Super-macroporous poly(ethoxytriethyleneglycol acrylate)hydrogels for sustained delivery of hydrophilic drugs

Petar Petrova,⁎, Denitsa Momekovab, Bistra Kostovab,Georgi Momekovc, Natalia Toncheva-Monchevaa,Christo B. Tsvetanova, Nikolay LambovbaInstitute of Polymers, Bulgarian Academy of Sciences, Akad. G. BonchevStreet 103A, 1113 Sofia, BulgariabDepartment of Pharmaceutical Technology and Biopharmaceutics,Faculty of Pharmacy, Medical University-Sofia, 2 Dunav Street, 1000Sofia, BulgariacDepartment of Pharmacology, Pharmacotherapy and Toxicology, Faculty ofPharmacy, Medical University- Sofia, 2 Dunav Street, 1000 Sofia, Bulgaria⁎Corresponding author.E-mail: ppetrov@polymer.bas.bg.

Abstract summaryWe report on the synthesis of novel biocompatible temperature-

responsive super-macroporous hydrogels (cryogels) via UV-assistedcrosslinking using hydrogen peroxide as initiator. The capability ofsuch materials for encapsulation and sustained release of hydrophilicdrugs at physiological temperature is demonstrated.

IntroductionHydrogels have beenwidely used in biomedical and pharmaceutical

applications due to their high water content, rubbery nature which issimilar to natural tissue, and biocompatibility [1]. In particular,hydrogels based on synthetic polymers have attracted attention becausedesirable chemical, physical, and biological properties can be impartedto the material. Currently, there is an increasing interest in super-macroporous polymer hydrogels due to their unique heterogeneousopen porous structure which significantly increases their equilibriumsorption properties and allows unhindered diffusion of solutes, nano-and even micro-particles [2]. Moreover, the temperature-responsivesuper-macroporous hydrogels can undergo an ultra-fast reversiblevolume phase transition upon changes of temperature [3]. Here, wereporton the synthesis and application of novel temperature-responsivesuper-macroporous hydrogel for sustained release of hydrophilic drug.

Experimental methodsSyntheses and characterization:The monomer, ethoxytriethyleneglycol acrylate (ETEGA), was

synthesized as described elsewhere [4]. Cryogels were synthesizedby UV irradiation of moderately frozen (−20 °C) aqueous solution ofETEGA, initiator H2O2 (5 wt.%), cross-linking agent, poly(ethyleneglycol) diacrylate (PEGDA, 10 or 30 wt.%), and Verapamil hydro-chloride (10 wt.%) with a “Dymax 5000-EC” UV curing equipment for5 min (Irradiation dose rate=5.7 J/cm2min) [3]. Gel fraction (GF)yield and degree of swelling (DS) of cryogels were determinedgravimetrically.

Cytotoxicity assessment:Тhe cytotoxicity was evaluated using a modification of the USP in

vitro biological reactivity elution test in a panel of human cell lines.Extracts of the tested materials were freshly prepared in RPMI-1640medium (200 mg/ml) for 24 h at 37 °C. The cells were exposed todilutions of the stock extract (200, 100, 50, 25 and 12.5 mg polymer/ml medium) and after 72 h the cellular viability was determinedusing the standard MTT-dye reduction assay.

Drug release:Drug release profiles were evaluated using a dissolution test

apparatus (Erweka DT 600, Germany) and the USP paddle method.The quantity of Verapamil was analyzed by UV absorbance at 278±2 nm (Hewlett Packard 8452A spectrophotometer).

Result and discussionPETEGA cryogels were synthesized by a facile procedure involving

preparation of homogeneous aqueous solution of monomer, initiator,and crosslinking agent, followed by freezing at a defined negativetemperature, anextremely shortUV irradiation andsubsequent thawing.Gels of good quality and nearly quantitative monomer conversion wereobtained from 2 to 10 wt.% aqueous solutions of monomer. PETEGAcryogels are opalescentmaterialswith anopenporous structure (Fig.1a),which significantly increases the rate ofwater uptake due to the capillaryeffects. DS of cryogels depends on the monomer concentration andtemperature (Fig.1b andc). A significant shrinkage, corresponding to thevolume phase transition of PETEGA from swollen to deswollen state isobserved in the 28–37 °C temperature range. Noteworthy, the cryogelsposses an ultra-rapid response to changes in the temperature of mediaand reversibly reach their nearly equilibrium states at a giventemperature within 10s in a reproducible way.

Fig. 1. Morphology (a) and swelling behavour at different temperatures (b,c) of PETEGAcryogels.

The biocompatibility of PETEGA cryogels was tested on humancell lines, representative for different cellular populations, namelyHT-29 (colon epithelium), BV-173 (pre-B-cell), SKW-3 (T-cell) andHL-60 (myeloid). The cellular viability after 72 h exposure toextracts of the tested materials in RPMI-1640 medium was assessedusing the MTT-dye reduction assay. Throughout the panel of humancell lines the tested polymers proved to be devoid of cytotoxicactivity.

In order to localize the hydrophilic drug into the polymer matrix,Verapamil was dissolved in water and mixed with ETEGA, H2O2 andPEGDA before freezing and crosslinking, respectively. In this approach,due to the cryostructuration effect [2,3] drug molecules are locatedpredominantly into the liquid microphase (cryogel walls). Themethod of drug incorporation in combination with the hydrophobicnature of PETEGA matrix at physioligical temperature allows precisecontrol of drug release rates. The results from the dissolution

Abstracts / Journal of Controlled Release 148 (2010) e74–e84 e81

experiments (Fig. 2) clearly demonstrate that after slight initial burstrelease (within the first 2 h) the drug release is sustained at a constantrate (zero-ordered kinetics with correlation coefficient R=0.998).

Fig. 2. Release kinetics of Verapamil hydrochloride from PETEGA cryogel at 37 °C.

ConclusionThe method for synthesis of temperature-responsive PETEGA

cryogels allows incorporation of hydrophilic drugs predominantlyinto the cryogel walls. This fact in combination with the hydrophobicnature of PETEGA network at physioligical temperature provides asustained drug release from the polymer carrier within 8 h.

AcknowledgementThe authors acknowledge the financial support by the National

Science Fund of Bulgaria (National Centre for New Materials UNION,contract no. DO-02-82/2008).

References[1] R. Langer, N.A. Peppas, AIChE Journal 49 (12) (2003) 2990–3006.[2] V.I. Lozinsky, I.Y. Galaev, F.M. Plieva, I.N. Savina, H. Jungvid, B. Mattiasson, TrendsBiotechnol 21 (10) (2003) 445–451.[3] P. Petrov, E. Petrova, C.B. Tsvetanov, UV-assisted synthesis of super-macroporouspolymer hydrogels Polymer 50 (5) (2009) 1118–1123.[4]X.G. Jiang, C.A. Lavender, J.W. Woodcock, B. Zhao,MultipleMicellization andDissociationTransitions of Thermo- and Light-Sensitive Poly(ethylene oxide)-b-poly(ethoxytri(ethyleneglycol) acrylate-co-o-nitrobenzyl acrylate) in Water Macromolecules 41 (7) (2008)2632–2643;[5] Ph. Dimitrov, N. Toncheva, P. Weda, S. Rangelov, B. Trzebicka, A. Dworak, C.B. Tsvetanov,Macromol Symposia Nano-Templates from Thermoresponsive Poly (ethoxytriethyleneglycolacrylate) for Polymeric Nano-Capsules 278 (1) (2009) 89–95.

doi:10.1016/j.jconrel.2010.07.017

Charged dextran hydrogels for post-loading and release of proteins

Joris P. Schillemans⁎, Wim E. Hennink, Cornelus F. van NostrumDepartment of Pharmaceutics, Utrecht Institute for PharmaceuticalSciences (UIPS), Utrecht University, PO Box 80.082, 3508 TB Utrecht,The Netherlands⁎Corresponding author.E-mail: j.p.schillemans@uu.nl.

Abstract summaryProteins were efficiently loaded to pre-formed chemically cross-

linked dextran hydrogels containing charged units, on the basis ofelectrostatic interaction in low ionic strength buffer. Diffusioncontrolled release of proteins from the hydrogels was accomplishedby the increase of the ionic strength to physiological conditions.

IntroductionChemically crosslinked Dextran hydrogels have great potential for

the controlled release of therapeutic proteins. However, damage tothe protein can occur as a result of exposure to free radicals duringthe crosslinking of the hydrogel. Therefore, in this study we proposepost-loading, i.e. loading of proteins after the formation of thehydrogel, on the basis of protein-hydrogel charge interaction as anattractive alternative.

Experimental methodsDextran hydrogels were prepared by co-polymerization of an

aqueous mixture of methacryloyl substituted Dextran (Dex–MA) andhydroxyethyl methacrylate (HEMA) supplemented with negativelycharged methacrylic acid (MA) or positively charged 2-N,N-dimethy-laminoethyl methacrylate (DMAEMA). APS and TEMED were used asinitiator and catalyst, respectively. Model proteins were chosen onthe basis of their charge at physiological pH; Bovine serum albumin(BSA, negatively charged), myoglobin (neutral), and cytochrome C(positively charged). Gels were loaded with protein by incubation inprotein solution at low ionic strength (10 mM hepes pH 7.4, or 10 mMMES pH 5.0) at 37 °C. Loaded gels were transferred to buffer withhigher ionic strength (Hepes Buffered Saline, HBS, pH 7.4) and releasewas followed in time.

Result and discussionCytochrome C (positive charge at pH 7.4) was efficiently loaded (up to

95%) into negatively charged Dex–MAhydrogels at pH 7.4. The amount ofloaded cytochrome C was dependent on the charge density in thehydrogel, and displayed an optimum at 90 μmol MA/g hydrogel. Increas-ing the charge density led to accumulation of the protein in the outer rimof the gel combinedwith a lower loading in total; the immobilized proteinin the rim prohibits the entry of additional protein to the gel.

Fig.1. The effect of network charge density on the loading efficiency of cytochrome C to pre-formed negatively charged Dex-MA hydrogels. Hydrogels (125mg) with varying networkcharge density were synthesized by co-polymerization of Dex–MA with different ratio's ofHEMAandMA.Hydrogelswere incubated in2 mlcytochromeCsolution (1.5 mg/ml inhepes,pH 7.4) at 37 °C. Values represent the average and standard deviation (n=3).

Similar data were obtained for the loading of negatively chargedBSA to positively charged hydrogels at pH 7.4. Loading of Myoglobin,which is neutral at pH 7.4, to negatively charged gels was negligible atpH 7. Higher loading was accomplished by lowering the pH of theloading medium (10% at pH 6 and 45% at pH 5). Transfer of theprotein loaded gels to HBS led to diffusion controlled release of theproteins. The release kinetics was independent of the charge densityof the gel. The amount of released protein reached up to 90% of theloaded amount, but decreased with increasing charge density of thehydrogel. The remaining part of the protein was released by furtherincreasing the ionic strength (1 M NaCl).

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