microcapsules. In addition the microcapsules with single/multi-layerswere dried at room temperature and sputter-coated with Au andexamined with a scanning electron microscope (SEM).

Ofloxacin was chosen as a model drug to investigate the drugrelease behavior of the microcapsules. The release was studied usingsolutions with different pH's (pH 4.84, pH 7.0 and pH 9.44) Therelease of ofloxacin from the microcapsules was monitored by a UV–Vis spectrophotometer U-3010 at a wavelength of 295 nm. Multi-layer chitosan/sodium alginate microcapsules of 5 walls and 9 wallswere studied to compare the release of ofloxacin from microcapsuleswith a different number of layers.

Results and discussionThe morphology of the microcapsules is shown in Fig. 1. The

diameter of the microcapsules was about 100 nm (Fig. 1a–c). Thesurface of the microcapsules was corrugated after dehydration.Further studies indicated that the diameters of the microcapsulescan be controlled via the parameters used in electrospraying, solutionconcentration or conductivities of the solution. Multi-layered micro-capsules showed different surface and microstructures from those ofsingle-layered microcapsules (Fig. 1d).

Fig. 1. Microscopic SEM photographs of chitosan/alginate microcapsules. (a–c) singlelayer, (d) multi-layer.

Chitosan and alginate are natural cationic or anionic polysacchar-ides, respectively, so they were sensitive to the pH of aqueoussolutions. However, the microcapsules prepared by chitosan andalginate showednopH sensitivity. Fig. 2 illustrates the release propertyof the chitosan/sodium alginatemicrocapsules in buffer solutions withdifferent pH values (pH4.84, 7.0, and 9.44). In the three different buffersolutions, the ofloxacin release rate and final ofloxacin releasepercentages were similar. They all showed fast drug release. Thereason for this may be that the carboxyl groups of alginate and theamino groups of chitosan formed ionic bonds independent of the pH.

Fig. 2. Cumulative release of ofloxacin from single-layeredmicrocapsules at different pHs.

Results of ofloxacin release from microcapsules with one layer,five layers and nine layers of microcapsules are plotted in Fig. 3 andcompared on the basis of the same ofloxacin loading. It appeared thatthe rate of drug release did not change with layer number.

ConclusionThe chitosan/sodium alginate multi-layered microcapsules should

have potential applications in controlled drug delivery. Electrospray-ing is a versatile method for incorporation of water-soluble bioactivemacromolecules such as proteins, growth factors and DNA, whichcould be released steadily from the microcapsules. This method hasthe advantages of being facile, high loading efficiency, mild prepara-tion conditions and relatively steady release characteristics. Thisstudy provides a basis for further design and optimization of core-shell nanostructures to achieve the desired release of bioactive agentsfor gene therapy and tissue engineering.

AcknowledgmentsThis study was supported by the National Science Foundation of

China (50803004). The author would like to thank the ChangzhouCity Youth Science and Technology Training Scheme (CQ20090001).

References[1] M. Ramadas, W. Paul, K.J. Dileep, Y. Anitha, C.P. Sharma, Lipoinsulin encapsulated

alginate–chitosan capsules: intestinal delivery in diabetics ratsJ. Microencapsul. 17(2000) 405–411.

[2] A.J. Ribeiro, C. Silva, D. Ferreira, F. Veiga, Chitosan-reinforced alginate microspheresobtained through the emulsification/internal gelation technique, Eur. J. Pharm. Sci. 25(2005) 31–40.

[3] J. Doshi, D.H. Reneker, Electrospinning process and applications of electrospun fibers, J.Electrostat. 35 (1995) 151–160.

[4] D.H. Reneker, I. Chun, Nanometre diameter fibres of polymer, produced byelectrospinning, Nanotechnology 7 (1996) 216–223.

[5] G.B. Sukhorukov, L. Dähne, J. Hartmann, E. Donath, H. Möhwald, Controlledprecipitation of dyes into hollow polyelectrolyte capsules based on colloids andbiocolloids, Adv. Mater. 12 (2000) 112–115.

[6] C.Y. Gao, E. Donath, H. Möhwald, J.C. Shen, Spontaneous deposition of water-solublesubstances into microcapsules: phenomenon, mechanism, and application, Angew.Chem. Int. Ed. 114 (2002) 3789–3793.

[7] L. Dähne, S. Leporatti, E. Donath, H. Möhwald, Fabrication of micro reaction cages withtailored properties, J. Am. Chem. Soc. 123 (2001) 5431–5436.

[8] F. Caruso, R.A. Caruso, H. Möhwald, Nanoengineering of inorganic and hybrid hollowspheres by colloidal templating, Science 282 (1998) 1111–1114.

[9] T. Okubo, M. Suda, Synchronous multilayered adsorption of macrocations andmacroanions on colloidal spheres: influence of foreign salt and basicity or acidity ofthe macro ions, Colloid Polym. Sci. 280 (2002) 533–538.

doi:10.1016/j.jconrel.2011.08.130

Hydrogel-based drug carriers for controlled release ofhydrophobic drugs and proteins

Ke Peng, Itsuro Tomatsu, Alexander KrosLeiden Institute of Chemistry, Leiden University, P.O. Box 9502,2300 RA Leiden, The NetherlandsE-mail address: a.kros@chem.leidenuniv.nl (A. Kros).

Fig. 3. Cumulative release of ofloxacin from microcapsules with different number oflayers at pH 4.84.

Abstracts / Journal of Controlled Release 152 (2011) e1–e132e72

Abstract summaryHydrogel-based drug carriers have been developed for the

controlled release of hydrophobic drugs and proteins. In the firstexample, a hydrogel was designed for the controlled release ofhydrophobic drugs. For this gel, maleimide-modified dextran (Dex-mal) was used as the backbone of an in situ forming hydrogelfunctionalized with β-cyclodextrins (CD). In the second example therelease of proteins from the hydrogel was controlled by light. In thiscase Dex-mal was functionalized with azobenzene (AB) and CD toconstruct a supramolecular hydrogel based on the inclusion complexbetween trans-AB and CD, which was used as a photo-switchablecrosslinker to control the release of physically entrapped proteins.

Keywords: Cyclodextrin, Dextran, Hydrogel, Hydrophobic drug, Protein

IntroductionHydrogel based biomaterials are of interest because of their

excellent biocompatibilities and the similarity between their highlyhydrated three-dimensional polymeric networks and hydrated bodytissues [1]. Particularly, hydrogel based drug delivery systems haverecently been paid much attention as promising drug carriers [2,3].

To advance applications of hydrogels in drug delivery systems,careful design of thehydrogelmaterials is critically important in order totune its drug release characteristics. In this contribution,wewill presentour recent development in hydrogel based drug delivery systems usingmaleimidemodified dextran (Dex-mal) and various thiol functionalizedcompounds (Fig.1). In the first system (Fig. 2) [4,5], we have used thiol-functionalized cyclodextrins (CD) to modify the hydrogel in order toincrease the affinityof hydrophobic drugswith the hydrogelmatrix. Thisis necessary because most hydrogels are typically hydrophilic, whichlimits their successful use as a drug delivery system for hydrophobicdrugs due to rapid and uncontrolled drug release in the initial phase. Inthe second example (Fig. 3) [6], we have used the inclusion complexformation between trans-azobenzene and CD as a photo-switchablecrosslinker to develop a photo-responsive supramolecular hydrogelresulting in a light controlled protein release system. This system canfacilitate anefficient protein delivery since it can transport the physicallyentrapped protein to the target site and the release is induced by light.

Fig. 1. Structures of the chemicals used in this work. Dex-mal is used as a backbone.PSCD plays two roles: a binding site for the hydrophobic drugs and a crosslinker,simultaneously. These functions are undertaken by MSCD and DSPEG separately in theother system for the hydrophobic drugs. MSCD and AB were used to modify Dex-malfor construction of a photo-responsive hydrogel system.

Experimental methodsDex-mal was prepared through DCC mediated esterification of the

hydroxyl group of the dextran with N-maleoylamino acid. Thiolfunctionalized compounds were prepared following reported proce-dures, see reference [4–6]. Thiol–maleimide reactions were used tofunctionalize and to crosslink the Dex-mal polymers into hydrogels.For hydrophobic drug release, all-trans retinoic acid (RA) was used asa model drug and the amount of RA released from the hydrogel wasdetected by the HPLC. For protein release, green fluorescent protein(GFP) was used as a model protein and the released amount of GFPwas monitored by the fluorescence.

Results and discussionDex-mal can be crosslinked to form a hydrogel by applying multi-

thiol compounds PSCD or DSPEG. In these systems, macroscopic

hydrogels were formed rapidly in situ via a Michael addition betweenmaleimide and thiol groups (Fig. 2a). As a result of CD functionaliza-tion, the CD-Gel showed an increase of the affinity for the modelhydrophobic drug RA, and a well controlled in vitro release profile ofRA was observed without initial burst effect. In the case of the CD-PEG-Gel, a sustained and constant release was observed after aninitial delay, comparable to the release from a CD-Gel. In contrast,when a hydrogel was prepared without CD moiety (i.e. PEG-Gel; Dex-mal was crosslinked with DSPEG), there was no strong bindingaffinity between the hydrophilic hydrogel network and the hydro-phobic drug. As a result, the release profile of RA from the PEG-Gel issimilar to many examples of reported hydrogels, which are generallybiphasic with an initial rapid release phase and followed by a slowerphase (Fig. 2b). By changing the concentrations of the polymersolutions during gel formation, nano-sized hydrogel particles(≤100 nm) were formed, which also showed a well controlledrelease profile of RA without an initial rapid release phase. In order toexamine the biocompatibility, the hydrogel particles were injected inzebrafish embryos at the one cell stage and it was found that CD-PEG-Gel was biocompatible and did not cause death or deformation of theembryos. Furthermore, in we also confirmed that RAwas released in acontrolled manner from these CD-PEG-Gel particles in zebrafishembryos [5].

Fig. 2. (a) Schematic representation of the drug loading processes. The hydrophobicdrug (RA) forms an inclusion complex either with PSCD or Dex-mal partially modifiedwith MSCD. Then hydrogels were prepared in situ via a Michael addition betweenmaleimide and thiol groups. In the resulting gel, the inclusion complexes of RA and CDare conjugated to the polymer network. (b) Release profiles of RA from the hydrogels.

Taking advantage of the efficient thiol–maleimide reaction, wealso designed a supramolecular hydrogel system (Fig. 3). For this wefunctionalized Dex-mal with either AB or CD moieties (AB-Dex andCD-Dex, respectively). These polymers were used as building blocksof a supramolecular crosslinked hydrogel for a light controlled releasesystem of proteins. Upon mixing of these two polymers, an inclusionof trans-AB in CD induces the formation of a hydrogel. The inclusioncomplexes dissociate upon trans-cis isomerization of the ABs afterirradiation with UV light (365 nm) resulting in the dissolution of thehydrogel. To investigate the potential of the hydrogel as a lightcontrolled protein release system, green fluorescent protein (GFP)was used as a model protein that was physically encapsulated insidethe hydrogel. The GFP carrying gel was placed in a PBS solution andthe fluorescence intensities of the solution part were measured as afunction of time (Fig. 3b). After 10 min of UV light irradiation, thefluorescence intensity in the PBS started to increase which waspronounced after 20 min. More GFP was released after prolongedirradiation; 40% of the loaded proteins were released in 60 min underUV irradiation and reached ca. 65% after 2 h. In contrast, hardly anyGFP was released when no UV light irradiation was applied to thehydrogel. Therefore we conclude that the release of GFP wascontrolled by the UV light which induced trans-to-cis isomerizationof AB resulting in the dissolution of the hydrogel.

ConclusionIn conclusion, maleimide modified dextran (Dex-mal) shows to be

a versatile building block for hydogel preparation and was used for in

Abstracts / Journal of Controlled Release 152 (2011) e1–e132 e73

situ forming hydrogels functionalized with cyclodextrins (CD), re-sulting in a controlled release system for retinoic acid. In the secondexample, Dex-mal was functionalized with azobenzene- and CD-moieties resulting in a photo-responsive supramolecular hydrogelwhich is able to control the release of proteins by light.

AcknowledgmentsWe are grateful to Wim Jesse and Jacques van der Ploeg for

technical assistance. The authors (A. K. and I. T.) acknowledge thesupport of the Smart Mix Programme of the Netherlands Ministry ofEconomic Affairs and the Netherlands Ministry of Education, Cultureand Science.

References[1] N.A. Peppas, J.Z. Hilt, A. Khademhosseini, R. Langer, Hydrogels in biology and medicine:

from molecular principles to bionanotechnologyAdv. Mater. 18 (2006) 1345–1360.[2] C. Hiemstra, Z.Y. Zhong, M.J. van Steenbergen, W.E. Hennink, J. Feijen, Release of model

proteins and basic fibroblast growth factor from in situ forming degradable dextranhydrogels, J. Control. Release 122 (2007) 71–78.

[3] F. van de Manakker, K. Braeckmans, N. el Morabit, S.C. De Smedt, C.F. van Nostrum,W.E. Hennink, Protein-release behavior of self-assembled PEG-beta-cyclodextrin/PEG-cholesterol hydrogels, Adv. Funct. Mater. 19 (2009) 2992–3001.

[4] K. Peng, I. Tomatsu, A.V. Korobko, A. Kros, Cyclodextrin-dextran based in situ hydrogelformation: a carrier for hydrophobic drugs, Soft Matter 6 (2010) 85–87.

[5] K. Peng, C. Cui, I. Tomatsu, F. Porta, A.H. Meijer, H.P. Spaink, A. Kros, Cyclodextrin/dextran based drug carriers for a controlled release of hydrophobic drugs in zebrafishembryos, Soft Matter (2010)doi:10.1039/c0sm00096e.

[6] K. Peng, I. Tomatsu, A. Kros, Light controlled protein release from a supramolecularhydrogel, Chem. Commun. 46 (2010) 4094–4096.

doi:10.1016/j.jconrel.2011.08.131

Radiopaque microspheres for improved transarterial chemicalembolization (TACE)

Ketie Saralidze, Menno L.W. Knetsch, Robbert G.M. van Berkel,Charlotte Mostert, Leo H. KooleDepartment of Biomedical Engineering/Biomaterials Science,Faculty of Health, Medicine, and Life Sciences, Maastricht University,P.O. Box 616, 6200 MD Maastricht, The NetherlandsE-mail address: K.Saralidze@bioch.unimaas.nl (K. Saralidze).

Abstract summaryNew improved X-ray visible microspheres for transarterial

chemical embolization (TACE) were prepared. The currently usedmicrospheres are not visible during and after the procedure bystandard X-ray techniques. Additionally these spheres have limitedcapacity for the anti-tumor drug doxorubicin. We have preparedradiopaque microspheres with micro- or macro-porosity that showincreased drug-loading capacity.

Keywords: Embolization, Microsphere, X-ray visibility

IntroductionTACE aims at blocking the feeding artery of a solid tumor and

subsequent local release of drugs, to shrink the size of the tumor. Theinterventional radiologist injects microspheres via a catheter into thefeeding artery of a tumor (Fig.1). This treatment is often applied for theminimally invasive treatment of hepatocellular carcinomas, or ofuterine fibroids inwomen.[1,2] The ultimate aim is to shrink the tumorwithout damage to the surrounding tissue, i.e. to conserve as muchliver function as possible or to conserve the uterus, respectively.

Fig. 1. Scheme of the embolization of a uterine fibroid, a solid tumor of the uterus wall.

There are several commercial embolization microspheres on themarket. These are composed of poly(vinyl alcohol) (Contour SE, BeadBlock, DC beads) or tris-acryl-gelatin (Embosphere). These are solid,hydrophilic spheres that are available in a wide range of sizes, and canbe loaded with the anti-tumor drug doxorubicin (DC Bead).[3] Thetumor is thus attacked in two ways: 1) by blocking arterial blood flowand 2) local release of doxorubicin. Microspheres used for uterinefibroid embolization are loaded with ibuprofen to combat post-operative pain.

Commercial embolization microspheres have some disadvantagesnamely they are radiolucent, i.e. X-ray invisible, and they have limiteddrug-loading capacity. Here we present preliminary data on a rangeof new microspheres that combine radiopacity, i.e. X-ray visibility,with increased drug loading capacity.

Experimental methodsMicrospheres were prepared by suspension polymerization as

described earlier [4]. Radiopacity of the microspheres was due to theincorporation of the iodine containing monomer 2-[4-iodobenzoy-loxy]-ethyl methacrylate (4IEMA) [5]. Porosity was introduced in themicrospheres by a method adapted from Jayakrishnan et al. [6]. Inshort, the monomer mixture was supplemented with pMMAdissolved in toluene, before polymerization of the microspheres.The pMMA could be dissolved from the solid microspheres, resultingin microporosity. The microspheres were loaded with rhodamine anddoxorubicin by either soaking or a vacuum technique.

Results and discussionThe synthesis of radiopaque, hydrophilic microspheres was

successful. The spheres displayed fine porosity (1–5 μm) as can beseen from Fig. 2. Radiopacity of the microspheres was confirmed byX-ray fluorimetry.

The microspheres could be easily filled with rhodamine or thedrugs (doxorubicin or ibuprofen). The release-profile was dependenton the medium in which the experiment was performed. Release inserumwas faster thanwhen determined in buffer. The release profilesof the drugs demonstrate an initial burst release followed by a slowrelease for up to at least 48 h. Since doxorubicin has limited (thermo)

Fig. 3. (a) Schematic representation of photoresponsive protein release from the gelcomposed of trans AB-Dex and CD-Dex. Upon the UV light irradiation AB isomerizesfrom trans to cis configurations, resulting in the dissociation of crosslinking points, andallowing the entrapped protein to migrate into the media. (b) Release of GFP from thesupramolecular hydrogel.

Abstracts / Journal of Controlled Release 152 (2011) e1–e132e74