Use of metal carboxylate glasses in the controlled release of bioactive molecules: Culex quinquefasciatus oviposition pheromone

journal of

Journal of ControlledRelease31 (1994) 145-149

ELSEVIER

controlled release

Use of metal carboxylate glasses in the controlled release of bioactive molecules: Culex quinquefasciatus oviposition pheromone J . A . B l a i r a, A . J . M o r d u e

( L u n t z ) b'*, J . A . D u f f y a, J . L . W a r d e l l a

Departments of Chemtstrf and Zoologyb, Umversttyof Aberdeen, Aberdeen, UK

Accepted28 January 1994

Abstract

Mixed metal carboxylate glasses have recently been developed, and it is shown that these are good hosts for Culex qumquefasciatus oviposition pheromone. The glasses degrade in a humid environment, releasing the volatile pheromone in a controlled fashion. Successful aerial release of the pheromone was demonstrated in an oviposition choice bioassay, showing both time and dose relationships. The glasses alone did not affect the behavioural responses of the mosquitoes. Keywords" Metal carboxylateglass, Bioactlvemolecule,Oviposmonpheromone

1. Introduction

The composition of glassy materials can be adapted to suit a variety of factors, including those which govern its solubility and hence rate of dissolution. Thus, some glasses (e.g. phosphate glasses) have great potential for controlled release applications, but to date only for materials capable of withstanding the high temperatures involved in preparation. It is possible, however, to produce glasses, from mixtures of metal carboxylates, which are liquid at much lower temperatures. [ 1-3]. For example, (CH3(CH2)6CO2)2Zn CH3(CH2) 6CO2Na (1:0.9 mole ratio) glass is formed by quenching the melt from a temperature just below 100°C. A large number of glasses, with a range of different properties, have been prepared using metal cations such as Na +, K +, Ca 2÷ and Zn 2÷ and numerous branched and straight chain carboxylate anions. We *Correspondingauthor 0168-3659/94/$07.00 © 1994ElsevierScienceB V All rights reserved SSDIOI68-3659(94)OOOI5-M

have recently reported the use of these novel low temperature glasses as hosts for thermally labile materials, having biological, optical and electronic applications [ 2 ]. Many thermally unstable biological molecules can be dissolved in the molten glass with little or no decomposition, and incorporation of 5 % by weight or higher, of solute has been shown not to impair glass formation. The higher carboxylate glasses are good hosts of thermally labile materials and have release rates which depend on (i) the chain length of the carboxylate and (li) the cation composition, for example, the Na ÷ :Ca2÷ ratio. The glasses can be stored indefinitely in an anhydrous environment without affecting the dissolved compound. In contact with water or a humid environment breakdown occurs resulting in products of the glasses which are harmless in the environment. The application for such glasses might be for the slow release of insecticides or nutrients into the soil or the release of insect pheromones into the air [4]. In this paper selected water-soluble, low softening-tempera-

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J.A Blatret al / Journalof ControlledRelease31 (1994)145-149

ture carboxylate glasses have been investigated with respect to the incorporation and controlled release of Culex quinquefasciatus (Diptera: Culicidae ) oviposition pheromone (erythro-6-acetoxy-5-hexadecanolide) [ 5 ]. The pheromone is released slowly from apical droplets at the apex of each egg in the Culex egg raft and attracts other gravid females to oviposit in the same area. Synthetic pheromone has demonstrated uses in mosquito control, both alone [6] and in combination with ajuvenoid type insecticide [7]. It also has potential to be combined with habitat-related cues in a control strategy [ 8].

2. Materials and methods

2.1. Preparation of controlled release matrices The glasses were prepared by grinding together the component carboxylate salts followed by melting in 10 g quantities and quenching to a glass between a bronze block and aluminium stamp. The prepared glass was then reground to a fine powder and remelted to the encapsulation temp, (1.e. when the glass was fluid enough to incorporate a material). C. quinquefasciatus oviposition pheromone (diluted m dichloromethane) was introduced into the melted glass by injection below the surface of the melt. After stirring the melt was quenched rapidly and poured into 10 nun diameter circular bronze moulds, producing glass "tablets" (3 mm thick, 0.8 g weight). These tablets were stored in sealed, dry flasks until required. Pheromone content of the glass was analysed by GC using a Perkin-Elmer 8320 capillary gas chromatograph and 25 m S.G.E. 0.25 /x bonded phase fused silica BP5 column (50°C, 2 min; 20°C, min-I; 230oc, 15 mm) with N2 at 15 psi as a carrier gas. The pheromone was soxhlet extracted in dichloromethane. A timed experiment measured the residual pheromone in the glass in a humid atmosphere. 2.2. Bioassay The bioassay was carried out in muslin-covered wooden framed cages (30 × 30 x 30 cm) at 27 + I°C and 12L: 12D cycle. Gravid female C. quinquefasciatus were used four days after feeding (c. 7-10 days old). They were offered the choice between two glass bowls

for oviposition: one test and one control. Test bowls contained either 200 ml distilled water plus pheromone alone, applied onto a glass coverslip, floated on a plastic vial cap, or pheromone encapsulated into a carboxylate glass, also on a plastic vial cap. Control bowls contained 200 ml distilled water plus a glass coverslip with solvent alone, or the relevant undoped glass. Control experiments of glass versus no glass were also run. The bowls were placed in diagonally opposite comers of the cage some 30 cm apart. Neither the relative position of the test and control bowls nor the density of gravid females (20-200 per test) significantly affected egg laying behaviour [3]. The trials were run overnight and assessed the following morning by counting the number of egg rafts in each bowl and expressing the results as percentage in the test bowl compared with the total number in both test and control bowls.

3. Results and discussion

3.1. Encapsulation of pheromone in glass The pheromone was successfully dissolved into and recovered from a number of carboxylate glasses with varying water solubility. The glasses alone, or the free acids present as breakdown products of the glasses, did not adversely affect the oviposition behaviour of the mosquitoes (Table 1). Recovery of the pheromone from the glasses showed it to be unaltered by the encapsulation procedure (Table 1): the lactone ring remained intact and the erythro and threo diastereomers were present in equal proportions (GC and13C NMR analyses, Blair, 1992; G. Dawson and J.A. Pickett IACR, Rothamsted Exp. Station, Harpenden, Herts, UK). The more hygroscopic glasses released pheromone at the fastest rates. The acetate glass broke down completely in 70-80% humidity, releasing most of the encapsulated pheromone within one month, whereas the more resistant propanoate glasses exhibited slower, and/or greatly reduced release rates (Fig. 1 ). Glasses which did not degrade significantly often became crystalline in appearance, which may have opened up channels through which the pheromone could diffuse out.

J.A. Blatr et al / Journal of Controlled Release 31 (1994) 145-149

147

Table 1 Propemes of selected carboxylate glasses as controlled release medm Glass Compos~tton

Ratio

Encapsulation temperature /°C

Solubihty ~

Density g cm 3

Mean % encapsulation of pheromone

Ovlpositional response of C. qumquefasctatus to glass alone 2

1'1 1

140

2 hours

1.478

841+376(5)

526+370(10)

1'1

180

8 hours

1.322

74.05-5 18(3)

51.95-3.02(9)

1 2.2

160

2 weeks

1 185

7245-1.94(5)

4955-3.46(5)

Acetate glass Sodmm acetate Potassmm acetate Calcmm acetate

Propanoate glass Sodium propanoate Calcium propanoate

Mtxed amon glass Sodium butanoate Calcmm propanoate Sodmm octanoate

IT~me for 1 tablet to lose half its weight m stall water 2Expressed as mean % egg rafts laid m test bowls vs total laid (control +test). No szgnificant differences were found between the glass controls and the blank control value (Blank control= 50.5 + 5 29(3) ) Figures m brackets refer to number of rephcates

1001 BO

1

~60t,0202 ~ 2~

~]O Time

Acefale glass

Propanoate glass

I0 gIO 6 (days]

reached significance (Fig. 2). Powdering the glass to increase surface area enabled lower pheromone loadl ngs to become significant (Table 2). The mixed-anion matrix, even at an initial loading of 4.2 mg/glass disc, showed no preferential response until the overall surface area was increased by powdering the tablet, (response for 250 /xg pheromone in powdered tablet was 66.4+2.6%; P < 0 . 0 1 ) (Table 2). Only minimal

100[/

Fig 1 The release of Culex qumquefasclatus ovlposltion pheromone from carboxylate glasses with time in a humid environment, when held over water, as measured by the residual pheromone in the glass (n = 3 ) Loading of the glasses was 8 mg g - 1

c o

3.2. Response of C. quinquefasc]atus to synthetic oviposition pheromone

:~ 60

1 Pheromone m a c e t a l e glass

~x

3 Pheromone in m)xed anion glass ~ ~//

Pheromone alone

50

Maximal behavioural response to pheromone was achieved at 0.1 /~g pheromone (Lawrence & Pickett (1985); Mordue (Luntz) et al., (1992)) with the threshold response at 0.05 /zg ( P < 0 . 0 5 ) (Fig. 2). When incorporated into carboxylate glasses the pheromone also brought about a positive response (Fig. 2). The threshold response for acetate glass was 75 /zg pheromone/glass disc (P <0.05). Propanoate glass, containing 1000 /zg pheromone/ glass disc almost

40

-3

-'1

;

i

I

3

Log [pheromone ](pg) Ftg 2. Response of Culex qu,nquefasctatus to ovlposition pheromone either alone or incorporated into different carboxylate glasses. Pheromone loading is expressed as log lpheromone] per glass disc Response is measured as the number of egg rafts in the test bowl agmnst total number of egg rafts laid (n = 5-11 ) Control response ( ac SEM) m the absence of pberomone is shown by continuous and dashed lines

148

J.A Blatr et al /Journal of Controlled Release 31 (1994) 145-149

Table 2 Formulation of the more resistant carboxylate glasses for the release of Culexqumquefasctatusoviposltmn pheromone Time (Weeks)

Pheromone per sample (/zg)

Means of application~

Mean % egg rafts laid in test bowl (+SEM)

No of rephcates

Range in n u m b e r of egg rafts lind

Slgmflcance 2 P

t t t t p

50 9 5:5 07 50 9 5:5 05 55 8 5:3 94 60.7 4-7.68 68 9 5:2 43

4 4 3 3 3

22-62 22-36 22-53 11-30 20-51

NSD NSD NSD NSD < 0 05

t t t p

48 3 +4 32 57 2 + 4 76 48 8 + 4 79 66.4 + 2 64

6 5 6 8

21-210 17-107 42-245 10-217

NSD NSD NSD < 0 01

A Propanoate glass

1 2 5 6 6

250 250 250 250 250

B Mixed-anion glass

1 2 3 3

4200 4200 4200 250

1 t = tablet; p = powdered glass 2 Test preference compared to glass alone, Students t-test NSD = No stgnificantdifference release of pheromone, however, occurred from this mixed-anion matrix, even up to 21d after exposure to a humid environment (Fig. 1), explaining the observed nil behavioural response. 3.3. Response to pheromone with time

With naturally-produced pheromone, no significant loss of activity occurred from the apical droplet within 24 hours of the egg being laid [9] and the apical droplet material remained active for up to 21 days at 21°C in light [ 10]. A total loss in response to 0.1 ~ g synthetic

9oI i

/ /

/-/.,

{

\\

~_ 7o= IO0~g Igtass ~* 6 o

so ~0

" I pheromoneatone 110

\~------~ 2~

7s,~g ,grass

310

Time l days} Fig 3. Controlled release of Culex qumquefasctatus ovlpositlon pheromone wdh time when incorporated into acetateglass Response measured as the number of egg rafts in the test bowl against the total

number of egg rafts laxd ( n = 6)

pheromone presented on glass occurred after one week with activity being lost irrespective of whether light was present or not. Encapsulation m glass, however, conferred long term stabihty of the synthetic pheromone as demonstrated with acetate glass (Fig. 3 ). Pheromone incorporated mto this glass at 75 and 1 0 0 / z g / glass disc was released for a significantly longer period of time when compared with 0.1 /zg free pheromone. The 75 and 100/zg loaded glasses, after 14 days, produced significant responses when compared with 0.1 /zg free pheromone after 7 days ( P < 0 . 0 5 and 0.02 respectively). The degree of loading of the glass was important in the overall effectiveness of the pheromone, with the response receding more quickly at the lower incorporation of 7 5 / ~ g per disc. After 21 days the 100 /xg loaded glasses produced a significant response compared with the 75 /~g loaded glasses (P<0.05). With propanoate and mixed-anion glasses significant behavioural responses occurred only after the tablets were powdered, confirming that these less hygroscopic glasses have the potential for long-term controlled release with the capacity to hold high concentrations of pheromone. Thus, the mosquito oviposition pheromone can be successfully incorporated into carboxylate matrices with release occurring by a mixture of erosion and diffusion. Repeated extraction from separate samples

J A Blatr et al. / Journal of Controlled Release 31 (1994) 145-149

of pheromone-doped glass revealed that the pheromone was evenly dispersed throughout the matrix. During encapsulation, some pheromone was lost to the atmosphere through evaporation; such losses might be reduced by recently developed carboxylate glasses with much lower softening temperatures [3]. Also, improved experimental techniques now lead to much improved encapsulation efficiency [ 3 ]. It is suggested that such matrices, with their large loading capacity, may be suitable hosting materials for a wide variety of thermally unstable molecules.

Acknowledgements W e thank Professor J.A. Pickett, IACR, Rothamsted Experimental Station, Harpenden, Herts., for the kind provision of synthetic oviposition pheromone and the~3C N M R analyses; also Dr M.M. Cameron, Department of Zoology, University of Oxford, South Parks Road, Oxford, for the provision o f mosquito eggs and advice on culture techniques. JAB was supported by an Aberdeen University Research Studentship.

References [ 1] Blmr, J.A., Duffy, J A, Harston, P. and Wardell, J L Acetate glasses for the slow release of thermally unstable materials Glass Tech, 30 (1989) 190-191.

149

[2] Blair, J.A, Duffy, J A. and Wardell, J L A new glass for hosting organic compounds with opUcal, electronic and biological applications Phys Chem. Glasses, 33 (1992) 191198 [3] Blmr, J A. Development and apphcatlon of novel metal carboxylate glass matrices, (1992) PhD thesis, Umverslty of Aberdeen. [4] Jutsum, A R. and Gordon, R.F S (Eds), (1989) Insect pheromones m plant protection, John Wiley and Sons, Chlchester [5] Laurence, B.R. and Pickett, J A Ervthro-6-acetoxv-5hexadecanohde, the major component of a mosquito oviposition attractant pheromone J Chem. Soc, Chem Commun, ( 1982)59--60 [6] Dawson, GW, Mudd, A, Pickett, J A , Pile, MM and Wadhams, L.J Convenient synthes~sof mosquito ovtposmon pheromone and a highly fluorinatedanalog retaining biological acUvity J Chem. Ecol., 16 (1990) 1779-1789 [7] OUeno, W A, Onyango, T O, lhle, M M, Laurence, B.R, Dawson, G W, Wadhams, L J and Pickett, J A A field trial of the synthetic ovtposttmn pheromone w i t h Culex qumquefasctatus Say (Diptera' Cuhcldae) in Kenya Bull ent Res., 78 (1988) 463--478 [8] Mordue (Luntz), A J., Blackwell, A, Hansson, B.S, Wadhams, L.J and Pickett, J.A. Behavtoural and electrophyslolog~calevaluation of oviposmon attractants for Culex qumquefasctatus Say (Diptera Cuhctdae). Expenentia, 48 (1992) 1109-111t [9] Laurence, B.R and Pickett, J A An ovlpostUon attractant pheromone in Culex qumquefasctatus Say (Diptera Cuhcldae) Bull ent Res., 75 (1985) 283-290. [ 10] Bruno, D W and Laurence, B R.. The influence of the apical droplet of Culex egg rafts on ovlposmon of Culex pipiens fangans (Diptera' Culicidae) J Med. Entomol, 16 (1979) 300-305. [11] Kydonieus, A.F. and Beroza, M (Eds), (1982) Insect suppression with controlled release pheromone systems, Vol 1, CRC Press, Boca Raton