Meltable Dextran Esters As Biocompatible and Functional Coating Materials

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  • Published: July 08, 2011

    r 2011 American Chemical Society 3107 dx.doi.org/10.1021/bm200841b | Biomacromolecules 2011, 12, 31073113

    ARTICLE

    pubs.acs.org/Biomac

    Meltable Dextran Esters As Biocompatible and FunctionalCoating MaterialsTim Liebert, Jana Wotschadlo, Peggy Laudeley, and Thomas Heinze*

    Institute of Organic Chemistry and Macromolecular Chemistry, Center of Excellence for Polysaccharide Research, Friedrich SchillerUniversity of Jena, Humboldtstr. 10, 07743 Jena, Germany

    bS Supporting Information

    INTRODUCTION

    Hybrid materials consisting of a scaffold and a biocompatiblecoating are in the focus of medical research because long-termimplantation of synthetic polymers or metallic compounds suchas stents or joints might provide a chronic inflammatory stimulus.This may lead to medial atrophy and may cause enhancedthrombotic response.1,2 Moreover, infection is a severe problemduring implantation.3 Biocompatible coating can overcome thisproblem. In addition, modern coatings can act as reservoirs andthereby as controlled release systems for bioactive substancessuch as antibiotics or growth factors imparting defined biologicalactivity to implants.4,5 Different polymeric coatings are used suchas poly(D,L-lactide) (PDLLA) as coating of titanium implantsthat serves as a local drug delivery system for gentamicin.6

    Polysaccharides and their derivatives are well suited for suchapplications because of their biocompatibility and their hightendency toward the formation of defined supramoleculararchitectures.79 Starting materials are today preferably chitosan,alginate, hyaluronic acid or carboxymethylated polysaccharides,most commonly carboxymethyl dextran.10 The polymers areusually applied as hydrogels. A major problem of these water-soluble or swellable hydrophilic materials is the proper fixation tothe carrier and undefined swelling. Symplex formation or acovalent cross-linking may be exploited to overcome suchshortcomings.1114 Nevertheless, these are irreversible processesand the cross-linking is usually combined with the use of toxicreagents limiting or avoiding the application of the resultingmaterial in the medical field.

    A new strategy could be the use of meltable coatings based onpolysaccharides. In this regard fatty acid esters of polysaccharides

    may be considered as promising substances15 but conventionalsynthesis of long chain fatty acid esters (LCE) of polysaccharidesinvolves the use of acid chlorides and bases such as pyridine ortriethylamine yielding derivatives with byproduct. This prohibitsbiomedical application and prevents proper melting of thesubstances. As can be seen in Figure 1, only dark brown meltsare obtained from such esters which form brittle and inhomoge-neous films upon cooling.

    In contrast, it has been shown that the esterification ofpolysaccharides with carboxylic acids after in situ activation is avery useful alternative for the preparation of very purederivatives.15,16 Even sensitive structures such as unsaturatedand heterocyclic acids can be bound to polysaccharides in a veryefficient and gentle manner leading to compounds withoutbyproduct. For the introduction of aliphatic ester moietiesiminium chlorides are well suitable. During the reaction mostlygaseous byproduct are formed and a simple aprotic, polar solvent(Figure 2) making this a proper reaction path for the manufac-ture of pure derivatives which should be applicable in thebiomedical field.

    Therefore, the synthesis of long chain fatty esters of dextranvia in situ activation of the carboxylic acids with iminium chloridewas studied to generate meltable and biocompatible coatingmaterials. The influence of the reaction conditions on the meltingbehavior, and their application for the preparation of homoge-neous, biocompatible and long-lasting films was investigated.

    Received: June 20, 2011Revised: July 6, 2011

    ABSTRACT: The conversion of dextran with in situ synthe-sized iminium chlorides of long chain carboxylic acids was usedto obtain pure and defined melting dextran esters in an efficientone-pot synthesis. The melting point of these esters can betailored by the degree of substitutions (DS), the molecularweight of the starting polymer, and the chain length of the estermoiety. The dextran esters give homogeneous and completelytransparent melts, which form stable films on a broad variety ofmaterials. Even complex geometries, such as implants, can beevenly coated by multiple melting steps. The films do notdisplay any inhomogeneity and have a very low surface roughness. Therefore, no unspecific protein binding is observed. Moreover,the dextran esters are biocompatible as demonstrated for the interaction with three types of cells namely human brain microvascularendothelial cell, primary human fibroblasts, and mouse myoblast cells.

  • 3108 dx.doi.org/10.1021/bm200841b |Biomacromolecules 2011, 12, 31073113

    Biomacromolecules ARTICLE

    In addition first results toward the surface characteristics and theinteraction of these surfaces with biological material such asproteins and cells will be presented.

    EXPERIMENTAL SECTION

    Materials. Dextran from Leuconostoc mesenteroides ssp. (1, Mw6000 g/mol, Fluka) was treated in vacuum at 105 C for 2 h prior to use.Oxalyl chloride,N,N-dimethylacetamide (DMAc),N,N-dimethylforma-mide (DMF), and the fatty acids were obtained from Fluka and wereused without further purification. Lithium chloride was supplied bySigma Aldrich and was treated in vacuum for 24 h at 100 C to guaranteethe absence of water.Dissolution of Dextran in DMAc/LiCl (2). A total of 1.0 g (6.2 mmol)

    of dried dextran (1), 1 g anhydrous LiCl, and 40 mL of DMAc wereheated to 100 C for about 30 min under stirring until completedissolution occurs. The solution became completely clear during coolingdown to room temperature under stirring.Preparation of Palmitic Acid Iminium Chloride (3; Typical Exam-

    ple). In a 100mL flask equipped with amagnetic stirrer, a thermometer, abubble counter, and a SUBA-SEAL septum 30 mL DMF were cooledwith a mixture of isopropanol and dry ice to20 C. At this temperature2.7 mL (31 mmol) oxalyl chloride were added very carefully. Gasformation and a white precipitate were observed. The reaction mixturewas kept at 20 C until the gas formation stopped. 7.9 g (31 mmol)palmitic acid was added to the mixture. After 20 min stirring undercooling the temperature was increased to 0 C. A clear solution of theacid iminium chloride is formed.

    Synthesis of Dextran Palmitate (E7; Typical Example). A solution ofthe iminium chloride (3) was carefully added to the dextran solution (2).This mixture was kept for 16 h at 60 C. After cooling to room tem-perature the product was precipitated by addition of 300mL isopropanol.The product was isolated by filtration and washed three times with50 mL isopropanol. After drying in vacuum at room temperature a puredextran palmitate (E7) was obtained. It is soluble in CHCl3, THF,toluene, and diethylether. The melting point is 46 C.

    Yield: 1.66 g (88.8%); DS (determined by 1H NMR afterperpropionylation): 2.7; FTIR (KBr; cm1): 3483 (OH), 2925, 2892(CH alkyl), 1742 (CdO ester), 1227 (COC ester); 13CNMR(DMSO-d6): (ppm) = 172.3 (CO), 100.9 (C-1), 95.8 (C-1),66.170.6 (C-2 to C-6), 34.1143.4 (alkyl C-atoms).

    Peracetylation of the Dextran Palmitate for 1H NMR Analysis(According to Ref 16). A mixture of 6 mL of pyridine, 6 mL of acetic acidanhydride, and 50mg 4-(dimethylamino)pyridine was added to 0.3 g dextranpalmitate (E7). After 24 h at 80 C, the reactionmixture was cooled to roomtemperature andprecipitated in50mLof ethanol. For purification the isolatedproduct was reprecipitated from chloroform in 50 mL of ethanol, filtered off,washed with ethanol, and dried in vacuum at room temperature.

    Yield: 0.31 g (86.1%); FTIR (KBr; cm1): no (OH), 2910, 2854(CH), 1758, 1737 (CdOester); 1H NMR (of the peracetylateddextran palmitate dissolved in CDCl3): (ppm) = 3.495.48 (HAGU),2.04 (CH3-acetate), 0.771.93, 2.122.24 (CH3 and CH2-palmitate).DSpalmitate = 2.7, DSacetyl = 0.3.

    Coating of Surfaces with the Meltable Dextran Esters. For thecoating of glass surfaces, the flat substrates were evenly covered with thesolid material and placed on a heating plate. Within a few minutes, thedextran ester didmelt and gave very uniform layers on the substrate. Afterannealing for 20min, the air bubbles disappeared. For objects with amorecomplex geometry, the coating was done by dipping the object in a melt.If necessary, a second heat treatment of the precoated material in a waterbath yields very homogeneous and even films.

    Cell Cultures. Different cell types were used for testing cell compat-ibility of the dextran ester coating in vitro, namely, endothelial cell lineHBMEC (human brain microvascular endothelial cell), primary humanfibroblasts, and mouse myoblast cells C2C12. Dextran ester coatedmicroscope slides were dipped into 96% ethanol and were flamedshortly. A total of 6 104 cells (primary human fibroblasts, C2C12)and 1 105 cells (HBMEC) per well (9.6 cm2) were grown on thecoated microscope slides in six-well plates (37 C in a humidifiedatmosphere of 5% CO2 in air) in phenol red-free DMEM or RPMI-1640supplemented with 10% heat inactivated fetal calf serum.

    Characterization of Actin Cytoskeleton. After growing for 3 days,slides with adherent cells were washed three times with phosphate

    Figure 1. Microscopic image of a dextran palmitate (degree of substitution = 1.7, synthesized according to the conventional esterification method withthe acid chloride and pyridine as a base) on glass with human fibroblasts after 2 days: (A) transmitted light microscopy image; (B) fluorescencemicroscopic image.

    Figure 2. Synthesis path for the preparation of dextran esters (in thisexample, a dextran palmitate) via iminium chloride.

  • 3109 dx.doi.org/10.1021/bm200841b |Biomacromolecules 2011, 12, 31073113

    Biomacromolecules ARTICLE

    buffered saline (PBS) and fixed in 4% neutral buffered paraformalde-hyde for 20 min. Subsequently, the cell membrane was permeabilized in0.1% Tween-20/PBS for 30 min. For visualization and microscopiccharacterization of cells, the cytoskeleton was fluorescently labeled withAlexa Fluor488 phalloidin (Invitrogen) for 90 min. The cell nucleus wasstained with 40,6-diamidino-2-phenylindole (DAPI, Vysis Inc.). Micro-scope slides were completely washed in PBS and prepared with Immu-Mount (Thermo Shandon) for fluorescence microscopy (Axiocam,Zeiss). All incubations were operated at room temperature.Protein Affinity to Dextran Esters. Dextran ester-coated glass slides

    were incubated for 24 h in a complete protein lysat solution of cellculture cells (breast cancer cell line MCF-7). After washing several timeswith PBS, staining for 15 min with a 0.1% coomassie brilliant bluesolution (5% acetic acid, 50% ethanol, 45% dest. water) and discolora-tion (10% acidic acid, 20% isopropanol, 80% dest. water) differences indye staining could be observed with a microscope.Measurements. FTIR spectra were recorded on a Nicolet Protege

    460 spectrometer with the KBr technique. KBr tablets were dried at100 C for 1 h to remove moisture prior to the measurement. NMRspectra were acquired on a Bruker AMX 250 spectrometer with 16 scansfor 1H NMR and 1500089000 scans for 13C NMR measurements(Bruker AMX 400, 50 mg sample/mL). 1H NMR spectra of the esterswere acquired in dimethyl sulfoxide (DMSO)-d6 after peracetylation ofthe unmodified hydroxyl groups.17,18 Elemental analyses were performedwith a CHNS 932 Analyzer (Leco). UVvis spectroscopy was carriedout with a Genesys 6 (Thermospectronic). AFM was conducted in thenoncontact mode with a DualScope C-21 (DME) and silicon nitride tips(60.0 N 3m

    1, 0.20 nN). Rheological characterizations of the melts werecarried out with a Haake MARS rheometer equipped with coneplategeometry (35 mm radius, cone angle 1). Differential scanning calorim-etry (DSC) measurements were performed with a Mettler Toledo DSC822e using a heating rate of 10 C/min in the range20 to 300 C afterdrying all the samples at 40 C for 24 h in vacuum (except samples E2andE4). All the fluorescence images were takenwith an Axioplan 2 imagingfluorescence microscope, transmitted light images with an Axiovert 25microscope and an AxioCam HRc (all from Zeiss). Images wereprocessed with an AxioVision 3.1 program (Zeiss). Objectives: Ph2Plan-NEOFLUAR 20/0.5 and Ph1 CP-ACHROMAT 10x/0.25.

    RESULTS AND DISCUSSION

    Iminium chlorides are simply formed by conversion of N,N-dimethylformamide (DMF) with a variety of chlorinating agentsincluding phosphoryl chloride, phosphorus trichloride or oxalyl

    chloride.11 In this study, oxalyl chloride was used. The formationof the iminium chloride and the conversion with the carboxylicacid were carried out as a simple one-pot-reaction, that is, DMFwas cooled to 20 C, oxalyl chloride was added very carefully,and after the gas formation has stopped, the carboxylic acid wasadded. The conversion occurred with quantitative yield at thistemperature. Esterification of the polysaccharide was simplyachieved by mixing the solution of the acid iminium chloridewith dextran dissolved in DMAc/LiCl. The purification of thedextran ester was rather easy because most of the products aregaseous and during the last step DMF is reformed making this avery efficient reaction path toward the preparation of highly puredextran esters (Figure 2).

    A summary of reaction conditions and results is given inTable 1. The DS values were determined by 1H NMR spectros-copy after peracetylation of the remaining hydroxyl groupsaccording to ref 16. The degree of substitution (DS) wascalculated by the following equation (DSLCE = 3 (7 IH,acetyl)/(3 IH,AGU).

    The esterification method is suitable for the synthesis of alltypes of aliphatic carboxylic acids (samples E1E10). Esterifica-tion succeeds with comparable efficiency for all acids applied. Arather good control of the DS values is possible via the amount ofreagent used. For the myristate even complete conversion of allOH moieties was observed. The esters prepared are whitesubstances, which are well soluble in DMSO, THF, acetone,toluene, or chloroform, depending on the DS values.

    No chlorine was determined by elemental analysis. In FTIRspectra (KBr) of the products, the typical absorption bands forthe polysaccharide backbone (3620, 2920, and 1140 cm1) andsignals for the carbonyl function of the ester moiety at17451760 cm1 were found. The representative 1H,1H COSYNMR spectrum (CDCl3) of a highly functionalized dextranpalmitate depicted in Figure 3 shows signals for the anhydroglu-cose unit (AGU) at = 3.45.5 ppm (H-1H-6) and for thealiphatic protons of the palmitoyl moiety at 0.82.3 ppm (H-7, 8).Assignment of the cross peaks gave no hints for side reactions orimpurities, which would lead to substructures such as deoxy-chloro functions. Elemental analysis confirmed the DS calculatedfrom the 1H NMR spectra.

    Further evidence for the purity of the dextran esters was gainedfrom 13C NMR spectra of the products. In a typical spectrum

    Ta...

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