Liposomes as an agrochemical tool: optimization of their production

INDUSTRIAL CROPS ANDPRODUCTS AN INTERNATIONAL

JOURNAL

Industrial Crops and Products 5 (1996) 203-208

Liposomes as an agrochemical tool: optimization of their production Miquel Pons, Joan Estelrich * Unitat de Fisicoquhica,

Facultat de Farmricia, Universitat de Barcelona, 08028 Barcelona, Spain

Received 29 February 1996; accepted 11June 1996

Abstract Liposomes are structures made of lipid bilayers entrapping an aqueous solvent. Their unique properties have triggered numerous applications in several scientific and technological fields. In agrochemicals, liposomes can be used to improve and to deliver some essential nutrients. For this purpose, liposomes have to be manufactured in large quantities by means of economical processes. Factorial design allows studying, with few experiments, the influence of any component present in the formulation. We have studied the influence of different component on the homogeneity and stability of liposomes produced by a method susceptible to be scaled-up. the efficacy of biocides

Keywords:

Liposome;

Controlled

delivery; Optimization;

Scaling-up;

1. Introduction

Liposomes are structures made of lipid bilayers forming one or more concentric spheres which entrap part of the solvent in which they freely float, into their interior. Their unique properties have triggered numerous applications in various fields of science and technology, from basic studies of membrane function to the use as carriers of very different substances. In agriculture, they can be used to improve the efficacy of different biocides and to deliver some essential nutrients (Lasic, 1993). Herbicides, fungicides and pesticides are rapidly washed from the leaves of plants and encapsulation in liposomes may prolong the action of these agents on plants and reduce the damage in soil cultures. In the case of oil or other hydrophobic liquid wastes spilled on soil, the addition of liposomes suspension can reduce the *Corresponding author. Fax: +34 (3) 402-I 886.

Stability

surface tension and allow oil droplets to detach from mineral surfaces and exit capillaries. Also, liposomes broaden the spectrum of microorganisms which can adhere to oil and other wastes and, therefore, facilitate their biodegradation. The use of liposomes in agriculture requires the manufacture of large quantities of them by an economical process. Furthermore, the final product may be stable in order to allow its storage and transport. We have described (Pons et al., 1995) a method for large scale production of liposomes. This method provided a suspension of vesicles with an average diameter that ranged from 280 to 350 nm and with a high physical stability. However, this method, as any manufacture process, is susceptible of optimization, that is, of determining the experimental conditions that give an optimal performance. Regarding the liposome production, the optimal performance can be defined in terms of homogeneity and stability of the vesicle population. The search for optimum

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M. Pans, J. Estelrich /Industrial

conditions of a process is usually done measuring the desired response on varying the casual variables one at time, while keeping other factors constant. This strategy is largely insufficient to explore the experimental domain, since the results obtained from such studies do not provide any information about the potential interactions and their significance, if any. Therefore, multivariate approaches are needed to evaluate the effects of all the variables as well as the effects of their interactions. These objectives can be fulfilled by using factorial design (Box et al., 1978). This statistical technique achieves the stated objective as accurately and efficiently (i.e. with few experiments) as possible. In the present work we have studied the effect of different formulation components on the homogeneity and stability of the liposomes produced by high speed dispersion method. In order to check the influence of four variables (lipid/cholesterol ratio, glycerin, poloxamer and lipid concentration) on size and polydispersity (as criteria of homogeneity) and stability a factorial design was used.

Crops and Products 5 (1996) 203-208

conditions for at least 6 months. In the emulsification step, the desired amounts of lipid and aqueous phases were warmed at 50°C. After reaching this temperature, the aqueous solution was added to the lipid mixture and homogenized at 3000 rpm for 2-3 min by an electrical homogenizer. Aqueous phase used was a solution of sodium chloride 0.9% with the desired amount of glycerin. This method provided a very viscous multilamellar vesicles (MLVs) liposome suspension. 2.3. Size determination

The lipid used was Lipoid S- 100 (Lipoid KG, Ludwigshafen, Germany), a mixture of lipids whose main component is soybean phosphatidylcholine (>94%). Cholesterol, Poloxamer 188 (Surfoxid 7068) and other reagents were of analytical reagent grade.

Diameter and polydispersity of vesicles were determined by photon correlation spectroscopy which analyzes the fluctuations in scattered light intensity generated by diffusion of vesicles in solution. An Autosizer IIc (Malvern Instruments, UK) consisting of a 5 mW, 623.8 nm, helium-neon laser irradiating the scattering cell placed inside a temperature regulated housing was used. Data acquisition was via a Malvem 7032-N, 72 channel multibit correlator. Experimental conditions were: temperature 25°C; reference angle 90; viscosity 0.899 x 10e3, 2.858 x 10e3 or 1.089 x lop2 Pa s; refractive index 1.330, 1.375 or 1.411, depending on the amount of glycerin in the sample. The exponential sampling method (Dahneke, 1983) was used for data analysis. Samples were diluted accordingly, prior to size analysis, with the same aqueous solution used to prepare them. Size and polydispersity were checked during 3 months.

2.2. Liposome production

2.4. Stability determination

The method involves two separate steps: the production of lipid phase and the emulsification process. To obtain lipid phase Lipoid SlOO, poloxamer 188 and cholesterol were dispersed in ethanol and warmed at 50°C with stirring until total dissolution. After solving the mixture, the temperature was kept at 50°C to evaporate the ethanol until the product looked like a gel. Then glycerin, previously warmed, was added and stirred at 3000 rpm to obtain an homogeneous mixture. Final glycerin concentration should be 70-75% (w/w). The final lipid mixture could be stored at 4°C under nitrogen atmosphere until its use. The lipid gel remains stable under this

As liposome suspensions are colloidal dispersions, their physicochemical behaviour is similar to that presented by the emulsions. In this way, the parameters checked to measure the stability of emulsions will be suitable for studying the stability of liposomes. Given that the first signs of unstability of an emulsion are the creaming and the sedimentation, such parameters have been chosen to determine the stability of different liposome preparations. Creaming has been studied 7 days after the preparation of liposome suspension by observation of macroscopic appearance, measurement of conductivity and stability to centrifugation.

2. Experimental procedures 2.1. Materials

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Crops and Products 5 (1996) 203-208

Conductivity was measured at the top and at the bottom of the liposome suspension sample keeping it right position for 1 week at 4°C. A significant difference between both values is a sign of unstability (Sagon, 1983). Measurements were made with a micro CM 2202 conductimeter (Crison, Switzerland). Stability to centrifugation was determined sequentially at 2000, 3000 and 4500 rpm for 15 min in a Sigma 301 centrifuge (Sigma, USA). The step in which creaming or sedimentation appeared was recorded (Fonteneau et al., 1976). 2.5. Evaluation

of the response

Size, polydispersity, creaming, conductivity and stability to centrifugation were the parameters checked for three months. In order to unify the individual responses, an arbitrary system of quantification was used. In this way to quantify the variation in size and polydispersity the scores shown in Table 1 were used. Data were averaged according to the following schedule: Block A: Average of data of ten first days Block B: Average of data of following twenty days Block C: Average of data of the second month Block D: Average of data of the third month Table 1 Score used to quantify

in size and polydispersity

Size (nm)

Polydispersity

Score

t300 300-400 400-500 500-750 750-1000 > 1000

o-o.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5-0.6 20.6

5 4 3 2 1 0

Table 2 Selected variables

and levels for the experimental

Variable

X1 X2 X3 &

The final score was obtained averaging the values of the four blocks. Creaming or sedimentation were checked a week after preparing the sample. The existence of creaming or precipitated implied a score of 0 points, while their absence gave 5 points. The stability against centrifugation was determined in this manner: nor creaming neither precipitation at any centrifugation step. 3 points; creaming or precipitation at third centrifugation, 2 points; at the second, 1 point, and at the first, 0 points. Finally, the following values were assigned for the conductivity differences: no differences or differences less than 0.05 mS/cm, 3 points; from 0.05 to 0.1 mS/cm, 2 points: from 0.1 to 0.2 mS/cm, 1 point, and higher than 0.2 mS/cm, 0 points. 2.6. Statistical treatment of the results Response (Y) of each individual liposome preparation is the result of the individual influence of the four variables, of their quadratic effects and of their interactions. Hence the response is modelled by the following polynomial model: Y=bo+blxl+b2X2+b3X3+b4Xq+bl,X: +

b22X;

+

b33X32+ bMx; + buxlx2

+ bwx1.a + bzxm the variation

Lipoid : cholesterol Glicerin in aqueous solution Poioxamer % (w/w to lipid) Final lipid concentration

205

+ bnxlx3

+ b24x2x4 +634x3x4

where Xi is the linear effect of ith variable; xi’ is the quadratic effect of ith variable; Xixj is the interaction of ith and jth variables; bo is the independent term, and bi, bii and bij are the regression coefficients associated with linear, quadratic and interaction equation variables. Experimental conditions of the four parameters tested are summarized in Table 2.

design

Level -1

0

+l

1oo:o 0% 0% 18 mh4

66~33 25% 7.5% 36 mM

50:50 50% 15% 72 mM

M. Pans, .I. Estelrich/Industrial

206

Table 3 Box-Behnken experimental design and response values obtained Run

Xl

I II

0

III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII XIX xx XXI XXII XXIII XXIV xxv XXVI XXVII XXVIII XXIX xxx xxx1 xxx11 XXXIII- 1 XXXIII-2 XxX111-3 XxX111-4

0 0

0

0

0 0 0

-1 +l -1 -1 +l +l -1 +l -1 +l -1 -1 +l +l -1 +l -1 +l -1 -1 +l +l -1 +l 0 0 0 0

X2

x3

x4

Resp.

-1 +l -1 -1 +1 +l -1 +I 0 0 0 0 0 0 0 0 -1 -1 +l -1 +l -1 +l +l -1 -1 +l -1 -1 -1 +l +l 0 0 0 0

-1 -1 +l -1 +l -1 +l +l -1 -1 +I -1 +1 -1 +I +l 0 0 0 0 0 0 0 0 -1 -1 -1 +1 -1 +1 +1 +1 0 0 0 0

-1 -1 -1 +l -1 +l +l +l -1 -1 -1 +l -1 +l +l +l -1 -1 -1 +I -1 +l +l +l 0 0 0 0 0 0 0 0 0 0 0 0

3 14 2 9 12

19 4 19 20 19 19 20 11 13 20 14 4 3 19 11 18 5 18 18 16 17 18 11 19 7 20 17 12 12 12 12

In order to determine the regression coefficients, the response Y has to be found by using different experimental combinations of the variables under consideration. Such combinations have been carried out according to the Box-Behnken factorial experimental plan (Box and Behnken, 1960) shown in Table 3. The experiments were performed in random order, not as they are reported in the table. The significance of each regression coefficient was estimated through a test of F-ratios (analysis of variance) of the model by means of the software Statgraphics 7.0 (Statistical Graphics Corp., USA).

Crops and Products 5 (1996) 203-208

Nonsignificant coefficients were omitted and recalculations were done. 3. Results Response values are listed in Table 3. As it can be seen, the preparations that afforded the best results were the following: IX, XII, XV and XxX1, their composition being shown in Table 4 Coefficients of multivariate regression were calculated from response values of the thirty three formulations checked. First analysis showed the significance of main effects, second order interactions and quadratic terms, and those effects with a low significance level (p > 0.05) were excluded and data reanalysed. The second ANOVA showed that, except the final lipid concentration, that is variable x4, all variables and the corresponding quadratic terms, exerted a marked influence on the liposome stability, while second order interactions were negligible (Table 5). Regression coefficients of the polynomial model were calculated using only the significant variables (Table 6). As an exception, final lipid conTable 4 Composition of optimum liposome formulations Formulation IX XII xv XXX1

Lipoid : CHOL

%

%

Glicerin

Poloxamer

Lipid concentration

100:0 100:0 100:0 100:o

25 25 25 50

0 0 15 15

18 mM 72 mM 72 mM 72 mM

Table 5 Analysis of variance corresponding to multiple regression of Box-Behnken experimental design Source

Degree of freedom

Mean square

F-ratio

Probability

1 1 1 1 1 1 1 28

48.1667 590.0417 37.5000 32.6667 128.0000 55.1250 40.5000 7.8839

6.11 74.84 4.76 4.14 16.24 6.99 5.14

0.0198 0.0000 0.0377 0.0514 0.0004 0.0133 0.0313

I&or

48.1667 590.0417 37.5000 32.6667 128.0000 55.1250 40.5000 220.7500

Total

1152.7500

35

Xl x2 x3

2 $

Sum of squares

M. Pons. J. Estelrich /Industrial

Crops dttd Products 5 (1996) 203-208

207

8,65 2

4

6

8

0

Relative effect Fig. 1. Pareto plot corresponding to multiple regression of Box-Behnken experimental design. Table 6 Regression coefficients of the multivariate analysis Variable

Coefficient

constant X1

11,1667 -1.4167 4.9583 -1.2500 1.1667 4.0000 -2.6250 2.2500

x2 x3

x4 3 3

centration was not excluded in spite of its significant level of 0.05 14, since it represents a main effect. Fig. 1 illustrates the Pareto plot corresponding to the multivariate regression analysis, showing the relative effect of variables. Among the studied parameters, glycerin concentration showed the major effect on stability. Glycerin acts increasing the viscosity and, thereby, decreasing liposome mobility. Consequently, the aggregation of vesicles is less likely to happen and creaming or sedimentation process slows up. On the other hand, although final lipid concentration did not exert an important effect according to the variance analysis carried out, it has been com-

monly stated than the higher the lipid concentration in a liposome suspension, the better stability is. This statement is confirmed by the positive sign of the corresponding coefficient in regression. Cholesterol concentration showed a slight effect on suspension stability, the corresponding regression coefficient having a negative sign. This fact implies a destabilization, so the cholesterol absence enhances the stability of liposome suspension. Other negative sign was found for poloxamer concentration, but with a lesser effect. In this way, liposomes of a well defined size under 200 nm can be produced in absence of nonionic surfactants, contrarily to the opinion of Matsumoto et al. (1986). In the present paper we have described the optimization of a cheap and easy method of preparing liposomes. Among the studied formulations there are some with a high stability and this property makes them very suitable to be used as an agrochemical product. References Box, G.E.P. and Behnken, D.W., 1960. Some new three levels designs for the study of qualitative variables. Technometrics, 2: 445-45 1.

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Box, G.E.P., Hunter, W.G. and Hunter, J.S., 1978. Statistics for Experimenters. An Introduction to Design, Data Analysis and Model Building. Wiley, New York, NY. Dahneke, B.E., 1983. Measurement of suspended particles by quasi-elastic light scattering. John Wiley and Sons, Inc., New York, NY. Fonteneau, A., Laquais, B. and Cotty, .I., 1976. Relation entre la composition d’une emulsion et sa viscosite. II Farmaco Ed. Pr., 31: 295-321. Lasic, D.D., 1993. Liposomes from physics to applications. El-

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sevier, Amsterdam, pp. 507-5 16. Matsumoto, K., Shinozaki, K. and Tabata, Y., 1986. Process for the preparation of liposome. European patent EP 0 220 797 A2. Pons, M., Lizondo, M., Gallardo, M., Freixas, J. and Estelrich, J., 1995. Enrofloxacin loaded liposomes obtained by high speed dispersion method. Chem. Pharm. Bull., 43: 983-987. Sagon, J., 1983. Controle et conditionement des emulsions. In: F. Puisieux (Editor), Agents de Surface et Emulsions. Technique et documentation, Paris, pp. 439-440.