Microspheres for protein delivery prepared from amphiphilic multiblock copolymers

Journal of Controlled Release 67 (2000) 249–260 www.elsevier.com / locate / jconrel

Microspheres for protein delivery prepared from amphiphilic multiblock copolymers 2. Modulation of release rate J.M. Bezemer a , R. Radersma a , D.W. Grijpma a , P.J. Dijkstra a , C.A. van Blitterswijk a,b , J. Feijen a , * a

Institute for Biomedical Technology ( BMTI), Polymer Chemistry and Biomaterials, Faculty of Chemical Engineering, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands b IsoTis BV, Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, The Netherlands Received 20 August 1999; accepted 24 January 2000

Abstract Amphiphilic multiblock copolymers, based on hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks were used as matrix material for protein-loaded microspheres. The efficiency of lysozyme entrapment by a double emulsion method was found to depend on the swelling behavior of the polymers in water and decreased from 100% for polymers with a degree of swelling of less than 1.8 to 11% for PEG–PBT copolymers with a degree of swelling of 3.6. The particle size could be controlled by varying the concentration of the polymer solution used in the microsphere preparation. An increase in the polymer concentration resulted in a proportional increase in the particle size. The in vitro release profiles of the encapsulated model protein lysozyme could be precisely tailored by variation of the copolymer composition and the size of the microspheres. Both a slow continuous release of lysozyme, and a fast release which was completed within a few days could be obtained. The release behavior, attributed to a combination of diffusion and polymer degradation, could be described by a previously developed model.  2000 Elsevier Science B.V. All rights reserved. Keywords: Block-copolymer; Controlled release; Protein; Microsphere; Swelling

1. Introduction Injectable controlled release systems for protein and peptide drugs receive much attention [1]. The application of biodegradable amphiphilic block copolymers as matrix material for such dosage forms *Corresponding author. Tel.: 131-53-489-2968; fax: 131-53489-3823. E-mail address: [email protected] (J. Feijen)

offers attractive possibilities in the design of systems with tailor-made properties. Matrix characteristics, such as swelling, permeability and degradation rate, can be precisely controlled by a proper combination of the different block copolymer segments [2–7]. As hydrophilic segment, usually poly(ethylene glycol) (PEG) is selected, because of its non-toxicity, lack of immunogenicity and its solubility in both organic solvents and water [8]. Various other polymers have been applied as biodegradable hydrophobic blocks,

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00212-1

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e.g., polylactide (PLA) [9], polyglycolide (PGA) [10], poly(lactide-co-glycolide) (PLG) [11], poly(´caprolactone) (PCL) [12] and poly(ethylene terephthalate) (PET) [13]. Amphiphilic block copolymer microspheres for protein delivery have been prepared from diblock [14] as well as from triblock [15–20] copolymers. Compared to the widely utilized PLA and PLG, protein delivery systems based on hydrophilic–hydrophobic block copolymers may have some important advantages. Since diffusion of high-molecular weight proteins through the hydrophobic PLA and PLGA polymers is negligible, release is mainly governed by transport through pores and by matrix degradation [21]. Frequently, this is associated with a discontinuous release of the protein [22]. Incorporation of hydrophilic blocks in a hydrophobic polymer can be utilized to modify the degradation rate as well as the permeability of the matrix, leading to improved release kinetics which can be readily modulated by adjusting the copolymer composition [18]. Furthermore, it has been observed that the PLG matrix can negatively affect the stability of the incorporated protein drug [23–29]. Degradation may generate a pH drop within the polymer matrices, which may cause protein instability problems [23– 25]. Furthermore, the hydrophobic nature of PLG is responsible for protein adsorption onto the polymer surface, denaturation and aggregation [26–29]. Using amphiphilic block copolymers as matrix material for a protein delivery system may prevent these problems. Nevertheless, it was observed that the pH within PLG–PEG–PLG triblock copolymer microspheres, immersed in phosphate buffer (pH 7.4), decreased to a value of 3.5 after 200 h [20]. This formation of acid degradation products may be responsible for the incomplete release of recombinant human erythropoietin (EPO) from PLG–PEG– PLG triblock microspheres [19]. Another factor which may negatively affect protein release kinetics is that degradation of di- and triblock copolymers may lead to relatively hydrophobic matrices. Rapid and preferential loss of PEG can be prevented by using block copolymers with multiple short hydrophilic and hydrophobic segments in one polymer [3,30,31]. Furthermore, we expect that such amphiphilic multiblock copolymers offer more flexibility in tailoring the properties of a

protein delivery system than di- or triblock copolymers. In this study we report on the entrapment of a model protein (lysozyme, 14.5 kDa) using biodegradable multiblock copolymers based on hydrophilic PEG and hydrophobic poly(butylene terephthalate) (PBT) segments. These copolymers are known to be biocompatible and biodegradable [32–38], and are under clinical investigation for a wide range of applications, including bone replacement [39], antiadhesive barrier [37], and artificial skin [35,36]. The first part of the study describes the effect of preparation parameters on particle characteristics and release behavior [40]. The aim of this paper is to modulate protein release kinetics by adjusting the copolymer composition and the size of the microspheres.

2. Materials and methods

2.1. Materials A series of poly(ethylene glycol)terephthalate– poly(butylene terephthalate) (PEG–PBT) multiblock copolymers was obtained from IsoTis (Bilthoven, The Netherlands). The poly(ether ester) copolymers used in this study vary in PEG–PBT weight ratio (80:20–30:70) and PEG segment length (600, 1000 and 4000 g / mol), and are indicated as aPEGbPBTc, in which a is the PEG molecular weight, b the wt% PEG-terephthalate and c the wt% PBT. The molecular weights (determined by gel permeation chromatography (GPC) relative to polystyrene) and the equilibrium swelling in PBS at 378C of the polymers were measured as described elsewhere [3]. Phosphate-buffered saline (PBS), pH 7.4, was purchased from NPBI (Emmercompascuum, The Netherlands). Polyvinylalcohol (PVA, Mw 522 000 g / mol, approximately 80–90% hydrolyzed) was obtained from ICN Biomedical, lysozyme from chicken egg white (33 crystallized, dialyzed and lyophilized) was purchased from Sigma (St. Louis, MO, USA). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) (1 M) was obtained from Fluka Chemika, NaHCO 3 , 1 M NaOH, HCl (37%), chloroform (CHCl 3 ), obtained from Merck (Darmstadt, Germany) and hexafluoroisopropanol, obtained from Aldrich, (Belgium) were of analytical grade.

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2.2. Preparation of PEG–PBT microspheres Lysozyme-loaded microspheres were prepared by a water-in-oil-in-water (w / o / w) emulsion method [40]. A lysozyme solution (0.6 ml, 55 mg / ml) in PBS (pH 7.4) was emulsified in a solution of 1 g PEG–PBT in chloroform using an ultra-turrax (T25 Janke & Kunkel, IKA-Labortechnik, 30 s at 20 500 rpm). The w / o emulsion was slowly poured into 50 ml PBS containing 4 wt% PVA (Mw 522 000). After 5 min stirring at 400 rpm, 200 ml PBS was added. This was kept at a constant stirring rate of 800 rpm for 2 h at ambient conditions. The microcapsules were collected by filtration, washed with PBS and lyophilized. The product was stored at 2208C. To study the influence of the copolymer composition on the protein release behavior, a series of polymers was used, with PBT contents varying from 20 to 60 wt% and PEG molecular weights of 600, 1000 and 4000 g / mol. Polymers were dissolved in chloroform, except for the polymer 1000PEG40PBT60, which was dissolved in 7 ml of a mixture of chloroform and hexafluoroisopropanol (6:1, v / v). To prepare microspheres of different sizes, the polymer concentration in chloroform was varied from 0.143 to 0.048 g / ml.

2.3. Determination of microsphere size The size (number and volume mean diameter) and the size distribution of microspheres suspended in water was determined with use of a Microtrac (X100, Leeds & Northrup).

2.4. Scanning electron microscopy ( SEM) A Hitachi S-800 field emission SEM was used to evaluate the surface characteristics of the microspheres. Samples were sputter-coated with a thin gold–palladium layer. The internal structure of the microspheres was observed after fracturing of the microspheres in liquid nitrogen.

2.5. Determination of the lysozyme content in the microspheres Microcapsules (20–30 mg) or 50 ml of protein standard solutions (0–30 mg lysozyme per ml) were

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incubated with 1 ml 6 M HCl for 24 h at 378C. Subsequently, 6 ml 1 M NaOH were added and the suspension was shaken for another 24 h at 378C. The protein concentration in the solution was determined using trinitrobenzenesulfonic acid (TNBS). In short, 50 ml portions of the samples were added to a 96-well microplate and mixed with 125 ml of a 4% (w / v) NaHCO 3 (pH 9) and 50 ml of a 0.5% (w / v) TNBS solution. After 2 h incubation at room temperature, the extinction at 450 nm was measured using a SLT 340 ATTC microplate reader. The extinction was related to the protein concentration using the lysozyme standard solutions. The method was validated by determination of the lysozyme content of PEG–PBT films containing a known amount of lysozyme. The entrapment could be determined in a reproducible and quantitative manner.

2.6. In vitro protein release Protein-loaded microspheres (15 mg) were immersed in 1.5 ml PBS (pH 7.4). Tubes were continuously shaken at 378C and samples were taken at various time points after the suspension was centrifuged (1000 rpm, 3 min). Protein concentration in the buffer was determined using a standard Coomassie Blue assay (Pierce). Buffer was refreshed after sampling.

2.7. Modeling of the effect of particle size on lysozyme release from 600 PEG77 PBT23 microspheres In order to describe quantitatively the effect of particle size on the rate of lysozyme release from PEG–PBT microspheres, experimental release curves were compared with calculated predictions. The relationship between particle size and the fraction of a drug released from a sphere at time t (Mt /M` ), can be described by well known equations for diffusion [41,42]: ]] M T 3T ]t 5 6 ]]2 2 ] M` pr r2

œ

for Mt /M` , 0.6 and

(1)

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252

S

M 6 p 2T ]t 5 1 2 ]2 exp 2 ]] M` p r2

D

(2)

for Mt /M` . 0.4, where r is the average radius of the spheres and T is a function of the time and the drug diffusion coefficient. As shown in a previous paper, the diffusion coefficient for diffusion of lysozyme through PEG–PBT matrices is not constant, but a function of the release time, due to the effect of polymer degradation [7]. The following empirical relationship for T has been found: t

E

1 1 T 5 D(t) dt 5 Dinitial (t 1 ]at 2 1 ]bt 3 ) 2 3

(3)

0

with Dinitial is the lysozyme diffusion coefficient through the PEG–PBT matrix at t50 and a and b are constants for a certain matrix–drug combination, representing the effect of polymer molecular weight and rate of degradation on the diffusion coefficient. In this study, the effect of the average size of 600PEG77PBT23 microspheres on the lysozyme release rate was investigated. The constants a and b in Eq. (3) for this particular system were obtained by curve fitting of the release of lysozyme from 600PEG77PBT23 films with known dimensions, molecular weight and initial lysozyme diffusion coefficient Dinitial (2.1310 213 cm 2 / s [7]). The best fit was obtained when a51310 27 s 21 and b51.13 10 211 s 22 . It is assumed here that the lysozyme diffusivity in microspheres is the same as in films, which was previously confirmed [7].

3. Results and discussion

3.1. Effect of copolymer composition on lysozyme entrapment Lysozyme-containing PEG–PBT microspheres were prepared by a multiple emulsion technique [40]. The first step in this process is the formation of a primary water-in-oil (w / o) emulsion from an aqueous protein solution and a polymer solution. This w / o emulsion is subsequently dispersed in an aqueous poly(vinyl alcohol) (PVA) solution, resulting in a w / o / w emulsion. Hardening of the microspheres is accomplished by evaporation of the or-

ganic solvent. To manipulate the release properties of the microspheres, a series of copolymers, with different composition, was used as matrix material. The multiblock copolymers used in this study vary in PEG–PBT weight ratio (80:20–30:70) and PEG segment length (600, 1000 and 4000 g / mol). The degree of swelling in PBS (Q) of the copolymers increases with increasing PEG content and with increasing molecular weight of the PEG segment. Within the composition range tested, the equilibrium swelling ratio Q varies from 1.26 for polymers with PEG segments of 600 g / mol and a PBT content of 60 wt% up to 3.64 for polymers with PEG segments of 4000 g / mol and a PEG–PBT weight ratio of 80 / 20 (Table 1). The weight average molecular weight of the polymers is in the range of 75–120 kg / mol, all with a molecular weight distribution of about 2, which is expected for polymers prepared by polycondensation reactions. As discussed in detail elsewhere [7], the permeability of the PEG–PBT block copolymers for lysozyme is strongly dependent on the degree of swelling of the copolymers. An increase in swelling from 1.48 up to 3.66 causes an almost 50 000-fold increase in the lysozyme diffusion coefficient (Table 1). It can be expected that this will influence the characteristics of lysozyme-containing microspheres to a large extent. Representative scanning electron micrographs of the surface of PEG–PBT microspheres are given in Fig. 1. Microspheres prepared from relatively hydrophobic block copolymers (600PEG40PBT60) present a smooth surface, with small surface pores (Fig. 1A), whereas the morphology of hydrophilic particles (1000PEG70PBT30) was much rougher (Fig. 1B). As already noticed by others [18], the rough surface structure of amphiphilic microspheres is the consequence of the w / o / w preparation process. The microparticles are formed in the swollen state, and subsequent dehydration by vacuum drying prior to SEM will cause collapse of the hydrogel structure. The internal structure of the microspheres was dense, with only some small pores (Fig. 2). A clearly different morphology was found for microspheres prepared from 1000PEG40PBT60 (Fig. 3). A large number of hollow microspheres was observed, as well as particles with a porous internal structure. This particular copolymer was dissolved in a mixture of chloroform and hexafluoroisopropanol (HFIP),

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Table 1 Molecular weights, equilibrium swelling and initial diffusion coefficient for lysozyme of poly(ether ester) copolymers in PBS at 378C (from Ref. [3,7]) ]a ]a Polymer Mw Mn Qb Dlysozyme c (kg / mol) (kg / mol) (310 212 cm 2 / s) 600PEG77PBT23 600PEG55PBT45 600PEG40PBT60 1000PEG70PBT30 1000PEG60PBT40 1000PEG40PBT60 4000PEG80PBT20 4000PEG55PBT45

77.6 114.3 116.0 99.3 94.2 99.5 88.1 97.0

40.8 53.2 54.9 45.0 45.0 45.6 36.7 45.5

1.65 1.35 1.26 1.96 1.83 1.48 3.64 2.63

0.2160.01 – – 5.460.2 0.3060.03 0.0860.01 39006300 710620

a

Determined by gel permeation chromatography (GPC). Q5(equilibrium swollen volume) /(dry volume). c Dlysozyme is the initial lysozyme diffusion coefficient through a PEG–PBT matrix of a particular composition and molecular weight, before degradation has a significant influence on the diffusion (see Section 2.7). b

while the other polymers were dissolved in chloroform. HFIP was needed as a cosolvent to dissolve the long crystallizable PBT blocks of 1000PEG40PBT60. HFIP is miscible with water, and will be rapidly extracted from the polymer phase in

the w / o / w emulsion, leading to fast precipitation of the matrix polymer and the formation of porous and / or hollow microspheres. Particle size and lysozyme entrapment efficiency of microspheres prepared from the various PEG–

Fig. 1. Scanning electron micrographs of the surface of lysozyme-containing microspheres prepared from 600PEG40PBT60 (A) and 1000PEG70PBT30 (B).

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Fig. 2. Scanning electron micrograph of the internal structure of lysozyme-containing microspheres prepared from 1000PEG60PBT40.

PBT copolymers are presented in Table 2. A large difference between number and volume mean particle size was observed, indicating that the particle size distribution was broad. The (volume mean) diameter of the swollen particles was usually between 100 and 150 mm. However, if 1000PEG40PBT60 or block copolymers with PEG segments of 4000 g / mol were used as matrix material, larger microspheres were produced with a volume mean diameter in the range of 200–415 mm. Concerning 1000PEG40PBT60 microspheres, the larger particles may result from the above-described rapid precipitation of this polymer, which prevents shrinkage of the microspheres during drying. Two factors may contribute to the large diameter of 4000PEG80PBT20 and 4000PEG55PBT45 microspheres. First, it has to be noted that the particle size was determined in the swollen state. The polymers with relatively long PEG segments of 4000 g / mol display a higher degree of swelling compared to the

Fig. 3. Surface morphology (A) and internal structure (B) of lysozyme-loaded PEG–PBT microspheres prepared from 1000PEG40PBT60.

polymer with PEG blocks of 600 and 1000 g / mol (Table 1). Second, it was observed that the w / o emulsions prepared from the block copolymers with PEG segments of 4000 g / mol were more viscous than emulsions based on copolymers containing shorter PEG segments. As is discussed below, a higher viscosity of the dispersed phase leads to larger particles. The entrapment efficiency of lysozyme was strongly dependent on the copolymer composition (Table 2). Fig. 4, which correlates the equilibrium swelling data from Table 1 and the entrapment

J.M. Bezemer et al. / Journal of Controlled Release 67 (2000) 249 – 260 Table 2 Characteristics of lysozyme-loaded poly(ether ester) microspheres prepared from polymers of different copolymer composition (concentration of the polymer solution is 0.143 g / ml chloroform) Polymer

dn a (mm)

dv b (mm)

Entrapment efficiency (%)

600PEG77PBT23 600PEG55PBT45 600PEG40PBT60 1000PEG70PBT30 1000PEG60PBT40 1000PEG40PBT60 4000PEG80PBT20 4000PEG55PBT45

24 64 41 34 38 48 88 117

108 153 142 150 128 215 414 214

8662 10265 9862 6465 103613 5665 1162 2268

a b

Number mean particle size. Volume mean particle size.

efficiency from Table 2, shows that with increasing degree of swelling of the polymers in PBS, the lysozyme entrapment efficiency decreased from 100% to only 11%. This can be ascribed to premature release of lysozyme during the 2-h solvent evaporation in PBS. The release increases with increasing equilibrium water content of the matrix due to a greater diffusivity. An exception is the entrapment in 1000PEG40PBT60 microspheres (indicated in Fig. 4 with a filled symbol). Although the swelling of this polymer is low (Q51.48), the lysozyme content of the microspheres was only 56% of the amount of lysozyme initially used for prepara-

Fig. 4. Efficiency of lysozyme entrapment in PEG–PBT microspheres as a function of the equilibrium swelling (n53; 6S.D.).

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tion (33 mg per g of polymer). This may be the result of the porous and hollow structure of these microspheres (Fig. 3), which will facilitate premature leakage of lysozyme into the external aqueous phase during microsphere hardening.

3.2. Effect of copolymer composition on lysozyme release Release profiles of the model protein lysozyme from PEG–PBT microspheres varying in copolymer composition are presented in Fig. 5. Not included is the release from microspheres prepared from polymers with PEG segments of 4000 g / mol, since the release could not be detected in a proper way, due to the low protein content of these microspheres. Fig. 5 shows that, irrespective of the copolymer composition, an initial protein burst did not occur, indicating that the protein was effectively encapsulated within the core of the microcapsules. The release rates were clearly related to the diffusion coefficient of lysozyme through the copolymers. A higher lysozyme permeability resulted in a faster release rate. Release from 1000PEG70PBT30 microspheres was completed within 10 days, whereas 600PEG77PBT23 continued for about 30 days. A very slow release was observed for most hydrophobic microspheres (600PEG55PBT45 and 600PEG40PBT60). However, 1000PEG40PBT60 microspheres released lysozyme substantially faster than expected on account of the lysozyme diffusion coefficient, which is again most likely due to the hollow and porous structure of these particles. Except for 1000PEG40PBT60 microspheres, near zero-order release of lysozyme was observed. Such a constant release of proteins from biodegradable amphiphilic block copolymer microspheres has been reported before for PLG–PEG–PLG triblock copolymers [15,18]. This release behavior of amphiphilic triblock copolymers was explained by a combination of protein diffusion and rapid mass loss [15]. We have found that protein release from PEG– PBT matrices was not governed by mass loss, but by an interplay of diffusion and chain scission [7]. As has been described elsewhere in detail, hydrolysis of PEG–PBT matrices in PBS results in a decrease of the polymer molecular weight during the release periods [3]. As a result, the permeability of the

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Fig. 5. Release of lysozyme from PEG–PBT microspheres. (A) Molecular weight of the PEG segment is 1000 g / mol and PBT wt% is 30 (h), 40 (n) and 60 (s). (B) Molecular weight of the PEG segment is 600 g / mol and PBT wt% is 23 (s), 45 (h) and 60 (n) (n53;6S.D.).

matrix for the incorporated protein may increase during the release period. This may compensate for the decline in release rate caused by the reduced drug concentration in the matrix, to yield an almost constant release rate [7]. An important characteristic of a protein or peptide release system is the activity of the released proteins. It has been observed that, for example, PLG matrices can negatively affect the stability of incorporated protein drugs [23–29]. In a previous publication we have shown that the activity of proteins, entrapped within PEG–PBT matrices by a w / o emulsion method, was preserved during the formulation, storage and release periods [7]. It can be concluded from this part of the study that the copolymer composition is an effective tool to modulate release of proteins from PEG–PBT microspheres over a prolonged period of time of 30 days.

3.3. Preparation of microspheres with different particle sizes Besides the copolymer composition, the particle size might be used to control the rate of protein release from PEG–PBT microspheres. The size of microspheres, prepared by emulsification techniques can be manipulated by several parameters, such as the mixing speed [43,44], the concentration of the

stabilizer in the external phase [36] and the viscosity of the dispersed phase [17,45–47]. In this study, the viscosity of the dispersed phase (the w / o emulsion) was varied by changing the concentration of the initial polymer solution which was used to prepare the w / o emulsion. Increasing the volume of the organic solvent used to dissolve a fixed amount of polymer (1 g 600PEG77PBT23 per batch of microspheres) resulted in a decrease of the particle size (Fig. 6). This may be due to the increased viscosity of the dispersed phase, leading to less efficient disruption of the w / o droplets by shear forces. Interestingly, over the range of concentrations used in this study, a linear relationship between particle size (number mean as well as volume mean) and polymer concentration was found. The ratio between volume and number mean particle size, which is a measure for the polydispersity, was approximately two for all batches, except for the microspheres prepared from the most concentrated polymer solutions (d v /d n 5 4.5). For microspheres produced using polymer solutions with concentrations lower than 0.14 g / ml, unimodal particle size distributions were found. However, as is shown in Fig. 7, a multimodal size distribution (bi- or tri-modal) was observed for microspheres prepared from the most concentrated polymer solution.

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is reported by others [17,41,44]. A lower viscosity of the dispersed phase leads to a more extensive disruption of the emulsion droplets. During disruption, the aqueous protein-containing domains inside the w / o droplet are exposed to the external aqueous phase, which causes protein leakage [48]. Apparently, a stable polymer film is formed around the water droplets of the w / o emulsion, which prevents exposure of these droplets to the external water phase. Because of their amphiphilic character, the copolymers may act as an intrinsic surfactant, thereby stabilizing the inner water droplets of the w / o emulsion.

3.4. Effect of particle size on lysozyme release Fig. 6. Volume mean (n) and number mean (s) particle size of lysozyme-containing PEG–PBT microspheres as a function of the concentration of the polymer solution used in the preparation of the w / o emulsion.

The efficiency of lysozyme entrapment varied between 86 and 67% for the various batches of 600PEG77PBT23 microspheres. No correlation was observed between the concentration of the initial polymer solution and the entrapment efficiency. It might be expected that the viscosity of the dispersed phase has an effect on the entrapment efficiency, as

Fig. 7. Particle size distribution of lysozyme-loaded PEG–PBT microspheres prepared from a polymer solution of 0.143 g / ml (dense line, volume mean particle size is 108 mm) or 0.091 g / ml (dotted line, volume mean particle size is 63 mm).

The release of lysozyme from microspheres of various sizes is presented in Fig. 8. With increasing microsphere size, a considerable decrease in release rate was found. Lysozyme was released within 10 days from the smallest microspheres (volume mean diameter of 29 mm), whereas release from the largest microspheres (volume mean diameter of 108 mm) continued much longer. Not only the release rate, but also the release kinetics varied with the microsphere size. An almost constant release rate was observed

Fig. 8. Release of lysozyme from 600PEG77PBT23 microspheres. Volume mean particle size of the microspheres is 108 mm (m), 81 mm (s), 63 mm (♦) or 29 mm (h) (n53;6S.D.). Lines are lysozyme release curves calculated by Eqs. (1)–(3) with fitting parameters a51310 27 s 21 and b51.1310 211 s 22 .

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for lysozyme release from batches of microspheres with volume mean diameters of 108 and 81 mm. Decreasing the average microsphere size from 108 to 29 mm causes the release kinetics to change gradually from zero-order release (cumulative release is proportional to the time) to release controlled by Fickian diffusion (cumulative release is proportional to the square root of time) (Fig. 8). In order to describe quantitatively the variation of both the release rate as well as the release profile with the average microsphere size, lysozyme release from the various 600PEG77PBT23 microsphere batches was calculated using a previously employed mathematical description [7]. This model describes the effect of polymer degradation on the diffusion of lysozyme in the polymer matrix. Due to chain scission during the release period, the permeability of the matrix for lysozyme increases, so the diffusion coefficient is time dependent. An empirical equation for the time-dependent diffusion coefficient is incorporated into the well-known equations for diffusion from spheres, as is shown in Section 2.7. Using the volume average diameters from Fig. 6 in Eqs. (1) and (2), the theoretical lysozyme release as a function of time can be calculated for these different microsphere batches. The curves in Fig. 8 show the results of the calculation of lysozyme release from the various microsphere batches. The theoretical curves closely follow the experimental data points, except for release from the largest microspheres. In the latter case, the predicted release was faster than experimentally observed. Probably, this deviation is caused by heterogeneous particle size distribution of the microspheres with mean volume diameter of 108 mm (see Fig. 7). Besides the release rate, also the drift in release profile with decreasing microsphere size (from a constant release rate to release which is determined by Fickian diffusion) can be explained by the model. For small microspheres, release is fast, and contribution of degradation to an increase of the permeability of the microspheres is only limited. In terms of Eqs. (1)–(3), Dinitial (1 1 at 1 bt 2 ) is approximately equal to Dinitial for relatively short release times, which leads to a square root of time dependence of the release. In the case of large microspheres, prolonged release periods and consequently a considerable influence of degradation on

lysozyme diffusion can be expected. Now, the increase of the diffusion coefficient due to polymer degradation may compensate for the decrease in release rate caused by the reduced drug concentration in the matrix, to yield an almost constant release rate.

4. Conclusion Amphiphilic multiblock copolymers, based on hydrophilic PEG blocks and hydrophobic PBT blocks were successfully applied as matrix material for protein-loaded microspheres. The in vitro release profiles of the encapsulated model protein lysozyme could be precisely tailored by variation of the copolymer composition and size of the microspheres. A slow continuous release of lysozyme could be obtained, as well as a fast release which was completed within a few days. The observed release behavior was attributed to a combination of diffusion and polymer degradation, and could be described by a previously developed mathematical model. This study shows that this class of amphiphilic multiblock copolymers has good potential as microparticulate delivery system for protein and peptide drugs.

Acknowledgements This research is financially supported by the Dutch Technology Foundation (STW). M. Smithers is acknowledged for performing the scanning electron microscopy studies

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