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Preparation of egg albumin microparticles for oral application
Journal of Controlled Release, 25 ( 1993 ) 107- 113 0 1993 Elsevier Science Publishers B.V. All rights reserved
107 016%3659/93/$06.00
COREL 00838
Preparation
of egg albumin microparticles
for oral application
Melinda M&a and JAnos Pat6 Central Research Institute for Chemistry of the Hungarian Academy of Sciences, Budapest, Hungary (Received 28 April 1992; accepted in revised form 24 December 1992)
Gentamicin sulfate, a mixture of aminoglycoside type antibiotics applied in veterinary practice, was entrapped in a protein cover in order to prevent its decomposition before absorption from the gastrointestinal system. Unpurified egg albumin was used for this purpose. For detection of the drug a fluorescence label was introduced. The microparticles were prepared by a heat stabilization method in a water/ oil system. The drug-release was checked in in vitro experiments. Since the drug retaining ability of the microspheres was not satisfactory after this procedure, further hardening was accomplished by chemical crosslinks which were induced with glutaraldehyde. We have examined the effect of changes in different parameters of the synthesis on the drug-retaining ability of produced microcapsules. We have produced microspheres which hold about 80% of entrapped material even after 4 h at pH 7,2, which corresponds to the conditions in saliva and rumen of animals but having reached the stomach they presumably release the total amount of the drug. Key words: Egg albumin; Oral application;
Gentamicin
Introduction Gentamicin (Fig. 1)) a mixture of aminoglycoside type antibiotics (Cl a, Cl, C2) is frequently used for medical pretreatment in cattle keeping. Because of the carbohydrate-degrading enzymes of the animals’ saliva and rumen only a part of the applied dose can be absorbed from the gastrointestinal system. This absorption can be improved by protecting the drug from premature dissolution. Microencapsulation seems to be a suitable way to achieve this goal. Gentamitin treatment needs a large amount of antibiotic, thus it is important, that the cost of the protective technique should be lower than the costs of Correspondence to: Dr M. M6ra, Central Research Institute for Chemistry of the Hungarian Academy of Sciences, p.o. box 17,1526 Budapest, Hungary.
sulfate; Microsphere
FRl W-RR,
Fig. 1. Structure of gentamicin.
the drug waste due to its metabolism. Numerous high molecular weight materials of synthetic as well as natural origin are used for microencapsulation. We have chosen a protein matrix for our purpose, which presumably could
108
protect gentamicin from the enzymatic hydrolysis in the mouth and the rumen, but will break down by the proteolytic enzymes of the gastrointestinal system. Serum albumins are widely used in microencapsulation [ 1-3 1, but because of the high price of these substances we tried to find other proteins suitable for this purpose. For oral application we expected cheap unpurified egg albumin to be suitable [4-61, therefore ovalbumin and egg albumin were used beside bovine serum albumin for the experiments.
Materials and Methods Materials Gentamicin sulfate was supplied by Chinoin Pharmaceutical Works, Hungary; sunflower oil was of feeding quality; other chemicals were of analytical grade. Ovalbumin was isolated according to Ref. [ 71. Unpurified egg-albumin was prepared as follows: a white of a hen’s egg was mixed with 200 ml of distilled water with a magnetic stirrer. After 10 min the solution was filtered three times on Whatman 1 paper, and freeze dried. The weight of the product was on average 2 g. Pepsin (EC 3.4.23.1, 165 U/g) and cr-chymotrypsin (EC 3.4.21.1, > 1000 E/mg) were purchased from Fluka AG. Methods
Preparation of dansyl-gentamicin 2.88 g (5 mmol) ofgentamicin sulfate was dissolved in 25 ml of 5% NaHC03 solution and mixed with acetone containing 27 mg (0.1 mmol) dansyl chloride. After standing overnight the acetone was evaporated from the reaction mixture and the remaining solution was freezedried. This product was used without any further purification. Drug concentrations were measured using an excitation wavelength of 335 nm and emission of 5 I4 nm on a Farrand MK- 1type spectrofluorimeter.
Preparation of microspheres (general procedure)
Heat stabilization Gentamicin
and albumin were dissolved in 1 ml of distilled water and after 20 min the solution was added dropwise to 100 ml of agitated oil at 40°C at a rate of 100 @/min. The reaction mixture was stirred further at a controlled rate at elevated temperature for a while. The heating was accomplished at a rate of 2 ’ C/min. After cooling to room temperature in an ice-water bath the reaction mixture was diluted with ether, filtered, the product thoroughly washed with ether, and dried in a vacuum (yield, 75-80%). This procedure was applied for all different types of albumins (ovalbumin, bovine serum albumin, egg-albumin) and oils (paraffin oil, sunflower oil ) .
Chemical crosslinking Two hundred mg of heatdenaturated microspheres were suspended in 50 ml of glutaraldehyde-containing diethylether and this mixture was shaken for 2 h. Then the solid phase was filtered off, washed with ether and dried. Size determination The size of the microparticles was determined in triplicate by a light microscope equipped with a calibrated scale. The lowest and highest values are indicated in the tables.
Release studies Gentamicin
release was measured in 0.02 M phosphate buffer (pH 7.2), in 0.01 M HCl solution (pH 2.0) in the presence of pepsin or in 0.1 M Tris-HCl buffer (pH 8.0) in the presence of chymotrypsin. Concentrations were: lo-50 mg microspheres and 1 mg of enzymes in 5 ml of buffer. After stirring for 2-4 h at 37°C the reaction mixtures were filtered and the fluorescence of the filtrates was measured (in case of HCl solutions the pH was adjusted to 7.0 before measurements).
Results and Discussion Considering the solubility and stability of gentamicin sulfate, the heat stabilization method
109 TABLE 1 The effect of stirring rate and temperature of microspheres
on physical data
No of MC
Stirring temperature (“Cl
Stirring speed (rpm)
Size km)
SE1 SE2 SE3 SE4 SE5 so1
80 80 80 80 120 80
1000 300 600 600 600 600
3-9 n.d. 3-10 5-12 10-21 21-16
nd., not determinable; MC, microcapsule; E, egg albumin; S, sunflower oil; 0, ovalbumin.
[ 81, in a water/oil system seemed to be appropriate for the synthesis of albumin microparticles. In order to lind the best parameters correlations between the size and the shape of microspheres as well as the release of the entrapped material and the conditions of preparation of microparticles were studied. It is important to emphasize that our purpose was to produce a coating which gives gentamicin a total protection in the rumen but which allows the release of its entire quantity all at once in the
Fig. 2. Microcapsules
from unpurified
stomach which is in contrast with the usual continuous drug release from microparticles. During the microencapsulation process and in vitro release experiments a simple and reproducible method was necessary for checking the amount of the drug. Unfortunately, microbiological evaluation was not available in our laboratory, thus a chemical determination had to be developed. Widely used calorimetric methods, e.g., ninhydrine reaction of the amino groups, periodic acid, sodium nitroprusside or HPLC were not applicable because of the simultaneous presence of a huge amount of protein. Fluorescence labelling is a very sensitive technique which proved to be adequate for our purposes. Dansylation was chosen because the preparation of dansyl-gentamicin is a simple process. Due to its very strong fluorescence the dansylated material can be diluted with the unmodified drug to a great extent. Results of release-measurements were evaluated according to a calibration curve. It was considered, of course, that the absorption and diffusion of the ‘labelled’ drug could differ from those of the original one. Therefore conlirmation by microbiological assay seems to be advisable before starting in vivo experiments.
egg albumin prepared in sunflower oil at 80°C for 30 min at 600 rpm; magnification, 290~;size, 5-12pm.
110
Factors affecting the size and shape of microparticles We have prepared ‘blank’ microspheres (MC) at first, which did not contain drug, in order to ensure that reproducible results can be obtained by the applied method. The reaction medium was sunflower oil (S), the coating protein material egg albumin (E) or ovalbumin (0). The speed of stirring and the temperature of heat treatment were changed during these experiments. The results are presented in Table 1. Bell-shaped microparticles occurred except for the SE2 batch even without using any emulsifier [ 91. The particle size of the SE2 sample could not be determined because the capsules were aggregated, consequently 300 rpm stirring rate is too low. The product became powderlike at 600 rpm, and there was no considerable change at 1000 rpm regarding the size and shape. This result has proved to be reproducible (Fig. 2), therefore we generally used 600 rpm in future experiments. In similar circumstances bell-shaped microparticles were obtained with ovalbumin as well but in larger size. No fractionation was performed, because we did not aim for a gradual release of gentamicin which would have been affected by the size distribution.
reaction time was 2 h for detecting the break of the cover and 4 h when checking its retaining ability. The effect of different parameters on the drug retaining ability of products was evaluated after drug-release measurements. Quality of oily medium and protein. In these experiments the effect of media (paraflin oil (P ) and sunflower oil ) on the properties of products were examined. According to Jun and Lai experiments [ 41, microspheres of larger size can occur when using mineral oil. This fact can affect the drug-retaining ability. The results are summarized in Table 2. In our experiments comparing paraffin oil with sunflower oil it can be stated that the size of particles is about two times larger, while their drug-retaining ability is only slightly higher in paraffin oil. It is to be noted that in paraffin oil the microspheres are formed in a sticky state and it is necessary to wash them thoroughly with ether and acetone in order to get a powderlike product. Because of the planned industrial realisation, cheaper sunflower oil was chosen. Bovin serum albumin (B), ovalbumin and unpurified egg albumin were used for the synthesis. It is also obvious from the data that TABLE 2
Factors affecting the drug-release
The effect of the type of proteins and oily media on drugretaining ability of microparticles
In the course of preparing drug-containing microparticles their gentamicin content was controlled fluorometrically after total destroying the microspheres’ walls by dissolution in buffers or by enzymatic digestion. The quantities of entrapped drug corresponded to the calculated values in the range of method’s error. Gentamicin was not found either in the oily reaction medium or in ethereal washing fluids according to the measurements. The in vitro drug-release from the microspheres was measured at 37°C in a buffer, pH 7.2, in 0.01 M HCI solution, pH 2, with pepsin and in a buffer, pH 8.0, with chymotrypsin. These conditions were intended to model the circumstances in the saliva and the rumen, in the stomach and in the small intestine, respectively. The
No of MC
Drug cant (mg/lOO mg prot )
Time of 80°C (min)
Size (pm)
Release [%] pH 7.2
Pep.
CT 1O-24 23-54 5-14 14-79
PEI PE2 SE6 SE7
10 100 10 100
30 30 30 30
61 63 79 81
PBI PB2 SBI SB2 SB3
400 400 400 400 400
60 120 30 180 270
66 56 69 74 70
100 91 84 95 99
77 63
23-85 31-77 3-2 I 7-21 8-19
PO1
400
120
63
58
67
n.d.
MC, microcapsule; P, petroleum ether; B, bovine serum albumin; E, egg albumin; S, sunflower oil; 0, ovalbumin; Pep, pepsin; CT, chymotrypsin: nd., not determinable.
111
the type of albumin does not have a significant effect on drug release as well, so our study concentrated on egg albumin.
took place ( (T,), the temperature at which the oil was heated after adding of the whole amount of the protein solution);
Quantity of drug incorporated Regarding the cost of production it is practical to make microspheres with a high drug-content. We have studied in this part if there is any change in drug-retaining ability with increasing drug content and protein concentration of the starting solution. The results concerning this problem are summarized in Table 3. Gentamicin content compared to protein quantity embraced a wide scale but had only a slight effect on drug-release rate, as had the protein concentration. Since it is easier to make a concentrated gentamicin solution in a diluted protein solution, we generally used an aqueous 10% w/v protein solution and dissolved 40% w/ v gentamicin sulfate in it.
(3) duration of the denaturation
Effect of temperature In these experiments we have examined the effect of te oil temperature during preparation on the properties of microparticles. Three parameters were studied: ( 1) oil temperature during the emulsion formation (( Tr,) while the protein solution was added ) ; (2) oil temperature
at which heat denaturation
The results of the first set of the experiments are summarized in Table 4. Although the release parameters improved when the addition was performed to a warmer oil bath, the size of microspheres was also growing, and at 100 oC even the spherical form of particles disappeared. Therefore dropping of the protein solution was generally performed in a 40’ C oil bath. From the data in Table 5 it can be concluded, that the alterations made in reaction parameters have not resulted in the desired improvement of retaining ability of microparticles in contrast with literature data [ 81. Checking heat resistance of gentamicin, a decrease in its optical rotation was found after 20 min at 120°C. Consequently we wanted to avoid exposing the microspheres to further heating. Another possibility for hardening of proteins is a chemical reaction with glutaraldehyde [ 8 1, which crosslinks the free amino groups of the protein yielding biodegradable azomethin bonds. Instead of the usual procedure of this method we decided a subsequent hardening of heat stabilized microspheres because we could achieve more reproducible results in this way avoiding adherence of microparticles to each other. How-
TABLE 3
TABLE 4
Correlation of the starting protein solution concentration and the entrapped drug quantity with the permeability of microcapsules.
Connection permeability
No of
MC
Drug cant (mg/lOO mg prot.)
Protein soln cone (w/v, O/o)
Release (%) pH 1.2
Size (pm)
SE8 SE7 SE9 SE10 SE11 SE12
400 100 70 10 10 1
10 40 30 50 33 33
80 84 100 100 79 91
3-24 14-79 7-14 l-17 5-14 3-38
MC, microcapsule;
S, sunflower oil; E, egg albumin.
( t).
between
the temperature
of dropping
and
No of MC
Drug cant (mg/lOO mg prot.)
Tn (“C)
Release (%) pH 7.2
Size (pm)
SE11 SE13 SE14
10 10 10
40 80 100
80 75 68
S-14 46-169 n.d.
Heat treatment after dropping in was 8O”C/30 min in the first two cases and lOO”C/30 min in the third case. S, sunflower oil; E, egg albumin; MC, microcapsule; rn, oil bath temperature when adding the protein solution; n.d., not determinable.
112 TABLE 5 The effect of heat treatment microparticles Noof MC
T,
t
(“C)
(min)
PBI 80 PB2 80 PO1 80 SE8 80 SE1 1 80 SE15 80 SE16 80 SE7 80 SE14 100 SE17 120
60 120 120 30 30 120 30 60 30 30
on the drug release from
Drug cant (m&100 mgprot.)
400 400 400 400 10 10 130 100 10 130
Release (O/o)
Size (pm )
pH 7.2
Pep
CT
66 56 63 77 79 76 73 84 68 110
110 91 58
77 63 67
23-85 31-77 n.d. 3-21 S-14 4-12 IO-17 14-79 n.d. 15-50
S, sunflower oil; E, egg albumin; B, bovine serum albumin; CT, chymotrypsin; Pep, pepsin; T,,oil temperature (stirring); n.d., not determinable; MC, microcapsule.
ever, formation of chemical bonds with gentamicin amino groups is also possible, but the Schiff bases formed are unstable in a biological environment and the drug can be released during the degradation of the protein cover. The results can be summarized as follows. During the treatment of the microspheres with commercially available 25% aqueous solution of glutaraldehyde a large amount of entrapped gentamicin was released. To avoid the waste of drug an ethereal solution of glutaraldehyde was made by extraction of the aqueous solution with ether and used for the chemical hardening. The concentration of glutaraldehyde in the ethereal solutions was determined by the hydroxylamine method [ lo]. During this process the size of particles did not change but their color went brownish. The release measurements demonstrated an increased retaining ability of these spherules as can be seen in Table 6. A treatment with an l-2 x 1O- ’ M/l solution (SE8g, glutaraldehyde-containing SE1 5b) resulted in microspheres which are suitable for in vivo experiments.
TABLE 6 Relationship between the glutaraldehyde the drug release No of Drug cant MC (mg/lOO mg prot.)
Glutaraldehyde cont. ( 1O-2 mM/ ml)
400 400 400 400 400 400 400 400 400 400 130 130 130 130 100 100
0 0,2 1,l 2,3 3,8 7,5 11,3 19,6 32,5 50,o 0 I I,3 16,l 32,5 0 22,3
MC, microcapsule; S, sunflower pepsin; CT, chymotrypsin.
and
Conclusions
Drug release (%) pH 7.2 120’
SESa SE8b SE8c SE8d SE8e SE8f SE8g SESh SE8i SE8j SElSa SE15b SEISc SE15d SE7a SE7b
concentration
83 61 61 61 72 41 22 13 18 18 92
Pep.
240’
66 59 80 87 75 19 14
12 26 16 84 10
CT
100 94 58 50 57 81 60 49
oil; E, egg albumin;
65 69 45 15 26 30
38 26
Pep,
Summarizing the results above it can be stated that we succeeded in producing gentamicin-containing egg albumin microspheres by heat stabilization and subsequent chemical treatment which full3 the intended requirements that the drug should be retained by the protein cover in order to be protected from the carbohydrate degrading-enzymes when in the rumen (a few hours) and that it should be released from the cover in the stomach in order for it to be absorbed in the gastrointestinal system. Further favorable in vivo results include reduction of expenses, accomplished by re-using vegetable oil and other solvents or omission of the freezedrying step when preparing the egg-albumin solution. The cheap egg albumin, when applied orally, is equivalent to other purified and more expensive proteins. It therefore gives an opportunity for economical application of microparticles in veterinary practice.
113
Acknowledgements The authors thank Chinoin Pharmaceutical Works (Hungary) for financial support, and Mrs. V. CsSlnyi and L. Ajler for skillful technical assistance. References Y. Morimoto, K. Sugibayashi and Y. Kato, Drug carrier property of albumin microspheres in chemotherapy. Chem. Pharm. Bull. 29 (1981) 1433-1438. R. Arshady, Albumin microspheres and microcapsules: methodology of manufacturing techniques, J. ControlledRelease 14 (1990) 111-131. Y. Nishioka, S. Kyotani, M. Okamura, Y. Mori, M. Miyazaki, K. Okazaki, S. Ohniski, Y. Yamamoto and K. Ito, Preparation and evaluation of albumin microspheres and microparticles containing cisplatin, Chem. Pharm. Bull. 37 (1989) 1399-1400.
4
5
6
7
8
9 10
H.W. Jun and J.W. Lai, Preparation and in vitro dissolution tests of egg albumin microcapsules of nitrofurantoin, Int. J. Pharm. 16 (1983) 65-67. R.M. Brophy and P.B. Deasy, Egg albumin microspheres containing sulphamethizole, J. Microencaps. 1 (1984) 157-168. T. Ishizaka, K. Endo and M. Koishi, Preparation of egg albumin microcapsules and microspheres, J. Pharm. Sci. 70(1981)358-363. E.O.P. Thompson, R.W. Sleigh and M.B. Smith, Amino acid sequence of the large plakalbumin peptide and the C-terminal sequence of ovalbumine, Austr J Biol. Sci. 24(1971)525-534. A.F. Yapel Jr., Albumin microspheres heat and chemical stabilization, Methods in Enzymology, Vol. 112, p. 3. Eds., S.P. Colowick, N.O. Kaplan, Academic Press, NY, 1985. K. Widder, Cl. Fluoret and A. Senyei, Magnetic microspheres, J. Pharm. Sci. 68 (1979) 79-82. J. Mitchell Jr., Determination of carbonyl compounds, in Organic Analysis, Vol. 1 p. 259 Eds., J. Mitchell, I.M. Kolthoff, E.S. Proskauer, A. Weissberger, Intersci. Publ., NY (1953).
107 016%3659/93/$06.00
COREL 00838
Preparation
of egg albumin microparticles
for oral application
Melinda M&a and JAnos Pat6 Central Research Institute for Chemistry of the Hungarian Academy of Sciences, Budapest, Hungary (Received 28 April 1992; accepted in revised form 24 December 1992)
Gentamicin sulfate, a mixture of aminoglycoside type antibiotics applied in veterinary practice, was entrapped in a protein cover in order to prevent its decomposition before absorption from the gastrointestinal system. Unpurified egg albumin was used for this purpose. For detection of the drug a fluorescence label was introduced. The microparticles were prepared by a heat stabilization method in a water/ oil system. The drug-release was checked in in vitro experiments. Since the drug retaining ability of the microspheres was not satisfactory after this procedure, further hardening was accomplished by chemical crosslinks which were induced with glutaraldehyde. We have examined the effect of changes in different parameters of the synthesis on the drug-retaining ability of produced microcapsules. We have produced microspheres which hold about 80% of entrapped material even after 4 h at pH 7,2, which corresponds to the conditions in saliva and rumen of animals but having reached the stomach they presumably release the total amount of the drug. Key words: Egg albumin; Oral application;
Gentamicin
Introduction Gentamicin (Fig. 1)) a mixture of aminoglycoside type antibiotics (Cl a, Cl, C2) is frequently used for medical pretreatment in cattle keeping. Because of the carbohydrate-degrading enzymes of the animals’ saliva and rumen only a part of the applied dose can be absorbed from the gastrointestinal system. This absorption can be improved by protecting the drug from premature dissolution. Microencapsulation seems to be a suitable way to achieve this goal. Gentamitin treatment needs a large amount of antibiotic, thus it is important, that the cost of the protective technique should be lower than the costs of Correspondence to: Dr M. M6ra, Central Research Institute for Chemistry of the Hungarian Academy of Sciences, p.o. box 17,1526 Budapest, Hungary.
sulfate; Microsphere
FRl W-RR,
Fig. 1. Structure of gentamicin.
the drug waste due to its metabolism. Numerous high molecular weight materials of synthetic as well as natural origin are used for microencapsulation. We have chosen a protein matrix for our purpose, which presumably could
108
protect gentamicin from the enzymatic hydrolysis in the mouth and the rumen, but will break down by the proteolytic enzymes of the gastrointestinal system. Serum albumins are widely used in microencapsulation [ 1-3 1, but because of the high price of these substances we tried to find other proteins suitable for this purpose. For oral application we expected cheap unpurified egg albumin to be suitable [4-61, therefore ovalbumin and egg albumin were used beside bovine serum albumin for the experiments.
Materials and Methods Materials Gentamicin sulfate was supplied by Chinoin Pharmaceutical Works, Hungary; sunflower oil was of feeding quality; other chemicals were of analytical grade. Ovalbumin was isolated according to Ref. [ 71. Unpurified egg-albumin was prepared as follows: a white of a hen’s egg was mixed with 200 ml of distilled water with a magnetic stirrer. After 10 min the solution was filtered three times on Whatman 1 paper, and freeze dried. The weight of the product was on average 2 g. Pepsin (EC 3.4.23.1, 165 U/g) and cr-chymotrypsin (EC 3.4.21.1, > 1000 E/mg) were purchased from Fluka AG. Methods
Preparation of dansyl-gentamicin 2.88 g (5 mmol) ofgentamicin sulfate was dissolved in 25 ml of 5% NaHC03 solution and mixed with acetone containing 27 mg (0.1 mmol) dansyl chloride. After standing overnight the acetone was evaporated from the reaction mixture and the remaining solution was freezedried. This product was used without any further purification. Drug concentrations were measured using an excitation wavelength of 335 nm and emission of 5 I4 nm on a Farrand MK- 1type spectrofluorimeter.
Preparation of microspheres (general procedure)
Heat stabilization Gentamicin
and albumin were dissolved in 1 ml of distilled water and after 20 min the solution was added dropwise to 100 ml of agitated oil at 40°C at a rate of 100 @/min. The reaction mixture was stirred further at a controlled rate at elevated temperature for a while. The heating was accomplished at a rate of 2 ’ C/min. After cooling to room temperature in an ice-water bath the reaction mixture was diluted with ether, filtered, the product thoroughly washed with ether, and dried in a vacuum (yield, 75-80%). This procedure was applied for all different types of albumins (ovalbumin, bovine serum albumin, egg-albumin) and oils (paraffin oil, sunflower oil ) .
Chemical crosslinking Two hundred mg of heatdenaturated microspheres were suspended in 50 ml of glutaraldehyde-containing diethylether and this mixture was shaken for 2 h. Then the solid phase was filtered off, washed with ether and dried. Size determination The size of the microparticles was determined in triplicate by a light microscope equipped with a calibrated scale. The lowest and highest values are indicated in the tables.
Release studies Gentamicin
release was measured in 0.02 M phosphate buffer (pH 7.2), in 0.01 M HCl solution (pH 2.0) in the presence of pepsin or in 0.1 M Tris-HCl buffer (pH 8.0) in the presence of chymotrypsin. Concentrations were: lo-50 mg microspheres and 1 mg of enzymes in 5 ml of buffer. After stirring for 2-4 h at 37°C the reaction mixtures were filtered and the fluorescence of the filtrates was measured (in case of HCl solutions the pH was adjusted to 7.0 before measurements).
Results and Discussion Considering the solubility and stability of gentamicin sulfate, the heat stabilization method
109 TABLE 1 The effect of stirring rate and temperature of microspheres
on physical data
No of MC
Stirring temperature (“Cl
Stirring speed (rpm)
Size km)
SE1 SE2 SE3 SE4 SE5 so1
80 80 80 80 120 80
1000 300 600 600 600 600
3-9 n.d. 3-10 5-12 10-21 21-16
nd., not determinable; MC, microcapsule; E, egg albumin; S, sunflower oil; 0, ovalbumin.
[ 81, in a water/oil system seemed to be appropriate for the synthesis of albumin microparticles. In order to lind the best parameters correlations between the size and the shape of microspheres as well as the release of the entrapped material and the conditions of preparation of microparticles were studied. It is important to emphasize that our purpose was to produce a coating which gives gentamicin a total protection in the rumen but which allows the release of its entire quantity all at once in the
Fig. 2. Microcapsules
from unpurified
stomach which is in contrast with the usual continuous drug release from microparticles. During the microencapsulation process and in vitro release experiments a simple and reproducible method was necessary for checking the amount of the drug. Unfortunately, microbiological evaluation was not available in our laboratory, thus a chemical determination had to be developed. Widely used calorimetric methods, e.g., ninhydrine reaction of the amino groups, periodic acid, sodium nitroprusside or HPLC were not applicable because of the simultaneous presence of a huge amount of protein. Fluorescence labelling is a very sensitive technique which proved to be adequate for our purposes. Dansylation was chosen because the preparation of dansyl-gentamicin is a simple process. Due to its very strong fluorescence the dansylated material can be diluted with the unmodified drug to a great extent. Results of release-measurements were evaluated according to a calibration curve. It was considered, of course, that the absorption and diffusion of the ‘labelled’ drug could differ from those of the original one. Therefore conlirmation by microbiological assay seems to be advisable before starting in vivo experiments.
egg albumin prepared in sunflower oil at 80°C for 30 min at 600 rpm; magnification, 290~;size, 5-12pm.
110
Factors affecting the size and shape of microparticles We have prepared ‘blank’ microspheres (MC) at first, which did not contain drug, in order to ensure that reproducible results can be obtained by the applied method. The reaction medium was sunflower oil (S), the coating protein material egg albumin (E) or ovalbumin (0). The speed of stirring and the temperature of heat treatment were changed during these experiments. The results are presented in Table 1. Bell-shaped microparticles occurred except for the SE2 batch even without using any emulsifier [ 91. The particle size of the SE2 sample could not be determined because the capsules were aggregated, consequently 300 rpm stirring rate is too low. The product became powderlike at 600 rpm, and there was no considerable change at 1000 rpm regarding the size and shape. This result has proved to be reproducible (Fig. 2), therefore we generally used 600 rpm in future experiments. In similar circumstances bell-shaped microparticles were obtained with ovalbumin as well but in larger size. No fractionation was performed, because we did not aim for a gradual release of gentamicin which would have been affected by the size distribution.
reaction time was 2 h for detecting the break of the cover and 4 h when checking its retaining ability. The effect of different parameters on the drug retaining ability of products was evaluated after drug-release measurements. Quality of oily medium and protein. In these experiments the effect of media (paraflin oil (P ) and sunflower oil ) on the properties of products were examined. According to Jun and Lai experiments [ 41, microspheres of larger size can occur when using mineral oil. This fact can affect the drug-retaining ability. The results are summarized in Table 2. In our experiments comparing paraffin oil with sunflower oil it can be stated that the size of particles is about two times larger, while their drug-retaining ability is only slightly higher in paraffin oil. It is to be noted that in paraffin oil the microspheres are formed in a sticky state and it is necessary to wash them thoroughly with ether and acetone in order to get a powderlike product. Because of the planned industrial realisation, cheaper sunflower oil was chosen. Bovin serum albumin (B), ovalbumin and unpurified egg albumin were used for the synthesis. It is also obvious from the data that TABLE 2
Factors affecting the drug-release
The effect of the type of proteins and oily media on drugretaining ability of microparticles
In the course of preparing drug-containing microparticles their gentamicin content was controlled fluorometrically after total destroying the microspheres’ walls by dissolution in buffers or by enzymatic digestion. The quantities of entrapped drug corresponded to the calculated values in the range of method’s error. Gentamicin was not found either in the oily reaction medium or in ethereal washing fluids according to the measurements. The in vitro drug-release from the microspheres was measured at 37°C in a buffer, pH 7.2, in 0.01 M HCI solution, pH 2, with pepsin and in a buffer, pH 8.0, with chymotrypsin. These conditions were intended to model the circumstances in the saliva and the rumen, in the stomach and in the small intestine, respectively. The
No of MC
Drug cant (mg/lOO mg prot )
Time of 80°C (min)
Size (pm)
Release [%] pH 7.2
Pep.
CT 1O-24 23-54 5-14 14-79
PEI PE2 SE6 SE7
10 100 10 100
30 30 30 30
61 63 79 81
PBI PB2 SBI SB2 SB3
400 400 400 400 400
60 120 30 180 270
66 56 69 74 70
100 91 84 95 99
77 63
23-85 31-77 3-2 I 7-21 8-19
PO1
400
120
63
58
67
n.d.
MC, microcapsule; P, petroleum ether; B, bovine serum albumin; E, egg albumin; S, sunflower oil; 0, ovalbumin; Pep, pepsin; CT, chymotrypsin: nd., not determinable.
111
the type of albumin does not have a significant effect on drug release as well, so our study concentrated on egg albumin.
took place ( (T,), the temperature at which the oil was heated after adding of the whole amount of the protein solution);
Quantity of drug incorporated Regarding the cost of production it is practical to make microspheres with a high drug-content. We have studied in this part if there is any change in drug-retaining ability with increasing drug content and protein concentration of the starting solution. The results concerning this problem are summarized in Table 3. Gentamicin content compared to protein quantity embraced a wide scale but had only a slight effect on drug-release rate, as had the protein concentration. Since it is easier to make a concentrated gentamicin solution in a diluted protein solution, we generally used an aqueous 10% w/v protein solution and dissolved 40% w/ v gentamicin sulfate in it.
(3) duration of the denaturation
Effect of temperature In these experiments we have examined the effect of te oil temperature during preparation on the properties of microparticles. Three parameters were studied: ( 1) oil temperature during the emulsion formation (( Tr,) while the protein solution was added ) ; (2) oil temperature
at which heat denaturation
The results of the first set of the experiments are summarized in Table 4. Although the release parameters improved when the addition was performed to a warmer oil bath, the size of microspheres was also growing, and at 100 oC even the spherical form of particles disappeared. Therefore dropping of the protein solution was generally performed in a 40’ C oil bath. From the data in Table 5 it can be concluded, that the alterations made in reaction parameters have not resulted in the desired improvement of retaining ability of microparticles in contrast with literature data [ 81. Checking heat resistance of gentamicin, a decrease in its optical rotation was found after 20 min at 120°C. Consequently we wanted to avoid exposing the microspheres to further heating. Another possibility for hardening of proteins is a chemical reaction with glutaraldehyde [ 8 1, which crosslinks the free amino groups of the protein yielding biodegradable azomethin bonds. Instead of the usual procedure of this method we decided a subsequent hardening of heat stabilized microspheres because we could achieve more reproducible results in this way avoiding adherence of microparticles to each other. How-
TABLE 3
TABLE 4
Correlation of the starting protein solution concentration and the entrapped drug quantity with the permeability of microcapsules.
Connection permeability
No of
MC
Drug cant (mg/lOO mg prot.)
Protein soln cone (w/v, O/o)
Release (%) pH 1.2
Size (pm)
SE8 SE7 SE9 SE10 SE11 SE12
400 100 70 10 10 1
10 40 30 50 33 33
80 84 100 100 79 91
3-24 14-79 7-14 l-17 5-14 3-38
MC, microcapsule;
S, sunflower oil; E, egg albumin.
( t).
between
the temperature
of dropping
and
No of MC
Drug cant (mg/lOO mg prot.)
Tn (“C)
Release (%) pH 7.2
Size (pm)
SE11 SE13 SE14
10 10 10
40 80 100
80 75 68
S-14 46-169 n.d.
Heat treatment after dropping in was 8O”C/30 min in the first two cases and lOO”C/30 min in the third case. S, sunflower oil; E, egg albumin; MC, microcapsule; rn, oil bath temperature when adding the protein solution; n.d., not determinable.
112 TABLE 5 The effect of heat treatment microparticles Noof MC
T,
t
(“C)
(min)
PBI 80 PB2 80 PO1 80 SE8 80 SE1 1 80 SE15 80 SE16 80 SE7 80 SE14 100 SE17 120
60 120 120 30 30 120 30 60 30 30
on the drug release from
Drug cant (m&100 mgprot.)
400 400 400 400 10 10 130 100 10 130
Release (O/o)
Size (pm )
pH 7.2
Pep
CT
66 56 63 77 79 76 73 84 68 110
110 91 58
77 63 67
23-85 31-77 n.d. 3-21 S-14 4-12 IO-17 14-79 n.d. 15-50
S, sunflower oil; E, egg albumin; B, bovine serum albumin; CT, chymotrypsin; Pep, pepsin; T,,oil temperature (stirring); n.d., not determinable; MC, microcapsule.
ever, formation of chemical bonds with gentamicin amino groups is also possible, but the Schiff bases formed are unstable in a biological environment and the drug can be released during the degradation of the protein cover. The results can be summarized as follows. During the treatment of the microspheres with commercially available 25% aqueous solution of glutaraldehyde a large amount of entrapped gentamicin was released. To avoid the waste of drug an ethereal solution of glutaraldehyde was made by extraction of the aqueous solution with ether and used for the chemical hardening. The concentration of glutaraldehyde in the ethereal solutions was determined by the hydroxylamine method [ lo]. During this process the size of particles did not change but their color went brownish. The release measurements demonstrated an increased retaining ability of these spherules as can be seen in Table 6. A treatment with an l-2 x 1O- ’ M/l solution (SE8g, glutaraldehyde-containing SE1 5b) resulted in microspheres which are suitable for in vivo experiments.
TABLE 6 Relationship between the glutaraldehyde the drug release No of Drug cant MC (mg/lOO mg prot.)
Glutaraldehyde cont. ( 1O-2 mM/ ml)
400 400 400 400 400 400 400 400 400 400 130 130 130 130 100 100
0 0,2 1,l 2,3 3,8 7,5 11,3 19,6 32,5 50,o 0 I I,3 16,l 32,5 0 22,3
MC, microcapsule; S, sunflower pepsin; CT, chymotrypsin.
and
Conclusions
Drug release (%) pH 7.2 120’
SESa SE8b SE8c SE8d SE8e SE8f SE8g SESh SE8i SE8j SElSa SE15b SEISc SE15d SE7a SE7b
concentration
83 61 61 61 72 41 22 13 18 18 92
Pep.
240’
66 59 80 87 75 19 14
12 26 16 84 10
CT
100 94 58 50 57 81 60 49
oil; E, egg albumin;
65 69 45 15 26 30
38 26
Pep,
Summarizing the results above it can be stated that we succeeded in producing gentamicin-containing egg albumin microspheres by heat stabilization and subsequent chemical treatment which full3 the intended requirements that the drug should be retained by the protein cover in order to be protected from the carbohydrate degrading-enzymes when in the rumen (a few hours) and that it should be released from the cover in the stomach in order for it to be absorbed in the gastrointestinal system. Further favorable in vivo results include reduction of expenses, accomplished by re-using vegetable oil and other solvents or omission of the freezedrying step when preparing the egg-albumin solution. The cheap egg albumin, when applied orally, is equivalent to other purified and more expensive proteins. It therefore gives an opportunity for economical application of microparticles in veterinary practice.
113
Acknowledgements The authors thank Chinoin Pharmaceutical Works (Hungary) for financial support, and Mrs. V. CsSlnyi and L. Ajler for skillful technical assistance. References Y. Morimoto, K. Sugibayashi and Y. Kato, Drug carrier property of albumin microspheres in chemotherapy. Chem. Pharm. Bull. 29 (1981) 1433-1438. R. Arshady, Albumin microspheres and microcapsules: methodology of manufacturing techniques, J. ControlledRelease 14 (1990) 111-131. Y. Nishioka, S. Kyotani, M. Okamura, Y. Mori, M. Miyazaki, K. Okazaki, S. Ohniski, Y. Yamamoto and K. Ito, Preparation and evaluation of albumin microspheres and microparticles containing cisplatin, Chem. Pharm. Bull. 37 (1989) 1399-1400.
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H.W. Jun and J.W. Lai, Preparation and in vitro dissolution tests of egg albumin microcapsules of nitrofurantoin, Int. J. Pharm. 16 (1983) 65-67. R.M. Brophy and P.B. Deasy, Egg albumin microspheres containing sulphamethizole, J. Microencaps. 1 (1984) 157-168. T. Ishizaka, K. Endo and M. Koishi, Preparation of egg albumin microcapsules and microspheres, J. Pharm. Sci. 70(1981)358-363. E.O.P. Thompson, R.W. Sleigh and M.B. Smith, Amino acid sequence of the large plakalbumin peptide and the C-terminal sequence of ovalbumine, Austr J Biol. Sci. 24(1971)525-534. A.F. Yapel Jr., Albumin microspheres heat and chemical stabilization, Methods in Enzymology, Vol. 112, p. 3. Eds., S.P. Colowick, N.O. Kaplan, Academic Press, NY, 1985. K. Widder, Cl. Fluoret and A. Senyei, Magnetic microspheres, J. Pharm. Sci. 68 (1979) 79-82. J. Mitchell Jr., Determination of carbonyl compounds, in Organic Analysis, Vol. 1 p. 259 Eds., J. Mitchell, I.M. Kolthoff, E.S. Proskauer, A. Weissberger, Intersci. Publ., NY (1953).