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Colloids and Surfaces A: Physicochem. Eng. Aspects 391 (2011) 80– 87

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jou rna l h om epa ge: www.elsev ier .com/ locate /co lsur fa

ight triggered release of solutes from covalent DNA gels

iana Costaa,b,∗, Artur J.M. Valenteb, M. Grac a Miguelb, Björn Lindmanb,c

CICS - Health Sciences Research Center, Beira Interior University, 6201-001 Covilhã, PortugalDepartment of Chemistry, University of Coimbra, Coimbra, PortugalPhysical Chemistry 1, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-22100 Lund, Sweden

r t i c l e i n f o

rticle history:eceived 25 January 2011eceived in revised form 14 March 2011ccepted 22 March 2011vailable online 31 March 2011

eywords:

a b s t r a c t

DNA gels were prepared by cross-linking and were examined with respect to the release of DNA as wellas macromolecular cosolutes introduced into the gels. In particular the effect of exposure to ultravioletlight was examined for these photodegradable gels. Different cross-linker densities (0.5%, 1%, 3% and 5%ethylene glycol diglycidyl ether) of the DNA gels were used. Network mesh size and the extent of swellingduring degradation have been determined to characterize the effect of radiation on hydrogel degradationkinetics and simultaneous changes in gel structure on solute release profile. Modelling release kinetics,

NA gelsrug releasehotodegradation

using the Weibull function, can describe the DNA release pattern, which is dependent on both radiationwavelength and cross-linker concentration; the mechanism of desorption is found to be complex. Thekinetics release of cosolutes (lysozyme, BSA and FITC-dextran), initially loaded on DNA gels, were studiedand clear differences on the release pattern were obtained. From the cosolute mechanism release it canbe inferred that not only effective size plays an important role, but also interaction with the DNA network.

. Introduction

Polymer gel networks have been used in the pharmaceuticaleld, for several decades, as carriers for and the controlled deliveryf drugs, peptides and proteins, and the interest in suitable deliveryystems to be used therapeutically is large [1–6]. Hydrogels, bothatural and synthetic, are well suited for biomedical applicationsecause of their tissue compatibility, arising from the high waterontent, and soft and rubbery consistency. Their flexibility in tailor-ng physiochemical properties, such as permeability and swelling,he ability to load drugs and release them in a controlled fashion, forimes ranging from minutes to years, are also relevant issues [7]. Inarticular, hydrogels based on synthetic polymers are versatile, dueo the fact that the respective network properties can be carefullyontrolled through chemical and structural modification [8–10].oreover, the controllable mechanical and degradative properties

f hydrogels are critical in soft tissue engineering [11–14]. Con-erning the scaffold engineering, hydrogels can be used to deliverignals to the cells and act as support structures for cell growth and

unction [15–17]. Desired properties of hydrogel scaffolds includehysical parameters such as mechanical strength and controlledegradability, along with biological aspects including biocompat-

∗ Corresponding author at: Health Sciences Center, Beira Interior University,ovilhã, Portugal. Tel.: +351 965532132; fax: +351 239 82 7703.

E-mail address: dcosta@fcsaude.ubi.pt (D. Costa).

927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2011.03.046

© 2011 Elsevier B.V. All rights reserved.

ibility and the ability to provide a biological microenvironment.Hydrogel degradation rates may be designed in accordance to therate of new tissue formation and one can greatly manipulate thisproperty depending on the specific situation.

We succeeded in preparing DNA networks by cross-linking DNAwith ethylene glycol diglycidyl ether (EGDE), which is a bifunc-tional cross-linker that binds to the guanine bases of the DNAmolecules [18–21]. Furthermore, we reported on the swellingbehaviour of the covalently cross-linked DNA, with addition ofdifferent cosolutes that included inorganic salts with differentcation valency, polyamines such as spermine and spermidine,cationic macromolecules such as chitosan, lysozyme, poly-l-lysine,poly-l-arginine, and surfactants. We found that DNA gels werevery interesting as “responsive” systems, since dramatic volumechanges could be induced by very small, changes to the compo-sition of the swelling medium. The swelling of the gels appearedto be reversible, as exemplified by the deswelling/swelling processinduced by subsequent addition of cetyltrimethylammonium bro-mide (CTAB) and sodium dodecyl sulphate (SDS), or chitosan andNaCl.

The field of gene therapy attracts great and widespread inter-est due to its technological challenging characteristics and alsoby its potential therapeutic value related to the possibility of try-

ing to correct genetic diseases by providing replacement copiesof the defective genes. Knowing that synthetic, non-viral vectors,offer advantage when compared to the viral ones in terms of theirgreater nucleic acid packaging capacity, lower immunogenicity and

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

reater safety [22], several strategies have been developed in ordero deliver DNA into target cells [23–28].

In a previous work [29], we synthesized covalent DNA gels withifferent cross-linking densities (0.5%, 1%, 3% and 5% ethylene glycoliglycidyl ether, EGDE) and the disruption behaviour of these gels,ue to photodegradation of the cross-linker molecule, was demon-trated by DNA release. We also monitored the weight loss of theydrogels, and the changes in the storage modulus and the degreef swelling as a function of disruption time [29]. The drug releaseharacteristics of covalently cross-linked DNA gels were studiedsing the protein bovine serum albumin (BSA). The effects of theydrogel disruption for different degrees of cross-linking on theelease rate of the protein were also examined [29]. Moreover, inur group, for the first time, release studies from DNA gel parti-les, formed 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 [30,31].

Here, we try to gain a deeper understanding of the release ofifferent molecular weight solutes from photodegradable cross-

inked DNA gels. In a first approach, the gel mesh size wasetermined and the ultraviolet range that causes the maximumetwork degradation was characterized. Then, we investigated howhe disruption-dependent network swelling relates to the meshize and the latter to the solute release behaviour.

. Materials and methods

.1. Materials

Ethylene glycol diglycidyl ether (EGDE) was from Aldrich.eoxyribonucleic acid (DNA) (from salmon testes, sodium

alt; ∼2000 base pairs), N,N,N′,N′ tetramethylethylenediamineTEMED), sodium hydroxide (NaOH), sodium bromide (NaBr),

bicinchoninic acid (BCA) kit, bovine serum albumin (BSA),uoresceinisothiocyanato-dextran (FITC-dextran) and lysozymeere obtained from Sigma. Lysozyme has a molecular weight of

4 100 Da and a Stokes’ radius of ca 16 A, BSA has a molecular weightf 65 000 Da and the radius is around 34.8 A [11] while FITC-dextranas a molecular weight of 77 000 Da and a radius is around 55.0 A.

.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 adding

M NaOH and TEMED the sample was mixed and then transferredo 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 concentrationrom 9 wt%, at preparation time, to 1 wt%, after immersion of theels in the NaOH solution, was observed. Thus, the reference statef the experiments is the equilibrium swelling in 1 mM NaOH solu-ion. Some of the gels were cut into thin cylinders and dried (gelsith approximately 1 g, 1 cm length and 0.25 cm in diameter).

.3. Swelling experiments

To study the swelling behaviour of the DNA hydrogels they wereeighed after being pre-swollen in a NaOH 1 mM solution (refer-

nce state), and on several occasions during their disruption. The

ochem. Eng. Aspects 391 (2011) 80– 87 81

swelling ratio was calculated by dividing the weight of the gels atsteady-state swelling by their weight in the reference state.

2.4. Determination of the average gel mesh size

The network mesh size represents the distance between consec-utive crosslinking points and provides a measure of the porosity ofthe network. The mesh size of the DNA hydrogel was determinedas described by Canal and Peppas [32] and it is understood as anaverage value. The root-mean-squared end-to-end distance of thepolymer chain in its unperturbed state was calculated using Eq. (1):

(r02)1/2 = lC1/2n n1/2 (1)

where l is the length of the bond along the backbone chain, Cn is thecharacteristic ratio of the polymer, and n is the number of bonds inthe cross-link, determined by Eq. (2):

n = 2Mc

M0(2)

where Mc and M0 are the molecular weight of the polymer chainsbetween cross-links and the molecular weight of the repeatingunits making up the polymer chains, respectively. The mesh size inangstroms, �, of the swollen polymer network was then calculatedfrom Eq. (3):

� = �−1/3(r20 )

1/2(3)

where � is the polymer volume fraction in the equilibrium swollengel, which equals the reciprocal of the volume swelling ratio, Q; itis a measure of the amount of fluid that a hydrogel can incorporateinto its structure. This parameter is determined using equilibriumswelling experiments.

3. 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.

3.1. Incorporation of solutes and release

Lysozyme, BSA and FITC-dextran have been incorporated intoDNA gels by imbibition. Concentrated solutions of the mentionedsolutes (5 wt%) were prepared. The dried gels were saturated withsolute by placing two cylinders in 50 ml of each solute solution.The gels were left in the solutions for approximately 48 h. Solutecontaining gels were placed in a 500 ml bottle containing 150 mlof the HEPES buffer (pH = 7.4, 37 ◦C). Aliquots of the sample solu-tions were withdrawn at appropriate time intervals and the volumeof the medium was kept constant by replacement. The lysozymeconcentration was measured with the Bio Rad protein assay fol-lowing the microassay procedure. The Bio-Rad Protein Assay is adye-binding assay in which a differential colour change of a dyeoccurs in response to various concentrations of protein [33]. Thisprotein assay, based on the method of Bradford, is a simple andaccurate procedure for determining the concentration of solubi-

lized protein. It involves the addition of an acidic dye to proteinsolution, and subsequent measurement at 595 nm with a spec-trophotometer. The absorbance maximum for an acidic solution ofCoomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm

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geneous distribution would make difficult the diffusion from thenetwork interior to the bulk solution and explain, at least partly,the observed time lag. In the presence of sun light, the initial lagtime, which strongly depends on the amount of EGDE, i.e. cross-

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hen binding to protein occurs [34,35]. Comparison to a standardurve provides a measurement of protein concentration.

BSA was analyzed by determining the total mass of protein usinghe bicinchoninic acid (BCA) method [36]. Bicinchoninic acid forms

complex with Cu+ ions, producing a purple coloured solution thatan be quantitatively measured at 562 nm. The protein to be ana-yzed reacts with Cu2+ in an alkaline solution producing Cu+-ions.hese ions are then chelated by the BCA which converts the apple-reen colour of the free BCA to the purple colour of the copper–BCAomplex [36]. As the physical state of the released BSA could beffected by exposure to UV light and cross-linking agents, leadingo changes in its structure and stability, the BSA released from theel was analyzed using a wavelength of 214 nm to study possibleonformational changes. It was then observed that the prepara-ion of the gels, as well as the conditions of the release study, didot measurably alter the native protein structure. Considering theavelength of maximum absorbance for FITC-dextran, 494 nm, its

oncentration was determined using a UV–vis spectrophotometert this wavelength. To avoid interference of gel degradation prod-cts on the measurement of the solute concentration, the solutionsrom identical DNA gels were used as a blank in measuring theolute concentration as a function of degradation.

.2. DNA gels degradation by ultraviolet light irradiation

To explore and characterize the ultraviolet (UV) light photo-cissive mechanism and determine the wavelength range thatauses gel disruption, several wavelength ranges were used tonduce DNA gel photodegradation. The DNA gels (gels withpproximately 1 g, 1 cm length and 0.25 cm in diameter) wererradiated, at determined time intervals, with the following wave-engths: �1 (280–300 nm), �2 (300–320 nm), �3 (320–340 nm), �4340–360 nm), �5 (360–380 nm), �6 (380–400 nm); each gel sampleas irradiated with only one wavelength range.

.3. Modelling release kinetics

The release kinetics is an important issue in the study of aew formulation for, e.g., drug delivery. For that, it is necessaryo have reliable equations that can predict the behaviour of diffus-ng species and so to allow the development of better formulations.here have been numerous attempts for providing equations fromhose based on purely theoretical grounds [37] to semi-empiricalnes [38–40]. In addition, some of the most common classical equa-ions (e.g., power law equation [41]) can only model/predict ca. 60%f the entire release profile. Another approach to describe the entireet of the release data is based on the “empirical” use of the Weibullunction

t = M∞[1 − exp (−at)b] (1a)

here Mt and M∞ are the cumulative amounts of the materialeleased at time t and at infinite time, respectively, and a and bre constants. The introduction of M∞ ensures that the asymptoticalue for the release might be different from 100% [42].

Although the use of Eq. (2) to model release kinetics has beenriticized, due to the lack of a kinetic basis for its use and theon-physical nature of its parameters[43], Papadopoulou et al. [44]emonstrated that Eq. (1) allows an insight into the diffusionalechanism of release, once b is closely related with the mechanism

f diffusional release.In the present study, we have used a slightly modified version

f the Eq. (1), in order to take into account that in several occasions

two steps release mechanism is occurring; the modified versionf Eq. (1) takes the form of

t = M0 + M∞[1 − exp (−a(t − t0))b] (2a)

ochem. Eng. Aspects 391 (2011) 80– 87

where M0 and t0 represents the initial values for the released quan-tity and time, respectively. Of course, for b = 1, Eq. (2) describes afirst-order function.

4. Results and discussion

4.1. Network disruption

In degradable gels, if the release of solutes is controlled by solutesize then the relation between mesh size and polymer degrada-tion is a decisive factor. Control of the variations of mesh size withtime is then crucial for the design of appropriate solute-releasingdevices. During hydrogel degradation, the water content tendsto increase which further promotes the release of gel entrappedsolutes. Therefore, the nature of DNA gels degradation and theparameters affecting both, the network mesh size and the volumeswelling ratio, during the degradation process need to be investi-gated.

The first evidence of disruption of the covalent DNA gels comesfrom the release of DNA with time. To demonstrate the pho-todisruption of the network, experiments on DNA release wereperformed with and without sun light exposure. Although the exis-tence of a lag time is a common phenomenon in both cases, asignificant difference was observed, as illustrated in Fig. 1 [29]. Wehypothesize that the distribution of the cross-linker in the gel isnot uniform, with a higher concentration of EGDE in the surfaceregion and a lower one in the interior of the network. This inhomo-

Fig. 1. Cumulative release of DNA from cross-linked DNA gels with 0.5%, 1%, 3% and5% (w/v) EGDE, as a function of time. Studies were performed in the presence (A)and absence (B) of sun light.Source: From Ref. [29].

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

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Fig. 2. Cumulative release of DNA from cross-linked DNA gels with 0.5%, and1% (w/v) EGDE, as a function of time, after irradiation with �1 (280–300 nm),�(

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2 (300–320 nm), �3 (320–340 nm), �4 (340–360 nm), �5 (360–380 nm) and �6

380–400 nm); each gel sample was irradiated with only one wavelength range.

inking density, may be related to the number of crosslinks thatave to be degraded to permit the release of DNA [29]. The amountf DNA released was considerably different when the experimentsere conducted in the absence of exposure to sun light and, in this

ase, the release of DNA reached a well defined plateau (Fig. 1B).t is known that EGDE is a substance that degrades on exposureo sun light (photo-oxidation) [45]. This degradation leads to theemoval of the chemical cross-links and increases swelling, affect-ng the stability of the DNA network allowing the release of DNA

ith time. The higher the cross-linker concentration used in the gelreparation, the lower the amount of DNA released and the longerhe time needed to release it [29]. The disruption behaviour of theseydrogels was confirmed by weight loss, decrease of modulus, and

ncrease in the degree of swelling [29].In order to characterize the ultraviolet (UV) light photoscis-

ive mechanism we determined the wavelength range that causesel disruption. For this, several wavelength ranges were used tonduce DNA gel photodegradation (see more details in Section 2).he cumulative DNA release over time was quantified for eachavelength interval studied, for gels cross-linked with 0.5% and

% EGDE, as shown in Fig. 2. A quantitative assessment of the effectf both wavelength radiation and cross-linker concentration on theelease kinetics of DNA has been obtained by fitting Eq. (2) to thoseata (shown in Fig. 2); the corresponding fitting parameters areeported in Table 1. Although an analysis of the release profilesf DNA (shown in Fig. 2) apparently shows a similar behaviour

or both types of DNA gels, a detailed analysis shows that theelease profile of DNA is dependent on both radiation wavelengthnd EGDE concentration. In order to rationalize the discussion, the

ochem. Eng. Aspects 391 (2011) 80– 87 83

effect of those factors on the fitting parameters will be analyzedseparately.

It can be seen that the maximum DNA released when gelsare irradiated using �1 (280–300 nm), �2 (300–320 nm), and �3(320–340 nm) is less than one-half of that released when DNA gelsare irradiated with lower energy radiation. In fact, quite significantamounts of DNA are released from 0.5% and 1% EGDE cross-linkedgels when samples were irradiated with �5 (360–380 nm) and �6(380–400 nm), with the latter being the wavelength range moreefficient in promoting the release of DNA with time. It is worthnoting that the effect of cross-linker on the maximum amount ofdesorbed DNA can only be observed at the �5 and �6 wavelengths.Only at these wavelengths, M∞ values for 0.5% cross-linked DNAgels are higher than those found for more cross-linked gels, show-ing the effect of a more compact gel structure. It is, however notfully understood why the higher wavelength range, and thus thelower energy, gives rise to the larger gel disruption efficiency. Itseems that the cross-linker molecules are quite sensitive to thetype A of ultraviolet light (320–400 nm) and certain specificity isalso involved.

From the analysis of data shown in Table 1 also results that theDNA release is faster when gel matrices are irradiated with lowerenergy radiation. Looking to the release constants (a) it is possible toconclude that these values are higher for the less cross-linked DNAgels, suggesting that the swelling degree and, consequently, thewater-free volume plays an important role in the release process.As we will see later, the initial mesh size is 47 A and 38 A for DNAgels cross-linked with 0.5% and 1% EGDE, respectively.

Other important information, coming out from the analysis ofthe release profile of DNA from DNA–EGDE gels, is related with themechanism of desorbed DNA. Again, it is clear that the mechanismis dependent on both wavelength radiation and EGDE concentra-tion and, not less important, is rather complex.

Considering the gels cross-linked with 0.5% EGDE, the releasestarts with a pure first-order kinetic model (b ≈ 1), at �1, indi-cating that the DNA release is diffusion-controlled. However, at�2, b becomes higher than 1 (b = 1.41), corresponding to a S-shaped, sigmoidal, release profile, characteristic of a complexmechanism, normally related to a super Case II transport [46].This mechanism occurs when the role of the surface on the trans-port process becomes predominant [47]. However, in contrastto the transport mechanism occurring in 1% EGDE gel (see dis-cussion below), the b value is only slightly higher than 1, andconsequently the sigmoidal profile is not well defined for initialtimes. For lower wavelengths the DNA release profile becomessimilar and follows a well-defined two-step mechanism. In thefirst step, during around 35 h, the release of DNA is diffusion-controlled, with release constants of ca. 8 (±3) h−1; after 38 h,the profile becomes S-shape and the release constant drasticallydecreases to values around 1.1 (±0.1) h−1. On the basis of thesedata we can hypothesise the following mechanism: with lowenergetic radiation, there is no effect on the gel matrix and, con-sequently, the transport of DNA is only concentration-dependent;however, by increasing the energy of radiation, it seems that someinteraction with DNA and/or EGDE occurs preferentially into thesurface, leading to a alteration in the surface with the formationof a coating-like structure, contributing for the overall slow raterelease and acting as obstacle preventing the way out of moreDNA.

Such a hypothesis is also supported by the analysis of dataobtained with 1% cross-linked DNA. In fact, the release of DNA athigher wavelengths shows a different mechanism than that found

can be fitted by the Weibull function, with good correlation coef-ficients, a detailed analysis for the release at �5 and �6, shows alinear relationship of Mt with time as expected for a first-order

84 D. Costa et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 391 (2011) 80– 87

Table 1Fitting parameters of Eq. (2) to experimental data shown in Fig. 2.

M0/% t0/h M∞/% a/(10−2 h−1) b R2 Notes

0.5% EGDE380–400 0 0 68.3 (±0.9) 3.4 (±0.1) 0.93 (±0.04) 0.9925 1st order

0 0 68.3 (±0.9) 3.45 (±0.1) 1.0 0.9925360–380 0 0 44.5 (±0.3) 2.64 (±0.05) 1.41 (±0.05) 0.9973340–360 0 0 1.14 (±0.03) 7.0 (±0.4) 1.12 (±0.03) 0.9995 Removed points t = 6 and 8

2.4 38 21.9 (±0.3). 1.18 (±0.04) 1.45 (±0.07) 0.9938320–340 0 0 1.16 (±0.04) 6.8 (±0.5) 1.11 (±0.05) 0.9992 Removed points t = 6 and 8

2.0 38 17.7 (±0.4) 2.0 (±0.1) 1.3 (±0.2) 0.9698300–320 0 0 1.03 (±0.08) 9 (±2) 1.1 (±0.2) 0.9852 Removed t = 8

1.3 38 16.5 (±0.4) 1.07 (±0.06) 1.14 (±0.07) 0.9916280–300 0 0 1.1 (±0.1) 7 (±2) 1.1 (±0.1) 0.9889 Removed t = 8

1.3 38 16.6 (±0.7) 0.60 (±0.04) 1.20 (±0.06) 0.99621.0% EGDE380–400 0 0 51.9 (±0.6) 2.04 (±0.05) 1.82 (±0.09) 0.9948

– – – 48.8 (±0.9)a 0 0.9970 From t = 0 to t = 35 h22.5 38 29.5 (±0.4) 4.5 (±0.2) 1 0.9917

360–380 0 0 33.5 (±0.4) 1.84 (±0.05) 1.54 (±0.06) 0.9957– – – 37 (±1)b 0.9925 From t = 0 to t = 35 h

12.9 38 20.6 (±0.3) 3.34 (±0.2) 1 0.9893340–360 0 0 46 (±11) 1.89 (±0.06) 1.10 (±0.06) 0.9965320–340 0 0 19 (±2) 0.32 (±0.03) 2.0 (±0.1) 0.9959300–320 0 0 14.3 (±0.5) 0.38 (±0.01) 2.47 (±0.09) 0.9977280–300 0 0 16 (±1) 0.32 (±0.01) 2.38 (±0.09) 0.9978

Values without standard deviation indicate that they have been locked in the fitting procedure.

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change in the order a(lysozyme) > a(BSA) > a(FITCdextran). Lookingto the fitting parameters reported in Table 2, we infer that bothlysozyme and BSA are efficiently released upon gel degradation,with the first solute exhibiting a faster release rate, probably due

0

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a Rate constant obtained by using the equation Mt = at.b Rate constant obtained by using the equation Mt = a(t − 2).

elease function. The differences in the mechanism of DNA releaseor 0.5% and 1% cross-linked DNA can be understood from theffect of cross-linking; a higher cross-linking will lead to a situationhere the rate of release is much faster than the polymer relaxationrocess [48].

However, when the gels are irradiated at a wavelength below,r equal, to 340 nm the release of DNA is well fitted by the Weibullunction in the entire range of data, with a b value higher than. This characterizes a very slow release at the beginning of therocess, suggesting that the release takes only place after a longime-lag. Under these conditions, the surface as well as the cross-inker has an important effect on the release of DNA; in fact,y increasing the amount of cross-linker by a factor of two theate constant decrease by one order of magnitude; furthermore,t should be stressed that these rate constants are quite similaro those obtained for the release of DNA from DNA gel parti-les [31], where the effect of surface on the DNA release is alsomportant.

In a previous report [29], the changes in the degree of swellingith sun exposure time were analyzed for the DNA gels cross-

inked with different cross-linker densities (0.5%, 1%, 3% and 5%GDE) in order to further understand the disruption profiles. Gelsross-linked with 0.5% and 1% EGDE presented an increased extentf swelling with increasing time of disruption, while those cross-inked with 3% and 5% EGDE showed only small changes in thewelling ratio [29]. As mentioned in Section 2, knowledge of thequilibrium degree of swelling allows for the calculation of matrixtructural parameters such as the molecular weight between cross-inks and the mesh size of the gel. Thus, the evolution of gel

esh size upon gel disruption was examined for all DNA gelsrepared. The results are shown in Fig. 3. The initial mesh size

s 47 A, 38 A, 35 A and 32 A for DNA gels cross-linked with 0.5%,%, 3% and 5% EGDE, respectively. As expected, the initial meshize of 0.5% EGDE cross-linked gels was the largest and upon gelegradation, the variation in mesh size was significantly influenced

y the cross-linking density used in the gel synthesis. The meshize increased with disruption time as EGDE degrades and thehemical cross-links are removed, changing the integrity of DNAetwork.

4.2. Release of solutes

Upon DNA gel degradation, both the network mesh size and thevolume swelling ratio increase, as discussed previously. To evalu-ate the dependence of the release rate on the size of an entrappedsolute, three different solutes were incorporated into 0.5% EGDEcross-linked DNA gels. The chosen solutes were lysozyme, BSAand FITC-dextran with molecular weights ranging from 14 100 to77 000 Da, and the hydrodynamic radius being 16, 34.5 and 55 A,respectively [39]. The effect of radiation at �disruption (380–400 nm),on the release profiles of these molecules has been studied. Fig. 4shows the release kinetics of the different molecules, from DNAmatrices irradiated at 380–400 nm. By fitting those data to Eq. (2),see Table 2, we can conclude that: (a) the cumulative release slightlydecrease from lysozyme to FITC dextran; and (b) rate constants (a)

300250200150100500Time (h)

Fig. 3. Gel mesh size of 0.5%, 1%, 3% and 5% EGDE cross-linked DNA gels as a functionof time, after the irradiation of gels with �disruption (380–400 nm).

D. Costa et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 391 (2011) 80– 87 85

Fig. 4. Cumulative release of lysozyme (�), BSA (�) and FITC-dextran (�) fromDNA gel matrices as a function of time, when gels were irradiated with �disruption

(380–400 nm).

Table 2Fitting parameters of Eq. (1) to experimental data of the release kinetics of differentmolecules from DNA gels irradiated at 380–400 nm (Fig. 4).

M∞/% a/(10−2 h−1) b R2

tamstlhtgifdd

Fdgtd

3002502001501005000

20

40

60

80

100

Cum

ulat

ive

Rel

ease

(%)

Time (h)

Lysozyme BSA FITCdextran

Lysozyme 0.97 (±0.02) 29 (±2) 0.70 (±0.08) 0.9910BSA 0.95 (±0.05) 6 (±1) 0.65 (±0.07) 0.99898FITCdextran 0.903 (±0.004) 0.915 (±0.007) 2.22 (±0.04) 0.9996

o its small size (16 A). In the framework of a hydrodynamic model linear relationship between the hydrated radii of the macro-olecules and the natural logarithm of the rate constants (ln(a))

hould be expected. This model is based on the assumption thathe molecules can be considered as hard spheres, which are mucharger than the solvent molecules [49,50]. Fig. 5 shows that theydrated radii of lysozyme, BSA and FITCdextran play an impor-ant role in the release of these different molecules through DNAel matrices. However, from the analysis of b values, which give

nformation on the release mechanism, it is worth noting that otheractors than rh are affecting the release; furthermore, there is aifference between the mechanism for lysozyme and BSA, and FITC-extran release; the latter, shows a b value higher than 1 (2.22),

ig. 5. Dependence of the hydrated radius [39] of lysozyme (�), BSA (�) and FITC-extran (�) on the natural logarithm of the rate constant (a) for the release from DNAel matrices (�disruption: 380–400 nm). a values have been calculated by fitting Eq. (3)o experimental data shown in Fig. 2. Solid line represents the fitting of experimentalata to a straight line equation: ln(a) = 0.2–0.089rh (R2 = 0.9997).

Fig. 6. Cumulative release of lysozyme, BSA and FITC-dextran from 0.5% EGDE cross-linked DNA gels, as a function of gel mesh size.

indicative of a sigmoid curve and a complex release mechanism,where the rate of release increases up to the inflection point andthereafter declines [44]. On the other hand, lysozyme and BSA showb values lower than 0.75, indicating that the release mechanismis predominantly Fickian [44]. Because of the limited number ofexperimental data points for the release of 60% of the diffusing par-ticles, the Fickian mechanism has been confirmed by fitting thosedata to the Korsmeyer–Peppas equation for the first 60% of therelease curve [51]. A similar mechanism has recently been reportedfor the release of lysozyme and BSA through peptide hydrogels [52].

To gain further insight into these mechanisms, the effect of gelmesh size on solute release was examined, as summarized in Fig. 6.These results suggested that there is a clear correlation betweenthe solute size, the gel mesh size and the global release profile.Although all the incorporated solutes were able to diffuse out fromthe gels cross-linked with 0.5% EGDE, when they suffer degradation,their release patterns are completely different due to their differentsizes. Initially, for 0.5% EGDE DNA gel mesh size is around 47 A andboth, incorporated lysozyme and BSA can rapidly diffuse out fromthe network in consequence of their size, 16 A and 34.8 A, respec-tively. These solutes were almost totally released at an early stage,probably with the high concentration gradient contributing to thissituation. This is in close agreement with the b value obtained byusing the Weibull function. In contrast, negligible amounts of FITC-dextran have been released in the first 24 h, which is referred toits larger size compared to the initial gel mesh size. Upon degrada-tion, the network mesh size is continuously increasing approachingthe dextran size, and so, the dextran began to diffuses slowly. At acertain time, the gel mesh size was larger than the solute size andthe dextran release rate greatly increases, as demonstrated by thesteeper curve in Fig. 6.

In the absence of light, the release profile of these moleculesis significantly different (Fig. 7 and Table 3). Lysozyme release isquite similar to that occurring in the presence of sun light, becausethe initial gel mesh size is larger than its size allowing its releasefrom the network. However, it is worth noting that the releaseof lysozyme from cross-linked DNA gels shows a different releasepattern from that found in DNA-lysozyme particles [31].

In contrast, and under the same conditions, only approximately11% of BSA can be released at the maximum release, due to the factthat the gel maintains its stability without additional changes inits initial mesh size (47 A). Similarly, negligible amounts of dextrancan be released (only 3.4%) under the dark conditions. This solute

has the highest molecular weight and due to the initial mesh size,the solute remains incorporated in the 0.5% EGDE cross-linked gelsover the time considered. It can be noted that both these molecules(BSA and FITC dextran) show a three-step released mechanism.

86 D. Costa et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 391 (2011) 80– 87

Fig. 7. Release kinetics of lysozyme (�), BSA (�) and FITC-dextran (�) from DNA gels in the absence of sun light. The lines correspond to the best fits of experimental data toEq. (2); different colour lines correspond to independent fits (for further information c.f. Table 3).

Table 3Fitting parameters of Eq. (2) to release data of lysozyme, BSA and FITC dextran from DNA gels, shown in Fig. 7.

M0/% t0/h M∞/% a/(10−2 h−1) b R2

Lysozyme 0 0 96(±2) 32 (±3) 0.8 (±0.1) 0.9918BSA 9.99 2 1.27 (±0.09) 1.7(±0.3) 0.9 (±0.1) 0.9888

.1)

0.01)

.2)

Tisftiboo

5

lafacTabEe

11.22 192 1.3 (±0FITCdextran 8.64 × 10−3 24 1.04 (±

1.06 144 2.3 (±0

he latter two steps are rather similar for both molecules, show-ng a Super Case II transport with the second rate constant beingmaller than the first one (see Table 3). However, the initial stepor both molecules shows a different behaviour, in agreement withhe previous arguments that the size of the desorbed particles is anmportant parameter. In the case of the BSA, during the first 2 h aurst release (around 10% of the loaded material, for a total releasef 12.4%) is measured, while in the case of FITC-dextran a time-lagf 24 h occurs before a small fraction being release (3.4%).

. Conclusions

Degradable DNA gels, cross-linked by 0.5%, 1%, 3% and 5% ethy-ene glycol diglycidyl ether, were used to control the release of DNAnd different molecular weight hydrophilic solutes. The Weibullunction can describe the DNA release profiles and shows that theyre dependent on both radiation wavelength and cross-linker con-entration; the mechanism of DNA desorption is rather complex.here is no effect on the gel matrix with low energetic radiation

nd the diffusion of DNA is controlled by its concentration, whereas,y increasing radiation energy some interaction with DNA and/orGDE takes place at the surface acting as an obstacle. The differ-nces in the DNA release pattern between 0.5% and 1% cross-linked

0.9 (±0.1) 1.3 (±0.2) 0.98664.0 (±0.1) 0.98 (±0.06) 0.99881.16 (±0.03) 2.0 (±0.1) 0.9963

gels can be understood with the effect of cross-linking related withthe volume swelling degree and, consequently the gel mesh size.Solute release from DNA gels is strongly affected by the relativevalue of the network mesh size and the hydrodynamic radius of thesolute. In the presence of light, the release mechanism for lysozymeand BSA follows a Fickian diffusion, while FITC-dextran releasepresents a sigmoid curve indicating a more complex mechanism.In the absence of light, the solute release behaviour is significantlydifferent. Although, lysozyme release is quite analogous to thatoccurring in the presence of light, BSA and FITC-dextran show athree-step released mechanism. The latter two steps are similar forboth molecules, presenting a Super Case II transport, while the ini-tial step indicates a different behaviour, probably related to the sizeof the desorbed particles.

Acknowledgments

We are grateful for financial support from Fundac ão para a Ciên-cia e a Tecnologia (FCT), (SFRH/BPD/47229/2008).

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