J. MICROENCAPSULATION,1990, VOL. 7, NO. 2, 155-165
Preparation, characterization and performance evaluation of neomycin-HSA microspheres S U B H A S H P A N D E , SURESH VYAS and V I N O D DIXIT
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Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya Sagar (M. P.) 470 003 India (Receiwed 23 January 1989; accepted 2 May 1989) Human serum albumin microspheres containing neomycin sulphate were prepared using emulsion polymerization and polymer dispersion techniques. The many variables which may affect the shape, size, stability, release of the drug from the microspheres such as internal phase to external phase volume ratio, human serum albumin content, stirring rate, polymer content and stabilizing agent concentration, were studied. Unlike the microspheres prepared by the emulsion polymerization technique, polymer dispersion stabilised microspheres were uniform in size and shape with a narrow range of size distribution. In w i t y o release of neomycin sulphate from albumin microspheres was studied using the dialysis cell method. The drug release from microspheres followed Q versus ( t ) - ” * linear relationship. The in wiwo distribution studies on prepared microspheres revealed that the localization takes place preferably in lung tissues, liver, spleen and kidney and is found to be dependent on the microsphere size. On administration of microspheres of 3-6pm size, approximately 5 5 per cent of administered drug could be localized in the lungs.
Introduction T h e main objective of modern chemotherapy is to minimize the side effects of the administered drug, which should reach the specific site where the drug response is required, with a dose as low as possible. T h e concept centres mainly around cancer chemotherapy and other localized diseases. Amongst the various drug carriers tried, the magnetic, non-magnetic and biodegradable polymeric albumin microspheres have attracted the attention of scientists. Yet a lot remains to be evaluated as regards to their clinical performance. Albumin microspheres were first prepared for the detection of abnormalities in the reticulo-endothelial system and blood circulation (Rhodes et al. 1968, Rhodes et al. 1969, Zolle et al. 1970a). These investigators produced radiolabelled microspheres with diameters ranging from 5 to 65 pm. Based on ultrasonication, the techniques were developed to produce microspheres of size 1 p m in diameter (Pasqualini et al. 1969, Zolle et al. 1970 b). Magnetic and non-magnetic albumin microspheres were studied for their potential as drug carriers for targeting drug(s) in cancer chemotherapy (Kramer 1974, Sugibayashi et al. 1977, Widder et at. 1978). With the basic method of preparation (Scheffel et al. 1972), optimization of various process variables and the successful localization of magnetic responsive microspheres in the tail segment of the rat were demonstrated (Widder et al. 1978, 1979). Poly alkylcyanoacrylate nanoparticles were prepared by adsorbing the drug on a polymer surface. T h e later study on poly alkylcyanoacrylate nanoparticles revealed a 0265-2048/90 83.00 0 1990 Taylor & Francis Ltd.
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reduction in the toxicity of doxorubicin (Couvreur et al. 1979). T h e use of polystyrene divinyl benzene for the preparation of microspheres has been reported (Motoko et al. 1980). Pectin gelatin microglobules of size 1-50 pm were prepared and their dissolution was studied (McMullen et al. 1982). Using polymethyl methacrylate and polyoxyethylene polymers as dispensing agent hydrophilic albumin microspheres were prepared (Longo et al. 1982). In the present work neomycin sulphate was used as a model drug which is mainly used for the treatment of staphylococcal infections and in the management of infective hepatitis of bacterial origin as well as in hepatic coma. I n conventional therapeutically effective intravenous dose administration, neomycin sulphate is reported to damage kidney and the eighth cranial nerve. Therefore it is preferable to localize the drug at the site of action, thus preventing damage to other organs. Two processes of preparation of microspheres, namely emulsion polymerization and polymer dispersion, were adopted and a comparative study was carried out to optimize the process variables, in vitro release pattern and in vivo localization of prepared microspheres.
Materials and methods Materials Human serum albumin (Serum India Ltd., Pune, India), formaldehyde, acetone, ether (E. Merck (India) Pvt. Ltd, polymethylmethacrylate (Aldrich Chemicals, U.S.A.), cotton seed oil (Sigma Chemical Co., U.S.A.) were used. Neomycin sulphate was a gift from Roussel Pharmaceuticals (Bombay, India). Method Preparation of microspheres ( a ) Emulsion polymerization method. The method of Widder et al. (1979) was adopted for the preparation of microspheres. 50 mg neomycin sulphate was dissolved in 5 per cent human serum albumin aqueous solution, 0.5ml of this solution was mixed with cotton seed oil and homogenized, resulting in an emulsion. This emulsion was then resuspended dropwise with high shear in 100 ml of cotton seed oil. A stabilizing agent was added. T h e mixture was centrifuged for 3min (2000 g) supernatant was discarded and microspheres were further washed three times with ether to remove any trace of oil. After drying the washed microspheres were suspended in distilled water and stored frozen until used for further studies. (b) Polymer-dispersion method. A 5 per cent aqueous solution of human serum albumin (pH 7.0) containing 50 mg of neomycin sulphate was added dropwise to a solution of polymethylmethacrylate in chloroform. T h e mixture was stirred at high speed for IOmin to produce aqueous phase dispersion in the polymer solution. Formaldehyde was added as stabilizing agent and microspheres were stirred at room temperature at 100 rpm for 2-3 h. T h e microsphere suspension was then centrifuged (2000 g)for 3 min. The supernatant was discarded, and the sediment (microspheres) were washed four times each with chloroform, acetone and distilled water. Between each washing the microspheres were centrifuged and the supernatant was discarded. Finally, the washed microspheres were resuspended in water and stored frozen till used for further studies.
Neomycin-HSA microspheres
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Eflect of process variables Various factors which may affect the physical characteristics and in vitro release rate of the microspheres such as internal phase to external phase volume ratio, human serum albumin content, stirring rate, stabilizing agent concentration and polymer concentration, were studied. While studying the effect of one of the factors all others were kept constant at their optimum values. T h e shape and size of the prepared microspheres were determined by optical microscopy and scanning electron microscopy. Estimation of drug Neomycin sulphate in microspheres was estimated by polarimetric method (reported by de Rossi 1975) using Bendix-NPL automatic polarimeter with microcell of 22nm pathlength and retention volume 11Op1, when used with a polarimeter set for full scale deflection of 0.125". In vitro dissolution rate studies T h e in vitro release rate of the drug from the microspheres was determined following the modified method of Samuelov et al. (1979 using a hollow Nessler's cylinder of 50 ml capacity, to the bottom of which a circular piece of cellophane was attached by adhesive. A known quantity of microspheres suspended in water were placed in 500 ml of buffer solution of p H 6.4 maintained at 37 f1°C that provided an absolute sink condition. 5 ml aliquots were withdrawn at hourly intervals for 24 hrs. At each sampling time the volume in the receptor compartment was replaced with an equal volume of fresh buffer. T h e drug content in withdrawn samples was determined using the polarimetric method. Cumulative per cent drug release (Q) was plotted against 0.5 function of time i.e. In vivo performance evaluation T h e HSA neomycin microspheres of composition HSA: drug ratio (4: 1) prepared using dispersion stabilization technique (at 1000 r.p.m., stabilized using formaldehyde 0.2 M and of 3-1 1 pm average size range) were studied for their in vivo distribution. Microspheres suspension equivalent to 1mg of neomycin sulphate was injected through the caudal vein in six albino rats weighing between 270-300 g. After 3 h of intravenous administration three out of six rats were sacrificed by giving potassium chloride as an intracardial infusion. T h e rats were dissected and visceral organs were removed and washed to remove any debris. T h e isolated organs were then sliced and macerated. From macerated tissues the drug was extracted by multiple washing and centrifugation. T h e supernatants were pooled and the drug was estimated polarimetrically. T h e remaining three rats were sacrificed similarly after 24h and the drug content in various organs was determined. I n the same manner the study was also conducted for the pure drug. T h e percentage distribution of the drug in different organs was calculated.
Results and discussion Eflect of internal phase to external phase volume ratio T h e ratio of phase volume was varied in both the methods of preparation i.e., microspheres prepared by emulsion polymerization and polymer dispersion method, while all other variables were held constant. It was noticed that with an
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increase in internal phase volume particles of bigger size were obtained. However, the effect was not pronounced in the case of polymer dispersion method. At 15 : 85 internal :external phase ratio, discrete microspheres of average size 13.5 pm could be produced under moderate stirring whereas with 2.0 : 98.0 ratio 13.0pm sized microspheres were obtained by the emulsion polymerization method. T h e findings indicate better and substantive stabilization of the dispersed phase in polymer dispersion media (figure 1; table 1). Effect of human serum albumin (HSA) content T h e amount of human serum albumin was changed keeping other variables unchanged. It was observed that stability of microspheres increases with an increase in HSA concentration. At low HSA concentration the prepared microspheres were found to be unstable. In the case of the emulsion polymerization method it was not possible to increase the HSA content beyond 250mg as it caused frothing on the surface, whereas no such frothing was observed with increasing HSA content in the polymer dispersion method (figure 2; table 1). The latter behaviour exhibited in the polymer dispersion technique could be due to the structural vehicle-like action of polymer which could have retarded coalescence and offered more stabilization of dispersed globules. Stirring rate The size of the microspheres was observed to decrease with an increase in stirring rate up to 1200r.p.m. At 1600r.p.m. the microspheres prepared by emulsion polymerization method showed non-uniformity in shape and were observed to form 70
1
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Figure 1 . Size frequency distribution plot at different internal to external phase volume A 1 :99 per cent, 0 B 1.55:98.5 per cent, W C 2:98 per cent (emulsion ratio. polymerizationmethod; A D 5 :95 per cent, 0 E 10 :90 per cent, V F 1 5 : 85 per cent (polymer dispersion method).
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I *
0
z
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Figure 2. Size frequency distributionplot at different HSA concentrations. A 200 mg, B 250 mg (emulsion polymerization method); A C 200 mg, A D 250 mg, 0 E 300 mg (polymer dispersion method).
70
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MICROSPHERE SIZE
~
pm
Figure 3. Size frequency distributionplot at different rates of stirring. A 1000, B 1200, El C 1600 (emulsion polymerization method); A D 1000, 0 E 1200, V F 1600.
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2
Figure 4.
4
6
8 10 12 MICROSPHERE S I Z E ptn
14
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Size frequency distribution plot at different polymer concentration. 0 A 1 5 per cent, A B 20 per cent, I I C 25 per cent (polymer dispersion method).
agglomerates on storage. In polymer dispersion method uniform discrete particles were obtained even at high rates of stirring, with considerably small size (average size 7.0pm) (figure 3; table 1). This could be attributed to the stabilization of dispersed microspheres at an initially comminuted size. Effect of polymer content T h e effect of polymer content (in dispersion media) on the shape and size of microspheres prepared by the polymer-dispersion method was studied keeping all other variables constant. T h e polymer content was found to contribute to the stability of dispersion and thus of microspheres. Microspheres prepared using polymer content of less than 20 per cent were unstable. With increasing polymer content the stabilization was recorded u p to 35 per cent w/v concentration only, beyond which the increase in concentration exhibited no significant effect on the particle size. T h e findings suggested that with increasing polymer content, the increased viscosity of the media could have prevented the coalescence of the particles, which ultimately resulted in discrete particles. Twenty five per cent polymer concentration was recorded to be the optimum (figure 4; table 1). Eflect of stabilizing agent concentration T h e concentration of formaldehyde used as stabilizing agent was changed keeping all other variables constant. T h e stabilizing agent concentrations ranged from 0.1 M solution to 0.3 M solution. It was observed that the size of the microspheres increased with increase in formaldehyde concentration and invariably an irreversible agglomeration occurred in microspheres prepared by either of the methods. 0 . 2 0 ~formaldehyde concentration was found to be optimum for both the methods (figure 5 ; table 1).
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a
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30 20
10
0 4
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MICROSPHERE SIZE p m
Figure 5 . Size frequency distributionplot at different concentrations of stabilizing agent. El AO.1 M, 0 B 0 2 M, 0.3 M (emulsionpolymerization method); A D 0 1 M, 0E 0 2 M, V F 0 3 M (polymer dispersion method).
c
21.5
7.7
9.5
10.9
134
15.5
17.2
19
24.5
HOURS'.'
Figure 6 . In oitro release pattern.
29
32.8 35.3 39.5
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In vitro release study The in witro release rate from all the HSA microspheres prepared followed a linear relationship which is in agreement with the findings of Higuchi (1963, figure 6). As neomycin sulphate is a hydrophilic drug, the release of such compounds is described to be dependent on the 0.5-06 exponent of time for the linear relationship with Q (cumulative drug release). However, the rate was noted to be dependent on the size of microspheres and HSA content. With an increasing particle size a slower drug release rate was recorded. This could be accounted for by the difference in total surface area of the microspheres. Similarly, increasing HSA concentration in microspheres, especially in the case of those prepared by polymer dispersion method, resulted in a slower rate. This behaviour could be ascribed to the amount of HSA that forms a gel diffusion hydrodynamic barrier. T h e microspheres prepared by either of the methods i.e. emulsion polymerization or polymer dispersion were stabilized using formaldehyde. T h e stabilizing agent showed a substantial effect on the release rate. With maximum molar strength the slowest release rate was recorded which could be attributed to a higher degree of crosslinking and reorientation of HSA molecules resulting in a higher tortuosity and decreased porosity. In vivo distribution study In vivo distribution of neomycin sulphate HSA microspheres was studied in albino rats and compared with the plain drug after intravenous administration (figures 7 and 8). It was observed that 55 per cent of the administered drug in the form of HSA microspheres could be recovered from the lungs. Equivalent to SO per cent of the administered particles in size range 6-12 pm were retained by the lungs, possibly by a filtration mechanism. Thus drug from localized microspheres and that supplied after the distribution of free drug from systemic circulation may contribute to such a high concentration. T h e result is indicative of the successful localization of neomycin sulphate via particle size control. Such localization shows great potential in the treatment of bacterial lung infections. Approximately 22 per cent of the administered drug was estimated to be in the liver indicating the uptake of drug above the normal level probably through phagocytosis, whereas in the case of plain drug only 6-8 per cent of the drug could be recovered in the liver in a similar manner while in the spleen 8-10 per cent of the drug was found to be distributed. From kidney and blood 8 and 5 per cent of the drug were recovered respectively which were comparable to the distribution levels that were obtained after administration of the plain drug. T h e 55 per cent of the drug obtained in lungs (estimated after renaturation of protein) revealed that immediately 5 per cent of the drug was in free form contributed by normal drug release whereas the remaining 50 per cent was obtained after renaturation of microspheres which otherwise could have remained for a prolonged period. These findings are in agreement with the results of Motoko et al. (1980). After 24 h the pattern of distribution remained the same, although the drug level decreased marginally it remained considerably higher in the lungs followed by liver and spleen. This was with the exception of kidney and blood where drug concentrations were found to be higher after 24 h. Thus it is concluded from the study that selective localization of drug in HSA microspheres can be achieved via particle size control. T h e particles bigger than 6 pm could be localized in the lungs. T h e concentration of drug can be increased in organs where phagocytosis predominates; thus particles in the size range 3-6 pm
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a c 20 0
K
w 10
0 LUNGS
LIVER
SPLEEN
KIDNEY
BLCOD
Figure 7 . In vivo drug distribution in different organs (drug in microspheres); El 3 h, Ed 24 h.
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LUNGS
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Figure 8. In viuo drug distribution in different organs (plain drug); E4 3 h,
24 h.
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Table 1. Effect of process variables. HSA concentration
Internal to external phase volume ratio
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Sample no. Product
1 2 3 4 5 6
A B C D
E F
Ratio
Average size (Prn)
1:99 1.5 :98.5 2.0:98 5:95 10 :90 15 : 85
9.32 99 13.0 8.1 11.3 13.5
Sample Conc. no. Product (rng)
1 2 3 4 5
-
A B C D E -
Average size (P-4
200 250 200 250 300
12.3 10.5 9.0 10.5 12.0
-
-
Stirring rate
Stabilization agent concentration
Sample Stirring rate Average size no. Product r.p.m. (Prn)
Sample Average size no. Product Conc. (pm)
1 2 3 4
A
5
E F
6
B C D
1000 1200 1600 1000 1200 1600
1 2 3 4 5 6
141 95 101 10-30 7.1 5 7.75
A B C D
E F
0 1 02M 0.3 M 0.1 M 02M 03M
~
125 8.7 148 12.4 9.2 15.4
Polymer concentration Sample no.
1 2 3
Product
A B
c
Conc. per cent
Average size (pm)
15 20 25
7.3 5 9.50 11.50
could be useful in the treatment of infective liver diseases of bacterial origin. Finally, the release rate of drug (if the microspheres are of a size that is not affected by phagocytosis) can be controlled or modified by varying HSA and formaldehyde content. It is important to note that HSA content could only be increased in the case of the polymer dispersion method, whilst in the emulsion polymerization method the increasing HSA content does not increase total HSA content of microspheres beyond a limit. Finally the polymer dispersion method is considered to be better in controlling particle size, shape and agglomeration.
References COUVREUR, P., KANTE,B., RONALD, M., and SPEISER, P., 1979, Adsorption of antineoplastic drugs to polyalkylcynoacrylate nanoparticles and their release in calf serum. Journal of Pharmaceutical Sciences, 68, 1521-1 523. DE ROSSI,P., 1975, A continuous flow cell for use with Bendix NPL automatic polarimeter application to neomycin analysis. Analyst, 100, 25-28. HIGUCHI, T., 1963, Mechanism of sustained action medication. Theoretical analysis of rates of release of solid drug dispersed in solid matrices. Journal of Pharmaceutical Sciences, 52,1145-1149.
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Newcastle on 09/08/14 For personal use only.
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KRAMER, P. A., 1974, Albumin microspheres as vehicles for achieving specificity in drug delivery. Journal of Pharmaceutical Sciences, 63, 1646-1 647. LONGO, W. E., IWATO, H., LINDHELMER, T. A., and GOLDBERG, E. P., 1982, Preparation of hydrophilic albumin microspheres using polymeric dispensing agents. Journal of Pharmaceutical Sciences, 71, 1323-1327. MCMULLEN, J. N., NEWTON,D. W., and BECKER, C. H., 1982, Pectin gelatin complex coacervates. I. Determinants of microglobules size morphology and recovery as water dispersible powders. Journal of Pharmaceutical Sciences, 71, 628-633. MOTOKO, K., SIMMONS, G. H., WEISS,D. L., BIRINS,B. A., and PATRICK, DELUCA, P., 1980, Clearance of 141Celabeled microspheres from blood and distribution in specific organs following intravenous and intraarterial administration in Beagle dogs. Journal of Pharmaceutical Sciences, 69, 755-762. PASQUALINI, R., PLASSIO,G., and SOSSIS, S., 1969, The preparation of albumin microspheres. Journal of Biological and Nuclear Medicine, 13, 80-84. RHODES, B. A., ZOLLE,I., BUCHANON, J. W., and WAGNER, H. N., 1969, Radioactive albumin microspheres for studies of pulmonary circulation. Radiology, 92, 1453-1460. RHODES, B. A., ZOLLE,I., and WAGNER, H . N. JR, 1968, Properties and uses of radioactive albumin microspheres. Clinical Research, 416, 245. SAMUELOV, Y., DONBROW, M., and FRIEDMAN, M., 1979, Sustained release of drugs from ethylcelldose-polyethylene glycol films and kinetics of drug release. Journal of Pharmaceutical Sciences, 68, 325-329. SCHEFFEL, U., RHODES,B. A., NATARANJAN, T. K., and WAGNER, H. N. JR, 1972, Albumin microspheres for study of reticuloendothelial system. Journal of Nuclear Medicine, 13, 498-503. SUGIBAYASHI, K., MORIMOTO, Y., NADIA,T., and KATO,Y., 1977, Tissue distribution of microspheres entrapped fluorouracil in mice. Chemical €9Pharmaceutical Bulletin, 25, 3433-3434. WIDDER, K. J . , SENVEI, A. E., and SCARPELLI, D. G., 1978, Magnetic microspheres: a model system for site specific drug delivery in vivo. Proceedings of the Society for Experimental Biology and Medicines, 58, 141-146. WIDDER, K. J., FLOURET, G., and SENYEI, A. E., 1979, Magnetic microspheres: synthesis of a novel parenteral drug carrier. Journal of Pharmaceutical Sciences, 68, 79-82. ZOLLE, I., RHODES, B. A., WAGNER, H. N. JR,1970a, Preparation of metabolizable radioactive albumin microspheres for studies of the circulation. International Journal of Applied Radiation and Isotopes, 21, 155-167. ZOLLE, I., HOSAIN, F., RHODES, B. A., and WAGNER, H. N. JR, 1970 b, Human serum albumin microspheres for studies of the reticuloendothelial system. Journal of Nuclear Medicines, 11, 379.
Preparation, characterization and performance evaluation of neomycin-HSA microspheres S U B H A S H P A N D E , SURESH VYAS and V I N O D DIXIT
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Newcastle on 09/08/14 For personal use only.
Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya Sagar (M. P.) 470 003 India (Receiwed 23 January 1989; accepted 2 May 1989) Human serum albumin microspheres containing neomycin sulphate were prepared using emulsion polymerization and polymer dispersion techniques. The many variables which may affect the shape, size, stability, release of the drug from the microspheres such as internal phase to external phase volume ratio, human serum albumin content, stirring rate, polymer content and stabilizing agent concentration, were studied. Unlike the microspheres prepared by the emulsion polymerization technique, polymer dispersion stabilised microspheres were uniform in size and shape with a narrow range of size distribution. In w i t y o release of neomycin sulphate from albumin microspheres was studied using the dialysis cell method. The drug release from microspheres followed Q versus ( t ) - ” * linear relationship. The in wiwo distribution studies on prepared microspheres revealed that the localization takes place preferably in lung tissues, liver, spleen and kidney and is found to be dependent on the microsphere size. On administration of microspheres of 3-6pm size, approximately 5 5 per cent of administered drug could be localized in the lungs.
Introduction T h e main objective of modern chemotherapy is to minimize the side effects of the administered drug, which should reach the specific site where the drug response is required, with a dose as low as possible. T h e concept centres mainly around cancer chemotherapy and other localized diseases. Amongst the various drug carriers tried, the magnetic, non-magnetic and biodegradable polymeric albumin microspheres have attracted the attention of scientists. Yet a lot remains to be evaluated as regards to their clinical performance. Albumin microspheres were first prepared for the detection of abnormalities in the reticulo-endothelial system and blood circulation (Rhodes et al. 1968, Rhodes et al. 1969, Zolle et al. 1970a). These investigators produced radiolabelled microspheres with diameters ranging from 5 to 65 pm. Based on ultrasonication, the techniques were developed to produce microspheres of size 1 p m in diameter (Pasqualini et al. 1969, Zolle et al. 1970 b). Magnetic and non-magnetic albumin microspheres were studied for their potential as drug carriers for targeting drug(s) in cancer chemotherapy (Kramer 1974, Sugibayashi et al. 1977, Widder et at. 1978). With the basic method of preparation (Scheffel et al. 1972), optimization of various process variables and the successful localization of magnetic responsive microspheres in the tail segment of the rat were demonstrated (Widder et al. 1978, 1979). Poly alkylcyanoacrylate nanoparticles were prepared by adsorbing the drug on a polymer surface. T h e later study on poly alkylcyanoacrylate nanoparticles revealed a 0265-2048/90 83.00 0 1990 Taylor & Francis Ltd.
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reduction in the toxicity of doxorubicin (Couvreur et al. 1979). T h e use of polystyrene divinyl benzene for the preparation of microspheres has been reported (Motoko et al. 1980). Pectin gelatin microglobules of size 1-50 pm were prepared and their dissolution was studied (McMullen et al. 1982). Using polymethyl methacrylate and polyoxyethylene polymers as dispensing agent hydrophilic albumin microspheres were prepared (Longo et al. 1982). In the present work neomycin sulphate was used as a model drug which is mainly used for the treatment of staphylococcal infections and in the management of infective hepatitis of bacterial origin as well as in hepatic coma. I n conventional therapeutically effective intravenous dose administration, neomycin sulphate is reported to damage kidney and the eighth cranial nerve. Therefore it is preferable to localize the drug at the site of action, thus preventing damage to other organs. Two processes of preparation of microspheres, namely emulsion polymerization and polymer dispersion, were adopted and a comparative study was carried out to optimize the process variables, in vitro release pattern and in vivo localization of prepared microspheres.
Materials and methods Materials Human serum albumin (Serum India Ltd., Pune, India), formaldehyde, acetone, ether (E. Merck (India) Pvt. Ltd, polymethylmethacrylate (Aldrich Chemicals, U.S.A.), cotton seed oil (Sigma Chemical Co., U.S.A.) were used. Neomycin sulphate was a gift from Roussel Pharmaceuticals (Bombay, India). Method Preparation of microspheres ( a ) Emulsion polymerization method. The method of Widder et al. (1979) was adopted for the preparation of microspheres. 50 mg neomycin sulphate was dissolved in 5 per cent human serum albumin aqueous solution, 0.5ml of this solution was mixed with cotton seed oil and homogenized, resulting in an emulsion. This emulsion was then resuspended dropwise with high shear in 100 ml of cotton seed oil. A stabilizing agent was added. T h e mixture was centrifuged for 3min (2000 g) supernatant was discarded and microspheres were further washed three times with ether to remove any trace of oil. After drying the washed microspheres were suspended in distilled water and stored frozen until used for further studies. (b) Polymer-dispersion method. A 5 per cent aqueous solution of human serum albumin (pH 7.0) containing 50 mg of neomycin sulphate was added dropwise to a solution of polymethylmethacrylate in chloroform. T h e mixture was stirred at high speed for IOmin to produce aqueous phase dispersion in the polymer solution. Formaldehyde was added as stabilizing agent and microspheres were stirred at room temperature at 100 rpm for 2-3 h. T h e microsphere suspension was then centrifuged (2000 g)for 3 min. The supernatant was discarded, and the sediment (microspheres) were washed four times each with chloroform, acetone and distilled water. Between each washing the microspheres were centrifuged and the supernatant was discarded. Finally, the washed microspheres were resuspended in water and stored frozen till used for further studies.
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Eflect of process variables Various factors which may affect the physical characteristics and in vitro release rate of the microspheres such as internal phase to external phase volume ratio, human serum albumin content, stirring rate, stabilizing agent concentration and polymer concentration, were studied. While studying the effect of one of the factors all others were kept constant at their optimum values. T h e shape and size of the prepared microspheres were determined by optical microscopy and scanning electron microscopy. Estimation of drug Neomycin sulphate in microspheres was estimated by polarimetric method (reported by de Rossi 1975) using Bendix-NPL automatic polarimeter with microcell of 22nm pathlength and retention volume 11Op1, when used with a polarimeter set for full scale deflection of 0.125". In vitro dissolution rate studies T h e in vitro release rate of the drug from the microspheres was determined following the modified method of Samuelov et al. (1979 using a hollow Nessler's cylinder of 50 ml capacity, to the bottom of which a circular piece of cellophane was attached by adhesive. A known quantity of microspheres suspended in water were placed in 500 ml of buffer solution of p H 6.4 maintained at 37 f1°C that provided an absolute sink condition. 5 ml aliquots were withdrawn at hourly intervals for 24 hrs. At each sampling time the volume in the receptor compartment was replaced with an equal volume of fresh buffer. T h e drug content in withdrawn samples was determined using the polarimetric method. Cumulative per cent drug release (Q) was plotted against 0.5 function of time i.e. In vivo performance evaluation T h e HSA neomycin microspheres of composition HSA: drug ratio (4: 1) prepared using dispersion stabilization technique (at 1000 r.p.m., stabilized using formaldehyde 0.2 M and of 3-1 1 pm average size range) were studied for their in vivo distribution. Microspheres suspension equivalent to 1mg of neomycin sulphate was injected through the caudal vein in six albino rats weighing between 270-300 g. After 3 h of intravenous administration three out of six rats were sacrificed by giving potassium chloride as an intracardial infusion. T h e rats were dissected and visceral organs were removed and washed to remove any debris. T h e isolated organs were then sliced and macerated. From macerated tissues the drug was extracted by multiple washing and centrifugation. T h e supernatants were pooled and the drug was estimated polarimetrically. T h e remaining three rats were sacrificed similarly after 24h and the drug content in various organs was determined. I n the same manner the study was also conducted for the pure drug. T h e percentage distribution of the drug in different organs was calculated.
Results and discussion Eflect of internal phase to external phase volume ratio T h e ratio of phase volume was varied in both the methods of preparation i.e., microspheres prepared by emulsion polymerization and polymer dispersion method, while all other variables were held constant. It was noticed that with an
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increase in internal phase volume particles of bigger size were obtained. However, the effect was not pronounced in the case of polymer dispersion method. At 15 : 85 internal :external phase ratio, discrete microspheres of average size 13.5 pm could be produced under moderate stirring whereas with 2.0 : 98.0 ratio 13.0pm sized microspheres were obtained by the emulsion polymerization method. T h e findings indicate better and substantive stabilization of the dispersed phase in polymer dispersion media (figure 1; table 1). Effect of human serum albumin (HSA) content T h e amount of human serum albumin was changed keeping other variables unchanged. It was observed that stability of microspheres increases with an increase in HSA concentration. At low HSA concentration the prepared microspheres were found to be unstable. In the case of the emulsion polymerization method it was not possible to increase the HSA content beyond 250mg as it caused frothing on the surface, whereas no such frothing was observed with increasing HSA content in the polymer dispersion method (figure 2; table 1). The latter behaviour exhibited in the polymer dispersion technique could be due to the structural vehicle-like action of polymer which could have retarded coalescence and offered more stabilization of dispersed globules. Stirring rate The size of the microspheres was observed to decrease with an increase in stirring rate up to 1200r.p.m. At 1600r.p.m. the microspheres prepared by emulsion polymerization method showed non-uniformity in shape and were observed to form 70
1
a
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0 1 2
I
I
I
I
1
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I
I
I
4
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MICROSPHERE SIZE pm
Figure 1 . Size frequency distribution plot at different internal to external phase volume A 1 :99 per cent, 0 B 1.55:98.5 per cent, W C 2:98 per cent (emulsion ratio. polymerizationmethod; A D 5 :95 per cent, 0 E 10 :90 per cent, V F 1 5 : 85 per cent (polymer dispersion method).
159
Neomycin-HSA microspheres
I *
0
z
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w
3
0
w
a
IL
I
o ! 2
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6
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pm
MICROSPHERE S I Z E
Figure 2. Size frequency distributionplot at different HSA concentrations. A 200 mg, B 250 mg (emulsion polymerization method); A C 200 mg, A D 250 mg, 0 E 300 mg (polymer dispersion method).
70
‘y 10
O f 2
I
1
I
I
I
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I
I
4
6
8
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18
MICROSPHERE SIZE
~
pm
Figure 3. Size frequency distributionplot at different rates of stirring. A 1000, B 1200, El C 1600 (emulsion polymerization method); A D 1000, 0 E 1200, V F 1600.
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160
2
Figure 4.
4
6
8 10 12 MICROSPHERE S I Z E ptn
14
16
18
20
Size frequency distribution plot at different polymer concentration. 0 A 1 5 per cent, A B 20 per cent, I I C 25 per cent (polymer dispersion method).
agglomerates on storage. In polymer dispersion method uniform discrete particles were obtained even at high rates of stirring, with considerably small size (average size 7.0pm) (figure 3; table 1). This could be attributed to the stabilization of dispersed microspheres at an initially comminuted size. Effect of polymer content T h e effect of polymer content (in dispersion media) on the shape and size of microspheres prepared by the polymer-dispersion method was studied keeping all other variables constant. T h e polymer content was found to contribute to the stability of dispersion and thus of microspheres. Microspheres prepared using polymer content of less than 20 per cent were unstable. With increasing polymer content the stabilization was recorded u p to 35 per cent w/v concentration only, beyond which the increase in concentration exhibited no significant effect on the particle size. T h e findings suggested that with increasing polymer content, the increased viscosity of the media could have prevented the coalescence of the particles, which ultimately resulted in discrete particles. Twenty five per cent polymer concentration was recorded to be the optimum (figure 4; table 1). Eflect of stabilizing agent concentration T h e concentration of formaldehyde used as stabilizing agent was changed keeping all other variables constant. T h e stabilizing agent concentrations ranged from 0.1 M solution to 0.3 M solution. It was observed that the size of the microspheres increased with increase in formaldehyde concentration and invariably an irreversible agglomeration occurred in microspheres prepared by either of the methods. 0 . 2 0 ~formaldehyde concentration was found to be optimum for both the methods (figure 5 ; table 1).
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3 0 W
a
LL
30 20
10
0 4
2
6
8
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I
MICROSPHERE SIZE p m
Figure 5 . Size frequency distributionplot at different concentrations of stabilizing agent. El AO.1 M, 0 B 0 2 M, 0.3 M (emulsionpolymerization method); A D 0 1 M, 0E 0 2 M, V F 0 3 M (polymer dispersion method).
c
21.5
7.7
9.5
10.9
134
15.5
17.2
19
24.5
HOURS'.'
Figure 6 . In oitro release pattern.
29
32.8 35.3 39.5
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In vitro release study The in witro release rate from all the HSA microspheres prepared followed a linear relationship which is in agreement with the findings of Higuchi (1963, figure 6). As neomycin sulphate is a hydrophilic drug, the release of such compounds is described to be dependent on the 0.5-06 exponent of time for the linear relationship with Q (cumulative drug release). However, the rate was noted to be dependent on the size of microspheres and HSA content. With an increasing particle size a slower drug release rate was recorded. This could be accounted for by the difference in total surface area of the microspheres. Similarly, increasing HSA concentration in microspheres, especially in the case of those prepared by polymer dispersion method, resulted in a slower rate. This behaviour could be ascribed to the amount of HSA that forms a gel diffusion hydrodynamic barrier. T h e microspheres prepared by either of the methods i.e. emulsion polymerization or polymer dispersion were stabilized using formaldehyde. T h e stabilizing agent showed a substantial effect on the release rate. With maximum molar strength the slowest release rate was recorded which could be attributed to a higher degree of crosslinking and reorientation of HSA molecules resulting in a higher tortuosity and decreased porosity. In vivo distribution study In vivo distribution of neomycin sulphate HSA microspheres was studied in albino rats and compared with the plain drug after intravenous administration (figures 7 and 8). It was observed that 55 per cent of the administered drug in the form of HSA microspheres could be recovered from the lungs. Equivalent to SO per cent of the administered particles in size range 6-12 pm were retained by the lungs, possibly by a filtration mechanism. Thus drug from localized microspheres and that supplied after the distribution of free drug from systemic circulation may contribute to such a high concentration. T h e result is indicative of the successful localization of neomycin sulphate via particle size control. Such localization shows great potential in the treatment of bacterial lung infections. Approximately 22 per cent of the administered drug was estimated to be in the liver indicating the uptake of drug above the normal level probably through phagocytosis, whereas in the case of plain drug only 6-8 per cent of the drug could be recovered in the liver in a similar manner while in the spleen 8-10 per cent of the drug was found to be distributed. From kidney and blood 8 and 5 per cent of the drug were recovered respectively which were comparable to the distribution levels that were obtained after administration of the plain drug. T h e 55 per cent of the drug obtained in lungs (estimated after renaturation of protein) revealed that immediately 5 per cent of the drug was in free form contributed by normal drug release whereas the remaining 50 per cent was obtained after renaturation of microspheres which otherwise could have remained for a prolonged period. These findings are in agreement with the results of Motoko et al. (1980). After 24 h the pattern of distribution remained the same, although the drug level decreased marginally it remained considerably higher in the lungs followed by liver and spleen. This was with the exception of kidney and blood where drug concentrations were found to be higher after 24 h. Thus it is concluded from the study that selective localization of drug in HSA microspheres can be achieved via particle size control. T h e particles bigger than 6 pm could be localized in the lungs. T h e concentration of drug can be increased in organs where phagocytosis predominates; thus particles in the size range 3-6 pm
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N J
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J
2
30
K
a c 20 0
K
w 10
0 LUNGS
LIVER
SPLEEN
KIDNEY
BLCOD
Figure 7 . In vivo drug distribution in different organs (drug in microspheres); El 3 h, Ed 24 h.
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t
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LUNGS
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KIDNEY
BLOOD
Figure 8. In viuo drug distribution in different organs (plain drug); E4 3 h,
24 h.
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Table 1. Effect of process variables. HSA concentration
Internal to external phase volume ratio
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Sample no. Product
1 2 3 4 5 6
A B C D
E F
Ratio
Average size (Prn)
1:99 1.5 :98.5 2.0:98 5:95 10 :90 15 : 85
9.32 99 13.0 8.1 11.3 13.5
Sample Conc. no. Product (rng)
1 2 3 4 5
-
A B C D E -
Average size (P-4
200 250 200 250 300
12.3 10.5 9.0 10.5 12.0
-
-
Stirring rate
Stabilization agent concentration
Sample Stirring rate Average size no. Product r.p.m. (Prn)
Sample Average size no. Product Conc. (pm)
1 2 3 4
A
5
E F
6
B C D
1000 1200 1600 1000 1200 1600
1 2 3 4 5 6
141 95 101 10-30 7.1 5 7.75
A B C D
E F
0 1 02M 0.3 M 0.1 M 02M 03M
~
125 8.7 148 12.4 9.2 15.4
Polymer concentration Sample no.
1 2 3
Product
A B
c
Conc. per cent
Average size (pm)
15 20 25
7.3 5 9.50 11.50
could be useful in the treatment of infective liver diseases of bacterial origin. Finally, the release rate of drug (if the microspheres are of a size that is not affected by phagocytosis) can be controlled or modified by varying HSA and formaldehyde content. It is important to note that HSA content could only be increased in the case of the polymer dispersion method, whilst in the emulsion polymerization method the increasing HSA content does not increase total HSA content of microspheres beyond a limit. Finally the polymer dispersion method is considered to be better in controlling particle size, shape and agglomeration.
References COUVREUR, P., KANTE,B., RONALD, M., and SPEISER, P., 1979, Adsorption of antineoplastic drugs to polyalkylcynoacrylate nanoparticles and their release in calf serum. Journal of Pharmaceutical Sciences, 68, 1521-1 523. DE ROSSI,P., 1975, A continuous flow cell for use with Bendix NPL automatic polarimeter application to neomycin analysis. Analyst, 100, 25-28. HIGUCHI, T., 1963, Mechanism of sustained action medication. Theoretical analysis of rates of release of solid drug dispersed in solid matrices. Journal of Pharmaceutical Sciences, 52,1145-1149.
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KRAMER, P. A., 1974, Albumin microspheres as vehicles for achieving specificity in drug delivery. Journal of Pharmaceutical Sciences, 63, 1646-1 647. LONGO, W. E., IWATO, H., LINDHELMER, T. A., and GOLDBERG, E. P., 1982, Preparation of hydrophilic albumin microspheres using polymeric dispensing agents. Journal of Pharmaceutical Sciences, 71, 1323-1327. MCMULLEN, J. N., NEWTON,D. W., and BECKER, C. H., 1982, Pectin gelatin complex coacervates. I. Determinants of microglobules size morphology and recovery as water dispersible powders. Journal of Pharmaceutical Sciences, 71, 628-633. MOTOKO, K., SIMMONS, G. H., WEISS,D. L., BIRINS,B. A., and PATRICK, DELUCA, P., 1980, Clearance of 141Celabeled microspheres from blood and distribution in specific organs following intravenous and intraarterial administration in Beagle dogs. Journal of Pharmaceutical Sciences, 69, 755-762. PASQUALINI, R., PLASSIO,G., and SOSSIS, S., 1969, The preparation of albumin microspheres. Journal of Biological and Nuclear Medicine, 13, 80-84. RHODES, B. A., ZOLLE,I., BUCHANON, J. W., and WAGNER, H. N., 1969, Radioactive albumin microspheres for studies of pulmonary circulation. Radiology, 92, 1453-1460. RHODES, B. A., ZOLLE,I., and WAGNER, H . N. JR, 1968, Properties and uses of radioactive albumin microspheres. Clinical Research, 416, 245. SAMUELOV, Y., DONBROW, M., and FRIEDMAN, M., 1979, Sustained release of drugs from ethylcelldose-polyethylene glycol films and kinetics of drug release. Journal of Pharmaceutical Sciences, 68, 325-329. SCHEFFEL, U., RHODES,B. A., NATARANJAN, T. K., and WAGNER, H. N. JR, 1972, Albumin microspheres for study of reticuloendothelial system. Journal of Nuclear Medicine, 13, 498-503. SUGIBAYASHI, K., MORIMOTO, Y., NADIA,T., and KATO,Y., 1977, Tissue distribution of microspheres entrapped fluorouracil in mice. Chemical €9Pharmaceutical Bulletin, 25, 3433-3434. WIDDER, K. J . , SENVEI, A. E., and SCARPELLI, D. G., 1978, Magnetic microspheres: a model system for site specific drug delivery in vivo. Proceedings of the Society for Experimental Biology and Medicines, 58, 141-146. WIDDER, K. J., FLOURET, G., and SENYEI, A. E., 1979, Magnetic microspheres: synthesis of a novel parenteral drug carrier. Journal of Pharmaceutical Sciences, 68, 79-82. ZOLLE, I., RHODES, B. A., WAGNER, H. N. JR,1970a, Preparation of metabolizable radioactive albumin microspheres for studies of the circulation. International Journal of Applied Radiation and Isotopes, 21, 155-167. ZOLLE, I., HOSAIN, F., RHODES, B. A., and WAGNER, H. N. JR, 1970 b, Human serum albumin microspheres for studies of the reticuloendothelial system. Journal of Nuclear Medicines, 11, 379.
