Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 653658

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Transactions of the Royal Society of Tropical Medicine and Hygiene

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Microencapsulated citronella oil for mosquito repellent nishing of cotton textiles

M.M. Mir Specos a,b , J.J. Garca c,d , J. Tornesello c , P. Marino a , M. Della Vecchia b , M.V. Defain Tesoriero b , L.G. Hermida b,
Centro de Investigacin y Desarrollo en Textiles, Instituto Nacional de Tecnologa Industrial (INTI), Avenida General Paz 5445 (B1650WAB), San Martn, Buenos Aires, Argentina Centro Investigacin y Desarrollo en Qumica, Instituto Nacional de Tecnologa Industrial (INTI), Avenida General Paz 5445 (B1650WAB), San Martn, Buenos Aires, Argentina c Centro de Estudios Parasitolgicos y de Vectores (CEPAVE), Consejo Nacional de Investigaciones Cientcas y Tcnicas (CONICET), calle 2 n 584, (1900), La Plata, Buenos Aires, Argentina d Investigador Comisin Investigaciones Cientcas de la Provincia de Buenos Aires (CICPBA)
b a

a r t i c l e

i n f o

a b s t r a c t
Microcapsules containing citronella essential oil were prepared by complex coacervation and applied to cotton textiles in order to study the repellent efcacy of the obtained fabrics. Citronella released from treated textiles was indirectly monitored by the extractable content of its main components. Repellent activity was assessed by exposure of a human hand and arm covered with the treated textiles to Aedes aegypti mosquitoes. Fabrics treated with microencapsulated citronella presented a higher and longer lasting protection from insects compared to fabrics sprayed with an ethanol solution of the essential oil, assuring a repellent effect higher than 90% for three weeks. Complex coacervation is a simple, low cost, scalable and reproducible method of obtaining encapsulated essential oils for textile application. Repellent textiles were achieved by padding cotton fabrics with microcapsules slurries using a conventional pad-dry method. This methodology requires no additional investment for textile nishing industries, which is a desirable factor in developing countries. 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Article history: Received 26 January 2010 Received in revised form 22 June 2010 Accepted 22 June 2010 Available online 31 July 2010 Keywords: microencapsulation citronella oil mosquitoes repellency cotton textiles coacervation

1. Introduction Mosquitoes are insects of major public health concern because many species are vectors of diseases. Aedes aegypti is the primary vector of urban yellow fever and dengue, the most important arboviral human infection worldwide. Infected specimens are widely distributed in Argentina due to the trafc of people from affected neighbouring regions and to adequate climatic conditions.1 From1998 to the middle of 2007, a total of 4718 suspected cases of Dengue

Corresponding author. Tel./Fax: +54 11 4724 6289. E-mail address: lhermida@inti.gob.ar (L.G. Hermida).

Fever were reported in Argentina.2 A signicant outbreak has been taking place in several Argentine provinces since February 2009 with more than 20 000 clinical cases of Dengue fever, including ve cases of Dengue Hemorrhagic Fever, and four deaths up to April 2009.3 In recent years various essential oils have been reported as mosquito repellents due to their eco-friendly and biodegradable nature,4,5 in particular, the essential oil extracted from Cymbopogon nardus (citronella), one of the main aromatic crops grown in subtropical regions of Argentina such as Mesopotamia, Chaco and Formosa. Citronella has been found to possess effective repellent activity against A. aegypti females for 2 h after the direct application of pure oil on the skin of human subjects.6

0035-9203/$ see front matter 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2010.06.004

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A more durable effect has been found with synthetic repellents such as DEET, the most widespread mosquito repellent. Recent studies have shown that a formulation with 80% DEET provides almost 14 h of protection against Aedes aegypti while a 7% formulation provides protection for less than 4 h.7 The incorporation of insect repellents in textiles seemed an alternative way to provide longer lasting protection, although their direct application to fabrics has proven to be inefcient in prolonging the repellence for long enough. Microencapsulation technology has enabled an increase in the durability of the desired effect in versatile textile nishing. A microcapsule provides both space for storing a certain amount of a functional agent and a protective shield from the effects of sunlight, moisture and oxygen. The release of the functional agent occurs by diffusion through the capsule wall and/or rupture of the microcapsules. Therefore, the use of microcapsules could provide a durable repellent nish that could be applied to a multitude of bres.8 Fabrics with repellent activity have been previously achieved by polymer coating methods,9 by encapsulation of DEET in chitosan microcapsules,10 and by inclusion of DEET and permethrin in cyclodextrins grafted to textile substrates.11 However, to the best of our knowledge, the repellent efcacy of cotton fabrics with microencapsulated citronella oil nishes has not been investigated. The aim of this work is to study the repellency to female A. aegypti of fabrics treated with gelatin-arabic gum microcapsules containing citronella oil. These microsystems have been selected due to the low cost and the biocompatible nature of the ingredients involved, which is most desirable in fabrics intended for human use. Microcapsules containing citronella oil were characterized and applied to cotton textiles. The residual mosquito repellency of treated fabrics was determined by means of an in vivo assay and was compared to the repellency of fabrics sprayed with non-encapsulated citronella. 2. Materials and Methods 2.1. Materials Citronella essential oil (Fornasari Ltd., Buenos Aires, Argentina) was used as mosquito repellent agent. The fabric used was a bleached 100% cotton plain weave with a specic weight of 140 g/m2 . Type A gelatin (Rousselot Argentina Corp., Buenos Aires, Argentina) and arabic gum (Van Rossum Ltd., Buenos Aires, Argentina) were employed as wall materials. Glutardialdehyde 25% w/w (Merck & Co. Inc., Hohenbrunn, Germany) was used as hardening agent. All chemicals were reagentgrade. 2.2. Preparation and characterization of microcapsules Microencapsulation of essential oil was performed by complex coacervation as previously described.12 Briey, an o/w emulsion was prepared by homogenization of 1.5% w/v gelatin with citronella essential oil for ve minutes at 18 500 rpm with a high shear homogenizer Hei-

dolph 900 DIAX (Schwabach, Germany). Temperature was maintained at 40 C while 1.5% w/v arabic gum solution was added drop-by-drop (5:1 w/w oil to total polymer ratio). Coacervation was accomplished by dilution with distilled water at a rate of 20 ml/min. Temperature was then reduced to below 10 C and the pH was adjusted to 8 with sodium hydroxide. 0.6 ml of a 25% w/v glutardialdehyde aqueous solution, corresponding to 0.03 mmol glutardialdehyde / g protein, was slowly added upon mechanical stirring with a pitched blade impeller Heidolph RGL 500 (Schwabach, Germany). The suspension of gelatin-arabic gum microcapsules containing citronella was kept upon magnetic stirring at room temperature overnight. Size and morphology of microcapsules were determined by optical microscopy (OM) using a Zeiss Axioskop 40 microscope with a Nikon coolpix 8800 camera (Gttingen, Germany). Microcapsules slurry was spray dried in an AS Niroatomizer mobile minor unit (Soeborg, Denmark) using an inlet temperature of 120 C and an outlet temperature of 90 C. A free-owing powder was obtained and was further analyzed by scanning electron microscopy in a Philips SEM 505 (Philips/FEI, Eindhoven, Netherlands). 2.3. Textile treatment and evaluation Cotton fabrics were treated either with microencapsulated citronella or by spraying with an ethanolic solution of citronella. In the rst case, cotton fabrics were padded twice through an aqueous nish bath containing citronella microcapsules (16% solid in water), nipped to obtain a wet pickup of 100%, and dried in the tenter frame at 100 C for three minutes. In the second case, a 10% w/w solution of citronella oil in ethanol was prepared and sprayed onto fabrics until the samples were entirely wet. All samples were cut into 10 cm 10 cm pieces as specimens and stored for up to 44 days indoors at 22 4 C and 65 5% relative humidity. On each day of a predened series of time intervals, samples were taken and tested by two independent methods. First, a judge smelt the swatch after scratching an x on the specimen with a plastic stick. Samples were measured on a positive and negative scale about the presence of the fragrance.13 Second, a solvent extraction of citronella essential oil from textiles was performed as previously described.14 Briey, three specimens of each sample were incubated with ethanol for 24 hours at room temperature in closed vials with magnetic stirring. The three main components of citronella: citronellal, citronellol and geraniol were determined in the ethanolic extracts by gas chromatography (GC) coupled to a ame-ionization detector (Thermoquest, Milan, Italy) using an HP-5 column in the following conditions: 80 C initial temperature, 8 C/min temperature gradient, and 200 C nal temperature. Textiles sprayed with the ethanolic solution of citronella were extracted with ethanol as described and analyzed by GC in order to determine the initial content of citronella. The aforementioned GC conditions were also used to characterize the composition of the original citronella essential oil.

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2.4. Test animals Mosquitoes were reared in the laboratory using the methods described.15 Egg hatching was induced in dechlorinated water. Larvae were reared in plastic pans and fed with nely ground guinea pig pellets. Adult mosquitoes were maintained in cages (50 x 50 x 50 cm) and fed with a 10% sucrose solution. 2.5. Repellency test procedure Three different types of gloves were employed for the evaluation of repellent activity. Gloves were made with untreated fabrics (UF), fabrics sprayed with an ethanolic solution of citronella (CF), and fabrics treated with microencapsulated citronella (MCF). They were hung in the open-air during the period of the assay. The repellent activity was evaluated by inserting a human hand and arm covered with the gloves into a test chamber (50 x 50 x 50 cm), based on cage tests described in bibliography.16 The covered arm was kept for one minute in the test chamber containing approximately 200 A. aegypti adult females which had not been fed for three to ve days. The number of insects landing was counted independently by two observers. The same subjects uncovered arm and hand were also inserted as control. The trials were conducted in triplicate at 26 1 C and 80 5%RH with a 5 minute waiting period between replicates. The experiments were performed with the written consent of the participants according to the regulations of Bioethics and Biosecurity of the National University of La Plata and of the province of Buenos Aires, Argentina. 2.6. Data analysis The percentage of insects landing on a treated fabric (CF or MCF) was calculated against untreated fabrics using Eq.1: MTF 100 MUF

Figure 1. Optical microphotographs of microcapsules containing citronella essential oil magnied x100.

geraniol contents, which were 35%, 9% and 19% w/w respectively, according to Argentine standard IRAM-SAIPA N 18520. Microcapsules containing citronella oil were characterized by optical and electronic microscopy before and after spray drying respectively. Figure 1 shows the blackberry-like morphology of the microcapsules in a fresh slurry and their apparent diameter which varied from 25 to 100 m. SEM microphotographs of spray dried microcapsules clearly show two types of structures: small spherical units of less than 10 m and agglomerates ranging from 25 to 100 m (Figure 2). 3.2. Characterization of fabrics treated with encapsulated citronella Textile substrates were analyzed by SEM after impregnation with the microcapsule slurry. Figures 3a and 3b show the surface appearance of untreated and treated fabrics respectively. Individual particles were no longer observed on the surface of treated fabric (Figure 3b); a thin layer covered the bers instead. Odour evaluation on fabrics was performed as a complementary test to the determination of extractable citronella

% Insects landing =

where MTF and MUF correspond to the mean number of mosquitoes landing on treated and untreated fabrics respectively. The repellency was indirectly calculated from the percentage of insects landing on treated fabrics with Eq.2: % Repellency = 100 MTF 100 MUF

Media and standard deviations for replicates were obtained at each time and analyzed using ANOVA and the Tukey test for P = 0.05 to detect signicant differences among media. 3. Results 3.1. Characterization of free and microencapsulated citronella oil Gas chromatography analysis of free citronella conrmed Java type oil based on citronellal, citronellol and
Figure 2. Scanning electron microphotographs of spray dried microcapsules containing citronella essential oil magnied x 500.

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Figure 4. Relative amounts of extractable components from MCF samples as a function of time: citronellal (black circles), citronellol (white circles) and geraniol (black triangles). Results are expressed as the percentage of each component with respect to the initial content (time = 0). Bars show standard deviations for n = 3.

Figure 3. Scanning electron microphotographs of a) untreated cotton fabrics (UF) b) cotton fabrics treated with microencapsulated citronella (MCF) magnied x 1000.

oil. Citronella fragrance could be smelt in MCF without scratching them after 28 days hanging in the open-air. At longer periods fragrance could only be smelt upon scratching the fabrics. The rst signicant difference between CF and MCF was the initial amount of extractable citronella oil. Citronellal, citronellol and geraniol contents determined in ethanol extracts from sprayed fabrics were 691, 384 and 833 mg per 100 g of fabric respectively, while much lower values were obtained after extraction of fabrics impregnated with microencapsulated citronella, namely 35, 18 and 75 mg per 100 g of fabric. The oil release from MCF was indirectly studied by quantifying the extractable amount of citronellal, citronellol and geraniol as a function of time. Results are expressed as percentage of extractable component with respect to its initial content in MCF. Figure 4 shows a striking decrease in the amount of extracted components, especially geraniol, after the rst week. 3.3. Repellency Test Table 1 shows the results of the repellency test expressed as the mean number of insects landing on the arm covered with treated and untreated fabrics; the number of insects landing on the bare arm is included as a

control. Four different levels can be clearly differentiated: rst, the untreated fabric (UF) gave a certain degree of protection to the bare arm during the whole experiment, as evidenced by the reduced number of landing mosquitoes. The glove sprayed with citronella (CF) increased the protection with respect to untreated cotton for 16 days but the repellency was limited. In contrast, the arm covered with MCF presented the highest and longest lasting protection from insects. The number of mosquitoes landing on MCF in the rst 21 days was negligible, which means repellency higher than 90%. In order to compare these two repellent fabrics with a lower inuence of experimental variability, results were plotted as the percentage of insects landing (Eq.1) as a function of time (Figure 5). The initial percentage of mosquitoes landing was 33% and 8% for CF
Table 1 Number of insects landing after 1 min exposure on the bare arm, on the arm covered with untreated fabric (UF), with fabrics treated by spraying citronella (CF) or with fabrics treated with microencapsulated citronella (MCF). Day/Sample 1 5 7 9 12 14 16 19 21 23 26 30 33 37 40 44 Bare arm 36 46 72 49 59 34 74 81 93 87 96 101 97 63 59 31 5 6 8 6 4 7 5 6 3 5 9 6 7 7 8 5 UF 24 26 48 18 28 6 33 45 55 45 43 48 43 44 35 16 4 4 9 6 5 3 6a 5 6 4 6 5 11d 15 7 4 CF 8 20 22 11 16 4 19 36 49 42 35 21 27 48 33 12 2 4 4 3 5 0 7a 4 6 11 6c 8 8d 15 8 7 MCF 2 1 3 5 1 0 3 4 3 25 24 23 28 29 25 15 1 1 1 2 1 1 2 2 2b 5b 7c 4 8d 13 8 10

Results are expressed as mean standard deviation (n = 3). a,b,c indicate statistical differences between the two means (P < 0.05). d indicates no statistical differences among the three values (P < 0.05).

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Figure 5. Variation of the percentage of insects landing on fabrics sprayed with citronella oil (CF; black circles) or fabrics treated with microencapsulated citronella oil (MCF; grey triangles) referred to untreated fabrics. Bars show standard deviations for n = 3.

and MCF respectively and this difference persisted during the rst 21 days (P < 0.05). Then, the repellent efcacy of MCF decreased sharply but it was still signicantly different from CF until day 26. After 33 days no difference could be observed between any of the cotton samples. 4. Discussion Microcapsules obtained by complex coacervation were similar in size and morphology to those reported in the literature.12,17 However, once applied to textiles they lose their morphology and only a thin layer covering the treated fabrics is observed. This can be due to the well known lmforming properties of gelatin, an effect already observed after treatment of cotton fabrics with microparticles of poly(n-butyl acrylate) and chitosan, both described as lmforming polymers.18 The low level of citronella oil extracted from MCF was much lower than the one obtained for CF and signicantly lower than our previous results of extractable limonene from fabrics treated with microencapsulated lemon essential oil.12 This difference could result from one or all of the following: (i) the loss of citronella oil during the microencapsulation process and/or (ii) during the textile application due to a low resistance of the microcapsules to heat or pressure and/or (iii) an incomplete oil extraction from impregnated textiles. The rst option (i) seems quite improbable because a massive loss of oil during the microencapsulation process should be evidenced by visible oil droplets oating on the suspension surface. An incomplete extraction (iii) was also discarded because citronella was not detected after further extractions with ethanol or smelt on extracted fabrics. Therefore, considering the structural changes observed in microcapsules once applied to the fabrics (Figure 3b), (ii) seems the most probable option. The parameters of complex coacervation which have a clear inuence on the resistance of microcapsules to textile treatments, such as the core to wall ratio and the hardening agent concentration, will be more deeply studied in further experiments.

Regarding the release patterns of the oil components, the initial decrease can be explained by the release of the supercial oil. This phenomenon, known as burst effect, has frequently been described in release studies of microcapsules intended for the controlled release of drugs.19 The formation of a lm on the fabric could have also contributed to the oil loss and the initial sharp release observed for the three citronella oil components. Surprisingly, even though the initial extractable oil was much lower in MCF than in CF, fabrics treated with microencapsulated citronella (MCF) presented a signicantly higher repellent activity; over 90% for at least three weeks. These results are consistent with those obtained for odour evaluation without scratching the fabrics. This was expected since the repellent activity is an olfactory effect mainly dependent on the quick evaporation of the active products.11 Therefore, any increase in the oil content of treated textiles would represent an increase in the durability of the repellency. Further assays regarding the possible causes of the oil loss will be performed in order to improve the retention of citronella in cotton fabrics and consequently increase the durability of the repellent activity of the proposed system. Most of the current research on long lasting repellency has been done with microencapsulated DEET, the most widely used insect repellent. NGuessan et al.20 assessed the efcacy of a slow-release formulation of microencapsulated DEET applied to bed nets against malaria vectors. This DEET-treated mosquito netting proved to repel, inhibit blood-feeding and kill mosquitoes for a period of at least 6 months under laboratory conditions.20 However, natural products such as repellent essential oils are becoming increasingly popular because of their low toxicity and customer approval. Therefore, it is important to work on the development of long lasting repellent textiles based on impregnation of essential oils. Washing durability has not been specically tested in this study because our previous results show a very low laundering stability for textiles treated with gelatin arabic

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M.M.M. Specos et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 653658 3. PAHO. Number of Reported Cases of Dengue and Dengue Hemorrhagic Fever (DHF), region of the Americas by country and subregion. Washington: Pan American Health Organization; 2009. 4. Prajapati V, Tripathi AK, Aggarwal KK, Khanuja SPS. Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Bioresour Technol 2005;96: 174957. 5. Gillij YG, Gleiser RM, Zygadlo JA. Mosquito repellent activity of essential oils of aromatic plants growing in Argentina. Bioresour Technol 2008;99:250715. 6. Trongtokit Y, Rongsriyam Y, Komalamisra N, Apiwathnasorn C. Comparative repellency of 38 essential oils against mosquito bites. Phytother Res 2005;19:3039. 7. Webb CE, Russell RC. Insect repellents and sunscreen: Implications for personal protection strategies against mosquito-borne disease. Aust N Z J Public Health 2009;33:48590. 8. Li S, Lewis J, Stewart N, Qian L, Boyter H. Effect of nishing methods on washing durability of microencapsulated aroma nishing. J Text Inst 2008;99:17783. 9. Faulde M, Uedelhoven W. A new clothing impregnation method for personal protection against ticks and biting insects. Int J Med Microbiol 2006;296:2259. 10. Fei B, Xin JHN. N-diethyl-m-toluamide-containing microcapsules for bio-cloth nishing. Am J Trop Med Hyg 2007;77:527. 11. Romi R, Lo Nostro P, Bocci E, Ridi F, Baglioni P. Bioengineering of a cellulosic fabric for insecticide delivery via grafted cyclodextrin. Biotechnol Prog 2005;21:172430. 12. Mir MM, Escobar G, Marino P, Puggia C, Defain MV, Hermida L. Aroma nishing of cotton fabrics by means of microencapsulation techniques. J Indl Text Forthcoming 2010. 13. Li S, Boyter H, Qian L. UV curing for encapsulated aroma nish on cotton. J Text Inst 2005;96:40711. 14. Prata AS, Zanin MHA, R MI, Grosso CRF. Release properties of chemical and enzymatic crosslinked gelatin-gum Arabic microparticles containing a uorescent probe plus vetiver essential oil. Colloids Surf B Biointerfaces 2008;67:1718. 15. Gerber FJ, Barnard DR, Ward RA., Bulletin #5. Manual for mosquito rearing and experimental techniques. [place unknown]: American Mosquito Control Association; 1994. 16. Kweka EJ, Mosha F, Lowassa A, Mahande AM, Kitau J, Matowo J, et al. Ethnobotanical study of some of mosquito repellent plants in northeastern Tanzania. Malar J 2008;7:152. 17. Yeo Y, Bellas E, Firestone W, Langer R, Kohane DS. Complex coacervates for thermally sensitive controlled release of avor compounds. J Agric Food Chem 2005;53:751825. 18. Ye W, Xin JH, Li P, Lee KLD, Kwong TL. Durable antibacterial nish on cotton fabric by using chitosan-based polymeric core-shell particles. J Appl Polym Sci 2006;102:178793. 19. Wang J, Wang BM, Schwendeman SP. Characterization of the initial burst release of a model peptide from poly(D,L-lactideco-glycolide) microspheres. J Control Release 2002;82:289 307. 20. NGuessan R, Knols BGJ, Pennetier C, Rowland M. DEET microencapsulation: a slow-release formulation enhancing the residual efcacy of bed nets against malaria vectors. Trans R Soc Trop Med Hyg 2008;102:25962.

gum microcapsules.12 Even though this is still an unsolved problem, fabrics treated with microencapsulated citronella may have potential in some situations where protection is required for several weeks with no need of washing such as disposable cloths or repellency bands for people working in the forest or in urban risk zones. The development of suitable methods for the retreatment of fabrics will be a next step in research on repellent textiles. The improvement of laundering stability will be also considered in future studies. It must be stressed that the methodology described for achieving this kind of repellent fabric is simple, low cost, and reproducible and requires no additional investments for textile nishing industries, a desirable factor in developing countries. Authors Contributions: LGH, MMS, MVDT and PM conceived the study protocol; LGH and MMS prepared the microcapsules and the textile samples for further evaluations; LGH, MVDT and MMS analyzed and interpreted the data and wrote the manuscript; JJG and JT carried out the repellency evaluations and reviewed the literature; MDV carried out the GC evaluations as well as the interpretation of the GC results. All authors read and approved the nal manuscript. MMS and LGH are guarantors of the paper. Acknowledgements: The authors would like to thank Mr. Germn Escobar and Mrs. Cristina Zunino for technical assistance in textile characterization and application and Mrs. Laura Herrera for the nal correction of the manuscript. Funding: None Conicts of interest: None declared Ethical approval: Not required. References
1. Vezzani D, Carbajo AE. Aedes aegypti, Aedes albopictus, and dengue in Argentina: Current knowledge and future directions. Mem Inst Oswaldo Cruz 2008;103:6674. 2. PAHO. Number of reported cases of dengue & dengue hemorrhagic fever, region of the Americas by country and subregion. Washington: Pan American Health Organization; 2007.