Controlled macromolecule release from poly(phosphazene) matrices

journal of

controlled release

ELSEVIER

Journal of ControlledRelease 40 (1996) 31-39

Controlled macromolecule release from poly(phosphazene) matrices Sobrasua M. Ibim a, Archel A. Ambrosio b, Deidre Larrier a, Harry R. Allcock Cato T. Laurencin c,d,e,.

b,

a Department of Chemical Engineering, Massachusetts Institute of Technology. Cambridge, MA, USA b Department of Chemistry The Pennsyh,ania State University, UniuersiO, Park, PA, USA c Department of Orthopaedic Surgery, The Medical College of Pennsyh,ania and Hahnemann Universi~, Philadelphia, PA, USA Department of Chemical Engineering, Drexel Universit3'. Philadelphia. PA, USA e The Helen Moorehead-Laurencin Laborato~ for Biomaterials Research, Division of Health Sciences and Technology, Massachusetts Institute of Technology. Cambridge, MA. USA

Received 12 May 1995; revised 1 September 1995; accepted 8 September 1995

Abstract

Hydrolytically unstable poly(phosphazene) PPHOS matrices with 50% ethyl glycinato/50% p-methylphenoxy substitution were investigated as vehicles for the controlled release of macromolecules. Specifically, the effects of matrix pH environment and macromolecule loading were studied. 14C-labeled inulin was incorporated into matrices by a solvent casting technique at 1, l0 and 40% loadings (w/w). Degradation and release studies were performed at 37°C at pH 2.0, 7.4 and 10.0. The PPHOS polymer degraded relatively slowly in neutral and basic solutions (pH 7.0 and pH 10.0). In contrast, significantly ( p < 0.01) higher levels of degradation were seen in acidic solutions (pH 2.0) after 35 days. The presence of the hydrophilic macromolecule inulin in the polymer matrix resulted in increased degradation of PPHOS with time. Inulin release, like polymer degradation, was highest at pH 2.0 followed by pH 10.0 and 7.4. Inulin release appeared to be dependent on polymer degradation and inulin diffusion. High inulin loading increased the levels of initial drug burst release and resulted in higher levels of ultimate drug release as measured at 25 days. Environmental scanning electron microscopy (ESEM) demonstrated smooth surfaces on matrices without drug, rough and granular surfaces on matrices loaded with inulin before release, and surfaces possessing micropores and macropores after inulin loading and release. PPHOS polymers can predictably release macromolecules such as inulin. Release can be modulated through changes in pH environment and drug loading. Keywords: Polyphosphazene;Polymer; Macromolecule;Inulin; Biodegradation;Drug release

1. I n t r o d u c t i o n

* Corresponding author. The Medical College of Pennsylvania Hospital, 3300 Henry Avenue, 7th FI., Philadelphia, PA 19129, USA.

Folkman and Long [1] first demonstrated the use of silicon rubber as a vehicle for the sustained delivery of low molecular weight compounds. Their work prompted subsequent early studies by others

0168-3659/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0168-3659(95)00136-0

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S.M. lbim et al. / Journal of Controlled Release 40 (1996) 31-39

using various delivery devices [2,3]. Controlled release of a wide range of macromolecules (Mol. Wt > 1000) was first performed using ethylene vinyl acetate copolymers [4]. More recently, great attention has focused on the development of degradable, biocompatible polymers for drug delivery applications [5-11]. Among these polymers include poly(lactideco-glycolides), poly(anhydrides), poly(orthoesters), p o l y ( c a p r o l a c t o n e ) and p o l y ( p h o s p h a z e n e s ) (PPHOS). The latter class of polymers has been of great interest to our laboratory because of their possible applications in controlled release and tissue engineering [12-16]. Poly(phosphazenes) are a novel class of highmolecular-weight polymers consisting of alternating nitrogen and phosphorus atoms in the backbone of the polymer [12]. Some side chains on the polymer confer hydrolytic instability. When the polymer degrades, by-products are produced which include ammonia, phosphate and side chain groups [12-17]. Previous studies have demonstrated these polymers are tissue compatible [18-20] and can promote cell adhesion and proliferation [16]. However, few studies have examined this polymer for the controlled delivery of macromolecules. The purpose of this investigation was to evaluate the suitability of matrices of poly[(50% p-methylphenoxy) (50% ethyl glycinato) phosphazenes] (PPHOS) for the controlled release of macromolecules and provide information on the degradation and release characteristics of this polymer, specifically in regard to effects of pH environment and drug loading.

2. Materials and methods

2.1. Materials 2.1.1. Poly(phosphazene) synthesis Poly(phosphazene) (PPHOS) polymer containing 50% ethyl glycinato and 50% methylphenoxy substitution was synthesized as described elsewhere [21]. Briefly, polydichlorophosphazene (Ethyl Corp.) was dissolved in tetrahydrofuran (THF) (No. TX0282-1, EM Industries Inc, Gibbstown, NJ) and reacted with the sodium salt of p-methylphenol (Aldrich). The mixture was refluxed for 48 h. Ethyl glycinato-HCl

,<2>

N~"- P

.l._a.0A Fig. 1. The structure of poly[(50% p-methylphenoxy)(50% ethylglycinato)phosphazene].

(Sigma Chemical Co., St. Louis, MO) was suspended in toluene and triethylamine (TEA) and refluxed for 4 h. After cooling, the TEA-HC1 was filtered while the ethyl glycinato solution was added to the partially substituted polymer and the reaction was continued for 10 h at room temperature, concentrated and isolated by precipitation into heptane (5 × 1 (Omnisolv). The polymer was then dried under vacuum. The molecular weight of the polymer was estimated by gel permeation chromatography (GPC) The 50% ethyl glycinato/ 50% methylphenoxy substituted poly(phosphazenes) (Fig. 1) had a moleculal weight of 1 × 106 (Table 1). The structural composi. tion of the polymer was analyzed by 31p and 1H NMR spectrometry (Bruker 360 MHz spectrometer) Elemental analysis was obtained by Gallraith Labo ratories (Knoxville, TN). 14C-labeled inulin (MW = 5000, lot. no. 29757-7) was purchased from Sigm~ Chemical Co. All other reagents were research grade

2.2. Methods 2.2.1. [14 C]Inulin ~polymer fabrication [14C]Inulin was diluted as previously reporte, [22]. Next, 2.4 mg [14C]inulin was added to a: Erlenmeyer flask containing 50 ml of acetone an the solution was stirred. To this mixture was addec with continuous stirring, 72 mg of unlabeled inulir After stirring for 1 h, the solution was filtered usin a Buchner funnel fitted with Whatrnan filter no. 59.' The filtrate was dried in a desiccator and weighed. 1 g of PPHOS was dissolved in THF to make 10% solution. Diluted [14C]inulin (specific activit = 0.055 mCi/mg) was added to the polymer prep~ ration to obtain 1, 10 or 40% loading by weigl respectively. The solution was stirred for 1 h and tl~

S.M. lbim et al./ Journal of ControlledRelease 40 (1996) 31-39 polymer/inulin mixture was cast in Teflon-lined petri dishes and dried overnight at room temperature. The polymer was frozen at - 2 0 ° C for 3 h and lyophilized using a Lyoph-Lock 12 lyophilizer (Labconco Corp., Kansas City, MO) for 48 h. Circular discs (7.0 mm x 1.5 mm) were produced using a cork borer (no. 7). The average weight of the PPHOS discs was 96 _ 3 mg. Radioactivity was determined by dissolving the polymer discs in THF and 200 /zl was added to 10 ml of scintillation cocktail (Hydrofluor, no. LS-I 11, National Diagnostics, Atlanta, GA) and the mixture was counted using a scintillation counter. In addition, the total radioactivity released into the buffer solution and the amount retained in the polymer at the end of the study was also determined. The amount of [14C]inulin released was expressed as percent release over time (h).

2.3. Degradation and release studies 2.3.1. Degradation (mass loss) Studies were performed in distilled water with pH adjusted continuously to 2.0, 7.4 and 10.0, using HC1 or NaOH. Weighed polymer discs containing 1%, 10% and 40% inulin and control discs (without inulin) (average weight of discs = 96.0 + 3.0 mg, 7.0 mm diameter, X 1.5 thick) were placed in glass vials containing 10 ml of distilled water at the chosen pH values in triplicate and maintained at 37°C. At specific time points (0-35 days), the discs were removed, and rapidly frozen in liquid nitrogen. The samples were then lyophilized for 3 days and their weights determined. Rates are expressed as cumulative percent degradation over time (days). 2.3.2. Effects of pH The effects of pH on the release of [14C]inulin were studied. Discs in triplicate were placed in vials containing 10 ml phosphate buffer at pH 2.0, 7.4 or 10.0. The vials were placed in a water-bath/shaker (Precision, American Scientific Products, Long Island, NY) maintained at 37°C. At specific time points (0-25 days), the buffers were changed. Samples (200 /zl) of the released [14C]inulin solutions were placed in 10 ml of scintillation fluid and counted for radioactivity using the scintillation counter. At the end of the study, the residual polymer discs were

33

weighed, dissolved in THF and measured for residual radioactivity. The cumulative amount of inulin released was expressed as cumulative percent inulin released over time (days).

2.3.3. Effects of loading ~4C-labeled inulin was incorporated into PPHOS matrices using a solvent casting technique as previously described [12] to produce polymer films with 1, 10 and 40% inulin loading by weight. Release studies were performed as described earlier and the data obtained were expressed as percent (%) release over time (days). 2.4. Environmental scanning electron microscopy (ESEM) Polymer films with and without inulin were analyzed for changes in surface appearances using environmental scanning electron microscopy (ESEM) (Electro Co., Wilmington, MA) with a Trector Detector. Unlike scanning electron microscopy (SEM) which requires sample coating with gold or platinum prior to analysis, the samples for ESEM were not gold-coated, thus permitting visualization of degradable polymers in a relatively undisturbed state. Scanning was performed at 5.4 Torr vacumn and at an accelerating voltage of 30 kV. Polymer discs were rinsed with distilled water, blotted dry rapidly and frozen in liquid nitrogen and lyophilized for 3 days prior to analysis. The films were analyzed prior to and after degradation/release studies.

3. Results

3.1. Polymer degradation - pH effects and drug loading effects 3.1.1. Polymer degradation: pH and drug loading effects Fig. 2 shows the degradation of PPHOS in distilled water at various pH values during 35 days. Degradation of the matrices in general was characterized by highly reproducible mass loss during the period studied. The polymer matrix degraded fastest in strongly acidic media (pH 2.0), and relatively slower in neutral and basic media (pH 7.4 and pH

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S.M. lbim et al, / Journal of Controlled Release 40 (1996) 31-39

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Fig. 2. Percent mass loss of poly(phosphazene) (PPHOS) substituted with 50% ethyl glycinato/50% methylphenoxy PPHOS without inulin in water (pH 2.0) after 35 days. Points represent mean_+ SD, n = 3.0. Asterisks ( * ) indicate stafiscally significant differences ( p < 0,001) at pH 2.0 relative to pH 7.4 and pH 10.0. (O) pH 10.0; ( 0 ) pH 7.4; (D) pH 2.0;.

Fig. 4. Percent release of 1% loaded inulin from poly(phosphazene) (PPHOS) substituted with 50% ethyl glycinato/50% methylphenoxy at pH 2.0, pH 7.4 and pH 10.0 after 25 days. Error margin is less than 10%. Points represent mean_+SD, n = 3. (O) pH 10.0: (Q) pH 7.4; (m) pH 2.0.

10.0) respectively. Without inulin, PPHOS matrices degraded approximately 63% at pH 2.0 at 35 days, and significantly faster ( p < 0.01) than the matrices placed in pH 7.4 and 10.0 solutions which degraded at approximately 18 and 22%, respectively, during the same period.

Fig. 3 presents degradation kinetic data for PPHOS in the presence of inulin at 40% loading. Polymer degradation was found to increase with loading of drug in the matrix. At the highest inulin loading PPHOS matrix degradation was highly significantly greater ( p < 0.001) at pH 2.0 (80% degradation) than at 7.4 and 10.0 (30 and 35% degradation) after 35 days.

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Fig. 3. Percent mass loss of poly(phosphazene) (PPHOS) substituted with 50% ethyl glycinato/50% methylphenoxy containing 40% inulin in water at pH 2.0 after 35 days• Points represent mean_+ SD, n = 3,0• Asterisks (* * ) indicate statiscally significant differences ( p < 0.001) at pH 2.0 relative to pH 7.4 and pH 10.0. (O) pH 10.0: (O) pH 7.4, ([2) pH 2.0.

3.1.2. Inulin release pH effects Fig. 4 shows the release of HC-labeled inulin from PPHOS matrices containing 1% inulin in 0.1 M sodium phosphate buffer at pH 2.0, 7.4 and 10.0. In acidic media (pH 2.0), approximately 43% of inulin was released in 10 days and the release increased to 50% after 25 days. In contrast at pH 7.4 and 10.0, lower amounts of inulin were released with 28 and 35% released by 25 days, respectively. Inulin release, like polymer degradation was highest at pH 2.0 followed by pH 10.0 and 7.4. This phenomenon occurred regardless of initial drug loading. 3.1.3. Effects of loading Figs. 5-7 show release of inulin at 1%, 10% and 40% loading for pH values: 2.0 (Fig. 5), 7.4 (Fig. 63 and 10.0 (Fig. 7). Release was characterized by an initial burst followed by a period of relatively steady release. As loading increased, the burst release effecl

S.M. lbim et al. / Journal of Controlled Release 40 (1996) 31-39

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became more pronounced at all pH values with bursts of approximately 5% with 1% loaded inulin matrices, 10% with 10% loaded inulin matrices and 25% with 40% loaded inulin matrices. At all pH values, increased loading translated to increasing release with highest amounts of release at 40% loading and pH 2.0.

3.1.4. Environmental scanning electron microscopy (ESEM) Fig. 8 shows electron micrographs of P(PHOS) matrices before and after the release of inulin at 25 100

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Time (Days) Fig. 6. Percent release of 1%, 10% and 40% loaded inulin from 50% e t h y l / 5 0 % methylphenoxy poly(phosphazene) (PPHOS) at pH 7.4 after 25 days. Points represent mean + SD, n = 3. Error margin is less than 10%. ((3) 1%; ( [ ] ) 10%; ( 0 ) 40%.

Fig. 7. Percent release of 1, 10 and 40% loaded inulin from 50% ethyl g l y c i n a t o / 5 0 % methylphenoxy poly(phosphazene) (PPHOS) at pH 10.0 after 25 days. Points represent mean + SD, n = 3. Error margin is less than 10%. ((3) 1%; ( [ ] ) 10%; ( 0 ) 40%.

days. The surfaces of P(PHOS) matrices without inulin appear fairly smooth (Fig. 8a). In 1% inulinloaded polymeric films, the surfaces appear rough, dense and granular (Fig. 8b) while pores or cracks characterize the surfaces of films following degradation and subsequent release of inulin in distilled water at pH 2.0 (Fig. 8c), pH 7.4 (Fig. 8d) and pH 10.0 (Fig. 8e).

4. D i s c u s s i o n

Folkman and associates [1] demonstrated the feasibility of macromolecule transport using silicon rubber as a vehicle, thus paving the way for subsquent development of biomaterials as delivery devices for high molecular weight compounds and proteins. Several biomaterials since then have been developed and used as vehicles for the delivery of macromolecule [4-17]. Biodegradable polymeric materials have several advantages over non-degradable materials for use in drug delivery. These materials obviate the need for surgical removal of the implanted devices [9-11]. Some biodegradable materials are hydrolytically unstable in aqueous environments, and drugs may be released from these matrices by hydrolytic polymer degradation, drug diffusion or a combination of both mechanisms.

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S.M. lbim et al. / Journal of Controlled Release 40 (1996) 31-39

S.M. lbim et al. / Journal of Controlled Release 40 (1996) 3 1 - 3 9

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Fig. 8. Environmentalscanningelectron micrographs (ESEM) showingsurface appearance of poly(phosphazene)(PPHOS) matrices with or without inulin before and after release or degradation in various solutions. The surfaces of the matrices without inulin are smooth and generally devoid of cracks or pores (a). The surfaces of inulin-loaded matrices before release (b) are granular and coarse. Matrices containing 1% inulin are high porous after degradation after 840 h in distilled water at pH 2.0 (c), while the surfaces of matrices at pH 7.4 and 10.0 have rough appearances (d) and (e), respectively, with limited pores relative to matrices at pH 2.0 (c).

Poly(phosphazenes) in general have mainly been studied as vehicles for the transport of low molecular weight molecules. A few studies have examined PPHOS materials containing imidazolyl methylphenoxy, phenylalanine ethyl ester and glutamic acid diethyl ester for the controlled release of naproxen, phenylalanine mustard (melphalan), progesterone and p-nitroaniline [I1,18,19]. There has been considerable interest in the synthesis, characterization and degradation characteristics of hydrolytically unstable

poly(phosphazenes) containing amino acids [22-24] More recently, studies have examined release of low molecular weight species from amino acid containing matrices [25]. We have utilized inulin in this study as a model macromolecule [26,27]. Inulin is a polysaccharide with a molecular weight of 5000 and has been extensively studied. It is not metabolized and thus completely excreted allowing for assessment and comparison of release rates in vitro and in vivo [28-30].

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S.M. lbim et al. / Journal of Controlled Release 40 (1996) 31-39

PPHOS matrices containing amino acids have been shown to undergo a slow decrease in molecular weight in solution and in the solid state. A decline in molecular weight from 8000 repeat units to less than 1000 units in 15 days in solid state at 25°C has been reported [17,21]. The pH of the surrounding environment influenced the rate of polymer degradation in this study. Levels of degradation were greatest in acidic environments, followed by basic environments and then neutral environments. In acidic, neutral and basic solutions, the effects of pH on PPHOS degradation may involve protonation-type reactions as previously suggested [17,21]. Protonation of skeletal nitrogen favors rapid hydrolysis. However, protonation of the side group nitrogen followed by nucleophilic attack of water at phosphorus may also be an important route to degradation. Crommen et al. [23] reported increased hydrolysis of these polymers in solutions containing acetic acid and in contrast to slower hydrolysis in basic solutions. The quantitative findings in this study are in agreement with the qualitative observations of their study and also with previous reports by Allcock [17,21]. Degradation of this polymer has been found to be dependent on the initial content of the hydrolysis inducing side chain group [16]. Increasing the initial content of ethyl glycinato side chain results in increased rates of degradation [16]. (This increase in degradation rate has been found to directly increase drug release [11].) At relatively high content, the polymer is completely bioerodible [16]. At lower levels of hydrolysis inducing side chain, the polymer may not be completely bioerodible, but may completely resorb in an in vivo environment. This is the subject of current study. Macromolecule release from the polymer matrix was characterized by a burst effect followed by a period of near constant release kinetics. Increased loading resulted in an increase in both the burst and in amounts of inulin released. Release appears to proceed through a combination of erosion and diffusion mechanisms. Increasing the loading in the matrix probably results in increased matrix hydrophilicity translating to increased matrix degradation. The environmental scanning micrographs showed pores of various sizes after 35 days in acidic or basic media. These pores appeared larger at pH 2.0 rela-

tive to pH 7.4 and 10.0 and it is interesting to postulate whether these differences in pore formation relate to differences in degradation phenomena seen. The present study evaluated the suitability of the PPHOS materials as transport devices for macromolecules under various loading levels and in a range of pH. Degradation of the matrices was reproducible at all pH values and found to increase with loading of inulin. In buffered solution at neutral pH (7.4), PPHOS matrices continued to release inulin through 25 days and beyond. Of the pH values studied, amounts of inulin release were highest in strongly acidic environments and next highest in the basic solutions with amounts of release the lowest at neutral pH. The effects of pH on drug release were consistent with findings of differential levels of polymer degradation at different pH values. At a broad range of loading, the PPHOS matrix reproducibly released inulin in a controlled fashion. In general, increased loading of macromolecules in the matrix increased amounts of drug released. This report suggests that degradable PPHOS matrices containing amino acids are suitable candidates for the delivery of macromolecules. Further studies involving the polyphosphazenes are underway in a number of areas. While local histological studies examining p-methylphenoxy-substituted polyphosphazenes have been performed [11] more extensive cytotoxicity testing of polymer degradation products is to be performed. Mechanisms specifically involving the relase of drugs from these polymeric systems are to be studied. Finally, the performance of these systems in in vivo, clinically relevant environments are to be carried out.

Acknowledgements This investigation was supported by NIH grant no. AR41972.

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