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225Preparation, modification, and applications of chitin nanowhiskers: a review

© 2012 Advanced Study Center Co. Ltd.

Rev. Adv. Mater. Sci. 30 (2012) 225-242

Corresponding author: V. Ostafe, e-mail: [email protected]

PREPARATION, MODIFICATION, AND APPLICATIONS OFCHITIN NANOWHISKERS: A REVIEW

M. Mincea1,2,3 , A. Negrulescu1,2 and V. Ostafe1,2

1West University of Timisoara, Department of Chemistry, Pestalozzi 16, Timisoara 300115, Romania2West University of Ti]isoara, Multidisciplinary Research Platfor] “Nicholas Georgescu - Roengen”,

Advanced Environmental Research Laboratories, Oituz 4, Timisoara 300086, Romania3 te]porally affiliated to “Alexandru Ioan Cuza” University, Ro]ania

Received: January 20, 2012

Abstract. This paper provides an overview of the most up-to-date information available relatingto chitin nanowhiskers. This paper presents aspects about chitin nanowhiskers, includingmethods of extraction and preparation, chemical modification and applications. Chitinnanowhiskers can be obtained by hydrochloric acid hydrolysis, TEMPO-mediated oxidation, partialdeacetylation with NaOH by fibril surface cationization, ultrasonication, electrospinning, aqueouscounter collision treatment, a simple grinding treatment and gelation with 1-allyl-3-methylimidazolium bromide. An introduction into the methods used to prepare chitin nanowhiskersis given. The chitin nanowhiskers applications are used mainly as reinforcing polymernanocomposites, but also to prepare scaffolds, hydrogels and wound dressings, as adsorbentsin industry, water purification, for protein immobilization, transformation of bacteria by exogenousgenes, stabilization of oil-in-water emulsion and nematic gels, formation of CaCO3/chitin-whiskerhybrids and as carbon precursors.

1. INTRODUCTION

Chitin is a natural, renewable and biodegradablepolymer, the second most abundant natural polymerafter cellulose [1]. Large amounts of this structuralmaterial can be found in animals, in exoskeletonshells of arthropods (crabs, shrimps and beetles),internal flexible backbone of cephalopods, worms,webs of spiders, cell walls of fungi and yeasts [2-4].Despite its easy accessibility, chitin is still anunderutilized resource because of its bulk structureand insolubility in water and common organicsolvents [5].

Chitin is considered to be the main biomassresource [6], with more than 1011 tones per yearproduction in nature [5]. The shellfish processingindustry (shrimp or crab shells) generates greatamounts of waste from shells, which contain about30% in chitin [4].

Chitin is non-toxic, odorless, biocompatible withliving tissues, biodegradable [2], presentingantibacterial, moisture retaining and healingcharacteristics [7]. Chitin and chitosan (partiallydeacetylated chitin) can be utilized in waterpurification [8], additives in cosmetics [9,10],antibacterial agents [11,12], pharmaceuticaladjuvants [11,13], paper production, textile finishes,photographic products, cements, heavy metalchelating agents, membranes, hollow fibers, andwaste removal [14-16] and biomedical applicationssuch as tissue engineering scaffolds, wounddressings, separation membranes, antibacterialcoatings, stent coatings, and sensors [13,15- 18],since they are harmless for the human body [19].

Chitin is a high molecular weight linear polysac-charide consisting of copolymer repeated units of-(1 4)-2-acetamido-2-deoxy- -D-glucose and -

(1 4)-2- amino-2-deoxy- -D-glucose [20], being

226 M. Mincea, A. Negrulescu and V. Ostafe

highly basic. The -(1 4)-N-acetyl glycosaminogly-can structure with two hydroxyl groups and an ac-etamide group makes chitin very crystalline withstrong hydrogen bonding [21].

Chitin is a semicrystalline biopolymer whichforms microfibrillar arrangements in living organisms.Native chitin fibers are made up of thin filaments,crystalline fibrils called microfibrils, tightly bondedto each other through a large number of hydrogenbonds. These fibrils are typically embedded in aprotein matrix and their diameters range from 2.5 to2.8 nm, depending on their biological origins [14].Chitin microfibrils are constituted of alternatingcrystalline and amorphous domains. Submitting thechitin to a strong acid hydrolysis treatment, causesthe longitudinal cutting of these microfibrils, allowingdissolution of amorphous domains [22]. The]icrofibrils consist of nanofibers with 2–5 n]diameters [23,24]. Each chitin crystallite (otherwisecalled whisker i.e. highly crystalline chitin nanofibril)is composed of about 20 linear chains of N-acetyl-glucosamine, based upon the rod diameter andcrystalline network dimensions [25].

Chitin is predominantly present as a fibrillar crys-talline material. Based on infrared spectroscopy andX-ray diffraction data, native chitin can occur in oneof the three crystalline forms [26]: -chitin, -chitin,and -chitin, respectively, depending on its origin.The molecules in -chitin are arranged in an anti-parallel fashion, with strong intermolecular hydro-gen bonding. -chitin is the most abundant formexisting in crabs, lobsters, krill and shrimps shells,insect cuticle, and fungal and yeast cells walls [3],having a crystallinity higher than 80% [27]. In -chitin, present in squid pens and tube worms [28,29],the chains are arranged in a parallel fashion, while-chitin is the form in which the molecules are ar-ranged in both parallel and anti-parallel manner. Dueto the molecular packing, intermolecular interactionsin -chitin are weaker than those in -chitin, mak-ing -chitin more susceptible to dissolution in somesolvents, and more reactive. -Chitin is more abun-dantly present in nature compared with squid pen-chitin and -chitin, purified -chitins being com-

mercially more readily available [30].This paper details preparation methods and

chemical modification, as well as applications ofchitin nanowhiskers.

2. PREPARATION OF CHITINNANOWISKERS

According with Watthanaphanit et al., whiskers orcrystalline nanofibrils are substances that can bemade from the breaking down of crystalline materi-

als into nanocrystalline entities with specific shapesor self-assembling of basic building blocks [31].

Chitin nanowhiskers occur in biological tissues,according to structural hierarchies, jointly withproteins and inorganic compounds [32]. Thepurification step of chitin has to be optimized inorder to remove remaining proteins and minerals thatare present in the animal raw material and to takethe best possible advantage of interwhiskersinteraction, by favoring the development of a rigidchitin nanowhiskers network [28]. Chitin can beextracted from the biological tissues and dispersedin aqueous media to form colloidal suspensions [25].

Various methods have been employed for thepreparation of chitin nanowhiskers (nanocrystals)or nanofibers including acid hydrolysis[7,14,22,28,29,31,33-42], TEMPO-mediatedoxidation [27,43,44], ultrasonication [45],electrospinning [46], mechanical treatment[21,47,49], and gelation [6]. Acid hydrolysis wasused to dissolve regions of low lateral order so thatthe water-insoluble, highly crystalline residue maybe converted into a stable colloidal suspension bysubsequent strong mechanical shearing action [40].Chain cleavage occurring at random locations alongthe microfibrils form these partially acetylatedwhiskers that are rod-like or spindle-like particlesthat tend to align cooperatively and to develop rigidstructures. Protonation of amino groups on the chitinnanowhiskers provide positive charges at theirsurface and stabilize the dilute colloidal dispersionsof chitin nanowhiskers due to repulsive forcesbetween crystallites [50]. It has also been reportedthat when such colloidal dispersions of acid-degraded chitin are dewatered to a critical increasedconcentration they can suffer an isotropic-anisotropicnematic transition [14,39,42].

Chitin nanowhiskers have been prepared fromcrab shells [21,22,35,36,40,42,47,51,52], squidpens [28], prawn shells [48,53], tubes of Riftiapachyptila worms [29] and shrimp shells [38,54].The procedure used for the purification of chitin andthe preparation of the suspensions of chitinnanowhiskers is quite similar in publishedprocedures [14,28,29,34,50] with little modificationdue to the source of chitin.

The method for the preparation of chitinnanowhisker reported by Morin [29] was as follows:(i) biological tissues (fragments of Riftia pachyptilatube worms) were suspended in a 5 wt.% aqueousKOH solution and boiled for 6 h under stirring inorder to remove contaminating proteins. Then, thedispersion was rinsed with distilled water and filtered,the resulting paste being kept at room temperature


overnight under agitation. Subsequently, the boilingstep was performed again, repeating also thewashing and filtering steps. (ii) the chitin samplesobtained after the above procedure, were bleachedwith a NaOCl2 solution (17 g of NaOCl2 in 1 L of 0.3M sodiu] acetate buffer, pH 4.0 , heated at 80 °Cfor 2 h under stirring. This procedure was repeatedthree times with rinsing. The resulting dispersionwas centrifuged for 15 min. (iii) chitin was hydrolyzedwith HCl boiling for 90 min under stirring and theproduct washed thoroughly with deionized water,followed by centrifugation (for 15 min) and decantingthe supernatant. This process was repeated threetimes with the residue. The nanowhiskers wereobtained by eliminating the amorphous parts of themicrofibrils. Afterward, the suspensions of chitinnanowhiskers were transferred to a dialysis bag anddialyzed for 2 h in running water and, then, keptovernight in distilled water. (iv) the product was furthersubjected to a supplementary dialysis for 12 h,changing the distilled water every 2 h; the dialysiswas performed until a pH=6 was reached. (v) thedispersion of nanowhiskers (0.15 wt.%) wasaccomplished further by three successive 2 minultrasonic treatments. The dispersions were,subsequently, filtered to remove residual chitinnanocrystal aggregates. Next, appropriate volumesof HCl solution were added until a pH of about 2.5was reached. The suspension, constituted fromcrystalline fragments of chitin, displayed a colloidalbehavior, stabilized by positive charge (NH3

+) at thesurface of the whiskers resulting from the protonationof amino groups [42]. The particles wereconcentrated by dialysis against poly-(ethyleneglycol). The solid fraction (0.22%) of this aqueoussuspension was deter]ined. It was kept at 6 °C ina refrigerator until used after adding chloroform toavoid microorganisms development [29].

The origin of chitin, namely the type ofcrystallinity, determines the structure andmorphology of the chitin nanowhiskers [20]. Thenanoparticles occur as rod-like or spindle-likenanowhiskers [27,43] with properties comparableto perfect crystals [4]. The structural characteristicof the chitin nanowhisker extracted by differenttreatments from various chitin sources aresummarized in Table 1. Chitin nanowhiskersextracted from various sources by a multi-stageche]ical%]echanical separation are generally 4–80 nm in width and 50-10.000 in length. Thedimensions for chitin whiskers obtained from crabshells are much shorter in length when comparedwith the chitin whiskers from Riftia tubes. The aspect

ratio is an important parameter, especially whenchitin nanowhiskers are used to reinforce polymers;a higher aspect ratio usually results in greaterreinforcement [55].

Chitin nanowhiskers dispersed in water wereprepared by 2,2,6,6-tetramethylpiperidine-1-oxylradical (TEMPO) mediated oxidation of -chitin inwater at pH 10 with ultrasonic treatment. NaClOwas added as co-oxidant in the reaction. Chitin wastransformed into water-soluble polyuronic acid andwater-insoluble chitin nanowhiskers. The TEMPO-oxidized chitin had crystallinity as high as that ofthe original -chitin. A significant factor that affectsthe transparency of the dispersions, the resultantweight ratio of water-insoluble fractions, shape,length, and width of the chitin nanowhiskers obtainedis the carboxylate content in the TEMPO oxidizedchitins or the amount of NaClO added in the oxidationof -chitin. The addition of 5.0 mmol of NaClO pergram of chitin seems to be optimal for preparationof mostly individualized nanowhiskers with highaspect ratios. The TEMPO-oxidized chitinnanowhiskers, prepared with 5.0 mmol of NaClO,had fiber widths smaller than 15 nm, and the averagewidths 8 nm. The anionic C6 carboxylate groupsformed by TEMPO-mediated oxidation present onlyon the chitin whiskers surfaces may increase theindividualization of the whiskers by simplemechanical treatment, such as sonication.Interwhisker linkages could be formed to someextent by electrical repulsion interactions betweenanionic C6 carboxylate groups and cationic C2ammonium groups on the surfaces of chitin whiskersand/or through the osmotic effect [27].

Fan et al. [43] reported preparation of chitinnanowhiskers from squid pen -chitin by TEMPO-mediated oxidation of native chitins, and subsequentmild mechanical agitation in water, at pH 3-4.8.Mostly individual and highly crystalline chitinnanowhiskers, 3–4 n] in width and few ]icrons inlength were successfully prepared. Cationization ofthe C2 amino groups in the -chitin at pH 3-4 iskeeping the stable dispersion state by interfibrillarelectrostatic repulsion in water. This simpledisintegration method for preparation ofnanowhiskers is valuable in terms of safety issues,because the protocol involves no chemicalmodification. Nanowhiskers obtained by this methodcan be used in functional foods and cosmetics fields.The relatively low crystallinity of the nanowhiskersfrom squid pen is an important drawback. In addition,the biomass quantity of the pen is considerably lowerthan those of crab and shrimp shells.


Comparing with hydrochloric acid hydrolysis,TEMPO-mediated oxidation method is morecontrollable by the amount of NaClO and the yieldof chitin nanowhiskers can reach 90%. N-deacetylation does not occur on the TEMPO-oxidized chitins [27].

Individual chitin nanowhiskers with average widthand length 6.2 ± 1.1 and 250 ± 140 n], respectively,have been obtained from partially deacetylated

-chitin by fibril surface cationization. Chitinnanowhiskers with N-acetylation values 0.74–0.70were mechanically disintegrated in water at pH 3 to4. In this method chitin nanowhiskers surfaces arepositively charged, leading to electrical repulsion andnanowhiskers individualization [44].

Zhao et al. [45] developed a simple, versatile andenvironmentally friendly approach for extractingbionanowhiskers from various natural materialsbased on an ultrasonication technique.Bionanowhiskers, among which chitin nanowhiskershave been fabricated from various materials (chitinfibers, spider and silkworm silks, collagen, cotton,bamboo, and ramee and hemp fibers), with uniformdiameters in the range of 25 to 120 nm, a usefulsize range for many tissue engineering and filtrationapplications. The ultrasonication causes the naturalfibers to disassemble into nanowhiskers in water.The micron sized natural fibers are graduallydisintegrated into nanowhiskers by the ultrasonicshock waves that cause erosion of the surface ofthe fibers which split along the axial direction. Thedisassembly rapidity depends on the intensity andfrequency of the ultrasonic wave.

Min et al. [46] used an electrospinning methodto fabricate chitin nanowhiskers. Electrospinningprocess is a technique that can produce polymernanowhiskers with diameter in the range from severalmicrometers down to tens of nanometers, dependingon the polymer and processing conditions. Toimprove chitin solubility, previous to electrospinningthe chitin powder (100~500 nm) was depolymerizedbeing packed in polyethylene bags and Co60 gammairradiated, for 3 days. Chitin solutions withconcentrations in the range from 3-6% wereobtained. The electrospinning of chitin was performedwith 1,1,1,3,3,3-hexafluoro-2-propanol as a spinningsolvent. The electrospun chitin nanowhiskers werecollected on a target drum which was placed at adistance of ~7 cm from the syringe tip and on whicha voltage of 15 kV was applied to the collecting targetby a high voltage power supply. The chitinnanowhiskers had a broad fiber diameter distribu-tion, which ranged from 40 to 640 nm, the averagediameter was 110 nm.

Kose and Kondo [49] prepared chitinnanowhiskers in an aqueous dispersion state fromcrab -chitin powder, using an aqueous countercollision treatment (ACC). The ACC system isejecting a liquid suspension of the sample from apair of nozzles under a high pressure of 200 MPa,forming a pair of jets. The chitin fibers are not solublein water, but chitin nanowhiskers can be dispersedin water by ACC method, that is able to cleave thefacial interaction only by water jets without chemicalmodification of the molecules. The number ofejecting steps and ejecting pressure is adjusted tosubject the sample to an appropriate degree ofpulverization. Before the ACC treatment, theaqueous dispersion containing -chitin powder wasphase-separated. After the ACC treatment at 0, 1,5, 10, 30, 60, and 120 ejecting steps, respectively,the chitin samples became turbid. Changing thenumber of ejecting steps and the desired pressure,the sample is supposed to be more downsized. Chitinparticles having micro-size diameter were observedin polarizing light microscopy in the range from 0 to10 ejecting steps. Chitin nanowhiskers havingnanoscale diameter were observed in the range from1 to 120 ejecting steps, their amount increasing withgrowing the ejecting steps number from 1 to 30.Also, the chitin nanowhiskers were homogeneouslydispersed in water with increasing the ejecting stepsnumber. The ACC treatment provided homogeneousaqueous dispersion of chitin nanowhiskers with 10–20 n] in width. The width of 10–20 n] was notsignificantly changed; by varying the treatment con-dition further pulverization did not take place anymore. After chitin samples obtained by ACC treat-ment with 60 ejecting steps were kept for 3 months,the chitin nanowhiskers exhibited a favorable ag-gregation as a three-dimensional network formation.

Ifuku et al. [21,47,48] used mechanical disas-sembly of chitin to obtain highly uniform chitinnanowhiskers. Chitin nanowhiskers with 10–20 n]in width were prepared from wet and dried chitinobtained from crab shells [21,47] and prawn shells[48] by a simple grinding treatment after the removalof proteins and minerals. Because the exoskeletonof prawn has a finer structure, the nano-fibrillation ofprawn shells is easier than that of crab shells,allowing the preparation of chitin nanowhiskers fromprawn shells under neutral pH conditions, while thenanowhiskers from crab shells are obtained underacidic conditions. The cationization of small amountsof C2 amino groups (NH3

+) on the chitinnanowhiskers surface, by the addition of an acid,promote the braking of the strong hydrogen bondsbetween the nanowhiskers, by electrostatic repul-


sions. The degree of substitution of the amino groupsof only 3.9% was enough to break strong hydrogenbonds that protect the rigid chitin structure [47].

The suspensions of crude -chitin were treatedwith a domestic blender. Then the slurry of 1%purified chitin was passed through a grinder. Themechanical treatment was used to isolate naturala-chitin nanowhiskers prepared in a never-driedstate. These chitin molecules are aligned in anantiparallel manner that gives rise to -chitin crystalsin the form of bundles of highly uniform crystallinechitin nanowhiskers having a width of 2-5 nm.Furthermore, these nanowhiskers are wrapped inprotein layers resulting chitin-protein fibers of around100 nm diameter. Because strong hydrogen bondingbetween the bundles of dried chitin nanowhiskers,leads to difficulty in achieving thin and uniformnanowhiskers, the material was kept wet after theremoval of the matrix [21].

Chitin nanowhiskers with a uniform structure anda long length were obtained by Ifuku et al. [48] fromthe cell walls of five different types of mushrooms,commonly used for human consumption Pleurotuseryngii, Agaricus bisporus, Lentinula edodes, Grifolafrondosa, and Hypsizygus marmoreus. The chitinnanowhiskers were isolated by removing glucans,minerals, and proteins, and subsequent simplegrinding treatment under acidic conditions.Depending on the species of mushroom, the widthof chitin nanowhiskers was in the range 20–28 n].It was noticed that the -chitin structure wasmaintained and that on the chitin nanowhiskerssurface a complex with glucans was formed.

Beside the excessive costs, the previouslypresented preparation methods based on chemicaltreatment of the raw materials have several otherdrawbacks, among which the low yield of theprocesses, dangerous handling of boiling HCl,disposal of the colored HCl solution, recovery ofenormous quantities of slightly acidic water, difficult

Fig. 1. Chemical modification of chitin nanowhiskers with isocyanate groups.

adjustment of the pH value because of the strengthof HCl, and so on [57].

Chitin nanowhiskers were easily produced by thegelation of a commercial chitin powder with 1-allyl-3-methylimidazolium bromide (AMIMBr), by soakingit in the ionic liquid at room temperature, followedby heating at 100 °C. Soaking the resulting gel inmethanol and subsequent sonication gave chitindispersion in which chitin nanowhiskers formationswere regenerated [6]. This processing technique forthe preparation of the chitin nanowhiskers isconsidered to have great advantages compared tothe methods presented above because specialequipments and chemical modifications are notnecessary.

3. CHEMICAL MODIFICATION OFCHITIN NANOWHISKERS

Chitin nanowhiskers possess a reactive surfacecovered with hydroxyl groups, which provides thepossibility of modification through chemical reaction.The purpose of chemical modification is to contributeto specific functions and to expand the applicationsof chitin nanowhiskers. A method for the processingof chitin nanowhiskers-based nanocomposites istheir transformation through long chain surfacechemical modification. The nanoparticles aremodified based on the use of grafting agents bearinga reactive end group and a long compatibilizing tail[58].

Surface chemical modification of chitinnanowhiskers is a method to decrease their surfaceenergy and disperse them in organic liquids of lowpolarity. Nair and Dufrense [52] investigated thesurface chemical modification of chitinnanowhiskers with different reagents. The surfaceof chitin nanowhiskers - prepared by acid hydrolysisof chitin from crab shells - was chemically modifiedusing a small molecule chemical reaction between


Fig. 2. Chemical modification of chitin nanowhiskers with alkenyl succinic anhydride.

Fig. 3. TEMPO oxidation of chitin nanowhiskers.

hydroxyl groups (–OH and isocyanate groups(–NCO fro] phenyl isocyanate (PI and isopropenyl-

, ’-di]ethylbenzyl isocyanate (TMI (see Fig. 1 .Alkenyl succinic anhydride (ASA) consisting of

a mixture of oligomers of different sizes centeredaround C18 (Mn = 300) was used for the modificationof chitin nanowhiskers [52] (see Fig. 2). Theacylated whiskers can be dispersed in medium andlow-polarity solvents. Nanowhiskers with differentdispersibility could be obtained by controlling thetime of the heating step [58]. FTIR spectroscopy,TEM, and contact angle measurements wereperformed to prove the occurrence of the surfacemodification without any major morphologicalchanges [52].

The C6 primary hydroxyl groups on the chitinnanowhiskers surfaces are selectively oxidized tocarboxylate groups via the aldehyde structure byTEMPO-mediated oxidation (see Fig. 3). The totalcontents of carboxylate and aldehyde groups at 5.0and 10 mmol of NaClO per gram of chitin were 12%and 24% of the C6 primary hydroxyl groups of theoriginal chitin to be oxidized to either carboxylateor aldehyde groups.

New -(1 4)-linked polyuronic acids (e.g.chitouronic acid) consisting of repeating units of thesodium salt of N-acetylglucosaminuronic acid, areobtained quantitatively by TEMPO oxidation. In orderto oxidize only the C6 primary hydroxyl groupspresent on the chitin nanowhiskers surfaces theamount of NaClO added should be controlled. Whena sufficient amount of NaClO is added to the chitin/water slurries in the oxidation, chitin can beconverted to the corresponding water-solublepolyuronic acid with partial depolymerization at pH10–11 [59,60].

4. APPLICATION OF CHITINNANOWHISKERS

Since large quantities of crab and shrimp shells areproduced annually as food waste, further utilizationof chitins as functionalized materials is desired [27].The chitin nanowhiskers are currently obtained asaqueous suspensions which are being studied andused as reinforcing additives for high performanceenvironment-friendly biodegradable nanocompositematerials, as biomedical composites for drug/genedelivery, nanoscaffolds in tissue engineering andcosmetic orthodontics [6,17,27,29,40,41,51,52,61,62]. The reinforcing effect of chitin nanowhiskersresults from the formation of a percolating networkbased on hydrogen bonding forces [58].The chitinnanowhiskers with fungal origin can be use inantitumor application and immuno-modulatingactivity [48]. Current research on chitinnanostructures is important for preparing materialsfor medical and veterinary applications, as well [57].

4.1. Nanocomposites

Most of chitin nanowhiskers investigations havefocused on hydrosoluble or latex-form polymers.Using surfactants or chemical grafting it is possibleto disperse these nanowhiskers in non-aqueousmedia.

The addition of fillers to process a compositematerial is a standard method to improve themechanical behavior of a material [29]. In thenanocomposite industry, a reinforcing particle isdefined as having at least one of its linear dimen-sions smaller than 100 nm. Because of the recentrising of nanotechnology, chitin nanowhiskers havegot significant attention, being promising reinforc-


ing materials for nanocomposites, due to their highstiffness and strength [31,63]. Nanocomposites area class of materials that has aroused much interestin the last years since they exhibit ultrafine phasedimensions, they are renewable and ecologicallyfriendly materials [27]. A large amount of the cur-rent nanofillers used to prepare nanocompositeswith synthetic polymeric materials are inorganic [64].The use of nanowhiskers from renewable resourcesas natural fillers, instead of traditional inorganic re-inforcement materials (e.g. glass fibers, carbon, andtalc) would be a much better choice providing nu-merous advantages including easy availability,nontoxicity, renewability, low density, low cost, goodspecific mechanical properties, biodegradability,good biocompatibility, reproducibility, and easychemical and mechanical modification [65,66].

Chitin whiskers have been used as an innovativetype of nanofillers as a reinforcing material in bothnatural [22,31,33,38,40,51,54] and syntheticpolymeric matrices [6,21,28,29,41,47,48,67-72]. Forobtaining of a good level of distribution of the fillerswithin the polymer matrix the use of either anaqueous suspension or an aqueous solution of thepolymer is required. The reinforcing effect dependson the aspect ratio of the chitin whiskers [29].

Polymer/chitin nanowhiskers nanocompositeprocessing techniques that are usually employedare (i) casting and evaporating technique[22,28,29,31,38,40,50-52,54,56,72-74] in whichpolymer aqueous solution or dispersion is mixedwith chitin nanowhiskers aqueous suspensionresulting a homogenous dispersion. The dispersionis cast into a container and, by evaporation of water,a nanocomposite with chitin nanowhiskersincorporated is obtained, (ii) freeze-drying and hot-pressing technique [29,40,51] in which well-dispersedaqueous mixtures, of thermoplastic polymer andchitin nanowhiskers are freeze-dried to givenanocomposite powders, which are, subsequently,processed into specimen by hot-pressing, (iii)polymer grafting [69], a solvent free technique thatuse “graft fro]” strategy by which long chain sur-face chemical modification of polysaccharidenanoparticles consisting in grafting agents bearinga reactive end group and a long “co]patibilizing”tail are taking place [58], and (iv) nonaqueous sol-vent dispersion technique [52] in which the hydroxylgroups fro] the chitin nanowhisker’s surface aremodified by chemical reactions improving their hy-drophobicity. Hydrophobic chitin nanowhiskers canform a good dispersion in nonaqueous solvents,such as toluene and form stable suspensions.

Nanocomposite films are obtained by removal oftoluene.

4.1.1. Nanocomposites with NaturalPolymers as Matrix

A particular group of biocomposites is greencomposites, in which polymers from renewableresources (bio-based polymers), representing thematrix of the composite, are reinforced by naturalfibers. The study of “green co]posites” is ane]erging area in poly]er science. ‘‘Greenco]posites’’ [75,76] is a ter] that indicates thatboth matrix and reinforcement material of thecomposite originate from renewable resources [77],their use being advantageous not only from aneconomical viewpoint, but also environmentallyfriendly.

Nair and Dufresne [40,51] investigated the effectsof processing methods on the mechanical propertiesof natural rubbers (NR) reinforced with chitinnanowhiskers from crab shells nanocomposites.Chitin nanowhiskers reinforced NR nanocompositeswere obtained from a colloidal suspension of chitinwhiskers as the reinforcing phase and latex of bothunvulcanized and prevulcanized natural rubber asthe host matrix. The aqueous suspensions of chitinwhiskers and rubber were mixed and stirred, solidcomposite films were obtained either by freeze-drying and hot-pressing method or by casting andwater evaporation method. The samples preparedby casting and evaporation method showed higherreinforcing efficiency. Using this method thereinforcing effect of chitin nanowhiskers stronglydepended on their ability to form a rigid three-dimensional network in the NR matrix, resulting fromstrong interactions such as hydrogen bondsbetween the whiskers during the evaporationmethod. The disruption of the network resulted inlowering or loss of this ability. Among the evidencesfor the existence of a three-dimensional rigid chitinnetwork is better resistance of evaporated samples,than the hot-pressed ones, against swelling in anorganic solvent medium, the values of diffusioncoefficient, bound rubber content, and relative weightloss [40]. Latex from rubber trees (Heveabrasiliensis) is the source of all commercial naturalrubber (cis-1,4- polyisoprene), one of the mostimportant elastomers, an important and irreplaceablematerial in applications such as automotive (tiresand engine mounts) and constructions [77,78], inindustrial and technological areas [51].

Nair and Dufrense [52] investigated the incorpo-ration of surface chemical modified chitin


nanowhiskers into natural rubber to obtain compos-ite materials with enhanced mechanical propertiesand to enlarge the number of potential polymermatrixes. The surface of chitin whiskers from crabshells, was chemically modified with PI, ASA, andTMI. PI and ASA were used to improve adhesionbetween the NR used as a matrix and the chitinwhiskers. The TMI was used to copolymerize withthe unsaturations present in NR matrix. Stable sus-pensions of these chemically tailored chitin whis-kers were obtained in toluene as a replacement forusing aqueous suspensions. Nanocomposites wereprepared using a toluene natural rubber solution inwhich the chitin nanowhiskers were dispersed. Thevarious chemical treatments improved the adhesionbetween the filler and the matrix, but mechanicalperformances of the composites decreased after thechemical modification. The loss of mechanical per-formance, more obvious for the isocyanate treat-ments, could be owing to the partial or total de-struction of the three-dimensional network of chitinwhiskers after the surface modification which re-duces the hydrogen bonding.

For unvulcanized NR latex basednanocomposites two processing methods wereused, casting and water evaporation or freeze-dryingand hot-pressing, whereas for vulcanized materialsonly the former method was used, in order toinvestigate the effect of the processing method onthe properties of the material. The most importantaspect that governs the mechanical behavior of thechitin whisker reinforced NR nanocomposites is theprocessing technique. Dynamic mechanicalanalysis showed the existence of a small percentageof crystallinity in unvulcanized NR prepared by theevaporation method, whereas no such evidence ofcrystallinity was detected either in unvulcanized NRprepared by hot pressing or in vulcanized rubberprepared by evaporation methods. The evaporationmethod is a slow step procedure, in which the chitinnanowhiskers get enough time and mobility toestablish a rigid filler-filler network within the hostmatrix [51].

Jacobs et al. [33] have studied the introductionof chitin nanowhiskers as functional componentsinto chitosan for nanocomposites preparation byelectrospinning. Mostly individualized chitinnanowhiskers with spindle-like morphology anddiameter ranges of 10-20 nm were developed asnanocomposite materials for the reinforcement ofchitosan. The chitin nanowhiskers content in theresultant composites ranged from 1.25% to 5%. Theaverage fiber diameter of neat chitosan nanofibreswas 213 nm, and the chitin nanowhiskers loading

increased for 1.25 to 5% inducing a significant re-duction in fibre dia]eter, down to a range of 143 –171 nm. The results indicated a good interactionbetween the matrix and the chitin nanowhiskers thatled to enhanced structural morphology, fibre diam-eter and thermo-mechanical properties ofelectrospun nanofibres.

The successful preparation and characterizationof chitosan/ -chitin whiskers with or without heattreatment has been reported. Films were cast fromchitosan solutions containing dispersed -chitinnanowhiskers in the range between 0 and 2.96%.Thermal stability and the apparent degree ofcrystallinity of the chitosan matrix were not muchaffected by the addition of -chitin nanowhiskers.The tensile strength of -chitin nanowhisker-reinforced chitosan films was greater from that ofthe pure chitosan film, achieving a maximum at thenanowhisker content of 2.96%. Water resistance ofthe nanocomposite films - marked by decreasedweight loss and swelling in an aqueous ]ediu] –was improved by the addition of -chitin whiskersand heat treatment [38].

Chitosan has been used in a film form formultiple applications, such as tissue engineering,wound dressing, controlled release, and foodpackaging [79-82]. The real utilization of chitosanfilms is limited due to its solubility in water and otheraqueous solutions. The presence of -chitinwhiskers and heat treatment decreased the affinityto water of the chitosan/ -chitin whiskersnanocomposite films, being more stable when usedin an aqueous environment [38].

Glycerol plasticization was used to incorporatechitin nanowhiskers from crab shells as a filler toreinforce soy protein isolate (SPI) matrix forproducing a class of environmentally friendlynanocomposites. SPI and different content of chitinwere mixed and stirred to obtain a homogeneousdispersion. The dispersion was freeze-dried and 30%glycerol was added, then the resulting mixture washot-pressed and, then, gradually cooled to roomtemperature. The SPI/chitin nanowhiskercomposites, with thickness of about 0.4 mm, werethus obtained. Nanocomposites with lower whiskercontent displayed a relatively homogeneous disper-sion in the SPI matrix than those with increasedchitin whisker content. The composites show greaterwater-resistance as the chitin whiskers increase inthe SPI matrix. Strong intermolecular hydrogenbonding interactions among different chitin whiskersand between filler and SPI matrix play a significantrole in the improvement of mechanical propertieswhich restrict the motion of the matrix and decrease


water sensitivity of the SPI-based nanocompositeswithout interfering with their biodegradability [22].

Biodegradable plastics, such as plastics fromSPI can have important applications in rubbish andcompost bags, mulch films, and disposable diapers[83,84]. Plastics from SPI have very high strengthand good biodegradability. Their applications arelimited because they are brittle and water sensitive[85]. By chitin nanowhiskers incorporation into SPI,the thermo-mechanical properties have beenimproved and the composites have shown greaterwater-resistance.

Glycerol plasticized-potato starch was mixedwith chitin nanowhiskers to obtain fully naturalnanocomposites by casting and evaporation. Theresults showed improvements in tensile strength,storage modulus, glass transition temperature, andwater vapor barrier properties of the composite.However, at >5% loading, aggregation of the chitinnanowhiskers took place with negative effects [73].

4.1.2. Nanocomposites with SyntheticPolymers as Matrix

The possible applications of the chitin nanowhiskerswith synthetic polymers, such as poly(vinyl alcohol)(PVA) and polycaprolactone (PCL) to givecorresponding composite materials depend on theprocedures that assure the compatibility betweenchitin and the polymeric matrix. Synthetic polymersare flexible materials with many industrialapplications because of their excellent physicalproperties and chemical resistance. Somedrawbacks of synthetic polymers are high cost, non-biocompatibility, poor mechanical or thermalperformance of some polymers [86], and limitedprocessability, biocompatibility, and biodegradability[41].

In some studies the preparation of compositesor blends composed of chitin nanowhiskers andpoly(vinyl alcohol) (PVA) have been accomplished[30,38,87-90]. The chitin nanowhiskers prepared byacid hydrolysis were incorporated in the compositeswith PVA [30]. Sriupayo et al. [38] prepared andcharacterized -chitin whisker-reinforced chitosannanocomposite films with or without heat treatment.The nanocomposite formed with PVA reinforced with

-chitin whisker presented enhanced thermalstability, tensile strength and water resistance. Thecontents of the nanowhiskers were less than 30%.Junkasem et al. [41] also obtained chitin whisker-PVA reinforced nanocomposite. The nanocompositeswere fabricated by electrospinning a mixture betweenan aqueous solution of PVA and a suspension of -

chitin whiskers prepared from chitin flakes from shellsof Penaeus merguiensis shrimps. Kadokawa et al.[6] described a method completely different fromthe previous techniques. Composite materials ofchitin nanowhiskers with PVA were easily preparedby the gelation of a commercially available chitinpowder with AMIMBr, followed by the regenerationwith methanol. The SEM images of the composite(weight ratio of chitin to PVA = 1:0.3) illustrated thatthe nanowhisker-like morphology was preserved. Arelative immiscibility of chitin and PVA in thecomposite was specified, PVA components probablyfilled in spaces between the whiskers. Chitinnanowhisker and PVA might be partially miscible atthe interfacial area between the two polymers in thecomposites due to formation of hydrogen bondingbetween them or by the presence of a little amountof AMIMBr.

Ifuku et al. [47] examined the fibrillation propertiesof dry chitin and nanowhisker homogeneity bypreparation of optically transparent nanocompositesusing chitin nanowhiskers and acrylic resin. Chitinnanowhiskers with a uniform width and a high aspectratio were prepared from dried chitin, extracted fromcrab shell. The suspension, that contained 0.1%fibrillated chitin nanowhiskers dispersed in water,was vacuum filtered obtaining chitin nanowhiskersheets, which were cut into 2 - 3 cm fragments andwere impregnated with neat acrylic resin with 2-hydroxy-2-methylpropiophenone photo-initiator. Theresin-impregnated sheets were UV treated resultingchitin nanowhisker composite sheets. The opticallosses of the transparent composites after the dryingprocess were smaller than 2%, showing that driedchitin was fully fibrillated.

Nge et al. [67,68] studied the incorporation ofchitin nanowhiskers in poly(acrylic acid) forpreparation of composites, with unique optical prop-erties. The chitin crystallites presented uniplanarorientation; the X-ray diffraction data revealed thatmolecular long axes were perpendicular to thedirection of the magnetic field.

Using free-radical photopolymerization of acrylicacid in an unidirectional shearing alignedmesophase, a liquid chitin nanowhiskers/poly(acrylic acid) composite was fabricated, withunique optical properties. The composite, coatedwith a calcium fluoride substrate, was transparent.Its alignment was depending on the mesophasecomposition of the ternary dispersion composed ofchitin microfibrils, water and acrylic acid. Themesophase behavior strongly influenced the degreeof orientation and the molecular interactions [68].


One important application of PCL, a semicrys-talline thermoplastic polymer, is to make suturethread. In order to keep the ]aterial’s biodegrad-ability, chitin whiskers are used as reinforcing phasefor PCL. Morin and Dufrense [29] preparednanocomposite materials from an aqueous suspen-sion of high aspect ratio -chitin whiskers, as thereinforcing phase, and a latex of PCL as the matrix.The colloidal microcrystalline dispersion was mixedwith the suspension of latex in different amounts inorder to obtain composite films with a weight frac-tion of chitin ranging between 0 and 10%. After mix-ing and stirring the two aqueous suspensions, solidfilms were attained by either freeze-drying and hot-pressing or casting and evaporating the preparations.The results showed that at high temperature and apercentage of above 5% nanowhiskers in the mix-ture, these nanowhiskers formed a rigid networkassumed to be governed by a percolation mecha-nism, which stabilized the mechanical properties ofthe composite.

Squid pen chitin nanowhiskers were introducedas filler into an acrylic polymer matrix (averagediameter of the particles around 150 nm), acopolymer of styrene and butyl acrylate,poly(styrene-co-butyl acrylate) and poly(S-co-BuA).The aqueous suspension of chitin microcrystals wasmixed with an aqueous suspension of polymer (latex)containing spherical particles of poly(S-co-BuA) indifferent amounts in order to obtain nanocompositefilms with a good level of dispersion and with a weightfraction of chitin ranging from 0 to 20 wt.%. Usingvacuum, the air from the suspension was removedand the sample was casted in a Teflon mold,obtaining homogeneous, 1 mm thick films after waterevaporation. These whiskers bring a reinforcing effectand improve the thermal stability of the composite.Dynamic mechanical analysis showed that themechanical properties of these composites weresubstantially improved by increasing the amount offiller. For low whiskers content the filler-matrixinteraction is the main phenomenon involved in thereinforcing effect of chitin whiskers. When the chitinwhiskers were smaller than 10 wt.%, the films didnot show consistent improved mechanical propertiesover the whole temperature range (between 200 and425K) [28]. Chitin nanowhiskers can be used asenvironmentally friendly particulate fillers forthermoplastic nanocomposites films, which can beused for the processing of stiff small-size products.

Chitin whisker-graft-polycaprolactone (CHW-g-PCL) was prepared by ring-opening polymerizationof the caprolactone monomer onto the chitinnanowhiskers surface under microwave radiation. It

involved surface chemical modification of the chitinnanowhiskers, based on the use of grafting agentswith a reactive end group and a long compatibilizingtail, as the key of thermoforming. In order to preservethe mechanical properties instead of small moleculespolycaprolactone long chains are grafted. Thisnanoco]posite prepared using the “grafting fro]”strategy were thermoformed to fabricate CHW-g-PCLmolded sheets with good mechanical properties. Theincrease of the PCL content in CHW-g-PCL, con-ducted to elevated strength and elongation, as wellas high hydrophobicity of the nanocomposites [69].

Huang et al. [70] obtained waterborne polyure-thane-based nanocomposites by casting and evapo-rating a mixture with waterborne polyurethane asmatrix and small quantities of chitin nanowhiskersas nanofillers. The strength and Young’s ]odulusof the nanocomposites were simultaneouslyimproved and maintained ca. 500% elongation. Themaximum tensile strength (28.8 MPa) and enhancedYoung’s ]odulus (6.5 MPa that were ca. 1.8- and2.2-fold over those of neat polyurethane were attainedat chitin nanowhiskers loading of 3%. The activesurface and stiffness of chitin nanowhiskers facili-tated the interface for stress transferring formationand gave endurance to stress.

Environmentally-friendly, organic, solvent-freepolyurethane (waterborne polyurethane) with lowvolatile organic compound levels and non-toxicitycan be applied to leather and textile finishing, floorcoverings, adhesives and pressure sensitiveadhesives [91-95]. Chitin derivative [96] has beenincorporated into waterborne polyurethane forreducing costs, better biodegradability, andincreased mechanical performance.

Zeng et al. [74] prepared two series ofnanocomposite films from waterborne poly(ester-urethane) and chitin nanowhiskers with and withoutultrasound treatment. The effects of ultrasonicationtreatment and chitin nanowhisker content on thechemical composition, crystallization behavior andmiscibility were studied. When chitin nanowhiskercontent was > 30% both nanocomposite filmsexhibited a certain degree of miscibility, resulting inhigher thermal stability and tensile strength than inthe case of the pure waterborne poly(ester-urethane)film. The nanocomposite films subjected toultrasound treatment possessed better miscibilityand mechanical properties (storage modulus, ther-mal stability and tensile strength) than those with-out ultrasound treatment over the entire composi-tion range studied. The difference can be attributedto the relatively higher dispersion level of


nanowhiskers within poly(ester-urethane) matrixresulting in stronger interaction between bothcomponents. The structure, miscibility andmechanical properties of the nanocomposite filmsdepended significantly on the preparation method.

Rizvi et al. [71], blended chitin nanowhisker withpolylactide to form melt-blended polylactide-chitincomposites and Li et al. [72] prepared chitosan/chitin whisker/rectorite ternary films, both organicand inorganic composites being suitable for food-packaging applications.

4.2. Biomedical materials

Chitin nanowhiskers were incorporated in somepotential biomedical materials, such as scaffolds,hydrogels and wound dressing in which the materialshave to be cytocompatible. Hydrogels are physicallyor chemically cross-linked polymer networkscapable to absorb large amounts of water. Hydrogelsbased on natural polymers present variousapplications in the field of tissue engineering [97].

Wongpanit et al. [54] studied the influence ofchitin whiskers incorporation as nanofiller to silkfibroin sponge in order to improve its dimensionalstability and to increase its compression strength.Silk fibers from the silkworm Bombyx mori have beenused commercially as biomedical sutures fordecades. Regenerated silk fibroin is biodegradable[98], and cells like L929 cells [99], endothelial cells[100], keratinocytes, osteoblasts fibroblasts [101],bone marrow stromal cells [102], and bone marrow-derived mesenchymal stem cells [103] grow wellon its surface. The highly cytocompatibleregenerated silk fibroin is an attractive scaffoldingbiomaterial applicable for a wide range of targettissues. Nanocomposite sponges filled with chitinwhiskers were prepared by using a freeze-dryingtechnique. All samples possessed an intercon-

Fig. 4. Preparation of chitosan nanoscaffold from chitin flake.

nected pore network with an average pore size of150 mm. Mouse fibroblast L929 cells were seededonto the nanocomposites surfaces. Results indicatedthat silk fibroin sponges, both with and without chitinwhiskers, were cytocompatible, making them pos-sible materials for tissue engineering applications.The incorporation of chitin whiskers into the silk fi-broin matrix was found to improve the dimensionalstability and to promote cell spreading on thenanocomposite materials [54].

The scaffolds are used to obtain products withnano/micro pore structure appropriate for advancedapplications, such as artificial extracellular matrices[104,105], micro-particles for drug delivery, andmedical implants [106,107]. Functional scaffoldingmaterials are used as templates for the attachmentof cells for consequent tissue development in theprocess of tissue regeneration. Materials used forfabrication into scaffolds are synthetic polymers,such as polylactide, polyglycolide, and theirrespective copolymers, and natural polymers, suchas collagen, gelatin, and alginate [108,109], as well.Because scaffolds made from natural polymers aremechanically weak blending, crosslinking, andcompositing means are used to arrive at functionalscaffolds with improved physical, mechanical, andbiological integrities.

Phongying et al. [61] report for the first time thatchitosan nanoscaffold can be directly prepared fromthe chitin nanowhisker (see Fig. 4), by deacetylationin a highly concentrated alkaline solution for 21 h,yielding high amount of chitosan nanoscaffolds,because no organic solvents or chemicals wereinvolved. The colloidal solution of nano-sizedchitosan, with a degree of deacetylation of 95%,presented a fibrous network with a nanoporousstructure and the pore diameter of ~ 200 nm.Although chitin whiskers showed a molecular weight


of 62,838 Da, the molecular weight of a chitosannanoscaffold increased to 137,262 Da, probably dueto the scaffold structure. This biocompatible materialcould potentially be used in scaffolding of a tissue-engineered vessel.

Efficient deacetylation of chitin nanowhiskers toa chitosan nanoscaffold in the form of a colloidalsolution was obtained with a 60% alkaline solution,using a microwave technique, under a N2

atmosphere, for only 3 h, seven times shorter thanthe treatment time of the conventional method. Thedegree of deacetylation of chitosan nanoscaffold wasabove 90%. The amorphous chitosan was obtainedfrom the highly crystalline chitin whiskers. Theaggregation of branched chitin whiskers initiates thefor]ation of nanoscale “scaffold” ]orphology [20].

-chitin-whisker-reinforced hyaluronan–gelatinnanocomposite scaffolds with enhanced physical,mechanical and biological performances wereprepared with a 50 : 50 w/w blend of hyaluronan andgelatin, using -chitin whisker as the reinforcing filler.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide wasused as crosslinker. The CW-reinforced HA/Gelscaffolds were fabricated by a freeze-drying tech-nique. The weight ratios of the chitin nanowhiskersto the blend were 0–30%, but the scaffolds with 10%chitin nanowhiskers sowed the greatest cell viabil-ity, being the finest for supporting the proliferation ofcultured human osteosarcoma cells, even higherthan the native scaffolds. The inclusion of 2% chitinnanowhiskers in the scaffolds doubled their tensilestrength. The average pore size of the scaffolds var-ied between 139 and 166 m, regardless of the chitinnanowhiskers content. Most of the incorporatedchitin nanowhiskers improved the thermal stabilityand the resistance to biodegradation. A lowproportion of the chitin nanowhiskers increased thetensile strength and enhanced the biocompatibility,conducting to attachment and proliferation of thecultured human osteosarcoma cells of the resultingscaffolds. Although the scaffolds that contained 10%chitin nanowhiskers showed great promise assubstrates for bone cell culture, their actual utilizationcould be limited to a low-stress bearing area, suchas the socket of a dental root [56].

Alginate-based materials (hydrogel and fibrousproducts) are widely used in wound-careapplications. Some of their advantages arebiocompatibility, haemostatic capability and gel-formability upon subjected to an aqueous environ-ment [110]. Watthanaphanit et al. [31] prepared cal-cium alginate nanocomposite yarn (30 fibers) con-taining 0.05–2% chitin whiskers obtained fro] shellsof Penaeus merguiensis shrimp, by wet spinning

process. Alginate is a biopolymer resulting from cellwalls of some brown algae [111]. Between the algi-nate molecules and the homogeneously dispersedchitin whiskers specific interactions are created,such as hydrogen bonding and electrostatic inter-actions. Apart from the nanocomposite fibers con-taining 2% whiskers that presented whisker aggre-gates on the fiber surface, in most of thenanocomposite fibers the chitin whiskers were em-bedded inside the fibers. The incorporation of a lowamount (between 0.5 and 2%) of the whiskers inthe nanocomposite fibers considerably improved themechanical and thermal properties of the fibers andaccelerated the biodegradation process of the fibersin the presence of lysozyme. The presence of Ca2+

ions in the Tris-HCl buffer solution improved the te-nacity of the nanocomposite fibers.

Zhang et al. [112] introduced chitin nanowhiskersinto supramolecular hydrogels based oncyclodextrin/polymer by inclusion, in order to improvemechanical strength and regulate drug releasebehavior. The in vitro cell viability of the extractedleached media from the nanocomposite and nativehydrogels was assessed by the MTT method usingthe L929 cell line. The elastic modulus of thenanocomposite hydrogels raised due to thereinforcing function of the polysaccharidenanowhiskers. The presence of polysaccharidenanocrystals increased the stability of the hydrogelframework and inhibited the diffusion of bovine serumalbumin (BSA). BSA served as a model protein drugin the nanocomposite hydrogels and showedimportant sustained release profiles. The resultsshowed that incorporation of chitin nanowhiskersdid not increase the cytotoxicity in comparison withthe native hydrogel. The nanocomposite hydrogelscould be used as injectable biomaterials due to theirinherited shear-thinning property.

Muzzarelli et al. [17] integrated highly crystallinechitin nanowhiskers into wound dressings made ofchitosan glycolate and dibutyryl chitin. Non-wovendibutyryl chitin was used as a biocompatible support.The obtained products were tested in murine woundmodels being applied in various traumatic woundsresulting in very good final healing.

4.3. Adsorbents in industry and waterpurification

Ma et al. [113] employed ultrafine chitinnanowhiskers with 5–10 n] dia]eters as barrierlayers in a new class of thin-film nanocompositemembranes for water purification. Thenanocomposite membranes presented high virus


adsorption capacity as demonstrated by MS2 bac-teriophage testing, due to the very high surface-to-volume ratio. The low cost of raw chitin, the environ-mentally friendly fabrication process, and the im-pressive high flux indicate that such ultrafinenanofibril-based membranes can surpassconventional-membranes in drinking waterapplications.

The study of Dolphen and Thiravetyan [114]showed that chitin nanowhiskers prepared fromshrimp shell waste is very promising for adsorptionof melanoidins and other pigments from sugar syrup,being appropriate for application in sugar industry.The maximum adsorption capacities of melanoidinsby chitin nanowhiskers at 20, 40, and 60 °C were131, 331, and 353 mg/g, respectively. Chitinnanowhiskers presented an elevated affinity formelanoidins when compared to other chitin-derivedadsorbents. The interaction between melanoidinsand chitin nanowhiskers involved both electrostaticand chemical adsorption.

4.4. Protein immobilization

Na Nakorn [115] carried out protein immobilizationwith chitin nanowhisker (the average diameter 420nm with Nanosizer) and chitosan nanoparticles (theaverage diameter 215 nm with Nanosizer) in orderto find suitable materials to fabricate an efficientbiosensor, using for the optimization BSA as modelprotein. Before immobilization, chitin whiskers andchitosan nanoparticles were resuspended indeionized water and sonicated for 10 s. Theimmobilization time was 1 min and 15 min for BSAand chitin whiskers and chitosan nanoparticlesincubation, followed by centrifugation. Theconcentration of remaining BSA was determined byUV absorption at 280 nm. BSA immobilization withchitosan nanoparticles for 15 min, pH 6 (acetatebuffer) and 20-25 °C were the opti]al conditions,because they provided the least remaining BSA. Inthis study, chitosan nanoparticles showed betterBSA immobilization characteristics than chitinnanowhiskers, subsequently being used for thedevelop]ent of a glucose oxidase – chitosanelectrode for the detection of glucose for a biosen-sor application.

4.5. Bioengineering

Mera et al. [7] investigated the frequency of plas-mid uptake in penetration-intermediate E. coli cellsthrough Yoshida effect [116] produced by chitinnanowhiskers. The optimum conditions required tocreate penetration-intermediates capable of

acquiring plasmid DNA for creating a genetic trans-formation method in E. coli dependent on chitinnanowhiskers and the Yoshida effect weredetermined. The chitin nanowhiskers surfacespossess amino groups due to acid hydrolysis-induced deacetylation. The protonation of theseamino groups imparts a positive surface charge thatinteracts with plasmid DNA due to the negativecharge of the nucleic acids to form ionic complexes.Chitin nanowhiskers were mixed with DNAoligonucleotides tagged at the 5’ end. Underfluorescence microscope important amounts of chitinnanowhiskers aggregated and acicular crystallinematerials were observed, concluding that the nucleicacid was adsorbed onto the chitin nanowhiskerssurface. Similarly mixing pUC18 plasmid DNA withchitin nanowhiskers resulted in adsorption of pUC18onto the surface of the nanowhisker. The colloidalsolution consisting of nano-sized acicular crystallinechitin containing pUC18 plasmid DNA and cells ofEscherichia coli was placed on an agar hydrogeland stimulated by sliding friction at the interfacebetween the agar hydrogel and polystyrene stir stick,facilitated the transformation of the E. coli cells toantibiotic resistant. The genetic transformation rep-resents the effect of both chitin nanowhiskers andsliding friction. The formation of E. coli cells pen-etration-intermediates was a result of Yoshida ef-fect induced by the chitin nanowhiskers. Chitinnanowhiskers form a complex that penetratedbacterial cells due to the driving force resulting fromthe sliding friction. Plasmid pUC18DNA wasadsorbed onto the chitin nanowhiskers and thenintroduced into E. coli cells through the perforations.Intracellular uptake of exogenous pUC18 leaded toresistance of E. coli towards ampicillin. Thetransformation efficiency varied with both the numberof recipient cells and amount of chitin nanowhiskers.Induction of the Yoshida effect with chitinnanowhiskers represents a simpler and efficientlycomparable alternative to conventional chemicalmethods for introducing genes into bacteria.

4.6. Stabilization of oil-in-wateremulsion and nematic gels

Chitin nanowhisker aqueous dispersions shift to-wards a nematic gel-like behavior with increasingthe solid particles concentration. Chitinnanowhiskers being charged rod-like colloids, at pH3, form parallel alignments of anisotropic particleson entropic terms, as predicted by Onsager [117].Between these nanowhiskers associative interac-tions occur, mostly of van der Waals type, which


could be responsible for sol-gel transition. Strongergels are formed when these associative interactionsare enhanced with increasing ionic strength, pH,temperature and time [34], or by adding whey pro-teins [35].

Tzoumaki et al. [118] studied the stabilizingproperties of chitin nanowhiskers, colloidal rod-likeparticles obtained from crab shells, in oil-in-water(o/w) emulsions under varying conditions. Emulsionspresent broad occurrence in food, cosmetics andpharmaceutical industries [119]. An oil-in-wateremulsion was prepared by homogenizing chitinnanowhiskers stock dispersion with corn oil and anaqueous solution, using an ultra-sonic homogenizer.The chitin nanowhiskers were effective in stabilizingo/w emulsions against coalescence, for one monthperiod, even when the droplets were of relatively largesize, due to the adsorption of the nanowhisker atthe oil-water interface. The increase in chitinnanowhiskers concentration leaded to networkformation in the emulsions, increased stability tocreaming, and a gel-like behavior. Pronouncedemulsion elastic responses and creaming stabilitywas obtained by raising the temperature, NaClconcentration or pH (from 3.0 to 6.7). The chitinnanowhiskers adsorption at the o/w interfaces,resulted in the formation of inter-droplet network anda chitin nanowhiskers network in the continuousphase, representing a potential mechanismresponsible for the o/w emulsion stabilization.

4.7. Formation of CaCO3/chitin-whisker hybrids

Yamamoto et al. [36] prepared CaCO3/chitin-whiskerhybrids with hierarchical structures using the liquid-crystalline suspension of the chitin whiskers.Suspensions of chitin nanowhiskers, obtained byacid hydrolysis of chitin powder displayed lyotropicliquid-crystalline behavior. The casted suspensionwas immediately transformed into a gel whenexposed to ammonium carbonate vapor. CaCO3

crystals were created for 30 days in chitin gels, thecrystals being deposited in the gels matrices toproduce hybrids with three dimensional structures.This approach inspired by biomineralization is usefulfor the design of mechanically stable inorganic/organic hybrid materials.

4.8. Chitin nanowhisker aerogels,used as carbon precursors

Nogi et al. [53] prepared nanowhisker aerogels fromnanowhiskers water suspensions by subjecting the

suspensions to solvent exchange and freeze-dry-ing. Chitin nanowhiskers with reduced diameter didnot aggregate with each other in carbon precursoraerogels and presented high thermal stability. Thesenanowhisker aerogels with fine and individualnanowhisker network were used as carbon precur-sors. After the carbonization of prawn chitinnanowhisker, the original fine nanowhisker networkwas preserved in the chitin carbon. Nanofibrillar chitincarbon is activated by physical or chemical treat-ment (H2O or NaOH) attaining chitin carbon with alarge surface area, with potential applications in fil-ter media, electric double-layer capacitors and highlyefficient catalysts.

5. CONCLUSIONS AND OUTLOOK

Chitin can be extracted from biological tissues anddispersed in aqueous media to form colloidal sus-pensions of chitin nanowhiskers. The utilization ofchitin nanowhiskers contributes to a healthy eco-system, chitin being a renewable resource. Chitinnanowhiskers have drawn attention in various appli-cations due to their properties like nanosized di-mensions, high surface area, high absorptivity, bio-degradability, nontoxicity, renewability, low densityand easy modification. The intrinsic rigidity of chitinnanowhiskers, special rod-like and spindle-likemorphology, strong interfacial interactions, and thepercolation network organized by nanowhiskerscontribute to optimized mechanical performance,thermal properties, solvent absorption, and barrierproperties. One key advantage is their high surfacearea that enables chitin nanowhiskers to interacteffectively with cells, factors, proteins and othercompounds. Chitin nanowhiskers possess a reac-tive surface covered with hydroxyl groups, giving theopportunity of chemical modification. Realization offunctional modifications of chitin nanowhiskers un-der mild reactive conditions would greatly improvetheir practical utility for the future.

Chitin nanowhiskers can be used as environmen-tally friendly particulate biofillers being compoundedwith many different kinds of polymer matrices. Thereinforcing effect strongly depends on the aspectratio of the chitin whisker, and therefore on its ori-gin, as well as on the processing technique of thecomposite. Chitin nanowhiskers can form viablematerials for biomedical areas, such as scaffoldingand immobilizing biomolecules in the process ofproducing novel biosensors by using waste andcheap materials obtained from other industries. Theyalso have applications in the cosmetics industry,


mainly for the ordered regeneration of wound tis-sues and as dermal fillers.

ACKNOWLEDGEMENTS

The author Mincea M. acknowledges the financialsupport from strategic grant POSDRU/89/1.5/S/63663, Project “Transnational network of integratedmanagement for postdoctoral research in the fieldof Science Communication. Institutionalconstruction (post-doctoral school) and fellowshipprogra] (Co]]Scie ” financed under the SectoralOperational Programme Human ResourcesDevelopment 2007-2013.

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