Journal of Colloid and Interface Science 379 (2012) 130–140

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Journal of Colloid and Interface Science

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Formation and characterization of natural polysaccharide hollow nanocapsulesvia template layer-by-layer self-assembly

Liu Yuxi a, Yang Jing a, Zhao Ziqi a, Li Junjie a,b, Zhang Rui a, Yao Fanglian a,⇑a School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, Chinab Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences,Beijing 100850, China

a r t i c l e i n f o

Article history:Received 29 January 2012Accepted 20 April 2012Available online 1 May 2012

Keywords:CarrageenanChitosanNanocapsulesLayer-by-layer self-assemblypH sensitive

0021-9797/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jcis.2012.04.058

⇑ Corresponding author. Fax: +86 22 27403389.E-mail address: yaofanglian@tju.edu.cn (F. Yao).

a b s t r a c t

With natural polysaccharides carrageenan (Car) and chitosan (Cs) as the polyanion and polycation,respectively, multilayer hollow nanocapsules have been fabricated via sequential layer-by-layer (LbL)electrostatic self-assembly from the sacrificed templates nanospheres (SiO2–NH2). The LbL assembly pro-cess with the polysaccharides on SiO2–NH2 core was followed by f-potential and size analysis. The fab-rication of (Car/Cs)x nanocapsules and the removing of the SiO2–NH2 core templates were confirmed byTGA and EDS analysis. The morphology of SiO2(Car/Cs)x nanospheres and (Car/Cs)x nanocapsules wereobserved by TEM analysis. The size analysis of (Car/Cs)x nanocapsules indicated that the cyst wall thick-ness and cavity volume of the nanocapsules are pH and ionic strength dual responsive. Due to the bio-compatibility of the natural polysaccharides carrageenan and chitosan and the responsiveness ofnanocapsules to pH and ionic strength, the (Car/Cs)x multilayer nanocapsules are expected to be usedas nanoreactors or nanocontainers to control the synthesis, encapsulation, and releasing behaviors of bio-active molecules.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

From the whole organism to a tiny cell, each compartment inhuman body can be considered as a reactor or a container.Synthetic hollow capsules, similar to a tiny cell, permit the studyof the influence of spatial confinement on chemical reactions,provide an opportunity to perform chemical reactions in ‘‘exotic’’environments, and have attracted much attention recently [1–5].An important feature of mirco- and nanocapsules is spatial controlof individual reaction steps in various complex reaction cascadesand its potential application in biomedical science.

Layer-by-layer (LbL) microcapsule is one type of such reactors orcontainers which composed of thin polymer shells that are assem-bled from multiple polyelectrolyte layers. Hollow microcapsuleshave been used as size-constrained reaction spaces to synthesizepolymers and inorganic materials [6]. The frequently-used rawmaterials as shell of LBL microcapsules are synthetic polymers suchas polystyrene sulfonate (PSS) and polyallylamine hydrochloride(PAH) [7,8]. The low biocompatibility of these synthetic polymermicrocapsules is the main barrier for their applications in biomed-ical field [9]. However, the introducing of natural polysaccharides inLbL technique represents an attractive alternative for biomedical

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applications because of their favorable biocompatibility and biode-gradability [10]. Another challenge for the microcapsules builtthrough layer-by-layer technology is their size limitation. Althoughthe preparation process of layer-by-layer approach is quite simple,it seems that this method provides a technical limitation to the sizeof the capsules (diameter between 5 and 10 lm) [1]. The most oftenused templates in LBL self-assembly are inorganic crystal (calciumcarbonate, 5 lm) [11,12], polystyrene (PS, 1 lm) [13,14] includingcopolymer with the second component, melamine–formaldehyde(MF, 5 lm) [15,16] resin spheres, and biological units (erythrocyte,5 lm; enzyme crystal, 20 lm) [17–19]. The hollow capsules pre-pared using these templates were all in the scale of micrometer.This may explain their relatively limited use as true nanoreactorsand even as vessels for delivery purposes, as nanoscale dimensionsare highly desirable in both cases.

Some nature polysaccharides, such as carrageenan and chitosan,have been widely studied for applications such as drug deliverycarriers [20–22], hydrogel [23] and porous tissue engineering scaf-folds [24–26]. Carrageenan, which can form hydrophilic colloid, isextracted from red algae and seaweed. It is a high weight anionicpolysaccharide made up of repeating galactose units and 3,6 anhy-drogalactose (3,6-AG), both sulfated and nonsulfated. The units arejoined by alternating alpha 1–3 and beta 1–4 glycosidic linkages[27,28]. Chitosan, which is the only natural alkaline polysaccha-ride, is a linear cationic polyelectrolyte composed of randomlydistributed b-(1–4)-linked D-glucosamine (deacetylated unit) and

Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140 131

N-acetyl-D-glucosamine (acetylated unit) obtained by the deacety-lation of chitin derived from shellfish [29]. Both carrageenan andchitosan are similar to Glycosaminoglycans (GAGs) in structurethat is found largely in extracellular matrix (ECM) [30]. From thebiomimetic perspective, the nanoreactors or nanocontainers withcarrageenan and chitosan as the cyst wall will have perspectiveapplications in biomedicine, that is, for the synthesis or controlledrelease of bioactive molecule.

The silica spheres (SiO2) with nanosize could be employed assacrificed templates to obtain the tailoring cavity with a requiredvolume in nanoscale. Compared with those synthetic polymer col-loids and inorganic crystal [31,32], SiO2 cores have significantadvantage in many aspects. SiO2 sacrificial cores could be synthe-sized by the hydrolysis and condensation of tetraethoxysilane(TEOS) [33]. Moreover, the well-defined size in nanoscale can berealized by adjusting the concentration of reactants and catalyst.Furthermore, the SiO2 cores are easily decomposed and get out ofthe capsules interior by osmotic pressure. In addition, amount ofhydroxyl groups on the surface of the spheres provide abundantreaction sites for further modification.

In this paper, a facile method to obtain hollow nanocapsules isproposed. It avoids the use of organic solvent, and all the procedureis in water system. SiO2 nanospheres were used as sacrificial tem-plates for the fabrication of hollow polyelectrolyte capsules withthe size in the scale of nanometer. Iota-carrageen (Car) and chito-san (Cs) were selected as negatively and positively charged poly-electrolytes, respectively, for LbL assembly.

Our strategy here is assuming that the sulfate groups on carra-geenan associate with the protonated amino on chitosan throughelectrostatic interactions, resulting in the formation of the poly-electrolyte complex via the LbL assembly technique. The raw mate-rials of the cyst wall are all natural polysaccharides that havebetter biocompatibility and biodegradability in contrast to the nor-mally used synthetic polymeric materials. The resultant polysac-charide polyelectrolyte nanocapsules are nearly monodispersed,and the cyst wall thickness and cavity volume are tunable. ThepH responsibility of the hollow nanocapsules, polyelectrolyte/anti-polyelectrolyte behaviors, and swelling/deswelling perfor-mance collectively could be affected by the structure and propertyof the nanocapsule cyst wall. The combination strength between

Scheme 1. Schematic representation of formation process o

layers and the permeability of the cyst wall of the polysaccharidenanocapsules can be adjusted through the change of microenviron-ment. Based on the excellent biocompatibility, convenience to con-trol the capsule size in the scale of nanometer and diversity toadjust the structure and properties of the cyst wall, these chito-san–carrageenan nanocapsules might be promising candidates asnanoreactors or nanocontainers to control the synthesis, encapsu-lation, and releasing behaviors of bioactive compounds in the areaof biomedicine.

2. Materials and methods

2.1. Materials

Chitosan (Cs) with a mol. wt. of 80 kDa was purchased fromHaihui Biochemical Co. Ltd. (Qingdao, China), and the degree ofdeacetylation is 85%. Iota-carrageenan (Car, 200 kDa) was obtainedfrom TCI (Japan). Tetraethoxysilane (TEOS) was purchased fromJiangtian Chemical Co. Ltd. (Tianjin, China). 3-Aminopropyltri-eth-oxysilane (APTES, 98%) was obtained from Aladdin Chemical Co.Ltd. (Shanghai, China). All other reagents were of analytical gradewithout further purification.

2.2. Preparation of SiO2–OH nanospheres

Silica nanospheres with hydroxyl groups on their surface (SiO2–OH) were prepared using the modified Stober method as previousreports [33]. In brief, 4 ml of tetraethoxysilane (TEOS) was added toa mixture of 4 ml ammonium hydroxide and 100 ml of ethanol un-der ultrasonic dispersion. The reaction was carried out for 2 h andthen aged over night. The resultant SiO2–OH nanosphere disper-sion solution was uniform blue and kept stable without aggrega-tion for more than 2 weeks [34].

2.3. Amino-modification of SiO2–OH nanospheres

The surface modification of the SiO2–OH nanospheres was doneby quickly adding 0.1 ml of 3-aminopropyltri-ethoxysilane (APTES)to 108 ml ethanol dispersion solution of silica nanospheres with

f (Car/Cs)x hollow nanocapsules via LbL self-assembly.

Table 1Preparation condition and characteristics of SiO2–OH nanospheres.

Sample TEOS(mL)

Aqueousammonia (mL)

Ethanol(mL)

Time(h)

Ave-size(nm)

1 4 7 100 2 121.20 ± 1.702 4 4 100 2 114.60 ± 1.303 1 4 60 2 83.93 ± 1.104 1 4 80 2 48.80 ± 1.005 1 4 100 2 31.75 ± 0.80

Fig. 2. f-Potential of SiO2–NH2 nanospheres obtained with different mole ratio ofAPTES/TEOS.

Fig. 3. f-Potential of SiO2–NH2 nanospheres at different pH medium (the SiO2–NH2

nanospheres were fabricated with the mole ratio of APTES/TEOS of 0.0238).

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vigorous stirring for more than 12 h. The resulting amino-modifiedsilica (SiO2–NH2) nanospheres were purified by centrifugation andredispersion in ethanol for three times. Wet solid SiO2–NH2 nano-spheres were obtained finally after centrifugation (8000 rpm,18 min).

2.4. Preparation of SiO2(Car/Cs)x nanospheres through layer-by-layerself-assembly

A dispersion solution of SiO2–NH2 nanospheres in 1% (v/v) ace-tic acid was dropwise added in iota-carrageen solution (3 mg/ml in1% (v/v) acetic acid solution, pH 2.7) with magnetic stirring. Stir-ring was continued for another 15 min to ensure the conjugationsufficiently. The mixture was centrifuged at 12,000 rpm for15 min and washed using 1% (v/v) acetic acid solution. There mustbe at least three centrifugation–washing cycles so that the extrapolyelectrolyte could be separated entirely. The Car/Cs bilayerbuildup was carried out by dropping the nanosphere dispersionsolution into chitosan solution (2 mg/ml in 1% (v/v) acetic acidsolution, pH 2.7) as described above. Repeating this procedureformed Car/Cs multilayer coating silica core.

2.5. Preparation of (Car/Cs)x hollow nanocapsules

To obtain (Car/Cs)x hollow nanocapsules, the silica cores ofSiO2(Car/Cs)x nanospheres were dissolved by treatment withhydrofluoric acid (HF) solution at room temperature for 15 min,followed by centrifugation–washing cycles (20,000 rpm, 10 min).

2.6. Characterization of nanospheres and nanocapsules

The size of nanospheres and nanocapsules was measured byNanoSizer Measurement (Malven, UK) based on dynamic laser lightscattering principle (DLS) at 25 �C with a detection angle of 173�,and f-potential was investigated by the same instrument. The C,H, and N elemental analysis (Elementar Ger.) was used to deter-mine the chemical composition of SiO2–NH2 nanospheres. The

Fig. 1. TEM images of nanosphere templates. (a) SiO2–

morphologies and approximate sizes of the nanospheres and nano-capsules were observed with a transmission electron microscope(JEM100CXII). The composition of the nanospheres and nanocap-sules was identified by EDS analysis (an accessory of JEOL 2100F).The thermal gravimetric analysis (TGA) (ZRP-2P, Shanghai CHN)

OH nanospheres and (b) SiO2–NH2 nanospheres.

Scheme 2. Two pathways for the first deposition of carrageen on SiO2–NHþ3 nanosphere template. Path A, carrageen was added dropwise into SiO2–NHþ3 nanospheredispersion solution; Path B, SiO2–NHþ3 nanosphere was added dropwise into carrageen solution.

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was carried out from room temperature to 600 �C under air atmo-sphere to confirm the relative amount of the organic matter ofSiO2(Car/Cs)x nanospheres. The heating rate was 10 �C/min.

3. Results and discussion

SiO2 nanospheres were used as sacrificial templates for the fab-rication of (hollow) polysaccharide nanocapsules. Iota-carrageen(Car) and chitosan (Cs) were selected as negatively and positivelycharged polysaccharide, respectively, for LbL assembly. Scheme 1shows the formation procedure of (Car/Cs)x nanocapsule. In thefirst step, TEOS and aqueous ammonia are mixed in the presenceof ethanol, leading to the formation of silica nanospheres with alarge amount of hydroxyl groups on their surface (SiO2–OH). Inthe second step, AETPS was conjugated onto the SiO2–OH nano-sphere’s surface forming amino-modified silica nanospheres(SiO2–NHþ3 ) to change the nanosphere surface to net positivecharge. Thirdly, Car/Cs bilayers were deposited on the SiO2–NHþ3nanospheres via LbL self-assembly technology, and, finally, theSiO2 nanocores were dissolved by treatment with HF solutionand (Car/Cs)x hollow nanocapsules were obtained.

3.1. Fabrication of SiO2–OH nanospheres

SiO2–OH nanospheres have been a choice of the template forthe preparation of many polyelectrolyte nanocapsules systemsdue to the nanoscale size, facile reactive conditions, and easilyremovable [35]. In the present research, SiO2–OH sacrificial coreswere synthesized by the hydrolysis and condensation of TEOSmodifying the methods described by Huang [36]. Controlling theamount of aqueous ammonia was crucial to obtain predominantlySiO2–OH spherical cores with ideal size. Table 1 shows the prepa-ration condition on the size of the SiO2–OH nanospheres. The

aqueous ammonia, used as the catalyst arising the formation ofspherical particles, apparently influenced the morphology andspheres size, as shown in Table 1, and the diameter of SiO2–OHnanospheres increased with enhancing aqueous ammonia content.Oppositely, the average size of SiO2–OH nanospheres decreasedwith a growing amount of ethanol due to the drop of the reactantconcentration.

3.2. Amino-modified silica (SiO2–NH2) nanospheres and theirdispersion stability

To ensure the following electrostatic self-assembly of SiO2–OHnanospheres with hydrosoluble polysaccharides in aqueous solu-tion, there must be abundant net positive or negative charges onthe surface of SiO2–OH nanospheres cores. Due to the largeamounts of hydroxyl groups on the surface, it is convenient forthe SiO2–OH nanospheres to be modified or functionalized further.Amino groups, which were easily protonated in acidic solution,were introduced on the surface of SiO2–OH nanospheres via the si-lane coupling agent 3-aminopropyltri-ethoxysilane (APTES). Whenthe feed ratio of APTES to TEOS was 0.0238 (mol/mol), the masspercentage of N atoms in the whole SiO2–NH2 nanosphers was0.30 wt.% (obtained from elemental analysis). That means APTESwere successfully grafted onto the surface of silica nanosphereand accounting for 4.38 wt.% of the whole SiO2–NH2 nanospheres.The SiO2–NH2 nanospheres could be dispersed in 1% (v/v) aceticacid solution due to the electrostatic repulsion between positivecharges of the protonated amino on the nanosphere surface, andthe f-potential was more than 40 mV (Fig. 2). The dispersion solu-tions of SiO2–NHþ3 nanospheres could keep stable for more thantwo weeks without any deposition.

The TEM images of the SiO2–OH and SiO2–NH2 nanospherestemplates are given in Fig. 1. The nanospheres are spherical,

Fig. 4. The average hydrodynamic diameter and PdI of SiO2(Car/Cs)x nanospheres with layer number.

Scheme 3. Schematic illustration of the size decrease from SiO2(Car/Cs)x to SiO2(Car/Cs)xCar nanosphere.

134 Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140

smooth, and nearly monodisperse. The average diameter of nano-spheres is ca. 100 nm, which is a little smaller than that was mea-sured in liquid state by DLS (ca. 120 nm similar to SiO2–OH). Thisphenomenon may be caused by the evaporation of the bondingwater during TEM sample preparing.

As is shown in Scheme 1, APTES conjugated with SiO2–OHnanospheres through condensation between alkoxy of APTES andhydroxyl on the surface of silica nanospheres. Since one APTESmolecule has three ethoxyl groups, it is possible for each APTESmolecule to react with more than one hydroxyl groups from two

or three different SiO2–OH nanospheres, which might cause cross-linking among silica nanospheres. To avoid this crosslinking whilemaximize the amino-modification of the SiO2–OH nanospheres,the feed ratio of APTES to SiO2–OH nanospheres is the key factor[37]. Fig. 2 shows the relationship between f-potential of theSiO2–NH2 nanospheres with the feeding amount of APTES. f-Poten-tial of the SiO2–NH2 nanospheres quickly increased with theenhancing of amount of APTES at first, which indicated that moreamino groups were conjugated on the silica nanospheres. But thef-potential of SiO2–NH2 nanospheres leveled off when the feed

Fig. 5. f-Potential of SiO2(Car/Cs)x nanospheres with layer number.

Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140 135

ratio of APTES to TEOS was higher than 0.0238 mol/mol, which isthe optimum feed ratio of APTES/TEOS on the conjugating densityof APTES on SiO2–OH nanospheres.

In order to determine the most suitable pH value for the follow-ing electrostatic assembly of SiO2–NH2 nanospheres with carra-geenan and chitosan, the effect of pH value of the dispersionsolution on the f-potential of SiO2–NH2 nanosphere was investi-gated. The pH value (pH = 1.43, 2.40, 3.41, 4.67) was adjusted byHCl solution, and the nanospheres were dispersed by ultrasound.As shown in Fig. 3, when pH declined from 4.67 to 3.41, the f-po-tential increased from 38.0 mV to 51.5 mV. After that, the f-poten-tial decreased from 51.5 mV to 29.0 mV when pH value decreasedfurther from 3.41 to 1.43. At pH 4.67, amino groups can exist as—NHþ3 or –NH2 simultaneously. But all –NH2 would transform into—NHþ3 when pH value dropped to 3.41, which resulting in a risingof f-potential. After all –NH2 changed into —NHþ3 (pH < 3.41), theincreased acidity did no contribution to f-potential any more.Oppositely, the f-potential would decline due to the increase inionic strength of the dispersion solution in the process of adjustingpH values (more HCl was introduced).

3.3. Self-assembly of polysaccharide multilayer onto SiO2–NHþ3nanospheres

The layer-by-layer assembly technology is based on the electro-static interactions between various polyelectrolytes with oppositecharges. In the condition of this research (pH 2.7), the SiO2–NHþ3nanospheres templates have net positive charges. So the initialstep of polysaccharide multilayer construction was completedwith the negative charged carrageenan solution. As a result, theoriginal positively charged SiO2–NHþ3 nanospheres core surface be-came negative charge. This first assembly step was carried out intwo different path ways (Scheme 2). In path A, carrageenan solu-tion was dropwise added into the SiO2–NHþ3 nanospheres disper-sion solution at pH 2.7 under 70 �C. The initial light blue SiO2–NHþ3 nanospheres dispersion solution became white immediatelyafter carrageenan was added, which indicated the aggregation ofthe dispersed SiO2–NHþ3 nanospheres. The average diameter ofthe SiO2–Car aggregations was more than 350 nm. At the begin-ning of this way, deficient carrageenan molecules met the massiveoverdose of SiO2–NHþ3 nanospheres at the moment of carrageenansolution added. There is a great possibility for –OSO�3 groups of thelong carrageenan chain to couple with two or more SiO2–NHþ3 nan-ospheres. As a result, several SiO2–NHþ3 nanosphere cores aggre-gated via electrostatic adhesion with one carrageenan chainamong them. Apparently, path A is not a proper method to achievethe LbL procedure. To avoid the aggregation or precipitation duringthe first assembly procedure, path B with the opposite approach ofpath A was investigated. As shown in Scheme 2, the SiO2–NHþ3 nan-ospheres dispersion solution was dropped into carrageenan solu-tion with gentle magnetic stir. The original colorless carrageenansolution gradually turned light blue during the whole coursewithout any sedimentation. In this procedure, when the SiO2–NHþ3 nanosphere dispersion solution was added to carrageenansolution, the nanospheres were independent, and each of themwould be coated by carrageenan chains immediately. As a result,the outside layer of the nanospheres was covered with carrageenanwith negative charge, which could inhibit the aggregation amongthe nanospheres through the electrostatic repulsive forces, andthe net negative charges of extra carrageenan could help to remainthe system uniform. Compared with the resultant sphere size inPath A (>350 nm), the diameter of SiO2–Car nanosphere was muchsmaller in Path B (�117.9 nm), and the size distribution wasnarrower.

After a sufficient incubation of SiO2–NHþ3 nanospheres incarrageenan solution to achieve complete surface coverage, the

nanospheres were centrifuged and washed with water to removethe free carrageenan. These washings after every deposition stepwere crucial for minimizing artifacts (polyelectrolyte agglomeratesformed in solution rather than on the core template). Charge over-compensation results in the adsorption of oppositely chargedchitosan on SiO2–Car nanosphers. In the next step, the depositionof the positively charged layer was completed in chitosan aceticacid solution.

The carrageenan layer and the near outside chitosan layer wasdefined as a bilayer. As is shown in Fig. 4, the average diameterof SiO2(Car/Cs)x increased with bilayer number x almost linearly.The size of the SiO2(Car/Cs)x nanospheres presented a noticeableincreasing from 120 nm to 210 nm with the adsorption of elevenlayers. In addition, an interesting phenomenon could also beobserved from Fig. 4, and for each cycle from SiO2(Car/Cs)x toSiO2(Car/Cs)xCar, the following depositing of carrageenan will re-sult in the decreasing of the nanosphere size. Both chitosan andcarrageenan are macromolecules with polymer rigidity and needmore charges to be condensed each other. The deacetylation de-gree of chitosan here used is 85%. That means there are 0.85 posi-tive NHþ3 groups on each hexopyranosyl ring of chitosan. However,each hexopyranosyl ring of carrageenan only possesses less than0.4 negative –OSO�3 because the sulfonic acid esterification degreeof carrageenan is <40%. So, when the outside layer is chitosan,there will be much higher superfluous net positive charges. The ex-cess net charges make the chitosan molecular chains more exten-sion and connect loosely each other. The situation is just theopposite if the end layer is carrageenan. As shown in Scheme 3,when the carrageenan was added into SiO2(Car/Cs)x nanaospheredispersion solution, two main approaches were provided for it tojoin the SiO2(Car/Cs)x nanospheres. Some of the carrageenan mol-ecules inserted into the gaps of the loosing chitosan chains andcondense the former slack chitosan layer considerably throughthe interactions of positive and negative charge between the poly-saccharides. The other part of carrageenan chains were adsorbedon the outside surface of SiO2(Car/Cs)x nanospheres. Comparedwith the inner strong electrostatic repulsive force and loose chito-san layer, the carrageenan has a relatively lower density of nega-tive charges that diminish the repulsive forces amongcarrageenan chains. Moreover, the strong hydrogen bond betweenhydroxy of unesterified hexopyranosyl ring tightened the carra-geenan closely. To sum up, due to the combination of the electro-static condensation, low charge density and hydrogen bond ofcarrageenan, the diameter of SiO2(Car/Cs)x would decrease afterthe next deposition of carrageenan (forming SiO2(Car/Cs)xCar).

The Polydispersity Index (PdI) of SiO2(Car/Cs)x nanospheres wasalmost less than 0.1 (Fig. 4), which revealed the dispersion solution

Fig. 6. TEM images of SiO2(Car/Cs)5Car nanospheres.

Fig. 7. Tracking of the average hydrodynamic diameter of SiO2(Car/Cs)5Car nano-spheres being exposed to HF solution.

136 Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140

was absolutely monodisperse. It demonstrates that no sedimenta-tion or aggregation yielded during the assembly process.

The stepwise adsorption of carrageenan and chitosan was alsofollowed by inspecting the surface charge of SiO2(Car/Cs)x nano-spheres in the dispersion solution upon the addition of each poly-saccharide layer. The f potential of SiO2(Car/Cs)x nanosphereschanged from a negative to positive alternatively, indicating thesuccessful coating with polysaccharides on each layer deposition.Besides that, the f potential gives considerable information onthe stability of the nanosphere suspensions. Particles with f-poten-tial values higher than 30 mV or lower than �30 mV are generallyconsidered to be stable because of the strong repulsion forces thatprevent the occurrence of aggregation phenomena among the par-ticles. Fig. 5 showed the f-potential values of SiO2(Car/Cs)x nano-spheres versus the number of layers adsorbed, and one can findthat the f-potential of SiO2(Car/Cs)x nanospheres was more than50 mV or less than �30 mV when the outside layer was chitosanor carrageenan, respectively. All the absolute value of f-potentialis large enough to demonstrate that the nanospheres in the sus-pensions could be generally considered to be stable owing tostrong electrostatic repulsion forces among nanospheres. The f-po-tential values, together with the size and the PdI measurements,confirm the formation of stable SiO2(Car/Cs)x nanospheres. Thissystem can be stable for more than 2 months without aggregation.

The TEM images of the resultant SiO2(Car/Cs)5Car nanosphereswere shown in Fig. 6. After coated by carrageenan and chitosan,the surface of SiO2(Car/Cs)x nanospheres become obviously roughand irregular (Fig. 6b) compare with SiO2–NH2 nanospheres

(Fig. 1b). The diameter of the SiO2(Car/Cs)5Car nanospheres isabout 150 nm (Fig. 6a), which is larger than that of the SiO2–NH2

nanospheres core. The increase in diameter is contributed to acombination of Car/Cs cyst wall on the SiO2–NH2 nanospheres bythe electrostatic interactions. The results suggested that thesequential Car/Cs cyst wall were formed on the silica nanospherescores. The size of SiO2(Car/Cs)x nanospheres in the dry conditionobserved by TEM is a smaller than the size obtained by the lasersize instrument in liquid state. As can be seen (Fig. 6b), the poly-saccharide cyst wall is bonded with core tightly in spite of theshrinking under drying before TEM measurement. Drying can alsolead to a little adhesion between the polysaccharide cyst walls ofthe nanospheres.

3.4. Formation of (Car/Cs)x hollow nanocapsules

To increase the mechanical strength of the (Car/Cs)x cyst walland avoid collapsing of the hollow nanocapsules, 0.25 wt.% glutar-aldehyde solution was used to crosslink the chitosan layers for 5 h.SiO2(Car/Cs)5Car nanospheres with the end layer of carrageenanwere used to prevent the occurrence of the crosslinking and aggre-gation between the nanospheres. Because the crosslinked chitosanchains tightened the cyst wall, the diameter of the SiO2(Car/Cs)5Carnanospheres after being crosslinked (348.5 nm) revealed a smalldrop in comparison with that without crosslinking (354.6 nm).

HF solution was utilized to remove the silica cores to obtain thehollow nanocapsules. The dissolution process of the silica cores oc-curs via the following reaction:

SiO2 þ 4HF! SiF4 þ 2H2O ð1Þ

The process from SiO2(Car/Cs)5Car nanospheres to (Car/Cs)5Carhollow nanocapsules was dynamically recorded by the diameterchange of the nanospheres along etching time (Fig. 7). Once HFsolution was added into the SiO2(Car/Cs)5Car nanospheres disper-sion solution, the size of nanospheres obviously decreased due tothe dissolution of SiO2 cores. The disorganization of the inner sup-porting cores leads to a shrink of the cyst wall; hence, the diameterundulated in a small range during the continue etching procedure.After the core dissolved completely, the cyst wall of nanocapsuleswelled again to achieve the equilibrium state with the increasingof the hollow nanocapsule size. This post swelling behaviorsshould be resulting from the osmotic pressure of Si4+ in and out-side of the capsules.

Fig. 8 shows the typical TEM photographs of the polysaccharidenanocapsules of (Car/Cs)5Car. The cores dissolution efficiency withlower concentration HF solution (Fig. 8a and c) showed apparentdifference compared with those with HF of higher concentration(Fig. 8b and d). For lower HF concentration, the cores were etched

Fig. 8. TEM images of (Car/Cs)5Car hollow nanocapsules. (a) and (b) Without crosslinking with glutaraldehyde; (c) and (d) were crosslinked; HF concentration used to removethe SiO2 core was 0.141 M for (a) and (c), 0.565 M for (b) and (d); HF etching time was 5 min for (a) and (b), 30 min for (c) and (d).

Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140 137

incompletely, and the fragments cannot be diffused out of thenanocapsules (Fig. 8a and c). Before the SiO2 template, cores wereremoved completely, many small black particles could be observedin and outside of the nanocapsules (Fig. 8c). It suggested that theSiO2 cores were disintegrated into many small fragments firstlyand then diffused out of the cyst wall along with the etching con-tinually. However, the undissolved cores can be barely observed inthe (Car/Cs)5Car nanocapsules dealt with higher HF concentration(Fig. 8b and d), because SiO2 cores reacted with HF completelyaccording reaction formula (1).

After removing the SiO2 core template, the polysaccharidenanocapsules have a hollow cavity and thin polysaccharide wall.The dried nanocapsules without being crosslinked are nonspheri-cal in shape and present a number of creases with a rough surface(Fig. 8b). During the gradual drying process in air for the TEM sam-ple preparation, the polysaccharide nanocapsules will collapse andthe polysaccharide cyst wall will shrink and fold in some degree. Itis found out that the shape of dried nanocapsules after crosslinkingwill be less distorted from the spherical template (Fig. 8d). In fact,those nanocapsules, which have been crosslinked by glutaralde-hyde, are still spherical in shape and slight conglutination was oc-curred (Fig. 8d). Although the dried thin-shelled nanocapsules are alittle distorted in shape from the spherical core template, theiraverage size of (Car/Cs)5Car nanocapsules about 150 nm is closeto the size of the original SiO2(Car/Cs)5Car nanospheres.

EDS analysis was carried out to examine the content variation ofSi, C, and O element to clarify the fact that silica was removed to-tally from SiO2(Car/Cs)5Car nanospheres. Fig. 9 shows EDS spectraof the SiO2(Car/Cs)5Car nanospheres (a) and (Car/Cs)5Car nanocap-sules (b). In SiO2(Car/Cs)5Car nanospheres, Si and O are the mainelements contributed by SiO2. After being incubated in HF solution,the amount of Si and O decreases, but C from polyelectrolytes in-creases sharply. Actually, the content of Si in (Car/Cs)5Car nanocap-sules nearly arrived at zero. Therefore, it is revealed that the cores

of SiO2(Car/Cs)5Car have been decomposed and removed com-pletely, and only the chitosan and carrageenan build up the hollowcapsules (the peak of Cu derives from the Cu grid).

Fig. 10 shows the TGA curves of SiO2–NH2, SiO2(Car/Cs)5Carnanospheres and (Car/Cs)5Car hollow nanocapsules. For the SiO2–NH2 nanospheres (Fig. 10a), a weight loss of near 10% was occurredwhen the sample was heated from room temperature to 250 �C.This was due to the loss of adsorbed water and the condensationreaction of the hydroxyl groups on the sphere surface. Comparedwith the SiO2–NH2 nanospheres, there was an apparent weight loss(Fig. 10b) for SiO2(Car/Cs)5Car nanospheres after 250 �C as the re-sult of the decomposition of the polysaccharide. The 28% weightloss of SiO2(Car/Cs)5Car nanospheres reveals that the organic com-position accounts for 18% weight of the polyelectrolyte multilayernanospheres. Moreover, for (Car/Cs)5Car hollow nanocapsules, al-most all the sample was decomposed at the temperature beyond550 �C, and at that temperature, all the polysaccharide should bedecomposed. In other words, there is hardly any silica residual inthe nanocapsules after treating with HF.

3.5. Ionic responsivity of (Car/Cs)x hollow nanocapsules

The effect of ionic strength of the dispersion medium on the sizeand f-potential of the (Car/Cs)5Car hollow nanocapsules weredetermined, and the results were presented in Fig. 11. The ionicstrength was adjusted by the addition of NaCl solution at varyingconcentrations from 0.01 to 0.20 mol/L. The average hydrodynamicdiameters of the hollow nanocapsules increased from 346 to1295 nm with the increase in the salt concentration from 0.01 to0.20 mol/L. It was found that the increase in the size with theincrease in the salt concentration was almost linear. But at a highersalt concentration, the slope increased, which means that thehigher the ionic strength was, the faster the hydrodynamic diame-ters increased. In salt-free solution, the electrostatic attractions

Fig. 9. EDS spectra of the SiO2(Car/Cs)5Car nanospheres (a) and (Car/Cs)5Car nanocapsules (b).

Fig. 10. TGA curves of SiO2–NH2 nanosphere core, SiO2(Car/Cs)5Car nanospheresand (Car/Cs)5Car hollow nanocapsules.

138 Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140

between chitosan and carrageenan molecular chains lead to a con-traction of the chains and a decrease in hydrodynamic size. On thecontrary, association among the polysaccharide molecular chainsare shielded and destroyed by salt ions at higher concentration.As a result, molecular conformation becomes outstretched so thatthe diameter rises with an increase in salt concentration. On theother hand, the faster increase in the diameter with the higherionic strength might be caused by the somewhat of aggregation

of polysaccharide nanocapsules, and this aggregation wasirreversible.

With regard to the f-potential, the variation tendency withincreasing salt concentration was shown in Fig. 11b. At low con-centration of NaCl, the added Cl� would take the place of –OSO�3of carrageenan and combine with –NHþ3 of the chitosan. The re-lease and increase of the free –OSO�3 lead to the f-potential drop.On the contrary, at high concentration of NaCl, besides the disag-gregating of the bonded chains, part of chitosan molecular chainswould like to extend and expose to the outside layer of the capsulewall that leads to a rise of f-potential value. In fact, the percentageof free –OSO�3 decreased via binding with positive ions.

3.6. Controlling of the interlayer structures of (Car/Cs)x nanocapsules

Considering the chemical structures of polysaccharides buildingthe hollow nanocapsules (Car/Cs)x, both —NHþ3 of chitosan and—OSO�3 of carrageenan play key roles in the formation of the nano-capsules. As well known, –NH2 is a weak base, and –SO3H is astrong acid in an opposite position. It can be expected that the per-formance of (Car/Cs)x nanocapsules will be responsive to thechange of the dispersion media pH. In order to investigate the ef-fect of pH values on the multilayer polysaccharide hollow nano-capsules, (Car/Cs)5Car nanocapsules were dispersed in acidicsolution and auto-titrated from pH = 2 to pH = 12. NaOH solutionwas involved into adjust pH value to reduce the influence of ionicstrength on the diameter sizes (Fig. 12). With the increase in pHvalue of the dispersion media from 2 to 12, the diameter of the(Car/Cs)5Car nanocapsules enhanced from 344 to 757 nm. This

Fig. 11. Average hydrodynamic diameter (a) and f-potential (b) of SiO2(Car/Cs)5Car nanospheres and (Car/Cs)5Car hollow nanocapsules (non-crosslinking) dispersed insolutions with various ionic strength.

Fig. 12. pH dependence of average hydrodynamic diameter and f-potential of (Car/Cs)5Car hollow nanocapsules (non-crosslinking).

Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140 139

phenomenon can be explained by the following reasons. With theincrease in pH, more —NHþ3 transformed into –NH2. At the interfaceof chitosan and carrageenan in the nanocapsules, the electrostaticinteractions between —NHþ3 and —OSO�3 groups reduced or evendisappeared. In the layer inside of chitosan, the hydrogen bond be-tween NH2 and OH groups will lessen the gap among chitosanchains. In the carrageenan layer, however, the electrostatic repul-sion between contiguous carrageenan chains will enhance becauseof the releasing or increasing of free —OSO�3 groups. So, there aretwo opposite factors dealing with the cyst wall changes along withpH. One is the strong hydrogen bond between chitosan molecularchains that will make the chitosan layer more condense. The con-trary one is the dropping of electrostatic attraction (between—NHþ3 groups of and —OSO�3 ) and a building up of electrostaticrepulsion between —OSO�3 groups, which would bring to a muchmore loose connection between chitosan and carrageenan or carra-geenan chains themselves. The overall result of an increase innanocapsule size with pH was shown in Fig. 12, which suggestedthat the interspace between chitosan and carrageenan will be morebroaden (from 18 nm to 53 nm corresponding pH from 2 to 12).

This controllable interlayer structure provides a great opportunityfor (Car/Cs)x nanocapsules to be used as a nanoreactors or nano-containers in the area of biomedicine.

4. Conclusion

Natural polysaccharide multilayer hollow nanocapsules, com-prising chitosan and carrageenan, were successfully prepared vialayer-by-layer technique. Smooth SiO2–OH nanospheres with anaverage size of 100 nm were obtained through the reaction of TEOSand aqueous ammonia in the presence of ethanol. Followed byamino-modification with APTES at the optimum feed ratio ofAPTES/TEOS (0.00238 mol/mol), after that, five bilayers of chitosanand carrageenan were deposited on the SiO2–NHþ3 cores. Exposingthe SiO2(Car/Cs)5Car nanospheres to glutaraldehyde and HF solu-tion successively, hollow nanocapsules with a size of 150 nm wereobtained, which was characterized by TEM. The (Car/Cs)x nanocap-sules were found to have a dual responding with pH and ionicstrength. These properties would bring a bright perspective for

140 Y. Liu et al. / Journal of Colloid and Interface Science 379 (2012) 130–140

the application of this nanocapsule system in the field of biotech-nology and biochemical engineering.

Acknowledgments

This work is supported by National Nature Science Foundationof China 50773050, 51073119 and 31100674; International Coop-eration from Ministry of Science and Technology of China (MOSTGrant No. 2008DFA51170).

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