Fibrin-based drug delivery systems III: The evaluation of the release of macromolecules from microbeads

Fibrin-based drug delivery systems III: The evaluation of the release of macromolecules from microbeads

journal of controlled release ELSEVIER Journal of Controlled Release 34 (1995) 65-70 Short Communication Fibrin-based drug delivery systems III: ...

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journal of

controlled release

ELSEVIER

Journal of Controlled Release 34 (1995) 65-70

Short Communication

Fibrin-based drug delivery systems III: The evaluation of the release of macromolecules from microbeads Hsiu-O Ho

a

Chih-Chuan Hsiao a, Theodore D. Sokoloski b Chau-Yang Chen Ming-Thau Sheu a..

a

"Graduate Institute of Pharmaceutical Sciences. Taipei Medical College, Taipei. Taiwan. ROC h SmithKline Beecham Pharmaceutics, UW2913. P.O. Box 1539, King of Prussia. PA 19406. USA

Received 22 August 1994: accepted 21 November 1994

Abstract In this study, fibrin beads containing macromolecules of different molecular weight were prepared in two different size distributions by varying the ratio of oleic acid to mineral oil in the oil phase of the emulsion system. The release characteristics were examined and compared. A dependence on the size of the fibrin microspheres for macromolecule release from fibrin microspheres was demonstrated: the smaller the size of the fibrin microspheres, the faster the release rate of the macromolecules. In addition, the apparent diffusion coefficient was found to be a function of the molecular size of the macromolecules. As a result, the larger the molecular weight of the macromolecules, the smaller the apparent diffusion coefficient. However, the release of lysozyme and insulin dissolved in 0.1 N HCI solution was undetectable. Keywords: Fibrin; Microsphere; Macromolecule: Apparent diffusion coefficient

1. Introduction The fibrin polymer represents a natural, biocompatible and biodegradable matrix. An approach utilizing the biochemical reaction between fibrinogen and thrombin to yield the fibrin polymer has been proposed [ 1 ]. As a delivery system, drugs are either entrapped within, or coated with, the fibrin polymer to form systems for parenteral injection, or they can be entrapped within sheets for surgical implantation [2]. However, recent advances in biotechnology have made the production and use of proteins and peptides feasible, although such materials present a challenging formulation and drug delivery problem. The specificity of the fibrinogen/thrombin reaction is such that reactive mol* Corresponding author. 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIOI 6 8 - 3 6 5 9 ( 9 4 ) 0 0 1 26-X

ecules such as proteins and peptides can be incorporated without modification. It has been reported by numerous laboratories [3,4] that a relatively large area is involved in the interaction between thrombin and its substrate, suggesting that there would be limited interaction with added proteins. Furthermore, the biochemical reaction between fibrinogen and thrombin is mild enough so that denaturing of macromolecules added does not occur. The diffusion characteristics of fibrin films have been studied with macromolecules of different molecular weights [2]. Recently, microsphere drug delivery devices have gained much attention. Various biodegradable materials, such as albumin, fibrinogen, etc., have been used as carriers [ 5,6 ]. Previously, an emulsion method was developed to prepare fibrin beads of different sizes [7]. This was achieved by

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H.-O Ho et al. /Journal of Controlled Release 34 (1995) 6 ~ 7 0

varying the amount of oleic acid in the oil phase to lower the interfacial tension. Here, we demonstrate how adjustable release of macromolecules can be achieved by using fibrin beads. The release characteristics of macromolecules of different molecular weights from different size distributions of fibrin beads were evaluated.

2. Experimental Fibrinogen (Type IV, from bovine plasma), Thrombin (Bovine plasma), Lysozyme ( # L - 4 6 3 1 , Chicken egg white), Carbonic Anhydrase ( # C - 2 2 7 3 , Bovine Erythrocytes), Ovalbumin ( # A - 7 6 4 2 , E g g ) , Bovine Serum Albumin ( # A - 7 5 1 7 ) , Insulin and Plasmin (Bovine plasma) were obtained from Sigma Chemical Co. Tris(hydroxymethyl)-aminoethane, highly liquid Paraffin (Light mineral oil), Tween 80 (Polyoxyethylene sorbitan monooleate), Isopropanol, Glutaraldehyde and Sodium Azide were supplied by Merck Co. Diethyl ether was provided by Riedel-de Haen and nHexane was obtained from Mallinckrodt. Fibrin microspheres containing macromolecules were prepared based on the procedure described previously [7]. Macromolecules examined included insulin, lysozyme, carbonic anhydrase, ovalbumin and bovine serum albumin, and their molecular weights and isoelectric points (pI) are listed in Table 1 . 6 0 mg of desalted, freeze-dried fibrinogen and various amount of macromolecules being examined (8 mg insulin, 8 mg lysozyme, 4 mg carbonic anhydrase, 30 mg ovalbumin and 15 mg bovine serum albumin) were dissolved in one gram of tris buffer (0.05 M, pH 7.4). 100/zl thrombin solution (containing 5 activity units of thrombin in the same tris buffer) was placed in a syringe with a 22G × 1.5" needle. Fibrinogen solution was then drawn into the syringe and mixed thoroughly. Table 1 Molecular weight and isoelectric point values (pl) of proteins Protein

Molecular weight

pl value

Insulin Lysozyme Cabonic anhydrase Ovalbumin Bovine serum albumin

5 700 14 500 29 000 45 000 66 000

5.30-5.35 10.5-11.0 5.9-5.4 4.63 4.7

The resulting solution was emulsified in the oil phase consisting of a mixture of light mineral oil and oleic acid (400/1 or 100/1) and stirred at 480 rpm. After curing for 1 h, 400/xl of glutaraldehyde solution (0.5% v / v ) was added to minimize coagulation of the fibrin beads and stirring was continued for 30 minutes. The recovery of fibrin beads from the emulsion system was achieved by simply decanting the oil phase and washing the residue once with diethyl ether and then twice with a mixture of isopropanol and n-hexane ( 1:3 ) containing 0.2% ( w / v ) Tween 80. The mean size and standard deviation for this two batches of fibrin beads was determined to be 0 . 6 2 3 + 0 . 1 3 6 mm and 0.228 _+0.024 mm respectively by using a Zoom lupe (Peak Zoom Lupe 816, Japan) with a scaled eyepiece. A similar technique was used to prepare fibrin bead containing insulin. However, insulin was first dissolved in 200/xl of 0.1 N HC1 or 100/zl of 1% ( w / v ) Tween 80 and then added to the fibrinogen solution. These protein containing fibrin beads were then placed in a desiccator for one day and then were used in the release studies. Release studies of macromolecules from fibrin beads were conducted in 2 ml of tris buffer containing 0.5% sodium azide (0.05 M, pH = 7.4) and kept in a shaking water bath at 37°C. 60/zl of the medium was removed at appropriate intervals and replenished with the same volume of fresh buffer solution. An average of nine samples was reported. The concentration change of the macromolecules in the medium was determined using a gradient HPLC system. A synchropak C4 column ( 4 . 6 x 150 mm, 3 0 0 / z m ) was used and UV detection was set at a wavelength of 220 rim. The flow rate was 1.5 m l / m i n . For lysozyme, bovine serum albumin, carbonic anhydrase and ovalbumin, the mobile phase system contained a mixture of solution A, 0.05% trifluoroacetic acid/deionized water, and solution B, 0.05% trifluoroacetic acid/acetonitrile; 24-76% of B was mixed in A over a period of 20 min. For insulin, the mixture of 0.05% trifluoroacetic acid/deionized water and acetonitrile was eluted from an A:B ratio of 74:26 to 38:62 over 15 min. These HPLC systems were validated. The retention times of insulin, lysozyme, bovine serum albumin, carbonic anhydrase and ovalbumin determined at varying concentrations, were found to be 4.66, 6.70, 7.81, 9.32 and 10.97 min, respectively, with acceptable coefficients of variation.

H.-O Ho et al. / Journal of Controlled Release 34 (1995) 65-70

3. Drug release kinetics

67 C,

1 O0

,3

80

In monolithic devices the material to be released is possibly dissolved uniformly throughout the rate-controlling polymeric matrix. The release profile is then determined by the loading of the dispersed agent, the nature of the components, and the geometry of the device. For spheric systems, a late time approximation method (Mt/M~ >_0.4), represented by Eq. ( 1 ), is used to describe the release kinetics for the solute in a dissolved state [8].

Mt/M~= l - 6 / Tr~*exp( - D* Tr2*t/a2),

60 c ,

40

insulin

20 01 100 80

~J r/?

50

~

f

w

4O'

Carbonic Anhydrase

2O

(1)

where M, denotes the quantity of diffusing substance which has been released from the microspheres in time t, M~ is the corresponding quantity after infinite time, D is the diffusion coefficient in the matrix of the fibrin beads, and a is the radius of the spherical particles. By assuming a is a constant and equal to the mean size of the beads, Mt/M~ (>0.4) was regressed with respect to time t, according to Eq. ( 1 ). Then the exponential term, which is equal to ( - D * T f l / a 2 ) , was used to calculate the diffusion coefficient of the macromolecules in the fibrin beads.

100 BO 60 40

Ovalbumin

20

10

Heat denaturation or chemical crosslinking With glutaraldehyde has been applied in emulsion systems to prepare microbeads for many biopolymers such as albumin. A similar emulsion method was employed in this study to prepare the fibrin beads [7], but utilized a mild biochemical reaction between fibrinogen and thrombin. In this study, oleic acid was selected as the surfactant to prepare two different size distribution of the fibrin beads, which were prepared in the oil phase, having a mineral oil/oleic acid ratio of either 100:1 or 400:1. The percent release of insulin, lysozyme, carbonic anhydrase, ovalbumin and bovine serum albumin from these two different size batches of the fibrin beads was examined and the results are shown in Fig. 1. Among them, insulin could be released only when dissolved with 100 /xl of 1% ( w / v ) Tween 80 during preparation. On the other hand, the release of lysozyme and insulin that dissolved with 200 /xl of 0.1 N HC1 was undetectable. However, the release of lysozyme and insulin was possible after digesting the fibrin beads

30

40

50

,7.

80 60 40

Bovine S e r u m A l bumi n

2O

10

4. Results and discussion

20

100

20

30

40

50

Time (hours) Fig. 1. Release profiles of macromolecules from fibrin microspheres of two different sizes prepared in the oil phase having a light mineral oil/oleic acid ratio of 100:1 (C)) and 400:1 (O ). (n = 9 ) .

with plasmin, indicating that both were entrapped inside the fibrin beads without denaturation. Since the amount of these proteins added was completely dissolved in the fibrinogen solution, employment of Eq. ( 1 ) to describe the release of the solute in a dissolved state is appropriate. As shown in Fig. l, there is a clear dependence on the size of the fibrin microspheres for macromolecule release from fibrin microspheres; the smaller the size of the fibrin microspheres, the faster the release rate of the macromolecule. This complies with the theoretical prediction of Eq. ( 1 ). The solid lines in the plots of Fig. 1 are the regression results of the fractional release (Mt/M~) versus time according to Eq. ( 1 ) by assuming the radius a of the fibrin beads is equal to the mean size of distribution and using release data greater than 40%. Comparing the exponential terms in Table 2, it is also concluded

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H.-O Ho et al. / Journal of Controlled Release 34 (1995) 65-70 20

.-g Q)

o,'>. to

I0 9 8 7

o v-I

°,.,.i

tO

o

r.D o

°f.-i

IL .r,-I

09 08

07 06

t

05 0

10000

I

I

20000

30000

Molecular

k 40000

I 50000

I 60000

70000

Weight

Fig. 2. Relationship between logarithmic diffi]sion coefficients and molecular weight of proteins. Symbols as in Fig. 1.

that the smaller the size of the fibrin beads prepared in the oil phase with a 100:1 ratio of mineral oil to oleic acid, the faster the release of the macromolecules. This is due to the larger surface area available for drug release.

A plot of the logarithmic apparent diffusion coefficients versus the molecular weight of each protein is shown in Fig. 2 and indicates that the larger the molecular weight of the macromolecules, the smaller the apparent diffusion coefficients. This is possibly due to

Table 2 The regression results of protein release test according to late time approximation (n = 9) Mineral oil/oleic acid

Exponential term

Apparent diffusion coefficient ( × 10 9 cm2/s)

400/1

100/1

400/1

100/1

Insulin"

0.4732

1.1468

12.8480 (0.0991) b

4.2688 (0.2224)

Lysozyme Carbonic anhydrase

0.1987

0.4355

Ovalbumin

0.1196

0.3071

Bovine serum albumin

0.0780

0.2524

5.4074 (0.0374) 3.2541 (0.0148) 2.1242 (0.0093)

1.6211 (0.1228) 1.1431 (0.0737) 0.9393 (0.0068)

"Dissolved in 1.0% ( w / v ) Tween-80 aqueous solution. bStandard deviation.

H.-O Ho et al. / Journal of Controlled Release 34 (1995) 65-70

much more hindrance being encountered by the large size of the macromolecules. Basically, the structural properties of the fibrin beads prepared in different sizes were consistent and drug release from these fibrin beads showed similar diffusion characteristics. Therefore, the apparent diffusion coefficient of each macromolecule in different sizes of the fibrin beads should be similar. However, as indicated in Table 2, the apparent diffusion coefficient for each macromolecule in different sizes of the fibrin beads was quite different. A possible explanation for this is that all beads were assumed to be of the same size, thus resulting in the calculation of each diffusion coefficient deviating from the predicted value. The release of lysozyme from fibrin beads was inhibited without digesting with plasmin. This might be a result o f lysozyme being adsorbed inside the fibrin microspheres through charge interactions. Previously [2], we reported that lysozyme was adsorbed onto fibrin film through charge interaction, resulting in a partition coefficient that was several-fold larger than other macromolecules. Since the isoelectric point of lysozyme is 10.5-11.0 and that of fibrin is slightly greater than 6.3 [9], lysozyme is positively charged and fibrin is negatively charged in pH 7.4 Tris buffer. The same interaction was possible in the release of lysozyme from fibrin bead. There were two possible reasons for insulin not being released when dissolved in 0.1 N HC1 solution. The first is due to the same charge interaction as in the case of lysozyme. The other is due to the change in the structure of fibrin prepared in a medium of different pH value. As indicated by the pI of insulin and fibrinogen, the pH of the solution needs to be around 5.5-6.5 to result in a charge interaction between insulin and fibrinogen. The pH value of the mixture of the fibrinogen solution and insulin dissolved in 0.1 N HC1 was found to be 6.47-1-0.02. Therefore, a charge interaction between insulin and fibrin was possible but may be minor. On the other hand, a rigid appearance of the fibrin beads containing the acidic insulin solution was noticed. Possibly, a greater hindrance in the fibrin bead was encountered by insulin in this situation. It has been discussed that the properties of the fibrin microspheres are delicately dependent on a host o f factors operating during the polymerization of the fibrin: concentration o f fibrinogen, activity of thrombin, rate o f polymerization, temperature, pH etc. The polymerization rate is

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strongly dependent on pH, with higher pHs having an inhibitory effect. This seems to indicate that the possibility of the polymerization of fibrin in a weak acidic medium results in a rigid structure [ 10].

5. Conclusion The unique properties of the fibrin microsphere make it a viable system for drug delivery. Biodegradation of the fibrin microsphere follows a natural, physiologic process, making it ideal as a surgically implantable or injectable delivery device. In conclusion, the release behavior of proteins from fibrin microspheres is dependent mainly on the molecular size of the macromolecules, the medium used to prepare the fibrin beads and the resulting size distribution. In addition, charge interaction may also interfere with the release of macromolecules. Therefore, the desired release rate can only be engineered into the drug delivery systems by controlling these factors.

Acknowledgements We would like to express our sincere thanks for the financial support of the National Science Council of the Republic of China ( N S C 83-0412-B038-004).

References [ 1] R.I. Senderoff, M.T. Sheu, and T.D. Sokoloski, Fibrin based drug delivery system, J. Parenter. Sci. Technol. 45 ( 1991 ) 26. [2] H.-O. Ho and C.-Y. Chen, Diffusion characteristics of fibrin films. Int. J. Pharm. 90 (1993) 95-104. [3] R. Machovich, The Thrombin, CRC Press, Inc., Boca Raton, FL, 1984, pp. 46-49. [41 L. Graf, E. Barat, J. Borvendeg, I. Herman and A. Patthy, Action of thrombin on ovine, bovine and human pituitary growth hormones, Eur. J. Biochem. 64 (1976) 333-340, [5] S. Miyazaki, N. Hashiguchi, M. Sugiyama, M. Takada and Y. Morimoto, Fibrinogen microspheres as novel drug delivery systems for antitumor drug, Chem. Pharm. Bull. 34 (1986) 1370-1375. [6] M.T. Sheu, C.H. Liu and T.D. Sokoloski, Prolongation of drug release by covalent bonding of drugs to serum albumin microbeads, Drug Design Del. 7 ( 1991 ) 251-257.

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[7] H.O. Ho, C.C. Hsiao, C.Y. Chen, T.D. Sokoloski and M.T. Sheu, Fibrin-based drug delivery systems, II: The preparation and characterization of microbeads, Drug Dev. Indust. Pharm. 20 (1994) 535-546. [8] R. Baker, Controlled Release of Biologically Active Agents, WiLey, New York, 1987.

[9] T.M. Price, D.D. Strong, M.L. Rudee and R.F. Doolittle, Shadow-cast electron microscopy of fibrinogen with antibody fragments bound to specific regions, Proc. Natl. Acad. Sci. USA 78 (1981) 200. [10] J. Martinez, R.R. Holbum, S.S. Shapiro and A.J. Erslev, Fibrinogen, Philadelphia, J. Clin. Invest. 53 (1974) 600~604.