preparation and characterization of chitosan nanoparticles as the delivery system for tuberose...

Research Article

Received: 4 August 2012, Revised: 23 May 2013, Accepted: 25 May 2013 Published online in Wiley Online Library: 21 July 2013

(wileyonlinelibrary.com) DOI 10.1002/ffj.3174

22

Preparation and characterization of chitosannanoparticles as the delivery system fortuberose fragranceZuobing Xiao, Ting Tian, Jing Hu,* Mingxi Wang and Rujun Zhou

ABSTRACT: Chitosan (CS) exhibits non-toxicity, biocompatibility and antibacterial activity in the pharmaceutical field.Fragrance is obtained by blending a large number of different spices and odorous materials in a specific ratio. However, theirmain ingredients are labile and volatile. Most volatile fragrances are easily lost during manufacture, storage and use of theperfumes or the perfumed consumer products. In this study, tuberose fragrance (TF) was encapsulated inside CSnanoparticles (TC-NPs) via the ionic gelification in emulsion system. The influence of reaction conditions, such as weight ratioof CS to tripolyphosphate (TPP) as well as CS and TF concentration, on the properties of nanoparticles (particle size and zetapotential) was investigated in detail. TC-NPs were characterized with transmission electron microscopy (TEM), dynamic lightscattering (DLS), Fourier transformation infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC–MS) andthermogravimetric analysis (TGA). TC-NPs (174 nm) were obtained with the weight ratio of CS/TPP 5:1, 1.5 mg/ml CS and100% TF. FTIR demonstrated that TF can be functioned with CS via intermolecular forces and hydrogen bonds. TGA displayedthat the thermal stability of TC-NPs was improved compared with that of TF and that TF loading capacity was 29.5%. GC–MSshowed that 90% fragrances in TF had been encapsulated into TC-NPs. GC with flame ionization detector (FID) displayed thatthe contents of fragrant components released from NPs were much less than that of TF after heating for 30 h. The antimicro-bial activities of TC-NPs were higher than that of TF. TC-NPs displayed lower cytotoxicity to the cells. It is shown that CS-NPs isa multifunctional carrier material for fragrance. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: chitosan nanoparticles; antimicrobial; cytotoxicity; tuberose fragrance; encapsulation; alcohol polyoxyethylene ether(n = 9) (AEO-9)

* Correspondence to: JingHu, School of PerfumeandAromaTechnology, ShanghaiInstitute of Technology, Shanghai 201418, China. E-mail: [email protected]

School of Perfume and Aroma Technology, Shanghai Institute of Technology,Shanghai 201418, China

IntroductionIn recent years, biodegradable polymer nanoparticles, especiallypolysaccharide colloidal nanoparticles and nanocapsules, havereceived much attention for their excellent chemical stabilityand bioactivity in biochemical and pharmaceutical fields.[1–3]

Chitosan (CS) is the second most abundant polysaccharide innature, which is obtained from crustacean shells and composedof D-glucosamine and N-acetyl-D-glucosamine units linked byβ-(1, 4) glycosidic bonds.[4] CS exhibits non-toxicity, biocompati-bility,[5] biodegradability,[6] antibacterial activity[7,8] andmucoadhesive property in the pharmaceutical and nutritionalfields.[9–16] Generally, ionic gelification is a common methodfor the fabrication of CS nanoparticles, in which cationic CSand multivalent polyanions interact to form CS nanoparticlesunder simple and mild conditions. Among various polyanions,tripolyphosphate (TPP) is most investigated due to its quickgelling capability and non-toxic property.[17,18] Fu et al. fabri-cated ammonium glycyrrhizinate-loaded CS nanoparticles byionic gelation of CS with TPP. The effects of chitosan molecularweight, chitosan concentration, ammonium glycyrrhizinateconcentration and polyethylene glycol (PEG) on the physico-chemical properties of the nanoparticles were studied indetail.[19] Zhang et al. prepared CS nanoparticles with sizes from90 to 200 nm through ionic gelification between CS and TPP. CSnanoparticles loaded with 40% bovine serum albumin providedthe sustained release over a 6-day period in simulated intestinalfluid (pH 7.5).[20] Jang et al. reported that when loaded with

Flavour Fragr. J. 2014, 29, 22–34 Copyright © 2013 John

L-ascorbic acid CS nanoparticles with good stability andantioxidant effects were prepared via ionic gelification of CSwith TPP.[21]

Fragrance and flavour are chemical materials applied exten-sively in the fields of cosmetics, foods, medicine, tobacco,textiles, leather, papermaking and so on. The fragrance isobtained by blending a large number of different spices andodorous materials in a specific ratio. However, their mainingredients are labile and volatile. Most volatile fragrancematerials are easily lost during the manufacture, storage and useof the perfumes or the perfumed consumer products. Until now,encapsulation of fragrance via emulsion preparation,[22,23] molecu-lar inclusion into a host,[24–26] coacervation with various naturalmacromolecular materials[27–29] or emulsion/microemulsionpolymerization,[30–32] has received more and more attention, dueto the reduction of the volatilization rate of fragrances andimprovement of their stability.

Up to now, few studies about encapsulation of compoundedfragrance based on CS nanoparticles via the ionic gelificationmethod have been reported. Our previous research demon-strated only that polybutylcyanoacrylate was used to encapsulate

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Chitosan nanoparticles as the delivery system for tuberose fragrance

rose fragrance to form 67.3 nm NPs via anionic polymerization.The cotton treated with these NPs has an excellent resistanceagainst fragrance loss during washing. The loss of the fragrancereleased from cotton fabrics finished by 67.3 nm nanocapsuleswas obviously lower than that by 339 nm nanocapsules androse fragrance.[33] In addition, the cotton textile treated withchitosan nanoparticles loaded with osmanthus fragrance alsoshowed an excellent washing resistance.[34] The present studyis different from the above-reported research, and aimed todesign a compound tuberose fragrance (TF) encapsulated inCS nanoparticles (CS-NPs) via ionic gelification for applicationin cosmetics, foods, leather and so on. The influence of thereaction conditions, such as CS/TPP weight ratio as well as CSand TF concentrations, on the size and zeta potential oftuberose fragrance (TF) encapsulated inside CS nanoparticles(TC-NPs) was investigated in detail. Then the structure andsustained-release property of TC-NPs were studied by Fouriertransformation infrared spectroscopy (FTIR), gas chromatogra-phy–mass spectrometry (GC–MS), GC with flame ionizationdetector (GC–FID) and thermogravimetric analysis (TGA) indetail. In addition, the antimicrobial activities of TC-NPs againstbacteria (Staphylococcus aureus, Escherichia coli and Bacillussubtilis) and cytotoxicity were also explored.

Experimental

Materials

Chitosan (average molecular weight = 150 000, degree of deacetylation90%) was purchased from Golden-Shell Biochemical Co., Ltd. (Yuhuan,Zhejiang, China). TPP was purchased from Sinopharm Chemical ReagentCo., Ltd (Shanghai, China). Fatty alcohol polyoxyethylene ether (n= 9) (AEO-9) was obtained from Shanghai Jinshan Chemical Co., Ltd (Shanghai,China). Nutrient agarmediumwas obtained from Shanghai Hufeng BiotechCo., Ltd. (Shanghai, China). All the components of TF had low solubility inwater. The chemical structure and properties of TF are shown in Table 1.All other reagents used were of analytical grade.

Preparation of CS-NPs

Chitosan nanoparticles were prepared via the ionic gelification reportedby Calvo et al.[35] Chitosan aqueous solutions (0.5–2.5 mg/ml) wereobtained by dissolving in 1% (w/w) acetic acid under ultrasonictreatment at 800 W for 10 min. The pH of the CS aqueous solution wasadjusted to 5.2–5.3 with 0.1 mol/l sodium hydroxide aqueous solution.The weight ratio of CS to TPP was controlled from 3:1 to 7:1 by addingTPP aqueous solution (0.7 mg/ml) into 35 ml CS solution with the stirringspeed of 400 rpm at 25°C. Finally, the reaction was kept for 1 h.

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Preparation of TS-NPs

TF (50%, 100%, 150% and 200% (w/w)) based on the weight of CS-NPswas blended with AEO-9 (50% based on TF) under 1000 rpm stirringfor 5 min at 25°C. Then the mixture was dispersed in the CS solution toform an oil-in-water (o/w) emulsion under ultrasonic treatment at 800W for 5 min. After that, the pH of the emulsion was controlled to 5.3 with1 mol/L sodium hydroxide aqueous solution. The weight ratio of CS toTPP was controlled from 3:1 to 7:1 by adding TPP aqueous solution(0.7 mg/ml) into 35 ml CS solution with a stirring speed of 400 rpm at25°C. Then, the reaction was kept for 1 h. Finally, TC-NPs were purifiedby the centrifugation at 15 000 rpm for 30 min at 5°C.


Particle Micromorphology, Size and Zeta Potential

Transmission electron microscopy (TEM) was used to investigate the mi-cromorphology of nanoparticles. TEM analysis of CS-NPs and TF-NPs wasdetermined with a H-600 electron microscope (Hitachi Company, Tokyo,Japan). The nanoparticle suspension was dripped onto a 300-mesh cop-per grid coated with carbon film. The sample was air-dried at room tem-perature before TEM. The particle size, the polydispersity index (PDI) andzeta potential of nanoparticles in three replicates were determined byZetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Eachsample measurement was done via a solid state He–Ne laser of 633.0 nmat 25°C with an angle detection of 90°.

Chemical Structure Analysis

Fourier transform infrared spectroscopy exploits the fact that moleculesabsorb specific frequencies that are characteristic of their structure. Thechemical structures of CS, TPP, TF and TC-NPs were determined with aVERTEX 70 FTIR spectrophotometer (Bruker, Ettlingen, Germany) in therange from 4000 to 600 cm–1. The FTIR spectrum was used to determinethe structure of TC-NPs and the interaction between TP and CS. Determi-nation of TC-NPs by FTIR was performed after freeze drying.

Determination of Fragrance Loading Capacity and ThermalStability

In order to determine the thermal stability and the fragrance loading ca-pacity (LC) of TF, CS-NPs and TC-NPs, the latter two were controlled withthe same quality after freeze-drying, which were carried out via Q5000thermal analyser (TA Instruments, New Castle, USA). Then, the fragranceloading capacity should be calculated according to Eq. (1):

LC %ð Þ ¼ W1

W2x100% (1)

whereW1 is the weight of the encapsulated fragrance andW2 is the totalweight of the encapsulated fragrance and CS.

Determination of the Encapsulated Fragrance in TC-NPs viaGC–MS and GC–FID

In order to identify the main components of fragrance encapsulated inTC-NPs, TC-NPs were pretreated in the following manner. After freezedrying 0.5 g TC-NPs was added into 10 ml dichloromethane. The suspen-sion was treated with the ultrasonication at 800 W for 15 min untilthe shell of TC-NPs was destroyed and TF was dissolved in thedichloromethane. Then the suspension was centrifuged at 10000 rpmfor 10 min and the supernatant was collected and evaporated at 40°Cto diminish dichloromethane at reduced pressure. Then, the benzylpropionate of 50 μg/ml as an internal standard (ISTD) was added inthe solution after dichloromethane extraction to determine the content(%) of fragrance ingredients.

The analysis of the encapsulated fragrance in TC-NPs was performedwith GC–MS (Agilent Technologies Inc., New York, USA) and GC–FID(Agilent Technologies Inc.) in order to identify whether the maincomponents of TF were encapsulated into nanoparticles. Each purearomatic compound in the TF was also been determined as itsstandard. An Agilent 6890N gas chromatograph with a 5973C massdetection in the range 30–450 mass/charge and a FID were used witha DB-5MS nonpolar column (60 m× 0.25 mm i.d. × 0.25 μm film,Supelco, Sigma-Aldrich Biotechnology L.P., Saint Louis, MO, USA). Thecarrier gas was ultrapurified helium at a flow rate of 1.0 ml/min. Theinjection volume was 0.2 μl with a spilt ratio of 30:1. The initial columntemperature was held at 60°C for 2 min, programmed to ramp to 150°Cat a rate of 5°C/min for 5 min, then to 300°C at a rate of 5°C/min andheld at this temperature for 5 min. The detector ion source tempera-ture was set at 230°C. The FID temperature was maintained at 300°C.Electron impact ionization was performed at electron energy of 70

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Table 1. The properties of the main ingredients of TF

Number Fragrance components Mass quality (%) Boiling point (°C) logP

1 Linalool 4.22 199 2.55

2 Phenylethyl alcohol 5.63 218 1.32

3 Benzyl acetate 8.13 215 2.03

4 Phenethyl acetate 0.54 233.3 1.97

5 Styrallyl acetate 0.52 213 2.01

6 Methyl salicylate 0.32 223.3 2.72

7 Nerol 0.65 225 2.49

8 Linalyl acetate 1.83 220 2.78

9 Trans-Geraniol 4.62 230 2.49

10 4-Tertiary butyl benzeneethanal 1.04 291 3.09

(Continues)

Z. Xiao et al.

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Table 1. (Continued)


11 Hydroxy citronellal 2.20 241 1.42

12 Methyl anthranilate 0.38 256 1.05

13 γ-Decalactone 0.89 281 2.58

14 Cis-Jasmone 0.01 134 2.21

15 Methyl Eugenol 0.80 248 2.83

16 Isoeugenol 2.10 266 2.52

17 α- Isomethyl ionone 2.46 285.3 3.1

18 4-Tertiary butyl-α- methyl benzenepropanal 0.05 96 3.88

19 β-Methylionon 1.19 301 3.25

(Continues)


Flavour Fragr. J. 2014, 29, 22–34 Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj

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20 Methyl-γ-ionone 0.74 206 3.4

21 Lilial® 5.10 279 4.38

22 γ-Unsecalactone 0.66 286 2.99

23 Diethyl phthalate 14.5 298 2.41

24 Methyl dihydrojasmonate 7.81 300 2.2

25 Lyral® 1.25 120 1.85

26 n-Hexyl salicylate 4.77 278 2.71

27 Hexyl cinnamal 6.15 305 4.1

(Continues)

Z. Xiao et al.


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28 Benzyl benzoate 8.03 323 3.59

29 Galoxolide 5.48 304 5.02

30 Benzyl salicylate 5.21 320 3.2

31 Benzyl cinnamate 1.13 224 3.93

32 Verdantiol® 1.59 325 5.9


eV. Identification of the main components of TF was achieved by com-paring mass spectra with those in the NIST05 library. All measurementswere carried out in triplicate and an average value was reported.

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Determination of Sustained-Release Property of TC-NPs viaGC–FID

In order to determine the sustained-release property of TC-NPs, afterfreeze drying 2 g TC-NPs were heated at 90°C for 10 h, 20 h and 30 hseparately. Then, these TC-NPs were added into 10 ml dichloromethane.The other extract processes were the same as above. Each volatilecomponent in the rose fragrance mixture released from TC-NPs afterdifferent heating times and TC-NPs without heating was determinedby a gas chromatograph equipped with a FID (Agilent Technologies Inc.).

The samples were diluted to 50 μg/ml benzyl propionate, which wasused as an ISTD. An Agilent 7890A gas chromatograph was used witha DB-5 nonpolar column (60 m×0.25 mm i.d. × 0.25 μm film, Supelco,Sigma-Aldrich Biotechnology L.P., Saint Louis, MO, USA). The carrier gaswas ultrapurified helium at a flow rate of 1.0 ml/min. The injection volumewas 0.2 μl with a split ratio of 30:1. The initial column temperaturewas held at60°C for 2 min, programmed to ramp to 150°C at a rate of 5°C/min for 5 min,then to 300°C at a rate of 5°C/min and held at this temperature for 5 min.


The FID temperature was maintained at 300°C. All measurements werecarried out in triplicate and an average value was reported.

The influence of heating treatment on the fragrance released fromTC-NPs was investigated. The initial content of TF in TC-NPs withoutheating was used as the initial basis (M1). The contents of those withdifferent heating times were labelled as (M2). The loss ratio (L) wascalculated according to Eq. (2):

L %ð Þ ¼ M2 �M1

M1x100% (2)

Analyses were performed in triplicate.

Antibacterial Evaluation

TC-NPs and TF were tested against a panel of microorganisms includingStaphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 andBacillus subtilis ATCC 6633. Bacterial strains were cultured overnight at37°C in the nutrient agar plates. The nutrient agar medium in a Petri dishwas inoculated with 0.1 ml 107–108 cfu/ml bacteria. Then sterile filterpaper discs (6 mm diameter) were impregnated with 1 ml TC-NPs andTF separately. The plates were left 15 min at room temperature to allow


Z. Xiao et al.

28

the diffusion of TC-NPs and TF, and then they were incubated at 37°C for24 h. At the end of the period, the diameter of the zone of inhibition wasmeasured using a caliper. The tests were performed in triplicate.

Cytotoxicity Assay

The in vitro cytotoxicity was evaluated by performing a methyl thioazolyltetrazolium (MTT, Sigma-Aldrich) assay in HeLa cells. Cells growing in logphase were seeded into 96-well cell-culture plate at 1 × 104/well andthen incubated for 24 h at 37°C under 5% CO2. The TC-NPs and TF(100 μL/well) at different concentrations (400, 800, 1200, 1600 and2000 μg/ml, diluted in Roswell Park Memorial Institute culture (RPMI1640)) were added to the wells of the treatment group, and RPMI 1640was added to the negative control group. The cells were then incubatedfor 24 h at 37°C under 5% CO2. Thereafter, MTT (20 μL; 5 mg/ml) wasadded to each well, and the plate was incubated for an additional 4 hat 37°C under 5% CO2. After changing the culture medium to 100 μL di-methyl sulfoxide (DMSO), the assay plate was allowed to stand at roomtemperature for 15 min. An enzyme-linked immunosorbent assay (ELISA)reader (Infinite M200, Tecan, Männedorf, Austria) was used to measurethe OD570 (absorbance value) of each well with background subtractionat 690 nm. The following formula was used to calculate the viability ofcell growth: cell viability (%) = (mean of absorbance value of treatmentgroup/mean of A value of control) × 100. The results were expressed asan average over three nominally identical measurements.

Results and DiscussionThe influence of the reaction conditions, such as weight ratio ofCS to TPP as well as CS and TF concentration, on the particle size,PDI and zeta potential of nanoparticles was investigated in de-tail. The whole reaction is summarized in Table 2.

Micromorphology

Figure 1 shows the different micromorphology of CS-NPs andTC-NPs. The irregular spherical CS-NPs were about 150 nm asshown in Figure 1A. Figure 1B shows that TC-NPs still exhibitedirregularity in spherical shape and are dispersed uniformly, al-though some NPs adhere to each other. In comparison withCS-NPs, the average diameter of TC-NPs was increased to 200nm for the encapsulation of fragrance. The molecular chains ofCS with a rigid crystalline structure through inter- and intramo-lecular hydrogen bonding can extend through the function ofelectrostatic charge repulsion in dilute acetic acid aqueous solu-tion and form the cationic polyelectrolyte with the protonatedamino group on the CS molecule. As shown in Scheme 1, fra-grance can be emulsified in the micelle with the emulsifiersand the ultrasonic treatment. So the fragrance can be encapsu-lated into NPs via the ionic gelification between the positivelycharged protonated amino groups on CS and the negativelycharged phosphate groups on TPP.

Particle Size

Figure 2 demonstrates the influence of the weight ratio of CS toTPP on the mean size of TC-NPs at different TF concentration.The average size of TC-NPs rose with increasing weight ratio ofCS to TPP. When TF concentration went up from 50% to 100%,the size of TC-NPs primarily decreased. After that, when TF con-centration was more than 100%, the excessive fragrance led tothe enhancement of NPs’ size. This was attributed to the aggre-gation and adhesion of NPs caused by the reduction of particlesurface charge.

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Figure 3 shows the effect of CS concentration on the meansize of TC-NPs at different TF concentration. The size of TC-NPsincreased with the enhancement of CS concentration. Generally,the higher the CS concentration, the higher the viscosity of CSsolution, and the less the average energy exerted on chitosanmolecules.[36] The larger NPs could be obtained via the ionicgelification, with the larger CS molecular fragments dependingon lower cavitation energy and lower shearing energy.[36,37] Inaddition, the size tendency of TC-NPs with TF concentrationincrease was similar to the effect of TF concentration on particlesize shown in Figure 2. However, when CS concentration was 2.5mg/ml, the size of TC-NPs was enhanced with the growth of TFconcentration.

Zeta Potential

The influence of different TF concentrations and CS/TPP weightratio on the zeta potential of TC-NPs is indicated in Figure 4.The zeta potential of TC-NPs declined gradually while TF in-creased, which was attributed to that the encapsulation of TFdiminishing the positive surface charge of CS-NPs. Furthermore,with CS/TPP weight ratio rising, the zeta potential of TC-NPsgrew, associated with the increase of free amino groups on thesurface of CS-NPs.[20] However, when TF content was more than150%, the zeta potential of TC-NPs primarily improved, thenreduced.

Figure 5 displays the effect of CS concentration on zeta poten-tial of TC-NPs at different TF concentrations. The tendency of thezeta potential of TC-NPs with increments of TF is similar to thatin Figure 2, and again it can be inferred that the encapsulationof fragrance could decrease the surface charge of NPs. Huet al.[14] reported that the zeta potential of CS-TPP has a linearrelationship with CS concentration, associated with the less neu-tral –NH3

+ formed on the surface of NPs. However, an oppositetrend in zeta potential of NPs was found in present study. Theresults indicate that the zeta potential of NPs tend to declinewith the growth of CS mass concentration. This is probablydue to the increment of nanoparticle size leading to instabilityof the system.

Chemical Structure Analysis

Figure 6 displays the differences among the structures of TPP,CS, TF and TC-NPs. In the FTIR spectrum of TPP, the obviousabsorption peaks at 1140 cm–1 and 889 cm–1 belonged to theextending vibration and bending vibration of P =O. In the FTIRspectrum of CS, the absorption peak at 3367 cm–1 was affectedby the superposition of the extending vibration of –OH and –NH,due to CS intermolecular hydrogen bonds. The absorption peakat 2886 cm–1 is associated with the stretching vibration of the C–H group in the benzene ring. In addition, the absorption peaks at1647 cm–1 and 1590 cm–1 correspond to the vibration of the am-ide I and amide II bands, respectively. The absorption peak 1078cm–1 was affected by the stretching vibration of the C–O groupof CS. In the FTIR spectrum of TF, characteristic absorption peaksat 3449 cm–1, 2959 cm–1, 1726 cm–1 and 1273 cm–1 are due tostretching vibrations of the –OH, C–H, C =O and C–O groups.In the spectrum of TC-NPs, the amino group vibration bandwas shifted to 1670 cm–1 and 1629 cm–1, in addition, a newabsorption peak at 1630 cm–1 appeared, associated with thecross-linking between the phosphate groups of TPP and aminogroups of CS.[37] The presence of the P=O group was indicated

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Table

2.Overview

ofalle

xperim

ents

fortheform

ationof

TC-NPs

Run

m(CS):m

(TPP

)CS(m

g/mL)

TF(%

)Pa

rticle

size

(nm)

PDI

Zeta

potential(m

v)

13:1

1.5

5017

3.26

±2.14

0.23

3±0.02

516

.15±0.69

23:1

1.5

100

150.52

±2.45

0.20

9±0.04

315

.32±0.77

33:1

1.5

150

165.07

±2.02

0.21

8±0.02

113

.65±0.77

43:1

1.5

200

187.36

±2.11

0.23

8±0.01

812

.27±0.76

54:1

1.5

5019

0.28

±2.08

0.25

4±0.03

520

.77±0.57

64:1

1.5

100

165.75

±2.33

0.18

5±0.01

218

.98±0.82

74:1

1.5

150

182.10

±1.98

0.22

5±0.02

217

.34±0.90

84:1

1.5

200

208.09

±2.04

0.25

6±0.04

214

.25±0.65

95:1

1.5

5020

3.54

±2.61

0.21

5±0.01

822

.65±0.61

105:1

1.5

100

174.33

±2.28

0.13

8±0.01

620

.41±0.81

115:1

1.5

150

200.67

±2.34

0.14

7±0.02

116

.90±0.89

125:1

1.5

200

234.14

±2.77

0.22

3±0.03

312

.52±0.56

136:1

1.5

5021

1.30

±2.61

0.20

8±0.02

423

.81±0.59

146:1

1.5

100

198.15

±2.29

0.16

1±0.01

920

.53±0.65

156:1

1.5

150

223.18

±2.18

0.18

8±0.02

416

.92±0.76

166:1

1.5

200

268.20

±2.36

0.21

6±0.02

712

.56±0.67

177:1

1.5

5023

0.09

±2.49

0.21

2±0.02

923

.17±0.46

187:1

1.5

100

221.01

±2.07

0.18

2±0.03

019

.65±0.71

197:1

1.5

150

257.32

±2.43

0.20

4±0.04

516

.18±0.57

207:1

1.5

200

284.19

±2.91

0.24

6±0.01

510

.82±0.65

215:1

0.5

5017

3.51

±2.08

0.23

4±0.03

924

.74±0.67

225:1

0.5

100

133.26

±2.29

0.21

5±0.02

821

.55±0.83

235:1

0.5

150

161.13

±2.31

0.22

4±0.02

418

.62±0.66

245:1

0.5

200

206.54

±2.35

0.25

6±0.03

413

.85±0.64

255:1

150

187.22

±2.45

0.21

7±0.02

822

.82±0.48

265:1

110

014

9.13

±2.09

0.17

5±0.01

120

.33±0.51

275:1

115

017

4.65

±2.29

0.19

2±0.02

118

.22±0.59

285:1

120

021

9.38

±2.57

0.24

5±0.02

614

.31±0.50

295:1

1.5

5020

3.29

±2.48

0.21

2±0.03

422

.54±0.55

305:1

1.5

100

174.00

±2.13

0.14

0±0.01

920

.80±0.70

315:1

1.5

150

200.32

±2.08

0.15

3±0.02

018

.34±0.63

325:1

1.5

200

234.55

±2.15

0.22

8±0.02

113

.12±0.59

335:1

2.0

5023

2.62

±2.34

0.25

8±0.03

421

.87±0.57

345:1

2.0

100

228.41

±2.22

0.25

6±0.02

419

.18±0.76

355:1

2.0

150

258.27

±2.61

0.30

1±0.03

515

.85±0.73

365:1

2.0

200

306.15

±2.71

0.32

0±0.03

210

.25±0.63

375:1

2.5

5030

3.24

±2.19

0.29

8±0.04

619

.97±0.63

385:1

2.5

100

323.28

±1.99

0.31

8±0.04

216

.29±0.65

395:1

2.5

150

388.27

±2.08

0.32

5±0.03

911

.65±0.71

405:1

2.5

200

480.14

±2.33

0.39

8±0.05

38.87

±0.66


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29

Figure 1. TEM images of (A) CS-NPs and (B) TC-NPs: weight ratio of CS to TPP 5:1; 2 mg/ml CS; 100% TF

Scheme 1. The formation process of TC-NPs

Figure 2. Effect of weight ratio of CS to TPP on the size of TC-NPs Figure 3. Effect of CS concentration on the size of TC-NPs

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Figure 5. Effect of CS concentration on the zeta potential of TC-NPs

Figure 4. Effect of weight ratio of CS to TPP on the zeta potential of TC-NPs

Figure 6. FTIR spectra of (A) TPP, (B) CS, (C) TC-NPs and (D) TF

igure 7. TGA curves of TF, CS-NPs and TC-NPs: weight ratio of CS toPP 5:1; 1.5 mg/ml CS; 100% TF


by the stretching and flexural vibration at 1093 cm–1 and 895cm–1 respectively. When TF was encapsulated in CS-NPs, thestretching vibration of the C–O group disappeared, while thestretching vibration of C–H and C=O were shifted to lowerwavenumber peaks, appearing in CS-NPs at 2928 cm–1 and1722 cm–1, respectively. In addition, the absorption peaks at3367 cm–1 and 2886 cm–1 of –OH and C–H in the benzene ringof CS transferred to 3329 cm–1 and 2871 cm–1 respectively.

31

Fragrance Loading Capacity and Thermal Stability

Figure 7 shows the TGA results for TF, CS-NPs and TC-NPs. Onlythermal decomposition of 70–185°C was observed for TF. Whentemperature reached 185°C, the weight of TF was reduced by99.7%. Comparison of CS-NPs and TC-NPs below 70°C showsthat the weight loss of CS-NPs and TC-NPs was 10.1% and6.3% respectively, due to the vaporization of residual moisturein NPs. For CS-NPs, the main weight loss occurred at 200–400°C and the weight loss was about 37.5% due to the loss of hydro-gen bonds between the N-acetyl and free amino groups,[38,39]

and the depolymerization and decomposition of glucosamine


FT

units of CS.[40] The main weight loss of TC-NPs was observed at90–450°C. The first weight loss of 90–230°C was ascribed tothe decomposition of fragrance encapsulated in CS-NPs andthe weight-loss rate was decreased significantly compared withTF. The thermal stability of fragrance encapsulated in CS-NPswas improved. The second weight loss occurred from 230 to450°C, which was due to the thermal decomposition of CS struc-ture and residuary fragrance encapsulated in CS-NPs. The totalweight loss of TC-NPs was nearly 56.3%. CS-NPs and TC-NPswere prepared under the same conditions. According to thesame quality of CS in CS-NPs and TC-NPs, the fragrance LC wascalculated as 29.5% according to Eq. (3):

56:3%� LC1� 6:3%� LC

¼ 37:5%1� 10:1%

(3)


Z. Xiao et al.

32

Fragrance Encapsulation and Sustained-Release Property

Table 3 shows the GC–MS result of TF in TC-NPs. As shown inTable 1, there were 32 components in TF compounds, whichincluded 15 esters, five aldehydes, five ketones, four alcohols,two phenols and one musk. When TF was encapsulated intoCS-NPs, most fragrant ingredients of TF could be detected apartform styrallyl acetate, cis-jasmone and isoeugenol. It appearedthat more than 90% of fragrance components could be encap-sulated in CS-NPs.

GC–FID analysis of the volatile ingredients remaining in TC-NPs subjected to heating of different times (10 h, 20 h and 30h), was performed separately, as shown in Table 4. The loss offragrance released increased with enhancement of theheating time for both TF and TC-NPs. What is more, the lossof fragrant components released from NPs was much less thanthat of TF. After heating for 20 h, styrallyl acetate, cis-Jasmone,4-tertiary butyl-α- methyl benzenepropanal, methyl anthrani-late and Lyral® in TF have been released completely. Exceptstyrallyl acetate and cis-Jasmone, the loss ratios of 4-tertiary

Table 3. GC–MS results of main ingredients of TF in TC-NPs

Number RIa Standard RI

1 1105 11022 1126 11213 1172 11684 1183 11875 1193 11926 1204 12017 1226 12288 1248 12499 1254 125110 1276 1278 4-T11 1293 128812 1570 156713 1369 137114 1395 139115 1409 140816 1458 145717 1427 143118 1479 1477 4-Tertiar19 1484 148220 1507 150821 1541 153722 1580 157823 1586 158824 1661 166425 1682 168526 1692 168927 1761 176528 1790 178929 1863 186430 1895 189231 2092 209032 2098 2102

Weight ratio of CS to TPP 5:1; 1.5 mg/ml CS; 100% TF.ND, fragrance has not been detected.aThese standard Retention Index (RI) values have been measured


butyl-α-methyl benzenepropanal, methyl anthranilate andLyral® in TC-NPs were 88.09%, 68.57% and 78.61% respectively.The logP values of styrallyl acetate, cis-Jasmone and 4-tertiarybutyl-α-methyl benzenepropanal are 2.01, 2.21 and 3.09,respectively. The logP values of methyl anthranilate and Lyral®were <2, but their boiling points are lower. When the aromaticcompounds were heated for several hours, those with lowboiling points were easily lost. Generally, for the aromaticcompounds encapsulated into the NPs, Those with higherlogP, the more hydrophobic ones, have a greater tendency tobe lost in the process. When TF and TC-NPs were heated for 30 h,linalool, 4-tertiary butyl benzeneethanal, hydroxycitronellal,γ-decalactone and n-hexyl salicylate in TF were not detected.The relative amounts of linalool, 4-tertiary butyl benzeneethanal,hydroxycitronellal, γ-decalactone and n-hexyl salicylate ofTC-NPs were reduced to 89.74%, 52.54%, 79.23%, 88.45%and 69.42%, respectively, of the initial content. Thenanoencapsulation of TF via CS exhibited excellentsustained-release property.

Fragrance components Content (ug/mL)

Linalool 0.50Phenylethyl alcohol 0.28

Benzyl acetate 0.69Phenethyl acetate 0.01Styrallyl acetate NDMethyl salicylate 0.04

Nerol 0.07Linalyl acetate 0.20Trans-Geraniol 0.46

ertiary butyl benzeneethanal 0.03Hydroxy citronellal 0.25Methyl anthranilate 0.22

γ-Decalactone 0.01Cis-Jasmone ND

Methyl Eugenol 0.02Isoeugenol ND

α-Isomethyl ionone 0.07y butyl-α- methyl Benzenepropanal 0.02

β-Methylionon 0.02Methyl-γ-Ionone 0.02

Lilial® 2.88γ-Unsecalactone 0.19Diethyl phthalate 5.30

Methyl dihydrojasmonate 5.35Lyral® 0.29

n-Hexyl salicylate 1.60Hexyl cinnamal 7.30Benzyl benzoate 9.75

Galoxolide 6.05Benzyl salicylate 7.50Benzyl cinnamate 0.09

Verdantiol® 1.17

from the authentic samples.


Table 4. GC–FID comparison result of main ingredients of (A) TF and (B) TF in TC-NPs heated for 10 h, 20 h and 30 h

Fragrance components Loss ratio 10 h (%) Loss ratio 20 h (%) Loss ratio 30 h (%)

A B A B A B

Linalool 65.33 24.11 87.54 46.62 100 89.74Phenylethyl alcohol 45.23 18.32 87.87 37.42 82.54 55.54Benzyl acetate 58.45 19.74 78.65 27.54 96.45 49.65Phenethyl acetate 51.24 22.43 77.65 50.12 91.23 75.23Styrallyl acetate 73.54 ND 100 ND ND NDMethyl salicylate 39.24 22.31 66.17 48.23 80.13 60.13Nerol 46.82 25.41 77.32 39.92 85.41 47.25Linalyl acetate 55.13 27.03 80.12 43.30 95.23 63.41Trans-Geraniol 38.51 22.13 66.35 39.85 74.24 60.214-Tertiary butyl benzeneethanal 66.28 32.51 86.31 54.88 100 52.54Hydroxy citronellal 69.56 37.21 89.46 52.32 100 79.23Methyl anthranilate 69.26 38.26 100 68.57 ND 100γ-Decalactone 61.25 33.46 89.12 58.35 100 88.45Cis-Jasmone 89.56 ND 100 ND ND NDMethyl Eugenol 34.45 20.38 59.86 36.27 88.62 59.54Isoeugenol 51.32 ND 78.65 ND 100 NDα-Isomethyl ionone 46.23 25.42 70.12 52.73 98.23 63.434-Tertiary butyl-α-methyl benzenepropanal 83.42 36.73 100 88.09 - 100β-Methylionon 40.11 24.35 64.77 48.53 92.27 64.02Methyl-γ-Ionone 55.26 29.34 77.42 38.54 96.87 53.24Lilial® 33.10 20.28 54.47 31.29 80.36 45.25γ-Unsecalactone 42.24 25.31 71.35 45.32 79.38 56.43Diethyl phthalate 36.12 12.42 56.24 30.42 70.69 50.54Methyl dihydrojasmonate 40.34 17.13 59.85 39.28 76.36 49.94Lyral® 77.27 36.21 100 78.61 ND 100n-Hexyl salicylate 57.11 23.25 77.34 50.16 100 69.42Hexyl cinnamal 38.31 15.27 64.92 36.64 89.28 50.21Benzyl benzoate 29.15 13.35 40.77 18.63 57.09 26.32Galoxolide 36.57 23.12 57.32 37.65 98.24 82.14Benzyl salicylate 32.67 17.12 54.39 28.74 66.45 52.95Benzyl cinnamate 53.85 21.55 67.31 48.24 72.90 58.54Verdantiol® 24.32 17.22 37.15 25.21 54.43 36.43

Weight ratio of CS to TPP 5:1, 1.5 mg/mL CS, 100% TF.ND, fragrance has not been detected.


Antibacterial Activity

Table 5 displays the antimicrobial activities of TC-NPs and TFagainst bacteria (S. aureus, E. coli and B. subtilis), which were exam-ined qualitatively by agar diffusion tests. In general, three bacteriawere not responding to TF. TC-NPs showed a significantly higherantibacterial activity than TF. The antibacterial activity of TC-NPsagainst B. subtilis was the strongest among the three bacteria.The inhibitory zone of TC-NPs against S. aureus was larger thanthat against E. coli, showing an apparently stronger antibacterial

Table 5. Disc diffusion test of TC-NPs and TF against a panel of r

Sample Staphylococcus aureus

TF 0TC-NPs 4.4 ± 0.2aDiameter of zone inhibition are expressed in millimetres


effect on S. aureus than on E. coli. This observation was the sameas that in the work of No et al.,[41] in which the inhibitory effects ofthe chitosan with low molecular weight (MW) had stronger bacte-ricidal effects on S. aureus than on E. coli.

Cytotoxicity

Figure 8 displays the cell viability of TC-NPs and TF by an MTTassay using HeLa cells. There was significant cell death observedwhen the cells were treated with different contents of TF. When

eference bacterial strainsa

Escherichia coli Bacillus subtilis

0 02.2 ± 0.4 5.6 ± 0.2


33

Figure 8. Cytotoxicity of TF and TC-NPs according to theirconcentration

Z. Xiao et al.

34

TF was added into the HeLa cells, there was only 25% cell viabil-ity. In addition, with increasing TF content, there was no varia-tion of cell viability. Upon comparison with the same amountof TC-NPs, the latter showed lower cytotoxicity to cancer cells,which can be explained by the shell of CS being a non-toxic, bio-compatible and biodegradable biopolymer. When 400 μg/ml TC-NPs was added into the cancer cells, 68% of cancer cells werealive. With the content of TC-NPs increased, the cell viability ofTC-NPs declined. In brief, fragrance was encapsulated by CS,which showed lower cytotoxicity.

ConclusionIn this work, TC-NPs were successfully prepared in o/w emulsionvia the ionic gelification method. The sizes and zeta potentials ofTC-NPs were strongly dependent on the weight ratio of CS toTPP as well as CS and TF concentration. With the weight ratioof CS/TPP as 5:1 of 1.5 mg/ml CS and 100% (w/w) TF, 174 nmand 20.8 eV TC-NP were obtained. FTIR demonstrated that TPPand CS had been reacted via the ionic gelification based onthe function between the phosphate group of TPP and theamino group of CS. TGA displayed that the main thermal decom-position of TC-NPs and TF occurred at 90–450°C and 70–185°C,respectively. The thermal stability of TC-NPs was obviously im-proved in contrast to that of TF. The TF loading capacity wasabout 29.5%. GC–MS showed that 90% fragrance componentsin TF had been encapsulated into TC-NPs, except styrallylacetate, cis-jasmone and isoeugeno. The loss of fragrant compo-nents released from TF in TC-NPs was much less than that of TFafter heating. Linalool, 4-tertiary butyl benzeneethanal, hydroxycitronellal, γ-decalactone and n-hexyl salicylate have not beendetected in TF. The relative amounts of linalool, 4-tertiary butylbenzeneethanal, hydroxy citronellal, γ-decalactone and n-hexylsalicylate of TC-NPs were reduced to 89.74%, 52.54%, 79.23%,88.45% and 69.42%, respectively, of the initial content. Interest-ingly, TC-NPs showed a significantly higher antibacterial activitythan TF. The antibacterial activity of TC-NPs against B. subtilis wasthe strongest among the three bacteria. TC-NPs displayed lowercytotoxicity to cells. CS-NPs would be a promising controlled-release carrier of fragrance.


Acknowledgements

The support from National Natural Science Foundation of China(Nos 21076125 and 21106084), National Key Technology R&DProgram (No. 2011BAD23B01) and Innovation Program ofShanghai Municipal Education Commission (No. 12YZ158) wasappreciated.

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