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Biomaterials 26 (2005) 3311–3318

www.elsevier.com/locate/biomaterials

Self-curing controlled release systems for steroids. Application ofprednisolone-based polymeric systems to ear diseases

Mar Fernandeza, Juan Parrab, Blanca Vazqueza,�, Antonio Lopez-Bravob,Julio San Romana

aInstituto de Ciencia y Tecnologıa de Polımeros, CSIC. C/Juan de la Cierva, 3, 28006 Madrid, SpainbHospital Provincial de Avila. C/Jesus del Gran Poder, s/n, Avila, Spain

Received 29 June 2004; accepted 8 September 2004

Abstract

An injectable delivery system for prednisolone has been prepared based on a self-curing formulation comprised of poly(methyl

methacrylate) particles and hydroxyethyl methacrylate as monomer. The polymerisation reaction was initiated by the redox system

4,40-bis (dimethylaminobenzydrol)/benzoyl peroxide (BZN/BPO) and followed at 25 1C by measuring the time–temperature profile.

A maximum temperature of 53 1C and a setting time of 15min were obtained, calculated according to standard specifications. The

swelling of the cured system was studied in phosphate-buffered saline (PBS) at 37 1C giving a hydration degree at equilibrium of

20%. The swelling kinetics fitted a fickian behaviour at the initial stages of the experiments, with a diffusion coefficient of

0.72� 10�7 cm2/s. The release of the drug was sustained from the beginning without an initial drug burst. The study of the

wettability showed a rather hydrophilic character of the surface of the loaded system, and the biocompatibility evaluated through

MTT assay revealed the absence of cytotoxicity due to the release of toxic substances.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Drug delivery; PMMA/PHEMA; Prednisolone; Ear disease

1. Introduction

Steroids are widely used to treat inner ear diseases [1].Local administration of steroids has been applied inpatients for whom systemic steroid treatment has failedor who could not tolerate systemic steroid therapy [2]and also as a way to avoid systemic toxicity. Transtym-panic steroids have been applied through a ventilationtube placed with the patient under local anaesthesia [2]or by means of injection or osmotic minipumps [3]. Highdose delivery of methyl prednisolone via perfusion at thelevel of the round window membrane has given goodresults compared with standard treatment of SSHL [4].The use of this therapy has also been proposed for thetreatment of tinnitus [5] or in cases of intractable

e front matter r 2004 Elsevier Ltd. All rights reserved.

omaterials.2004.09.018

ing author. Tel.: 34 91 5622900; fax: 34 91 5644853.

ess: bvazquez@ictp.csic.es (B. Vazquez).

Meniere’s disease [6]. However, current methods do notallow for accurate drug delivery to the inner ear. Theadvantages of using a drug delivery system stem fromthe best control of the dose and lack of loss of the drugfrom the middle ear site. Delivery systems based onbiodegradable sodium hyaluronate gels [7] or systemsbased on absorbable gelatin sponges soaked in the drug[6] have been applied in the treatment of vertigo andMeniere’s disease, respectively. The approach presentedin this paper is the formulation of a corticoid self-curingdelivery system which would be able to provide asustained release of the drug over long period of time.Self-curing delivery systems are well documented in theliterature. Different types of self-curing delivery systemsare reported which have been mainly tested for therelease of antibiotics. Totally, biodegradable systems ofpoly(propylene fumarate) [8], or partially biodegradablesystems formulated with poly(methyl methacrylate)/

ARTICLE IN PRESSM. Fernandez et al. / Biomaterials 26 (2005) 3311–33183312

poly(e-caprolactone) beads have been demonstrated tobe effective in the release of vancomycin [9]. Likewise, asystem incorporating b-tricalcium phosphate (b-TCP),PEMA and MMA has been developed for the controlledrelease of analgesic/anti-inflammatory drugs [10].This paper reports on the preparation and character-

isation of an injectable prednisolone delivery systembased on a support of poly(methyl methacrylate)/poly(hydroxyethyl methacrylate). The free radical poly-merisation reaction was followed by measuring theexotherm of polymerisation from which the settingparameters were determined. Prednisolone release andswelling kinetics were evaluated in vitro in phosphate-buffered saline (PBS) at the physiological temperature.The wettability of the material was assessed by contactangle measurements and the cytotoxicity was deter-mined using several assays that monitor different aspectsof cellular activity.

2. Experimental

2.1. Materials

Poly(methyl methacrylate) (PMMA) beads (33 mm ofaverage diameter) were supplied by Industrias Quirur-gicas de Levante (IQL beads) and have earlier beencharacterised [11]. 2-Hydroxyethyl methacrylate(HEMA) (Fluka), prednisolone (Sigma) and 4,40-bis(dimethylaminobenzydrol) (BZN) (Sigma) were used asreceived. Benzoyl peroxide (BPO) (Fluka) was purifiedfrom fractional recrystallization from methanol,mp=104 1C. Thermanox (TMX) control discs weresupplied by Labclinics S.L. and plasticware by Sarstedt.Tissue culture media, additives, trypsin, PBS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) were supplied by Sigma.

2.2. Preparation and setting parameters of the self-curing

delivery system

The drug-loaded injectable system PMMA/PHEMA/PRED was based on a mixture of 68.5wt% PMMAbeads and 30wt% prednisolone with the inititator BPO(1.5wt%) as the solid component, and 79.7wt% 2-hydroxyethyl methacrylate, 20wt% water and 0.3wt%of BZN as activator of reduced toxicity [12] as the liquidcomponent. A solid:liquid ratio of 1:1 was used.Accordingly, a control formulation (PMMA/PHEMA)was prepared in the absence of the drug for comparisonpurposes. The exothermic polymerisation temperatureprofile was registered using a thermocouple connected toa high-sensitivity thermotester positioned within itsjunction in the centre of the mould at a height of3mm in the internal cavity. The mould (10mm indiameter and 15mm high) was placed in a thermo-

statically controlled bath described in a previous paper[11]. Curing parameters were calculated according to theinternational standard specification ISO 5833 [13].

2.3. In vitro behaviour

2.3.1. Swelling behaviour

Rectangular-shaped samples of 30mm� 10mm and1mm thickness were immersed in PBS at 37 1C. Wateruptake was determined gravimetrically at differentperiods of time. At appropriate times, the samples wereremoved, blotted quickly with absorbent paper toremove the water attached on its surface and weighed.In all the experiments, a minimum of three samples weremeasured and averaged. The percentage of hydrationdegree was calculated according to

% hydration degree ¼ ½ðWw � WdÞ=Wd� � 100; (1)

where Ww is the weight of swollen specimen at time t

and Wd is the initial weight of the dry specimen.

2.3.2. Prednisolone release experiments

Rectangular-shaped samples similar to those used inswelling experiments were employed. The samples weresoaked in 10ml of PBS and kept at 37 1C. Aliquots weretaken at different periods of time and the medium wastotally changed by fresh solution. The concentration ofprednisolone released was determined by visible–ultra-violet spectroscopy (UV-VIS, Perkin-Elmer Lambda 35)analysing the signal at 248 nm corresponding toprednisolone [14]. A calibrated curve of the glucocorti-coid was obtained previously by measuring the absorp-tion of the UV signal of solutions of knownconcentration in the same medium. A minimum ofthree samples were measured and averaged. The surfacesof PMMA/PHEMA/PRED samples before and afterthe release experiment were analysed by ESEM using aPhilips XL 30 microscope.

2.4. Surface characteristics

The contact angle measurements were performed ondry films of material using a Contact Angle MeasuringSystem G10 (Kruss). The surface free energy wascalculated by the approach introduced by Fowkes, inwhich the total surface tension is considered as a sum ofindependent terms, each representing a particularintermolecular force [15], and by the application of theequation of Owens and Wendt [16], which is anextension to a so-called ‘‘polar’’ component. The liquidsused for this purpose were methylene iodide and distilledwater. The dispersion force component and the polarforce component of the surface energy of water are 21and 51mN/m, respectively, and the dispersion forcecomponent of the methylene iodide is 50mN/m.

ARTICLE IN PRESSM. Fernandez et al. / Biomaterials 26 (2005) 3311–3318 3313

2.5. Evaluation of cytotoxicity of leachables from the

cured materials

2.5.1. Specimens and in vitro cell culture for

biocompatibility experiments

Discs of 10mm diameter and 1mm thickness of thecured systems and the controls were used for direct andindirect biocompatibility experiments. All specimenswere sterilised with poly(ethylene oxide). The negativecontrol was tissue culture plastic, Thermanox, aninternational standard, and the positive control (toxicagent) was polyvinylchloride (PVC). The cells used inthe primary cell culture were cells of African greenmonkey kidney (VERO) and were cultured at 37 1C. Theculture medium was minimal essential medium eagle(MEM), modified with HEPES (Sigma) and supplemen-ted with 10% fetal bovine serum (FBS), 200mM

L-glutamine, 100U/ml penicillin and 100 mg/ml strepto-mycin. The culture medium was changed at selected timeintervals with care to cause little disturbance to cultureconditions.

2.5.2. Microscopic examination. Environmental scanning

electron microscopy (ESEM)

The materials were placed in a 24-well plate (induplicate) and seeded with VERO cells at a density of14� 105 cells/ml. These were incubated at 37 1C. Thecells were fixed with 1.5% glutaraldehyde buffered in0.1 M phosphate buffer after a 24 h incubation period.The dried samples were sputter-coated with gold beforeexamination under an ESEM apparatus (Philips XL 30)at an accelerating voltage of 15KeV.

2.5.3. MTT assay

TMX, PVC and films of cured systems were set up in5ml of MEM, FCS-free. They were placed on a rollermixer at 37 1C and the medium was removed at differenttime periods (1, 2 and 7 days) and replaced with other5ml of fresh medium. All the extracts were obtainedunder sterile conditions. VERO cells were seeded at adensity of 11� 104 cells/ml in complete medium in asterile 96-well culture plate and incubated to confluency.Then, the medium was replaced with the correspondingeluted extract and incubated at 37 1C for 24 h. Asolution of MTT was prepared in warm PBS (0.5mg/ml) and the plates were incubated at 37 1C for 4 h.Excess medium and MTT were removed and dimethyl-sulphoxide (DMSO) was added to all wells in order tosolubilise the MTT taken up by the cells. This was mixedfor 10min and the absorbance was measured with aBiotek ELX808IU detector using a test wavelength of570 nm and a reference wavelength of 630 nm.

2.5.4. Statistical analysis of biocompatibility test

The statistical analysis of the extracts of cured systemswas made by analysis of variance (ANOVA). In all

statistical analyses po0:05 was considered as statisti-cally significant.

3. Results and discussion

An injectable delivery system for prednisolone,PMMA/PHEMA/PRED, has been developed withpotential application in acute acoustic trauma. Thesystem was prepared with PMMA beads in which theprednisolone powder was dispersed. The hydrophilicmonomer, HEMA, widely used to obtain hydrogelnetworks [17], was the monomeric component. Thecombination of this monomer with the PMMA particleswill produce a relatively flexible material which will beable to swell a certain amount of liquid in thephysiological medium, contributing to the release ofthe drug in situ. The chemical structures of theformulation components are shown in Fig. 1. The finalmaterial will be a soft and flexible network of PMMAparticles embedded into a polymerised matrix ofPHEMA. The setting reaction was followed by measur-ing the evolution of the temperature with time from theonset of the mixture of the components. The mixturewas very fluid during the first 10min, which allowed itsinjection into the corresponding cavity. The values ofcuring parameters were determined from the tempera-ture–time profile according to international standardspecifications. A maximum temperature of 5370.9 1Cand a setting time of 1571.1min were obtained. Thevalue of maximum temperature is rather lower than56 1C, which corresponds to the onset of coagulation ofalbumin [18]. In addition, this low temperature guaran-tees the activity of the drug after the setting of thecement. The reaction of the control, PMMA/PHEMA,carried out in the same conditions was slower and tookplace at a temperature not higher than 26 1C, indicatingthat the prednisolone molecule acts as an accelerator ofthe polymerisation reaction, although the mechanism isstill unclear.The swelling behaviour of the PMMA/PHEMA/

PRED and PMMA/PHEMA systems was studied inPBS at 37 1C by measuring the water uptake with time.Results are shown in Fig. 2. For both loaded andunloaded systems, water uptake increased rapidly withtime of immersion during the first 10 h to reach ahydration degree of 6% for the control and 15% for theloaded system. From then on, hydration degree in-creased at a lower rate to reach a constant value around10% for the unloaded system and 20% for the loadedsystem after 100 or 500 h, respectively. The higherhydration degree obtained for PMMA/PHEMA/PREDcan be attributed to the presence of prednisolone.Although the chemical structure of this molecule(Fig. 1) is mainly hydrophobic, the hydroxilic groupspresent may participate in polar interactions with water

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0 100 200 300 400 500 600 700 8000

3

6

9

12

15

18

21

0 3 6 9 12 15 18 21 24 2702468

1012141618

Hyd

rati

on

Deg

ree

(%)

Hyd

ratio

n D

egre

e(%

)

t (h)

t (h)

Fig. 2. Hydration degree versus time of the PMMA/PHEMA/PRED

system (’) and the control PMMA/PHEMA (K) in PBS at 37 1C.

0 200 400 600 800 1000 12000.0

0.2

0.4

0.6

0.8

1.0

Mt/M

inf

t1/2 (s1/2)

Fig. 3. Reduced sorption curve of the prednisolone charged delivery

system (+) and the control (� ) in PBS at 37 1C.

(CH2 C)n

COOCH3

CH3

C O O C

O O

CH2 C

CH3

NH3C

H3C

COOCH2CH2OH

C

OH

CH3H2O

O

CH3

HO

H

H

OH

O

OH

H

CH3

PMMA beads Prednisolone Benzoyl peroxide (BPO)

SOLID COMPONENT

LIQUID COMPONENT

2-Hydroxyethyl methacrylate (HEMA) 4,4'-bis(dimethylaminobenzydrol) (BZN)

CH3

CH3

N

Fig. 1. Chemical structures of the components of the self-curing delivery system.

M. Fernandez et al. / Biomaterials 26 (2005) 3311–33183314

molecules increasing the hydrophilicity of the system incomparison to that of the unloaded system. Thecorresponding reduced sorption curves are plotted inFig. 3. A straight line was obtained at the beginning ofthe experiment for both cases showing that, at the firststages, the mechanism of swelling obeys Fick’s secondlaw according to Eq. (2), which can be applied for thinspecimens where edge effects can be neglected:

Mt=M1 ¼ 4ðDt=pl2Þ1=2; (2)

where Mt is the water uptake at time t, M1 is theequilibrium water uptake, D is the diffusion coefficientand l is the average thickness of the film [19]. From thecorresponding slopes a value of D ¼ 0:32� 10�7 cm2=swas obtained for the PMMA/PHEMA system and avalue slightly higher, D ¼ 0:72� 10�7 cm2=s; for the

prednisolone loaded system. The diffusion coefficient ofthe control was in the range of those reported byMigliaresi et al. [20] for the water sorption of severalHEMA/MMA copolymers.The prednisolone release in PBS at 37 1C is repre-

sented in Fig. 4. No burst effect was observed in theinitial stages, but the release of the drug was sustainedfrom the beginning, with 5% of prednisolone released inthe first 24 h. The totality of the initial amount of drugwas delivered in 35 days. A long-lasting sustainedrelease is desirable and difficult to achieve as has beendescribed in other delivery systems proposed in theliterature for glucocorticoids [21,22]. The diagram ofFig. 4 shows a rather constant release rate of the drugduring the first 15 days considering a pure diffusionmechanism of the drug through the polymeric matrix. Itis noteworthy that a slight increase of the release rate

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seems to be observed in the period of 15–35 days thatcould be related with the migration of the PHEMAcomponent and the consequent increase of porosity ofthe original matrix. The ESEM photographs of thesurface of the PMMA/PHEMA/PRED system beforeand after the release assay are shown in Fig. 5. The drysurface shows the initial pores which were mainlyproduced by the entrapment of air due to the stirringof the mass during the mixture of the solid and liquidcomponents. In the photograph of the surface after therelease experiment, the PMMA spherical particles of the

0 5 10 15 20 25 30 350

20

40

60

80

100

Pre

dnis

olon

e re

leas

e (%

)

Time (days)

Fig. 4. Prednisolone release in PBS at 37 1C.

Fig. 5. ESEM photographs of the surface of the PMMA/PHEMA/PRED s

(down). Left (400� ) and right (800� ).

solid component can be clearly observed as a conse-quence of the migration of the PHEMA component ofthe matrix and the release of the prednisolone.It is known that the nature of the biomaterial surface,

such as wettability or surface free energy, is critical forbiocompatibility [23]. The wettability of the surface ofthe delivery system either in the presence or not of thedrug was analysed through contact angle measurements.Table 1 shows the results of the cured systems alongwith those values corresponding to the homopolymersPMMA [16] and PHEMA [24] separately, for compar-ison purposes. Water contact angles of the drug-loadedand unloaded systems were in between those measuredfor PMMA or PHEMA; however, the value obtainedfor the system containing the drug was lower. Watercontact angles measured in this study were comparableto those values reported for a series of HEMA/MMAcopolymers with different composition [25,26]. Thesurface energy of solid (SES) behaved accordingly, andthe polar component of SES for the loaded system wascloser to that of PHEMA, indicating the more hydro-philic character of this surface.The biological response of the PMMA/PHEMA and

PMMA/PHEMA/PRED systems was first analysed bydirect microscopic examination at 1 and 2 days afterseeding. The results are shown in Fig. 6 and they arecompared with those obtained for a PMMA-basedsystem, commonly used in acrylic bone cement formula-tions [27]. The cells were able to adhere and proliferate

ystem before (upper) and after immersion of sample in PBS at 37 1C

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Table 1

Wettability properties of the loaded and unloaded delivery system prepared in this work, along with those reported for pure PMMA and pure

PHEMA

PMMA/PHEMA/PRED PMMA/PHEMA PMMA PHEMA

Water contact angle 5974 6772 80 5073

Diiodomethane contact angle 4571 4772 40 3873

Surface energy solid, gs (mN/m) 51 46 40 58

Polar part, gps (mN/m) 15 10 4 18

Dispersive part, gds (mN/m) 37 36 36 40

Fig. 6. ESEM photographs (900� ) of VERO cells colonization on a PMMA-based system (upper), the PMMA/PHEMA control system (medium)

and the PMMA/PHEMA/PRED system (down) at 1 (left) and 2 (right) days.

M. Fernandez et al. / Biomaterials 26 (2005) 3311–33183316

on the PMMA material showing a normal cellularmetabolism, and in 2 days time they formed a layer withnumerous cells on the material. However, the cellsobserved on the PMMA/PHEMA system after 1 dayshowed spherical morphology indicative of a scarcematerial recognition and a limited adhesion. After 2

days (on the right hand) only rests of cells on thismaterial were observed. In the PMMA/PHEMA/PREDmaterial, no cells on the surface were observed at all atany time, which can be attributed to the anti-prolif-erative cellular character of the prednisolone whichinhibits cellular adhesion.

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There are many studies dedicated to investigate theeffect of surface wettability on the interactions ofbiological species with solid substrates, but the findingsare rather controversial. Some studies report thatmaterials with low surface energies showed low cellattachment [28]. Other studies, however, show thatmaximum adhesion of fibroblasts occurs on surfaceshaving moderate water wettability [29]. Our findings areconsistent with the latter assumption and support theresults reported for a series of HEMA/MMA copoly-mers, on which optimal adhesion and spreading ofhuman endothelial cells were found to be optimum onthe moderately wettable copolymer surfaces [30]. On theother hand, the biological response of the systemPMMA/PHEMA/PRED was strongly influenced bythe presence of the prednisolone, since glucocorticoidsare known to be potent inhibitors of the cellular growthand proliferation [31].MTT assay was used to measure cell metabolic

function. MTT results for the PHEMA/PMMA systemformulated in the presence or not of prednisolone areshown in Fig. 7 along with the results obtained for aPMMA cement cured in the same conditions. Asignificant drop in cell viability in the presence of theeluates of the cements with respect to the negativecontrol TMX was obtained after 1 and 2 days, but itreturned to normal in subsequent elutions after 7 days,indicating the absence of mitochondrial damage as aresult of ‘‘leachables toxics’’ in any of the experimentalformulations, and that all the formulations can beconsidered as non-toxic. It is noteworthy that any of thePHEMA/PMMA systems behaved similarly to that ofPMMA in the MTT assay, in spite of the fact of thedifferences found among the three systems in themicroscopy examination. One can think from theseresults that the no-adherence of cells to PMMA/PHEMA surfaces is not due to any toxic reason butdue to the difference in hydrophilicity as revealed in

Fig. 7. MTT cytotoxicity results for the control TMX and the test

materials. Results are the mean 7SD (n ¼ 8). The extracts were

collected over a 7 day period (po0:05).

contact angle measurements, and due to the presence ofprednisolone for the loaded system.

4. Conclusion

A sustained release of prednisolone can be achievedfrom an injectable delivery system which cures in situ atthe physiological temperature to give rise to a rather softand flexible material. The system absorbed liquidsmainly in the first 10 h according to a fickian behaviour;they released the drug without an initial burst, but witha sustained release over 35 days. The analysis of thewettability of the surface showed a rather hydrophilicbehaviour attributed to the polar groups of the drug,and the cytotoxic analysis showed no mitochondrialdamage due to the release of toxic substances; however,no cell adhesion was observed by microscopic examina-tion which was attributed to the anti-proliferativecharacter of the corticoid.

Acknowledgements

Financial support from the Comision Interministerialde Ciencia y Tecnologıa, CICYT (MAT2002-04147-C02-02) is gratefully acknowledged.

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