Journal of Microencapsulation Micro and Nano Carriers
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20
A contribution to the evaluation of microcapsules by light microscopy M. Dittrich & L. Melichar To cite this article: M. Dittrich & L. Melichar (1990) A contribution to the evaluation of microcapsules by light microscopy, Journal of Microencapsulation, 7:4, 527-540 To link to this article: http://dx.doi.org/10.3109/02652049009040476
Published online: 27 Sep 2008.
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Date: 07 November 2015, At: 09:46
J . MICROENCAPSULATION,
1990,
VOL.
7,
NO.
4, 527-540
A contribution to the evaluation of microcapsules by light microscopy
Downloaded by [Australian National University] at 09:46 07 November 2015
M. DITTRICH, L. MELICHAR and V. S E M E C K Y ? Faculty of Pharmacy, Charles University, Department of Pharmaceutical Technology, ak. Heyrovskeho 1203, 501 65 Hradec KralovC, Czechoslovakia t Faculty of Pharmacy, Charles University, Department of Pharmacological Propedeutics, ak. Heyrovskeho 1203, 501 65 Hradec KralovC, Czechoslovakia (Received 2 January 1990; accepted 13 February 1990)
Light microscopy has been used for the evaluation of the internal and external structure of dry microcapqules. The method involves surface and penetrative staining with various dyes after which the microcapsules were embedded in suitable optically translucent material. Using this method the core material, its shape and position within the microcapsules either in total or as subunits of the core are clearly distinguishable from the wall material. The surface characteristics of the microcapsules can be observed with either light or fluorescent microscopy after staining with a fluorescent dye. Furthermore, it is a relatively simple and inexpensive method by comparison with the scanning electron microscopy. The natural character of microcapsules, without any artificial structures, has been maintained. It could serve as a routine auxiliary method for complex evaluation or control of the microencapsulation process and its optimization.
Introduction Microcapsules, although small particles, comprise a wide variety of structures. By definition, they consist of a distinguishable core and wall, the former being variable in size, shape, position and number. T h e thickness and homogeneity of the wall is of primary importance for the quality control of microcapsules, as influences the kinetics of drug release. Though the overall dissolution of the drug from microcapsules is a statistical mean of individual distributions (Donbrow et al. 1986, Benita et al. 1988), the methods available for direct evaluation of this typical property of the structure have a meaningful place in their formulation. Scanning electron microscopy (SEM) is considered to be useful method for the observation of the surface of microcapsules. Samples of dry microcapsules are covered with a very thin layer of suitable metal. T h i s technique has been used to study the possible influence of various coacervating agents on the surface morphology of gelatin microcapsules (Nixon and Matthews 1975), for the shape of aggregated microcapsules prepared from theophylline and ethylcellulose (Lin and Yang 1985), after the microencapsulation of hormones by an emulsion/solventevaporation method, in order to reveal possible errors in the preparation procedure (Tice and Gilley 1985), to give evidence of wall integrity for microcapsules made from ethylcellulose after they have been subjected to dissolution studies in media of various acidities (Forni et al. 1988). T h e internal structure of microcapsules 0265-2048/90 $3.000 1990 Taylor & Francis Ltd.
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prepared from acrylic resins, with ketoprofen as core material, was observed after slicing with a blade (Goto et al. 1986). Transmission electron microscopy (TEM), on the other hand, has been used only rarely for screening the internal structure of microcapsules (Wright et a1 1988). Both the methods (SEM, T E M ) are time-consuming and sometimes of low information value. Light microscopy ( L M ) was used as an alternative method to give evidence that electron microscopy did not show artificial structures which could arise during the sample adjustment of gelatin microcapsules (Matthews and Nixon 1974). I t was concluded that both S E M , and L M are reliable methods. T h e complementary character of both methods was employed to study the integrity of the surface of polyacrylate microcapsules before and after dissolution (Benita et al. 1985). T h e internal structure of poly(D, L-lactic) acid microcapsules with a neuroleptic drug was screened after breaking them down and freeze etching. Photomicrographs obtained by SEM were compared with those obtained by polarization microscopy (Suzuki and Price 1985). T h e microscopic observation of sections of microcapsules prepared with a microtome was used to verify the results of the indirect (mathematical) method of computing the wall thickness (Si-Nang et al. 1973). T h e advantage of LM is in the relatively gentle manipulation of labile structures required. L M may be successfully used during optimization of the microencapsulation procedure. So-called ‘native’ microcapsules, with semisolid or swollen walls, have been observed and evaluated in coacervating media, both hydrophilic (Newton et al. 1977, Okada et al. 1985, Kaser-Liard 1985), and hydrophobic (Koida et al. 1986, Leelarasamee et al. 1988). L M has been used as a convenient tool for the observation of the dissolution process from single microcapsules in aqueous media. Coloured core materials are readily visible, but highly opaque wall material makes the method difficult. Even so, the method has been adapted for dark field phase contrast and fluorescent microscopy (FM) (Hoffman et al. 1986). Routine methods for converting biological tissues into permanent translucent material have been used to provide visualization. T h e finest structures are recognizable in such preparations, which are usually fully translucent. T h e present work deals with the method of making permanent preparations of microcapsules using the technique of embedding into a layer of rigidizable translucent material. T h e method of surface and penetration staining of microcapsules was developed.
Experimental Materials Gelatin (isoelectric point 5.1), Rousselot, France; cellulose acetate phthalate (CAP), Eastman Kodak, USA; ethylcellulose (Ethocel N 10) (EC), Dow Chemicals, USA; acrylic resin (Eudragit RS), Rohm-Pharma, FRG; Entelan, Merck, F R G ; sulphathiazol, Chemopharma, CSFR; potassium chloride and dyes, Lachema, CSFR. Dyes used were: Fluorescein Rhodamine B Eosine Y
C.1. 45 350 C.I. 45 170 C.I. 45400
Evaluation of microcapsules by LM Methylrosaniline chloride Methylene blue Malachite green
529
C.I. 42 510 C.I. 52015 C.I. 42000
Other chemicals were of analytical purity
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Methods Preparation of microcapsules. A. Three different methods of preparing microcapsules with a core of sulphathiazol were used. T h e wall material consisted of the same proportions of gelatin and CAP (used as potassium or triethanolamine salt).
1. Complex coacervation. Coacervation of equal amounts of 1.5 per cent aqueous solutions of both polymers was induced at 50°C by gradual addition of hydrochloric acid (0.1 moll-') to give a final p H of 3.8. 2. Simple coacervation. Desolvation of a 3 per cent total colloid concentration of equal amounts of both polymers in water was induced at 50°C by gradual addition of 20 per cent solution of sodium sulphate in water. T h e temperature was maintained far above the gelation point of gelatin. 3 . Emulsion method. This method is based on dispersion and encapsulation of the core material with the 1 5 per cent solution of both polymers in water in the continous phase (light mineral oil). T h e temperture was maintained at 55°C and the solidification of the wall material was induced by gradual cooling. Three variants of this method were investigated: (a) A suspension of the core material in the polymer solution was added dropwise to the stirred continuous phase. This produced a suspension in oil ( s - 0 ) . (b) A solution of both polymers was added dropwise into the stirred suspension of the core material in the continuous phase (w-s). (c) T h e suspension of core material in a part of the continuous phase was added dropwise to the vigorously agitated emulsion (s-e).
B. Microcapsules with EC were prepared using the method of phase separation of EC from cyclohexane solution at elevated temperature by gradual cooling. Potassium chloride was dispersed in the solution at elevated temperature (70°C) prior to the phase separation. C. Microcapsules with Eudragit R S were prepared according to the method of Goto et al. (1986). A 3 per cent concentration of magnesium stearate was used as emulsifier. Potassium chloride was suitable core material in this case. Staining of the microcapsules. A. Surface staining: Samples of microcapsules were washed on filter paper with a 0.5 per cent solution of fluorescent dye and rinsed immediately with purified water before drying at room temperature. T h e best results were achieved with an aqueous solution of eosin Y which also gave good results as to the permanence of the staining.
R . Penetration staining: Samples of dry microcapsules were immersed in stain for 10-60min depending on the size of microcapsules and the quality of the wall material. T h e best stains were 0.5 per cent aqueous solution of eosin Y or 0.3 per cent solution of eosin Y in 40 per cent aqueous ethanol. T h e use of desaturated solution of methylrosaniline chloride in a 5 per cent aqueous solution of sodium sulphate also
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gave good results. After staining the samples were washed and dried as for the surface treatment.
Theformation of permanent microscopic preparations. Stained microcapsules were embedded either in media with acceptable physical properties used in the L M method for biological samples, or media used for the first time in the present work. Entelan, plasticized solution of polystyrene in chloroform (12 per cent), solution of Eudragit RS in dimethylketone (15 per cent), and an aqueous solution of sodium silicate (40 per cent) were tested for this purpose. Photomicrographs were prepared using an ‘Amplival’ microscope (Carl Zeiss, Jena, GDR) at a magnification of 60 to 400. Medium light sensitivity photographic material (negative and inverse) was used for documentation.
Results and discussion Method of surface staining of microcapsules Surface stained samples of microcapsules were observed in the light and fluorescent microscopes. Microcapsules prepared b y complex coacervation were embedded in ‘Entelan’, but they were not sufficiently optically translucent because of the considerable heterogeneity of the complex coacervate (figure 1 ( a ) ) . Surface staining with eosin Y made the polymeric material easily distinguishable from the core. This showed clearly that coacervate did not adhere well to the crystals of sulphathiazol (figure 1 (b)). The colour of the coacervate and sulphathiazol in fluorescent light was more pronounced being yellow or orange, respectively. Simple coacervate, on the other hand, is optically translucent, though prepared from the same materials (figure 2 ( a ) ) .I n figure 2 (b) there is the same structure in the light microscope after the staining with methylrosaniline chloride and embedding into ‘Entelan’. With the use of penetration staining it was possible to determine the deposition of the wall material which resulted in the inner layer consisting of gelatin the outer layer mainly of polysaccharide.
Method of penetration staining of microcapsules Figure 3 ( a ) shows microcapsules photographed using light microscopy. It is possible to distinguish their shape only and, having a surface charge, they exist mainly in aggregates. After immersion in water they swell because of the hydrophilic nature of both polymers. Simultaneously they become translucent. In figure 3 (b) there are dark spots of core material and marginally more optically transmitting swollen wall material. This is the stage of partial restitution of the ‘native’ microcapsules. By embedding dry microcapsules in translucent polymeric material (e.g. ‘Entelan’) it is possible to observe the exact structure of the microcapsules (figure 3(c)). The position of the core, integrity and wall thickness could be evaluated. In the case of multinuclear microcapules, fusion of the core into one compact unit is seen. According to the depth of focusing it is possible to observe either the internal wall structure, or its surface. At the same time individual core units are recognizable. T h e slide was prepared by penetration staining prior to embedding in ‘Entelan’ (figure 3 ( d ) ) . Microcapsules made from ‘Eudragit RS’ and potassium chloride were much more transparent after embedding in ‘Entelan’ (figure 4 ( a ) ) . Because the optical density of potassium chloride is similar to that of the acrylic polymer, the core material was not distinguishable (figure 4 (b)). On the other hand, pores and cracks
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Figure 1 . Photomicrographs (LM) of micrographs prepared by complex coacervation (sulphathiazol, mixture of gelatin and triethanolamine salt of CAP). ( a ) Unprepared microcapsules (bar = 200pm). b) Microcapsules stained on surface with eosin Y (FM) (bar = 200 pm).
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(b) Figure 2. Photomicrographs (LM) of microcapsules prepared by simple coacervation (sulphathiazol, mixture of gelatin and triethanolamine salt of CAP). ( a ) Microcapsules stained on the surface with eosin Y (FM) (bar=200pm). ( b ) Microcapsules after penetration staining with saturated solution of methylrosaniline chloride in 5 per cent aqueous solution of sodium sulphate (bar = 100 pm).
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Figure 3. Photomicrographs (LM) of microcapsules prepared by emulsion method (sulphathiazol, mixture of gelatin and potassium salt of CAP). ( a ) Unprepared microcapsules (bar = 300 pm). (b) Swollen microcapsules dispersed in water (bar = 500 pm). (c) Microcapsules embedded in ‘Entelan’ (bar = 300 pm). ( d ) Microcapsules stained with eosin Y 0 5 per cent aqueous solution for 30min (bar=500pm).
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(c)
Figure 4. Photomicrographs (LM) of microcapsules prepared by the emulsion/solventevaporation method (potassium chloride, ‘Eudragit’ RS). (a) Unprepared microcapsules (bar =400 pm). (b) Microcapules embedded in ‘Entelan’ (bar = 500pm). (c) Microcapsules stained with eosin Y 0.3 per cent in 40 per cent aqueous ethanol for 20 min embedded into ‘Entelan’ (bar = 300pm).
Figure 5. Photomicrographs (LM) of microcapsules prepared by phase separation method (potassium chloride, ethylcellulose). Microcapsules embedded in sodium silicate glass (bar=400 pm).
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(4 Photomicrographs (LM) of microcapsules prepared by emulsion method-three variants of the procedure. (a)Variant s-o (bar =400pm).(6) Variant w-s (bar = 400 pm). (c) Variant s-e (bar = 400 pm).
Figure 6.
are visible on the surface. Penetration staining with eosin Y makes the core material clearly visible. We presume that salting out of the dye on to the surface of the core is the main mechanism for this effect. T h e precipitation of a layer of dye by the saturated solution of potassium chloride renders the surface of the core distinguisable (figure 4 ( c ) ) . An aqueous solution of sodium silicate was introduced as an embedding material in the case of ethylcellulose microcapsules (figure 5). T h e fluidity of the supporting material remained for several hours and is the main disadvantage. Evaporation of water during storage of the slides caused the formation of crystalline silicate and subsequent damage as well as a somewhat opaque preparation.
The influence of preparative variations of the emulsion method on the structure of the products We have used three variations for mixing the core and wall materials in the continuous phase. In the first case ( s - 0 ) there are numerous cavities in the wall of the microcapsules. They are not filled with light liquid paraffin as shown by analysis of the crushed product and of the crushed product, previously washed with hexane. They are probably bubbles of air that originated in the course of dispersion of sulphathiazol in the viscous solution of polymers (figure 6 ( a ) ) . An homogeneous wall without any bubbles and with a centrally arranged core is a typical feature of the second variant (w-s). Empty microcapsules also exist because of the poor adhesion of the wall material on the core (figure 6 ( b ) ) . The last variant of this method is characterized by dropwise addition of the suspension of the core material into a vigorously stirred emulsion of wall material in the continuous phase (s-e). Immediately after dispersion the velocity of stirring was
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slowed to approximately 400 r.p.m. As seen in figure 6 (c), the core material was dispersed over the whole interior of the particles. A large number of cavities existed filled with oil which was extractable with hexane.
Conclusions T h e above methods are restricted to the evaluation of microcapsules with a relatively low proportion of core material. Furthermore, the wall material has to be sufficiently optically homogeneous. T h e main advantage of light microscopy by comparison with scanning electron microscopy is its simplicity. Though having considerably lower differentiation ability, the observed structures are more reliable and during sample preparation no artificial structures are produced. According to the depth of focusing, either the shape and position of the core, or the structure of the wall and its surface characteristics may be observed. T h e method could be used as a routine for the control and optimization of the microencapsulation procedure. Moreover, actual observation with the microscope gives more information in comparison with photographs which are intended mainly for documentation purposes. References BENITA,S., HOFFMAN, A., and DONBROW, M., 1985, Microencapsulation of paracetamol using polyacrylate resins (Eudragit Retard), kinetics of drug release and evaluation of kinetic model. Journal of Pharmacy and Pharmacology, 37, 391-395. RENITA,S., BABAY,D., HOFFMAN, A,, and DONBROW, M., 1988, Relation between individual and ensemble release kinetics of indomethacin from microspheres. Pharmaceuticaf Research, 5 , 178-1 82. DONBROW, M., HOFFMAN, A., and BENITA,S., 1988, Variation of population release kinetics in polydisperse multiparticulate systems (microcapsules, microspheres, droplets, cells) with heterogeneity of one, two or three parameters in the population of individuals. Journal of Pharmacy and Pharmacology, 40, 93-96. FORNI, F., COPPI,G., IANNUCCELLI, V., and BERNABEI, M. T., 1988, Papaverine hydrochloride release from ethyl cellulose-walled microcapsules. Journal of Microencapsulation, 5 , 139-146. Gow, S., KAWATA, M., NAKAMURA, M., MAEKAWA, K., and AOYAMA, T., 1986, Eudragit R S and RI, (acrylic resins) microcapsules as pH insensitive and sustained release preparations of ketoprofen. Journal of Microencapsulation, 3, 293-304. HOFFMAN, A,, DONBROW, M., and BENITA,S., 1986, Direct measurements on individual microcapsule dissolution as a tool for determination of release mechanism. Journal of Pharmacy and Pharmacology, 38, 764-766. KOIDA,Y., KORAYASHI, M., and SAMEJIMA, M., 1986, Studies on microcapsules. IV. Influence of properties of drugs on microencapsulation and dissolution behavior. Chemical and Pharmaceutical Bulletin, 34, 3355-3361. KASER-LIARD, B., 1985, Herstellung von Gelatine-Mikrokapseln unter Anwendung der Emulsions- Induktions-Technik. Pharmaceutica Acta Helvetiae, 60, 326-333. LEEI-ARASAMEE, N., HOWARD, S. A,, MALANGA, C. J., and MA,J . K. H., 1988, A method of the preparation of polylactic acid microcapsules of controlled particle size and drug loading. Journal of Microencapsulation, 5 , 147-1 57. LINSHAN-YANG, and YANCJUEI-CHYI, 1985, Effect of ehtylene-vinyl acetate concentration on ethylcellulose-walled microcapsules: preparation and release kinetics of theophylline microcapsules. Journal of Microencapsulation, 2, 3 15-325. MATTHEWS, R. R., and NIXON, J. R., 1974, Surface characteristics of gelatin microcapsules by scanning electron microscopy. Journal of Pharmacy and Pharmacology, 26, 383-384. NEWTON, D. W., McMur.12EN,J . N., and BECKER, C. H., 1977, Characteristics of medicated and unmedicated microglobules recovered from complex coacervates of gelatin-accacia. Journal of Pharmaceutical Sciences, 66, 1327-1 330.
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NIXON, J . H., and MATTHEWS, R. R . , 1975, The surface characteristics of gelatin coacervate microcapsules by scanning electron microscopy. MicroencapsuZation, edited by J . R . Nixon (New York: Marcel Dekker), pp. 173-183. J., KIISAI, A , , and UEDA,S., 1985, Factors affecting microencapsulability in simple OKADA, gelatin coacervation method. Journal of Microencapsulation, 2 , 163-173. S I - N A N GL., , CARI.IER, 1’. l:., DEI~ORT, P., GAZZOLA, J., and LAFONT, I)., 1973, Determination of coating thickness of microcapsules and influence upon diffusion. Journal of Pharmaceutical Sciences, 62, 452455. SUZIJKI, K., and PRICE,J . C., 1985, Microencapsulation and dissolution properties of a neuroleptic in a biodegradeable polymer poly( I>, 1,-lactide). Journal of Pharmaceutical Srienres, 74, 21-24. ’I‘IcE, T. R . , and GIILEY,R. M., 1985, Preparation of injectable controlled-release microcapsules by a solvent-evaporation process. Journal of Controlled Release, 2 , 343-352. WRIGHT, K. C., WALLACE, S., MOSIER, B., and MOSIER, D., 1988, Microcapsules for arterial chemoembolization: appearance and in-vitro drug release characteristics. Journal of Microencapsulation, 5 , 13-20.
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20
A contribution to the evaluation of microcapsules by light microscopy M. Dittrich & L. Melichar To cite this article: M. Dittrich & L. Melichar (1990) A contribution to the evaluation of microcapsules by light microscopy, Journal of Microencapsulation, 7:4, 527-540 To link to this article: http://dx.doi.org/10.3109/02652049009040476
Published online: 27 Sep 2008.
Submit your article to this journal
Article views: 9
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=imnc20 Download by: [Australian National University]
Date: 07 November 2015, At: 09:46
J . MICROENCAPSULATION,
1990,
VOL.
7,
NO.
4, 527-540
A contribution to the evaluation of microcapsules by light microscopy
Downloaded by [Australian National University] at 09:46 07 November 2015
M. DITTRICH, L. MELICHAR and V. S E M E C K Y ? Faculty of Pharmacy, Charles University, Department of Pharmaceutical Technology, ak. Heyrovskeho 1203, 501 65 Hradec KralovC, Czechoslovakia t Faculty of Pharmacy, Charles University, Department of Pharmacological Propedeutics, ak. Heyrovskeho 1203, 501 65 Hradec KralovC, Czechoslovakia (Received 2 January 1990; accepted 13 February 1990)
Light microscopy has been used for the evaluation of the internal and external structure of dry microcapqules. The method involves surface and penetrative staining with various dyes after which the microcapsules were embedded in suitable optically translucent material. Using this method the core material, its shape and position within the microcapsules either in total or as subunits of the core are clearly distinguishable from the wall material. The surface characteristics of the microcapsules can be observed with either light or fluorescent microscopy after staining with a fluorescent dye. Furthermore, it is a relatively simple and inexpensive method by comparison with the scanning electron microscopy. The natural character of microcapsules, without any artificial structures, has been maintained. It could serve as a routine auxiliary method for complex evaluation or control of the microencapsulation process and its optimization.
Introduction Microcapsules, although small particles, comprise a wide variety of structures. By definition, they consist of a distinguishable core and wall, the former being variable in size, shape, position and number. T h e thickness and homogeneity of the wall is of primary importance for the quality control of microcapsules, as influences the kinetics of drug release. Though the overall dissolution of the drug from microcapsules is a statistical mean of individual distributions (Donbrow et al. 1986, Benita et al. 1988), the methods available for direct evaluation of this typical property of the structure have a meaningful place in their formulation. Scanning electron microscopy (SEM) is considered to be useful method for the observation of the surface of microcapsules. Samples of dry microcapsules are covered with a very thin layer of suitable metal. T h i s technique has been used to study the possible influence of various coacervating agents on the surface morphology of gelatin microcapsules (Nixon and Matthews 1975), for the shape of aggregated microcapsules prepared from theophylline and ethylcellulose (Lin and Yang 1985), after the microencapsulation of hormones by an emulsion/solventevaporation method, in order to reveal possible errors in the preparation procedure (Tice and Gilley 1985), to give evidence of wall integrity for microcapsules made from ethylcellulose after they have been subjected to dissolution studies in media of various acidities (Forni et al. 1988). T h e internal structure of microcapsules 0265-2048/90 $3.000 1990 Taylor & Francis Ltd.
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prepared from acrylic resins, with ketoprofen as core material, was observed after slicing with a blade (Goto et al. 1986). Transmission electron microscopy (TEM), on the other hand, has been used only rarely for screening the internal structure of microcapsules (Wright et a1 1988). Both the methods (SEM, T E M ) are time-consuming and sometimes of low information value. Light microscopy ( L M ) was used as an alternative method to give evidence that electron microscopy did not show artificial structures which could arise during the sample adjustment of gelatin microcapsules (Matthews and Nixon 1974). I t was concluded that both S E M , and L M are reliable methods. T h e complementary character of both methods was employed to study the integrity of the surface of polyacrylate microcapsules before and after dissolution (Benita et al. 1985). T h e internal structure of poly(D, L-lactic) acid microcapsules with a neuroleptic drug was screened after breaking them down and freeze etching. Photomicrographs obtained by SEM were compared with those obtained by polarization microscopy (Suzuki and Price 1985). T h e microscopic observation of sections of microcapsules prepared with a microtome was used to verify the results of the indirect (mathematical) method of computing the wall thickness (Si-Nang et al. 1973). T h e advantage of LM is in the relatively gentle manipulation of labile structures required. L M may be successfully used during optimization of the microencapsulation procedure. So-called ‘native’ microcapsules, with semisolid or swollen walls, have been observed and evaluated in coacervating media, both hydrophilic (Newton et al. 1977, Okada et al. 1985, Kaser-Liard 1985), and hydrophobic (Koida et al. 1986, Leelarasamee et al. 1988). L M has been used as a convenient tool for the observation of the dissolution process from single microcapsules in aqueous media. Coloured core materials are readily visible, but highly opaque wall material makes the method difficult. Even so, the method has been adapted for dark field phase contrast and fluorescent microscopy (FM) (Hoffman et al. 1986). Routine methods for converting biological tissues into permanent translucent material have been used to provide visualization. T h e finest structures are recognizable in such preparations, which are usually fully translucent. T h e present work deals with the method of making permanent preparations of microcapsules using the technique of embedding into a layer of rigidizable translucent material. T h e method of surface and penetration staining of microcapsules was developed.
Experimental Materials Gelatin (isoelectric point 5.1), Rousselot, France; cellulose acetate phthalate (CAP), Eastman Kodak, USA; ethylcellulose (Ethocel N 10) (EC), Dow Chemicals, USA; acrylic resin (Eudragit RS), Rohm-Pharma, FRG; Entelan, Merck, F R G ; sulphathiazol, Chemopharma, CSFR; potassium chloride and dyes, Lachema, CSFR. Dyes used were: Fluorescein Rhodamine B Eosine Y
C.1. 45 350 C.I. 45 170 C.I. 45400
Evaluation of microcapsules by LM Methylrosaniline chloride Methylene blue Malachite green
529
C.I. 42 510 C.I. 52015 C.I. 42000
Other chemicals were of analytical purity
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Methods Preparation of microcapsules. A. Three different methods of preparing microcapsules with a core of sulphathiazol were used. T h e wall material consisted of the same proportions of gelatin and CAP (used as potassium or triethanolamine salt).
1. Complex coacervation. Coacervation of equal amounts of 1.5 per cent aqueous solutions of both polymers was induced at 50°C by gradual addition of hydrochloric acid (0.1 moll-') to give a final p H of 3.8. 2. Simple coacervation. Desolvation of a 3 per cent total colloid concentration of equal amounts of both polymers in water was induced at 50°C by gradual addition of 20 per cent solution of sodium sulphate in water. T h e temperature was maintained far above the gelation point of gelatin. 3 . Emulsion method. This method is based on dispersion and encapsulation of the core material with the 1 5 per cent solution of both polymers in water in the continous phase (light mineral oil). T h e temperture was maintained at 55°C and the solidification of the wall material was induced by gradual cooling. Three variants of this method were investigated: (a) A suspension of the core material in the polymer solution was added dropwise to the stirred continuous phase. This produced a suspension in oil ( s - 0 ) . (b) A solution of both polymers was added dropwise into the stirred suspension of the core material in the continuous phase (w-s). (c) T h e suspension of core material in a part of the continuous phase was added dropwise to the vigorously agitated emulsion (s-e).
B. Microcapsules with EC were prepared using the method of phase separation of EC from cyclohexane solution at elevated temperature by gradual cooling. Potassium chloride was dispersed in the solution at elevated temperature (70°C) prior to the phase separation. C. Microcapsules with Eudragit R S were prepared according to the method of Goto et al. (1986). A 3 per cent concentration of magnesium stearate was used as emulsifier. Potassium chloride was suitable core material in this case. Staining of the microcapsules. A. Surface staining: Samples of microcapsules were washed on filter paper with a 0.5 per cent solution of fluorescent dye and rinsed immediately with purified water before drying at room temperature. T h e best results were achieved with an aqueous solution of eosin Y which also gave good results as to the permanence of the staining.
R . Penetration staining: Samples of dry microcapsules were immersed in stain for 10-60min depending on the size of microcapsules and the quality of the wall material. T h e best stains were 0.5 per cent aqueous solution of eosin Y or 0.3 per cent solution of eosin Y in 40 per cent aqueous ethanol. T h e use of desaturated solution of methylrosaniline chloride in a 5 per cent aqueous solution of sodium sulphate also
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gave good results. After staining the samples were washed and dried as for the surface treatment.
Theformation of permanent microscopic preparations. Stained microcapsules were embedded either in media with acceptable physical properties used in the L M method for biological samples, or media used for the first time in the present work. Entelan, plasticized solution of polystyrene in chloroform (12 per cent), solution of Eudragit RS in dimethylketone (15 per cent), and an aqueous solution of sodium silicate (40 per cent) were tested for this purpose. Photomicrographs were prepared using an ‘Amplival’ microscope (Carl Zeiss, Jena, GDR) at a magnification of 60 to 400. Medium light sensitivity photographic material (negative and inverse) was used for documentation.
Results and discussion Method of surface staining of microcapsules Surface stained samples of microcapsules were observed in the light and fluorescent microscopes. Microcapsules prepared b y complex coacervation were embedded in ‘Entelan’, but they were not sufficiently optically translucent because of the considerable heterogeneity of the complex coacervate (figure 1 ( a ) ) . Surface staining with eosin Y made the polymeric material easily distinguishable from the core. This showed clearly that coacervate did not adhere well to the crystals of sulphathiazol (figure 1 (b)). The colour of the coacervate and sulphathiazol in fluorescent light was more pronounced being yellow or orange, respectively. Simple coacervate, on the other hand, is optically translucent, though prepared from the same materials (figure 2 ( a ) ) .I n figure 2 (b) there is the same structure in the light microscope after the staining with methylrosaniline chloride and embedding into ‘Entelan’. With the use of penetration staining it was possible to determine the deposition of the wall material which resulted in the inner layer consisting of gelatin the outer layer mainly of polysaccharide.
Method of penetration staining of microcapsules Figure 3 ( a ) shows microcapsules photographed using light microscopy. It is possible to distinguish their shape only and, having a surface charge, they exist mainly in aggregates. After immersion in water they swell because of the hydrophilic nature of both polymers. Simultaneously they become translucent. In figure 3 (b) there are dark spots of core material and marginally more optically transmitting swollen wall material. This is the stage of partial restitution of the ‘native’ microcapsules. By embedding dry microcapsules in translucent polymeric material (e.g. ‘Entelan’) it is possible to observe the exact structure of the microcapsules (figure 3(c)). The position of the core, integrity and wall thickness could be evaluated. In the case of multinuclear microcapules, fusion of the core into one compact unit is seen. According to the depth of focusing it is possible to observe either the internal wall structure, or its surface. At the same time individual core units are recognizable. T h e slide was prepared by penetration staining prior to embedding in ‘Entelan’ (figure 3 ( d ) ) . Microcapsules made from ‘Eudragit RS’ and potassium chloride were much more transparent after embedding in ‘Entelan’ (figure 4 ( a ) ) . Because the optical density of potassium chloride is similar to that of the acrylic polymer, the core material was not distinguishable (figure 4 (b)). On the other hand, pores and cracks
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Figure 1 . Photomicrographs (LM) of micrographs prepared by complex coacervation (sulphathiazol, mixture of gelatin and triethanolamine salt of CAP). ( a ) Unprepared microcapsules (bar = 200pm). b) Microcapsules stained on surface with eosin Y (FM) (bar = 200 pm).
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(b) Figure 2. Photomicrographs (LM) of microcapsules prepared by simple coacervation (sulphathiazol, mixture of gelatin and triethanolamine salt of CAP). ( a ) Microcapsules stained on the surface with eosin Y (FM) (bar=200pm). ( b ) Microcapsules after penetration staining with saturated solution of methylrosaniline chloride in 5 per cent aqueous solution of sodium sulphate (bar = 100 pm).
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Figure 3. Photomicrographs (LM) of microcapsules prepared by emulsion method (sulphathiazol, mixture of gelatin and potassium salt of CAP). ( a ) Unprepared microcapsules (bar = 300 pm). (b) Swollen microcapsules dispersed in water (bar = 500 pm). (c) Microcapsules embedded in ‘Entelan’ (bar = 300 pm). ( d ) Microcapsules stained with eosin Y 0 5 per cent aqueous solution for 30min (bar=500pm).
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(c)
Figure 4. Photomicrographs (LM) of microcapsules prepared by the emulsion/solventevaporation method (potassium chloride, ‘Eudragit’ RS). (a) Unprepared microcapsules (bar =400 pm). (b) Microcapules embedded in ‘Entelan’ (bar = 500pm). (c) Microcapsules stained with eosin Y 0.3 per cent in 40 per cent aqueous ethanol for 20 min embedded into ‘Entelan’ (bar = 300pm).
Figure 5. Photomicrographs (LM) of microcapsules prepared by phase separation method (potassium chloride, ethylcellulose). Microcapsules embedded in sodium silicate glass (bar=400 pm).
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(4 Photomicrographs (LM) of microcapsules prepared by emulsion method-three variants of the procedure. (a)Variant s-o (bar =400pm).(6) Variant w-s (bar = 400 pm). (c) Variant s-e (bar = 400 pm).
Figure 6.
are visible on the surface. Penetration staining with eosin Y makes the core material clearly visible. We presume that salting out of the dye on to the surface of the core is the main mechanism for this effect. T h e precipitation of a layer of dye by the saturated solution of potassium chloride renders the surface of the core distinguisable (figure 4 ( c ) ) . An aqueous solution of sodium silicate was introduced as an embedding material in the case of ethylcellulose microcapsules (figure 5). T h e fluidity of the supporting material remained for several hours and is the main disadvantage. Evaporation of water during storage of the slides caused the formation of crystalline silicate and subsequent damage as well as a somewhat opaque preparation.
The influence of preparative variations of the emulsion method on the structure of the products We have used three variations for mixing the core and wall materials in the continuous phase. In the first case ( s - 0 ) there are numerous cavities in the wall of the microcapsules. They are not filled with light liquid paraffin as shown by analysis of the crushed product and of the crushed product, previously washed with hexane. They are probably bubbles of air that originated in the course of dispersion of sulphathiazol in the viscous solution of polymers (figure 6 ( a ) ) . An homogeneous wall without any bubbles and with a centrally arranged core is a typical feature of the second variant (w-s). Empty microcapsules also exist because of the poor adhesion of the wall material on the core (figure 6 ( b ) ) . The last variant of this method is characterized by dropwise addition of the suspension of the core material into a vigorously stirred emulsion of wall material in the continuous phase (s-e). Immediately after dispersion the velocity of stirring was
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slowed to approximately 400 r.p.m. As seen in figure 6 (c), the core material was dispersed over the whole interior of the particles. A large number of cavities existed filled with oil which was extractable with hexane.
Conclusions T h e above methods are restricted to the evaluation of microcapsules with a relatively low proportion of core material. Furthermore, the wall material has to be sufficiently optically homogeneous. T h e main advantage of light microscopy by comparison with scanning electron microscopy is its simplicity. Though having considerably lower differentiation ability, the observed structures are more reliable and during sample preparation no artificial structures are produced. According to the depth of focusing, either the shape and position of the core, or the structure of the wall and its surface characteristics may be observed. T h e method could be used as a routine for the control and optimization of the microencapsulation procedure. Moreover, actual observation with the microscope gives more information in comparison with photographs which are intended mainly for documentation purposes. References BENITA,S., HOFFMAN, A., and DONBROW, M., 1985, Microencapsulation of paracetamol using polyacrylate resins (Eudragit Retard), kinetics of drug release and evaluation of kinetic model. Journal of Pharmacy and Pharmacology, 37, 391-395. RENITA,S., BABAY,D., HOFFMAN, A,, and DONBROW, M., 1988, Relation between individual and ensemble release kinetics of indomethacin from microspheres. Pharmaceuticaf Research, 5 , 178-1 82. DONBROW, M., HOFFMAN, A., and BENITA,S., 1988, Variation of population release kinetics in polydisperse multiparticulate systems (microcapsules, microspheres, droplets, cells) with heterogeneity of one, two or three parameters in the population of individuals. Journal of Pharmacy and Pharmacology, 40, 93-96. FORNI, F., COPPI,G., IANNUCCELLI, V., and BERNABEI, M. T., 1988, Papaverine hydrochloride release from ethyl cellulose-walled microcapsules. Journal of Microencapsulation, 5 , 139-146. Gow, S., KAWATA, M., NAKAMURA, M., MAEKAWA, K., and AOYAMA, T., 1986, Eudragit R S and RI, (acrylic resins) microcapsules as pH insensitive and sustained release preparations of ketoprofen. Journal of Microencapsulation, 3, 293-304. HOFFMAN, A,, DONBROW, M., and BENITA,S., 1986, Direct measurements on individual microcapsule dissolution as a tool for determination of release mechanism. Journal of Pharmacy and Pharmacology, 38, 764-766. KOIDA,Y., KORAYASHI, M., and SAMEJIMA, M., 1986, Studies on microcapsules. IV. Influence of properties of drugs on microencapsulation and dissolution behavior. Chemical and Pharmaceutical Bulletin, 34, 3355-3361. KASER-LIARD, B., 1985, Herstellung von Gelatine-Mikrokapseln unter Anwendung der Emulsions- Induktions-Technik. Pharmaceutica Acta Helvetiae, 60, 326-333. LEEI-ARASAMEE, N., HOWARD, S. A,, MALANGA, C. J., and MA,J . K. H., 1988, A method of the preparation of polylactic acid microcapsules of controlled particle size and drug loading. Journal of Microencapsulation, 5 , 147-1 57. LINSHAN-YANG, and YANCJUEI-CHYI, 1985, Effect of ehtylene-vinyl acetate concentration on ethylcellulose-walled microcapsules: preparation and release kinetics of theophylline microcapsules. Journal of Microencapsulation, 2, 3 15-325. MATTHEWS, R. R., and NIXON, J. R., 1974, Surface characteristics of gelatin microcapsules by scanning electron microscopy. Journal of Pharmacy and Pharmacology, 26, 383-384. NEWTON, D. W., McMur.12EN,J . N., and BECKER, C. H., 1977, Characteristics of medicated and unmedicated microglobules recovered from complex coacervates of gelatin-accacia. Journal of Pharmaceutical Sciences, 66, 1327-1 330.
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NIXON, J . H., and MATTHEWS, R. R . , 1975, The surface characteristics of gelatin coacervate microcapsules by scanning electron microscopy. MicroencapsuZation, edited by J . R . Nixon (New York: Marcel Dekker), pp. 173-183. J., KIISAI, A , , and UEDA,S., 1985, Factors affecting microencapsulability in simple OKADA, gelatin coacervation method. Journal of Microencapsulation, 2 , 163-173. S I - N A N GL., , CARI.IER, 1’. l:., DEI~ORT, P., GAZZOLA, J., and LAFONT, I)., 1973, Determination of coating thickness of microcapsules and influence upon diffusion. Journal of Pharmaceutical Sciences, 62, 452455. SUZIJKI, K., and PRICE,J . C., 1985, Microencapsulation and dissolution properties of a neuroleptic in a biodegradeable polymer poly( I>, 1,-lactide). Journal of Pharmaceutical Srienres, 74, 21-24. ’I‘IcE, T. R . , and GIILEY,R. M., 1985, Preparation of injectable controlled-release microcapsules by a solvent-evaporation process. Journal of Controlled Release, 2 , 343-352. WRIGHT, K. C., WALLACE, S., MOSIER, B., and MOSIER, D., 1988, Microcapsules for arterial chemoembolization: appearance and in-vitro drug release characteristics. Journal of Microencapsulation, 5 , 13-20.
