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Colloids and Surfaces A: Physicochem. Eng. Aspects 354 (2010) 28–33

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l homepage: www.e lsev ier .com/ locate /co lsur fa

ross-linked DNA gels: Disruption and release properties

iana Costaa,b,∗, Artur J.M. Valenteb, Alberto A.C.C. Paisb, M. Graca Miguelb, Björn Lindmana,b

Physical Chemistry 1, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-22100 Lund, SwedenDepartment of Chemistry, University of Coimbra, Coimbra, Portugal

r t i c l e i n f o

rticle history:eceived 25 May 2009eceived in revised form 4 August 2009ccepted 9 August 2009vailable online 18 August 2009

eywords:ydrogelsrug release

a b s t r a c t

We report on the disruption of DNA gels cross-linked with ethylene glycol diglycidyl ether (EGDE), bysunlight exposure. The disruption over exposure time was characterized through the cumulative DNArelease, the evolution in dry weight and extent of swelling, and also rheologically. The network disruptionis shown to depend strongly on the degree of cross-linking density. Bovine serum albumin has beenincorporated into the DNA networks, resorting to two different methods, and protein release underdifferent conditions was investigated. The protein release rate was affected by both the gel cross-linkercomposition and the ultraviolet light exposure time. In the presence of light, the BSA desorption kineticsfollows a Fickian behaviour and the diffusion coefficients were determined. Diffusion coefficients of BSA

hotodegradation decrease by increasing cross-linker concentration and retention capacity, whereas in the absence oflight, the mechanism of desorption kinetics is rather complex. A desired release rate can be achievedby adjusting the mentioned parameters. Additionally, these gels can release both DNA and BSA, usingthe hydrogel disruption. This study allows us to characterize and rationalize the release mechanism of

om t

covalent DNA gels, and frin a controlled way.

. Introduction

Water-swellable networks have been used as drug carriers inontrolled drug delivery for several decades, and the interest inuitable delivery systems to be used therapeutically is still increas-ng [1–6]. Hydrogels, both natural and synthetic, are well suited foriomedical applications because of their tissue compatibility, aris-

ng from the high water content, and soft and rubbery consistency.heir flexibility in tailoring physicochemical properties, such asermeability and swelling, and the ability to load drugs are also rel-vant issues [7]. In particular, hydrogels constructed from syntheticolymers are versatile, due to the fact that the respective networkroperties can be carefully controlled through chemical and struc-ural modifications [8–10]. Moreover, the controllable mechanicalnd degradative properties of hydrogels are critical in soft tissuengineering [11–14].

The formation of network gels can be achieved both by chemicalnd physical crosslinking [15]. Chemical cross-linking is an efficient

ethod to produce hydrogels with high mechanical stability. In

hemically cross-linked gels, covalent bonds are present betweenifferent polymer chains but labile bonds are often introducedo permit biodegradability, an advantage in many applications.

∗ Corresponding author at: Departamento de Química, Universidade de Coimbra,004-535 Coimbra, Portugal. Fax: +351 239 82 7703.

E-mail address: diana.costa@fkem1.lu.se (D. Costa).

927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2009.08.009

hat, suggest the development of devices that interact with living systems

© 2009 Elsevier B.V. All rights reserved.

The labile bonds can be broken under physiological conditionseither enzymatically or chemically, in most cases by hydrolysis[6,16,17].

Ethylene glycol ethers are widely used as cross-linker moleculesin the preparation of covalently cross-linked gels [18–24]. It hasbeen found that these chemicals do not persist in the environ-ment, nor bioaccumulate in tissue, and are “practically non-toxic”to aquatic organisms [25]. These ethers photooxidize in the pres-ence of sunlight, although, the primary degradation process isthe biodegradation that will occur in surface and ground waters[25]. Their photodegradation rates suggest moderately rapid atmo-spheric degradation [25].

In previous work [26–28], we succeeded in preparing DNA net-works by cross-linking DNA with ethylene glycol diglycidyl ether(EGDE), which is a bifunctional cross-linker that binds to the gua-nine bases of the DNA molecules. We reported on the swellingbehaviour of the covalently cross-linked DNA, with addition of dif-ferent cosolutes that included inorganic salts with different cationvalency, polyamines such as spermine and spermidine, cationicmacromolecules such as chitosan, lysozyme, poly-l-lysine, poly-l-arginine, and surfactants. We found that DNA gels can functionas “responsive” systems, since dramatic volume changes could be

induced by very small changes in the composition of the swellingmedium. The swelling of the gels appeared to be reversible, asexemplified by the deswelling/swelling process induced by sub-sequent addition of cetyltrimethylammonium bromide (CTAB) andsodium dodecyl sulphate (SDS), or chitosan and NaCl.

Physic

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D. Costa et al. / Colloids and Surfaces A:

In the biomedical field, there is an increasing demand for suit-ble gene delivery system, where the goal is the delivery of aissing gene or a functional substitute of a defective gene. In our

roup, for the first time, release studies from DNA gel particles,ormed by interfacial diffusion, were carried out. These studiesevealed that DNA was released from particles for a long periodnder in vitro conditions, which may provide a basis for the intra-ellular sustained release of DNA in vivo [29].

In this work, we synthesized covalent DNA gels with differentross-linking densities (0.5%, 1%, 3% and 5% EGDE) and the dis-uption behaviour of these gels, due to photodegradation of theross-linker molecule, was demonstrated by DNA release. We alsoonitored the weight loss of the hydrogels, and the respective

hanges in the storage modulus and in the degree of swelling asfunction of disruption time. The drug release characteristics of

ovalently cross-linked DNA gels were studied using the modelrotein bovine serum albumin (BSA). The effects of the hydrogelisruption and the degree of cross-linker density on the releaseate of the protein were also examined.

Andreopoulos et al. [30] synthesized polyethylene glycol basedydrogels by photopolymerization in the absence of photosensitiz-rs and/or photoinitiators. These hydrogels exhibit photoreversibleehaviour and photodegrade upon exposure to UV light (254 nm).his photoscissive property was used as a trigger for the releasef drugs from the hydrogel matrix [30]. We also used such lightxposure to induce changes in the gel network properties. Manyractical applications of controlled delivery systems in biopharma-eutics require faster release, while others such as the controlledelivery therapeutic proteins (e.g., antigenic proteins for vaccina-ion [31] or insulin [32]), can require a more prolonged releasef several days or weeks. The release behaviour of the systemsescribed in this paper may have advantageous applications.

. Materials and methods

.1. Materials

Ethylene glycol diglycidyl ether (EGDE) was from Aldrich.eoxyribonucleic acid (DNA) (from salmon testes, sodium salt;2000 base pairs),N,N,N’,N’ tetramethylethylenediamine (TEMED),

odium hydroxide (NaOH), sodium bromide (NaBr), a bicinchoniniccid (BCA) kit, and bovine serum albumin (BSA) were obtained fromigma. BSA has a molecular weight of 65,000 Da and its dimensionsre approximately 4.2 nm in diameter and 14.4 nm long [11]. Itsolidispersity index is 2.01.

.2. Preparation of gels

Double stranded DNA, from salmon testes, was dissolved inater containing 3.7 mM NaBr, to a DNA concentration of 9 wt%.NA was chemically cross-linked by EGDE at pH 9. After addingM NaOH and TEMED the sample was mixed and then transferred

o test tubes and incubated for 2 h in a water bath at 50 ◦C. Freshlyynthesised gels were neutralised and rinsed with large amountsf 1 mM NaOH solution. The DNA gels swelled considerably in theaOH solution and due to this fact the DNA concentration in theels is lowered. The concentration of DNA in the gels equilibratedith 1 mM NaOH (reference state) was obtained by weighing gels

efore and after freeze-drying. A decrease in the DNA concentra-

ion from 9 wt%, at preparation time, to 1 wt%, after immersionf the gels in the NaOH solution, was observed. Thus, the refer-nce state of the experiments is the equilibrium swelling in 1 mMaOH solution. Some of the gels were cut into thin cylinders andried (gels with approximately 1 g, 1 cm length and 0.25 cm iniameter.)

ochem. Eng. Aspects 354 (2010) 28–33 29

2.3. Weight loss experiments

After formation, DNA gels cross-linked with different amountsof EGDE were dried, by freeze-drying, and weighed (referencestate). The mass changes of dry gels were monitored over lightexposure time and normalized to their initial values before networkdisruption.

2.4. Rheological characterization

For the mechanical characterization of the DNA gels, rheologicalmeasurements were performed on a Reologica Stress Tech rheome-ter equipped with an automatic gap setting. Prior to any oscillatorydeformation test, the linear viscoelastic region was determined bystress sweep tests. The bottom plate was replaced with a Plexi-glass plate that was fixed on the rheometer with clamps, so that,DNA gels could be removed together with the bottom plate. Allmeasurements were performed with an acrylic top plate (diameter2 cm) and to minimize evaporation a solvent trap system was used.Experiments were carried out in oscillation mode at 1 Hz.

2.5. Swelling experiments

To study the swelling behaviour of the DNA hydrogels they wereweighed after being pre-swollen in a NaOH 1 mM solution (refer-ence state), and on several occasions during their disruption. Theswelling ratio was calculated by dividing the weight of the gels atsteady-state swelling by their weight in the reference state.

2.6. DNA release measurements

To determine the amount of DNA released, the gels (around1 g) were suspended in 20 ml of pH 7.6 10 mM Tris–HCl buffer.The samples were incubated at 25 ◦C with gentle shaking (40 rpm).At certain time intervals, the supernatant was collected and gelswere re-suspended in fresh solution. DNA released into the super-natant was quantified by spectrophotometrically measuring theabsorbance at 260 nm using a Shimadzu UV–vis 2100 spectropho-tometer.

2.7. BSA incorporation and release

BSA has been incorporated into DNA gels by two different meth-ods. First, BSA (5 wt%) was added to an aqueous DNA solution(9 wt%) and the mixture was chemically cross-linked by using ethy-lene glycol diglycidyl ether (EGDE) at pH 9, following the procedureof gel formation described above. Second, the drug was incor-porated by imbibition. A concentrated BSA solution (5 wt%) wasprepared. The dried cylinders were saturated with BSA by plac-ing two cylinders in 50 ml of the BSA solution. The gels were leftin the protein solutions for approximately 48 h. BSA-containinggels were placed in a 500 ml bottle containing 150 ml phosphatebuffered saline (PBS) at 35 ◦C. Aliquots of the sample solutions werewithdrawn at appropriate time intervals and the volume of themedium was kept constant by replacement. BSA was analysed bydetermining the total mass of protein using the bicinchoninic acid(BCA) method [33]. Bicinchoninic acid forms a complex with Cu+

ions, producing a purple coloured solution that can be quantita-tively measured at 562 nm. The protein to be analyzed reacts withCu2+ in an alkaline solution producing Cu+-ions. These ions are then

chelated by the BCA which converts the apple-green colour of thefree BCA to the purple colour of the copper-BCA complex [33].

As the physical state of the released BSA could be affected byexposure to UV light and cross-linking agents, leading to changesin its structure and stability, the BSA released from the gel was

30 D. Costa et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 354 (2010) 28–33

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those cross-linked with 3% and 5% exhibited minimal weight losseven after 5 weeks of sunlight exposure. As can be seen, the weightloss data differs from the cumulative DNA release in Fig. 1. In DNArelease measurements we determined spectrophotometrically the

ig. 1. Cumulative release of DNA from cross-linked DNA gels with 0.5%, 1%, 3% anbsence (B) of sunlight.

nalyzed using a wavelength of 214 nm to study possible conforma-ional changes. It was then observed that the preparation of the gels,s well as the conditions of the release study, did not measurablylter the native protein structure.

. Results and discussion

.1. Network disruption behaviour

The first evidence of disruption of the covalent DNA gels comesrom the release of DNA with time. To demonstrate the pho-odisruption of the network, experiments on DNA release wereerformed with and without sunlight exposure. Although the exis-ence of a lag time is a common phenomenon in both cases, aignificant difference was observed, as illustrated in Fig. 1. Weypothesize that the distribution of the cross-linker in the gel isot uniform, with a higher concentration of EGDE in the surfaceegion and a lower one in the interior of the network. This inhomo-eneous distribution would make difficult the diffusion from theetwork interior to the bulk solution and explain, at least partly, thebserved time lag. In the presence of sunlight, the initial lag time,hich strongly depends on the amount of EGDE, i.e. cross-linkingensity, may be related to the number of crosslinks that have to beegraded to permit the release of DNA. After light exposure and ataximum release, DNA gels cross-linked with 0.5% EGDE released

9% of DNA in approximately 30 days, gels prepared from 1% EGDEeleased 54% of DNA in 40 days while gels synthesized using 3%nd 5% EGDE released 32% and 26% of DNA, respectively in 47 days.ll the release curves presented a biphasic release profile, in which

he release after 7 days, for 0.5% and 1% EGDE cross-linked gels,nd 9 days for 3% and 5% EGDE gels, was accelerated. The amountf DNA released was considerably different when the experimentsere conducted in the absence of exposure to sunlight. In this case,

els made with 0.5% EGDE release 32% of DNA, i.e. less than halff the DNA released with photoxidation of the network. Gels with%, 3% and 5% EGDE release 19%, 9% and 3% of DNA, respectively,ithin approximately 2 weeks. Moreover, in the absence of expo-

ure to sunlight the release of DNA reached a well defined plateauFig. 1B).

It is known that EGDE is a substance that degrades on expo-

ure to sunlight (photooxidation) [25]. This degradation leads to theemoval of the chemical cross-links and increases swelling, affect-ng the stability of the DNA network and thus allowing the releasef DNA with time. The higher the cross-linker concentration used inhe gel preparation, the lower the amount of DNA released and the

w/v) EGDE, as a function of time. Studies were performed in the presence (A) and

longer the time needed to release it. In the presence of sunlight,the release behaviour reaches a plateau, arguably due to the factthat the inhomogeneous distribution of the cross-linker leads tothe existence of very concentrated cross-linker/DNA regions, fromwhere the release of DNA is extremely difficult. To release DNA fromthese “domains” it is possible that a longer sunlight exposure timeis needed or irradiation with a stronger ultraviolet light source. Theobserved release of DNA in the absence of sunlight corresponds, ineach gel system, approximately to the free DNA in the network, i.e.,the DNA not cross-linked. The release of DNA is halted when allfree DNA is released, as observed from the plateau in Fig. 1 B. At thepreparation time, the relation between the DNA concentration andthe EGDE concentration allows the gel to maintain some of chainsin a non-cross-linked state.

The disruption of the covalently cross-linked DNA gels was alsomonitored, as shown in Fig. 2, by determining the weigh loss ofthe dried hydrogel, as a function of disruption time. The higher thecross-linking density used to synthesize the gels, the lower the dis-ruption rate. DNA gels cross-linked with 0.5% EGDE disrupt withinapproximately 2 weeks, those with 1% EGDE within 3 weeks, while

Fig. 2. Weight loss as a function of time of DNA gels cross-linked with 0.5%, 1%, 3%and 5% (w/v) EGDE.

D. Costa et al. / Colloids and Surfaces A: Physic

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ig. 3. Change with time of storage modulus (G’) of DNA gels cross-linked with 0.5%,%, 3% and 5% (w/v) EGDE.

mount of DNA released into the bulk solution, while the weightoss measurements take into account every smaller portion of theel that separates from the main part, portions of gel that haveeparated into the release medium.

To further characterize the disruption behaviour of the cova-ently cross-linked gels, we performed rheological oscillatory sheartress experiments. The degree of cross-linking is related to gel stiff-ess, which is represented by G’ (storage modulus); G’ was chosens the parameter to monitor the evolution of the mechanical prop-rties during disruption. The reduction in mechanical strength ofhe covalently cross-linked DNA gels with 0.5% and 1% EGDE, asresented in Fig. 3, corresponds to the loss of weight, although atslower rate. In contrast, gels cross-linked with 3% and 5% EGDEisplay very retarded disruption. After 7 weeks, they still presentignificant mechanical stiffness; it is reasonable to assume that theigh cross-linking density retards the disruption of the hydrogel.or the latter gels, there is a clear correspondence between the lossf mechanical strength and the loss of weight with disruption.

The changes in the degree of swelling over sun exposure timeere analyzed for each of the DNA gels cross-linked with different

ross-linker densities, so as to further understand the disruptionrofiles. The results are summarized in Fig. 4. The weight of the gels

ig. 4. Change of the degree of swelling of DNA gels cross-linked with 0.5%, 1%, 3%nd 5% (w/v) EGDE as a function of time.

ochem. Eng. Aspects 354 (2010) 28–33 31

pre-swollen in the 1 mM NaOH solution was taken as the first point(this swelling ratio given as unity) in Fig. 4 and, therefore, repre-sents the equilibrium degree of swelling of the “intact” covalentlycross-linked DNA gels. Gels cross-linked with 0.5% and 1% EGDEpresented an increased extent of swelling with increasing time ofdisruption, while those cross-linked with 3% and 5% EGDE showedonly small changes in the swelling ratio. The degree of swellingis intimately related to the mechanical elastic properties (i.e., thestorage modulus) and reflects the cross-linking density of the gel[34].

3.2. Release of BSA

A protein, bovine serum albumin (BSA), at an initial concentra-tion C0, was loaded into DNA gels of different cross-link densities,and the protein release was monitored over time. The methodof BSA incorporation into the network only slightly affects therelease profile, with the imbibition method displaying a some-what increased release of protein. Therefore, we will present thoseresults here. To study the effect of network photodisruption on BSArelease, experiments were performed in the presence and absenceof sunlight exposure. Fig. 5 gives the retention capacity, K (definedas = retained BSA concentration after release/initial BSA concen-tration), of BSA with different degrees of cross-linking. Exposure ofthe gels to UV light permitted the release of BSA and lead to a lowretention capacity of the gel. This is referred to an increase in themesh size. In DNA gels cross-linked with 0.5% and 1% EGDE, K valueswere only 0.08 and 0.1. As more cross-linker was used to preparethe gels, a marked increase in the amount of protein retained wasobserved, with a K of 0.34 and 0.44 for gels cross-linked with 3%and 5%, respectively. From Fig. 5, it is also possible to observe thatwhilst in the less cross-linked hydrogels the steady-state is attainedafter around 140 h; in more cross-linked gels, only after 216 h theBSA released reaches a maximum value.

In contrast, minimal amounts of BSA were released from gelswhen studies were performed in the absence of sunlight expo-sure. In this case, a very small increase of K with the cross-linkerconcentration was observed.

From this, we established that the retention capacity of BSAwithin the covalently cross-linked DNA gels was mainly controlledby the degree of disruption, which was dependent upon the relativecross-linker density.

Fig. 5. Effect of different percentages of cross-linker (EGDE) on the retention coeffi-cients, K, of BSA inside covalently cross-linked DNA gels in the presence (grey bars)and absence (white bars) of sunlight.

32 D. Costa et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 354 (2010) 28–33

Table 1Diffusion coefficients for BSA release from DNA gels at different light conditions.

% Cross-linker D/(10−9 cm2 s−1) �/hours

Light Dark

g

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ci

wtt

i

w

ttb

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0.5 17.1 ( ± 0.7) 2.9 ( ± 0.1) –1.0 13.0 ( ± 0.5) 2.8 ( ± 0.1) –3.0 5.4 ( ± 0.2) 7.0 ( ± 0.2) 60.6 ( ± 0.3)5.0 2.6 ( ± 0.1) 7.7 ( ± 0.3) 52.7 ( ± 0.5)

Desorption kinetics of BSA, from covalently cross-linked DNAels, can be quantified using Fick’s second law

∂C

∂t= ∂

∂x(D

∂C

∂x) (1)

here C is the concentration of the desorbed substance, x is thepace coordinate measured normal to the section, t is the time andis the diffusion coefficient.In the case of a cylinder of radius r, and the boundary and initial

onditions C(r,t≥0) = 0 and C(0 < x < r,0) = f(r) = constant, the analyt-cal solution of Eq. (1) [35] is

Ct

C∞= 1 −

∞∑

n=1

4

r2˛2n

exp(−D˛2nt) (2)

here Ct and C∞ are the concentrations of desorbed species at timeand at infinite time, respectively. The values of ˛n are the roots ofhe first species of Bessel’s function of order 0.

For short range times, for which the gel behaves as a semi-nfinite medium [35], Eq. (2) reduces to

Ct

C∞= 4√

��1/2(1 −

√�

4�1/2 − �

12+ . . .) (3)

here � (=Dt/r2) is the dimensionless time.From experimental data shown in Fig. 5, it is possible to obtain

he normalised concentration of BSA released (Ct/C∞) (Fig. 6). Inhe presence of light, the BSA desorption kinetics follows a Fickianehaviour for Ct/C∞ up to 0.7; that is Eq. (3) can be re-written as

Ct

C∞= 4

r√

�(Dt)1/2 (4)

rom Eq. (4) and taking the radius of the DNA gel as 0.125 cm, dif-usion coefficients, D, can be calculated (see Table 1). The Fickianehaviour can be justified by the fact that the BSA release occursith a shorter exposure time than the release of DNA (see previ-

us section), indicating that the disruption of the network startsefore the DNA release and before detectable changes in weight

oss, in storage modulus and in the degree of swelling (for gelsross-linked with 3% and 5% EGDE) are observed. Due to this facthe determination of the diffusion coefficients is not affected byetwork disruption.

Diffusion coefficients of BSA decrease by increasing cross-linkeroncentration as well as retention capacity. Assuming that theater volume fraction, ϕw, has a linear dependence on swellingegree, ϕw = f(Q) [36], the relationship between D and ϕw fol-

ows an equation of the type D = D0exp(−˛ + ˛ϕw), [37] whereis a constant related to the cross-sectional area of the diffus-

ng solute and the free volume of water in the polymer, with aorrelation coefficient of 0.992 with a 95% confidence interval,howing that both hydrodynamic and obstruction effects affecthe BSA release [38]. The results mentioned above are in agree-

ent with the photodisruption of the network detected in the

umulative DNA release, weight loss changes, decrease in the stor-ge modulus (G’) and the increased extent of swelling, especiallyor the gels with lower cross-linker density. As the degree ofwelling increases, the density of the network decreases and theiffusion of BSA in the gel increases. These effects become more

Fig. 6. Effect of the percentage of cross-linker in the DNA gel matrices on the kineticsof desorption of BSA as a function of time, in the presence (A) and absence (B) ofsunlight. (�) [EGDE] = 0.5% (w/v); (o) [EGDE] = 1% (w/v); (�) [EGDE] = 3% (w/v); (�)[EGDE] = 5% (w/v).

pronounced with time, as demonstrated by changes in the storagemodulus. The storage modulus should decrease if the network isdisrupted, even when the non-cross-linked chains remain in thegel.

It should be noted that the diffusion coefficient of BSA in the0.5% cross-linked DNA gel is almost two orders of magnitude lowerthan that in aqueous solution (D0 = 7.01 × 10−7 cm2 s−1) [39].

In the case of gels which have not been exposed to light (Fig. 6B),the BSA desorption kinetics are rather complex. For the less cross-linked DNA gels release follows a linear relationship with the squareroot of time, whilst for the highly cross-linked gels (3% and 5%)the desorption mechanism is more complex. At these cross-linkingconcentrations, we observed a delay time prior to notable BSAdesorption (more pronounced in the case of 5% cross-linking). How-ever, when such a “time-lag” (�) was reached, a fast increase in theflux occurred faster than for the less cross-linked gels. In order toquantify the desorption kinetics, we have used a modified versionof Eq. (4) [40]

Ct 4 1/2

C∞=

r√

�[D(t + �)] (5)

As pointed out above, the desorption mechanism of BSA fromcovalently cross-linked DNA gels depends on the percentage ofcross-linker. It should be noted that, under these experimen-

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[[38] B. Amsden, Macromolecules 31 (1998) 8382–8395.[39] K.B. Kosto, W. Deen, M. AIChE J. 50 (2004) 2648–2658.[40] F. Gouanvé, S. Marais, A. Bessadok, D. Langevin, M. Métayer, Eur. Polym. J. 43

D. Costa et al. / Colloids and Surfaces A:

al conditions, only small traces of the incorporated BSA wereeleased. We believe that the gels may retain the larger aggregatesnd selectively allow the release of the lower molecular weightonomers. It is also possible that the protein is non-specifically

dsorbed to the surfaces of the release vial or complexes withNA.

The diffusion coefficients can be related to a frictional factorwhich depends on the size of the diffusing molecules and vis-osity) and an equilibrium thermodynamic factor for the changen chemical potential with concentration [41]. In the DNA gelsross-linked with 0.5% and 1% EGDE, the diffusion is one orderf magnitude lower in the absence than in the presence of lightnd follows a Fickian behaviour during all the desorption. Thisan be explained by a small concentration gradient that existsn the dark conditions. In the highest cross-linked DNA gels, theesistance on the diffusing molecules from the hydrodynamicedium increases. This can be due to the rigidity of the gelatrix and the very small BSA concentration gradient; as a conse-

uence, a time-lag is observed, corresponding to the time necessaryor the free diffusing particles to reach the gel/water interface;s soon as the particles reach the interface the flux drasticallyncreases.

. Conclusions

We report on the development of covalently cross-linked DNAels that have controlled disruption rates upon exposure to UVight. The disruption behaviour of these hydrogels was confirmedy means of cumulative DNA release, weight loss, decrease of mod-lus, and increase in the degree of swelling.

A protein, BSA, was incorporated into covalently cross-linkedNA gels and subsequently released. Experimental results demon-

trate that the protein release behaviour from these gels was greatlyffected by the degree of network swelling and by variations in theetwork mesh size. In the presence of light, the BSA desorptioninetics followed Fickian behaviour whereas in the absence of lighthe mechanism was rather complex. Diffusion coefficients of BSAecrease by increasing cross-linker concentration and retentionapacity.

These initial studies on DNA gels give us an insight into theelease mechanism. They suggest that one can obtain networksith an appropriate release profile by tailoring light exposure and

ross-linker density. These hydrogels appear as new promisingehicles for the release of DNA, drugs and pharmacological solutesn controlled delivery devices.

cknowledgments

We are grateful for financial support from Fundacão paraCiência e a Tecnologia (FCT (SFRH/BD/16736/2004) and

TDC/Qui/67962/2006.

[

ochem. Eng. Aspects 354 (2010) 28–33 33

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