).

which should be desirable for bone tissue engineering [233]. Similarly, 12.1. CMCS in bone tissue engineering
Shi fabricated N-carboxyethyl chitosan/nanohydroxyapatite N-CECS/n-
HAP composites for tissue-engineered trachea and investigate its In recent years, CMCS based scaffolds, hydrogels and biocomposites
biomechanical and biocompatible properties. The N-CECS/n-HAP com- have gained importance in orthopedic research because of their poten-
posites exhibited satisfactory tensile strength and Youngs modulus tial to minimise surgical invasiveness. When engineering bone tissue,
values thereby conrming their potential for tissue-engineered trachea the tissue engineering device must meet a number of requirements
[250]. Also, recently, the effect of the n-HAP/CMCS composite with vas- like being biocompatible, biodegradable in suitable time window, its
cular endothelial growth factor (VEGF)-transfected bone marrow stro- degradation products should be non toxic and capable of being easily
mal cells (BMSCs) in a rabbit radial defect model was studied [234]. eliminated by the metabolic pathways, must support cell adhesion
This composite was biocompatible, nontoxic, promotes the inltration and growth and exhibit adequate mechanical stability [252,253]. In ad-
and formation of the microcirculation, stimulates bone defect repair dition to this, the biomaterial designed should allow new bone in-
and the degradation rate of the composite matched that of growing growths (osteoconductive) [254] and angiogenesis to supply the
bone thus demonstrating its potential for bone defect repair. newly formed tissue with nutrients while inducing bone formation
(osteoinductive) [255]. In this view, the synthesis, characterisation
and in vivo study of an enzymatically cross linked CMCS/gelatin/n-
11.4. CMCS injectable gels, lms and membranes HAP injectable in mice reported by Mishra and coworkers have been
demonstrated as promising biomaterials that may be used in treating ir-
Apart from popular tissue engineering devices like scaffolds, regular small bone defects with minimal clinical invasion as well as for
hydrogels and composites, injectable gels, lms and membranes have bone cell delivery. The in vivo injectability study of the i-Gels in murine
also been developed by different researchers for tissue repair and regen- models (Fig. 23) showed that the injected i-Gels were successfully re-
eration purpose. Development of gelatin and CMCS gels in situ in the trieved from the exact position of euthanised mice. At the site of implan-
presence of tyrosinase and p-cresol where presence of n-HAP does not tation no apparent sign tof inammation (redness or edema) was
hamper in situ gelation of the polymers in physiological pH and temper- observed which illustrated that the i-Gels were nonimmunogenic in na-
ature has been reported by Mishra and coworkers [2]. The results clearly ture. Also the yellowish colour instead of purple colour of the retrieved
indicate the potential of tyrosinase/p-cresol crosslinked CMCSgelatin i-Gels can be attributed to the limitation of ample molecular oxygen in-
gel as injectable gel matrix for cell based bone tissue engineering. side the human body. The in vivo study also demonstrated that the i-
Guangyuan et al. [72] prepared CMCS lms and carboxymethylation Gels will have lesser gel strength in vivo as compared to in vitro situation
and bimodal MW distribution were successfully combined to regulate [2]. Oliveira et al. reported high efciency of DOX-loaded CMCS/PAMAM
rate of degradation of the lms formed. The results displayed that
these CMCS lms with tunable degradation rates provide a powerful
material system for tissue engineering. In this view, the development
of CMCS-graft-D-glucuronic acid (CMCS-g-D-GA) membranes prepared
by grafting CMCS with D-glucuronic acid by using EDC catalyst in
water by Jayakumar and coworkers is worth mentioning. The synthesis
method of the CMCS-g-D-GA membranes is shown in Fig. 22 [235]. The
results of this investigation indicated that as the membranes were capa-
ble of having bioactivity, they were expected to be suitable for tissue en-
gineering purposes.

12. CMCS applications in different tissues/organs

As the inherent ability of body to self-repair from disease or injury


and to regenerate depends on the specic tissue or organ system, it
seems 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 [225]. In context of CMCS, its fabrication depends
greatly on the target tissue for which the restorative device is being Fig. 23. In vivo gel stability study. Representative macroscopic image of post-mortal mouse
made which may include different organs like bone [251], nerve, carti- showing the location and texture of iGel 24 h post-implantation (reproduced with permis-
lage [72], vascular tissue [239], tooth [238] and even trachea [250]. sion from [2]).
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 79

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 seeding
was carried out with 3104 cells/cm2. (f) shows the Ti-CMCS substrate which had been placed in cell culture medium for 14 days without cell seeding after Alizarin Red staining. Scale bar
200 mm (reproduced with permission from [240]).

dendrimer nanoparticles on being internalised by different cell types, differentiation of rat bone marrow stromal cells in the presence of
in vitro, promoting the osteogenic differentiation of rat bone marrow 0.01 mg ml 1 DOX-loaded CMCS/PAMAM dendrimer nanoparticles,
stromal cells in tissue culture polystyrene dishes [256]. Similarly, the in vitro has been reported by several research groups [257,258].
low generation poly(amidoamine) (PAMAM) dendrimers with CMCS,
CMCS/PAMAM dendrimer nanoparticles were surface engineered and
loaded with DOX with the expectation of exhibiting high drug loading 12.2. CMCS in cartilage tissue engineering
efciency and non cytotoxicity compared to amine-terminated
PAMAM dendrimers of high generation [257]. Xuefeng Hu carried out The choice of biomaterial is very critical for the success of tissue en-
a study on an in vitro assessment of titanium functionalised with dopa- gineering approaches, particularly in cartilage repair [259]. The struc-
mine followed by CMCS or hyaluronic acid catechol (HAC) conjugated tural similarity of chitosan with various glycosaminoglycans found in
with vascular endothelial growth factor (VEGF). It proved to be promis- articular cartilage has made it a suitable scaffolding biomaterial in artic-
ing alternative for enhanced osteointegration and inhibition of bacterial ular cartilage engineering [260,261]. Therefore, CMCS has been now
inhibition. Fig. 24 displays the results of mineralisation of cells after two experimented for different cartilage tissue engineering applications.
weeks culture on different substrates which was assessed by staining by Earlier, N,N-di-CMCS as delivery agent for bone morphogenetic protein
Alizarin Red. The degree of staining on the Ti-CMCS substrates (Fig. 24b) in the repair of articular cartilage has already been experimented [262].
and Ti-HAC substrates (Fig. 24d) is not substantially higher than pris- Guangyuan et al. [72] successfully fabricated nanobrous collagen-
tine Ti (a). Figure shows an absence of purplish red stains which was coated porous CMCS microcarriers for cultivating cells and for applica-
control experiment carried out with Ti-CMCS in cell culture medium tion in cartilage tissue engineering as injectable scaffolds for cell deliv-
without cells. As shown by the dense coverage of calcium deposits on ery. Recently, double-network complex hydrogel with signicantly
the Ti-CMCS-VEGF (Fig) and Ti-CMCS-HAC (Fig) substrates improved gelation temperature and mechanical properties have been
mineralisation is greatly enhanced which can be attributed to the pres- prepared by mixing oxidised gellan gum with CMCS which was found
ence of immobilised VEGF [240]. Also the development of hydroxyapa- to be promising material for cartilage tissue engineering [249]. Apart
tite scaffolds with macroporous structure and non-cytotoxicity which from these research studies, the ability of chitosan and CMCS to protect
could efciently support the adhesion, proliferation and osteogenic chondrocytes from apoptosis, signicantly suppress the degeneration of
80 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

cartilage in osteoarthritis and to protect chondrocytes from the IL-1beta other biomaterials has demonstrated potential of enhanced wound
induced catabolism have also been reported in literature [263,264] healing property. In fact, oligo-chitosan, N,O-CMCS and N,CMCS in
sheet and paste forms were evaluated in vitro for possible utilisation
12.3. CMCS in nerve tissue engineering in wound dressing applications for wound healing [67]. Similarly, N,O-
CMCS/Collagen matrixes containing chondroitin sulfate or an acellular
Researchers have investigated different biodegradable biomaterials, dermal matrix developed by Chen et al. [82] demonstrated potential
including collagen, polyglycolic acid, polylactic acid and co-polymers of as wound dressings for clinical applications. An in vivo experiment to
L-lactide and -caprolactone, for nerve repair [265,266]. A research study evaluate the wound healing effect of water soluble chitosan/heparin
on the degradation of covalently cross-linked CMCS in vitro for the rst complex on the full thickness skin excision performed on the backs of
time and its potential application for peripheral nerve regeneration has the rat displayed that the complex is most effective in wound healing
been carried out by Lu et al. [69]. The results revealed that the faster [271]. Also, in an in vivo animal experiment using a burn wound
degradation of EDC-crosslinked CMCS than chitosan and decrease in model water-soluble O-CMCS derivatives modied with furfuryl glyc-
the hydrophilicity and elastic modulus of CMCS lms are benecial for idyl ether (O-CMCS/FGE) displayed wound healing effects. The results
application of CMCS in nerve repair. The study also suggested that the of the study showed that O-CMCS/FGE would be a promising candidate
EDC cross-linked CMCS lms enhanced the spread and provided a as an anti-adhesion material for biomedical applications [31]. A study on
good proliferation substratum of Neuro-2a cells. Similarly the tunable subcutaneous implantation of the ornidazole loaded (PVA)/CMCS lms
degradation rates of CMCS have been experimentally demonstrated re- prepared by blending/casting method in the surgical wound did not
cently by Guangyuan et al. [72]. He described the ability of Neuro-2a promote any adverse effect. Over a long period of time, it is expected
cells to adhere and proliferate when cultured on binary and unary that these drug lm would be absorbed and the wound would be
CMCS lms was found to be comparable to those on non-modied cicatrised by new forming tissues eventually [34]. Angiogenesis of full-
unary chitosan lms which established CMCS as a promising material thickness burn wounds repaired with collagen-sulfonated CMCS porous
for neural tissue engineering. Recently, ability of CMCS to stimulate pro- scaffold encoding VEGF, DNA plasmids has been reported by Teng et al.
liferation of Schwann cells in vitro by activating the intracellular signal- [272]. Recently, Ag nanoparticles/(PVA)/CMCS nanobers prepared by
ing cascades of extracellular signal-regulated kinase (ERK1/2) and electrospinning technique by Zhao et al. showed potential for wound
phosphatidylinositil-3 kinase (PI3K/Akt) has been reported by Bin He dressing biomaterial. Fig. 25 shows the in vitro Ag released amount of
and coworkers [267]. crosslinks AgNPs/PVA/CMCS nanobers as a function of time. At the
third hour the release of Ag in both the samples was fast and after
12.4. CMCS in wound healing that became relatively slow. Finally in both the samples after 24 h the
concentration of Ag+ reached equilibrium [197]. Apart from enhanced
CMCS-based materials, produced in varying formulations, have been wound healing capacity, CMCS has also been experimentally proven ef-
used in a number of wound healing applications. The inherent acceler- cacious in preventing as well as reducing post-operative surgical adhe-
ative wound healing effects of CMCS on second degree burn models per- sions by several researchers [80,81,273].
formed in rats was demonstrated in vitro and in vivo by Peng et al. [268].
In another experimental study, N,CMCS was used as biomaterial to heal
deep second-degree burn wounds. The results demonstrated that the N, 13. Tissue engineering application is often combined with drug
CMCS was efcient in accelerating wound healing via activating delivery strategy
transforming growth factor-1/Smad3 signaling pathway [269]. The im-
proved wound healing ability of CMCS of different MW has earlier been In the area of regenerative medicine and reconstructive surgery sub-
experimentally investigated by Chen et al. [20] by using a cell culture stantial input has been made from developments in tissue engineering
which showed positive results. The wound dressings of CMCS devel- and drug delivery technologies. The regeneration of functional tissue re-
oped by Qin and coworkers evaluated for wound healing ability quires an appropriate microenvironment that closely mimics the host
in vitro also displayed promising results [270]. CMCS, not only in its in- site for desired cellular responses which is typically provided by 3-D tis-
herent form, but also in many modied forms or in combination with sue engineering scaffold that acts as an architectural template [274]. But
repair and reconstruction of diseased and/or damaged tissues/organs
demands therapy that cannot only provide mechanical and structural
integrity to the tissue but also maintains sustained/controlled delivery
of therapeutics and/or growth factors in order to enhance the healing
and regeneration process. While scaffold provides structural support,
diffusivity to enable cellular inltration and acts as substrate for tissue
differentiation and organisation, the drugs/bioactive molecules embed-
ded in it, cues for the surrounding tissues to heal and regenerate. Recent
advances in the eld 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 dened
period of time. In fact the control over the regenerative and repairing
potential of tissue engineering scaffolds has dramatically improved in
recent years, mainly by using drug releasing scaffolds or by incorpora-
tion of drug delivery devices in the tissue engineering scaffolds
[275277]. While previous approaches of developing drug delivery for-
mulations mainly focused on the encapsulation or embedment of drugs
within the bulk phase and targeted delivery, recent strategies of tissue
engineering open up the new possibility of constructing scaffolds that
can provide the control over the sequestration and delivery of specic
bioactive factors to enhance and guide the regeneration process [278,
Fig. 25. In vitro Ag release curves of AgNPs/PVA/CM-chitosan nanobers in PBS 279]. Fig. 26 shows the more efcient and effective approach of combin-
(reproduced with permission from [197]). ing tissue engineering applications with drug delivery strategy for
L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 81

Fig. 26. Shows the more efcient and effective schematic approach of combining tissue engineering applications with drug delivery strategy for enhanced repairing and regeneration of
damaged and/or diseased tissues/organs.

enhanced repairing and regeneration of damaged and/or diseased tis- used either as blends, co-polymers or composites to alter the physico-
sues/organs. chemical characteristics and degradation behavior. CMCS has been
Extensive overviews of recent research studies on development and trialled in almost all novel drug delivery systems of current drug deliv-
applications of three dimensional scaffolds with potential capabilities ery approach due to its compatibility with wide range of polymers and
for the controlled delivery of therapeutic drugs particularly osteogenic its suitability with drugs of different properties. A number of research
drugs for bone regeneration are reported in literature [280,281]. In works have been reported where same drug being encapsulated in dif-
fact, this paradigm shift that has taken place to utilise the tissue engi- ferent CMCS formulations. Therefore, during the process of formulation
neering and drug delivery approaches towards the regeneration of den- development for a particular drug, such information can be used for
tal, oral and craniofacial structures using matrices and scaffolds capable comparative efcacy improvement studies between two or more for-
of controlled drug release has earlier been exhaustively reviewed [282]. mulations. Also, owing to its enhanced bioactivity and its ability to pro-
In this context, the research work for development of scaffolds with po- mote osteogenesis, CMCS is being studied at preclinical and clinical level
tential of drug delivery for neural [283] and bone [284,285] tissue engi- ensuring its potentiality in tissue engineering. CMCS can chelate calcium
neering is worth mentioning. As far as CMCS is considered, not much from mineralizing solution containing calcium and phosphate and in-
research work has been done in 3D scaffolding delivery system. But duce calcium phosphate or HAP formation thereby making it suitable
CMCS/gelatin/n-HAP i-gels susceptible to tyrosinase/p-cresol mediated for bone and cartilage regeneration. But being polymer, the mechanical
in situ gelling at physiological temperature capable of treating irregular properties of CMCS are not compatible enough with the host tissues
small bone defects have been investigated to exhibit potential of bone particularly with bone which restricts its usage as such in tissue engi-
cell delivery [2]. Reves et al. [286] prepared microspheres cross linked neering applications. Thus, efforts to improve the mechanical properties
by two different methods which were incorporated successfully into of CMCS based formulations are essential for this type of application.
the composite scaffolds. The X-CMCS beads (obtained by carbodiimide Therefore, CMCS has often been exploited in the form of either compos-
chemistry cross linking) displayed good potential for use in bone tissue ites with ceramics or HAP or in combination with some other polymers
engineering applications in which degradation and local drug delivery with better mechanical strength in order to make it suitable for tissue
are desired as compared to Gen-X CMCS (cross linked by genepin) engineering. CMCS based 3D scaffolds mimic the extracellular matrix
which showed poor degradation and drug release proles. and have proved to be highly useful formulations for repair and recon-
struction of damaged organs in general and tissues in particular. But de-
14. CMCS based systems: current challenges and opportunities spite of its versatility, CMCS based formulations are not commercialised
widely in clinical drug delivery and tissue engineering practice. Also,
CMCS based nanocarriers have become one of the most extensively there is a paucity of studies regarding the development of technological
studied nanometric drug delivery platforms. But these trials are still lim- strategies to integrate and position drug delivery devices with a
ited to experimental purposes and are not implicated widely as submicrometric spatial resolution within the scaffolds. Nevertheless,
marketed formulation. Despite of its increased aqueous solubility as numerous studies involving study of CMCS as drug delivery carriers
compared to native molecule chitosan, its signicant hydrophobicity re- and tissue engineering devices are being patented and some are under
mains the major drawback responsible for its limited use in biomedical preclinical or clinical investigation. The main goals are to improve
eld. A drug requires compatible physicochemical properties of the ma- their stability in the biological environment, to mediate the bio distribu-
trix polymer for developing a formulation successfully. CMCS alone or in tion of active compounds, enhanced drug loading, targeting, transport,
combination with other polymers, metals, and metal oxides has been release, and interaction with biological barriers. The cytotoxicity of
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