Polylactide Stereocomplex-Based Electrospun MaterialsPossessing Surface with Antibacterial and Hemostatic

Properties

Mariya Spasova,† Nevena Manolova,† Dilyana Paneva,† Rosica Mincheva,‡

Philippe Dubois,‡ Iliya Rashkov,*,† Vera Maximova,§ and Dobri Danchev|

Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G.Bonchev str., bl. 103A, 1113 Sofia, Bulgaria, Laboratory of Polymeric and Composite Materials, Universityof Mons (UMONS), Place du Parc 23, B-7000 Mons, Belgium, Department of Gene Regulations, Institute

of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, and Department ofHaemostasis, Military Medical Academy, G. Sofijski Street 3, 1606 Sofia, Bulgaria

Received September 4, 2009; Revised Manuscript Received October 28, 2009

Novel fibrous materials of stereocomplex between high-molecular-weight poly(D- or L-)lactide (HMPDLA orHMPLLA) and diblock copolymers consisting of poly(L- or D-)lactide and poly(N,N-dimethylamino-2-ethylmethacrylate) blocks, respectively (PLLA-block-PDMAEMA or PDLA-block-PDMAEMA), were prepared bysolution electrospinning. Fibers with mean diameters ranging from 1400 to 1700 nm were obtained. Thestereocomplex formation was evidenced by differential scanning calorimetry (DSC) and X-ray diffraction (XRD)analyses. Annealing at 100 °C for 8 h resulted in the appearance of crystalline peaks at 2θ values of 12, 21, and24° for PLA stereocomplex. X-ray photoelectron spectroscopy (XPS) analyses revealed the gradient compositionof the fibers with a surface enriched in tertiary amino groups from PDMAEMA blocks. The availability of tertiaryamino groups imparts hemostatic and antibacterial properties to the stereocomplex fibrous materials, as indicatedby the performed tests on blood cells and on pathogenic microorganisms.

Introduction

During the past few years, aliphatic polyesters have becomeone of the most preferred polymers for developing newgeneration devices for biomedical applications.1 Up to now,many studies on electrospinning of aliphatic polyesters, poly-lactide (PLA), poly(ε-caprolactone) (PCL), and their copoly-mers, have been accomplished.2-10 Electrospinning is anextremely effective method for the preparation of micro- andnanofibrous materials. The good mechanical properties of PLA-based materials render them suitable for application as scaffoldsfor tissue regeneration, implants, wound-dressing materials, andso on.2,4,6,8 However, the high hydrolytic sensitivity of PLAcould be a disadvantage at prolonged storage under physiologicalconditions. One of the approaches to overcome this drawbackis the preparation of novel materials by stereocomplex formationbetween enantiomeric PLLA and PDLA.11 Up to date, moststudies on the formation of PLA stereocomplexes are devotedto the investigation of their properties (thermal and hydrolyticstability), the preparation of films or nanoparticles, or both.12-15

Studies directed toward the preparation of stereocomplex fibersby electrospinning are still scarce.16-18 The high degree oforientation caused by the high voltage during electrospinningenhances the formation of stereocomplex crystallites andsuppresses the formation of homocrystallites, even when high-molecular-weight PDLA and PLLA are used.16 Recently, in vitroand in vivo studies on the degradation of PDLA/PLLAstereocomplex fibrous materials have revealed that they degrade

more slowly and preserve their shape for longer time thanpoly(L-lactide) nanofibers.18 Moreover, the reported stereocom-plex nanofibers showed improved tissue compatibility.

To our knowledge, in the literature, there are no reports onthe preparation of PLA stereocomplex fibers using amphiphilicblock copolymers. Amphiphilic PLA-based copolymers contain-ing ionogenic block are extremely attractive for the preparationof materials with improved physicochemical properties andtargeted behavior on contact with cells and tissues as well.Recently, we have synthesized PLA-based block copolymerscontaining polycationic block of poly(N,N-dimethylamino-2-ethyl methacrylate) (PLA-b-PDMAEMA).19 The copolymerswere obtained combining ring-opening polymerization andATRP. It was shown that PLA-b-PDMAEMA contained opti-cally pure PLA enantiomers. PLLA-b-PDMAEMA and PDLA-b-PDMAEMA copolymers were used for the preparationof stereocomplex films. It was found that the presence ofPDMAEMA block did not hamper the stereocomplex formationand imparted hydrophilicity to the novel materials. PDMAEMApossesses inherent hemostatic and antibacterial activity.20-25 Thelast property is of great importance for PLA polymer materialsthat are a favorable medium for the growth of pathogenicmicroorganisms.7,26 Recently, we have shown that electrospin-ning of mixed solutions of high-molecular-weight PCL andcopolymers consisting of PCL and PDMAEMA blocks allowsone-step preparation of fibrous materials based on aliphaticpolyesters possessing surface enriched in tertiary amino groupsof the polycation.10 It is expected that using this approach PLLA/PDLA stereocomplex fibrous materials containing PDMAEMAsegments with antibacterial and hemostatic properties may beprepared.

The present study demonstrates the possibility of preparingnovel PLA stereocomplex fibrous materials containing PLA-b-

* To whom correspondence should be addressed. E-mail: rashkov@polymer.bas.bg.

† Institute of Polymers, Bulgarian Academy of Sciences.‡ University of Mons (UMONS).§ Institute of Molecular Biology, Bulgarian Academy of Sciences.|Military Medical Academy.

Biomacromolecules 2010, 11, 151–159 151

10.1021/bm901016y 2010 American Chemical SocietyPublished on Web 11/30/2009

PDMAEMA block copolymers by electrospinning. The fibermorphology was studied by scanning electron microscopy(SEM), and the thermal properties were studied by DSC. Thecrystalline structure of the novel materials before and afterannealing was investigated by X-ray diffraction (XRD). Thesurface chemical composition of the fibrous materials wasdetermined by X-ray photoelectron spectroscopy (XPS). Thebiological behavior of the obtained fibrous materials was studiedwith respect to human blood and pathogenic microorganisms.

Experimental Section

Materials. D-Lactide (PURAC, The Netherlands) and L-lactide(GALACTIC, Belgium) were recrystallized from dried toluene andstored in a glovebox under a dry nitrogen atmosphere before use.Aluminum triisopropyloxide (Al(OiPr)3, Acros, 98%) was distilled undervacuum, quenched in liquid nitrogen, rapidly dissolved in dried toluene,and then stored under a dry nitrogen atmosphere.

The Al(OiPr)3 concentration was determined by back complexometrictitration of Al3+ using ethylenediaminetetraacetic (EDTA) acid disodiumsalt and ZnSO4 at pH 4.8. DMAEMA (Aldrich, 98%) was passedthrough a column of basic alumina to remove the stabilizing agentsand stored under nitrogen at -20 °C. High-molecular-weight poly(L-lactide) (HMPLLA, Fluka, Mj n ) 99 000 g/mol, Mj w/Mj n ) 1.5),dichloromethane (Chem-Laboratory, 99.8%), heptane (Labscan, 99%),methanol (Chem-Laboratory, 99.8%), chloroform (Aldrich, 99.9%),fluorescein disodium salt (Fluka), R-bromoisobutyryl bromide (Br-iBuBr, Aldrich), triethylamine (Fluka, 99%), 1,1,4,7,10,10-hexameth-yltriethylenetetramine (HMTETA, Aldrich, 97%), and copper(I) bro-mide (CuBr, Fluka, 98%) were used without further purification.Toluene (Labscan, 99%) and tetrahydrofuran (THF; Labscan, 99%)were dried using an MBraun solvent purification system under N2.

A strain of Esherichia coli (E. coli) (LE 329) was obtained fromthe Collection of the Department of Gene Regulations, Institute ofMolecular Biology, Bulgarian Academy of Sciences (Sofia, Bulgaria).Staphylococcus aureus (S. aureus) (NBIMCC3359) was obtained fromthe National Bank for Industrial Microorganisms and Cell Cultures(NBIMCC, Bulgaria). E. coli growth media was Luria-Bertani (LB)containing bacto trypton and bacto yeast extract purchased fromScharlau (Barcelona, Spain) and bacto agar supplied by Difco. S. aureusgrowth media was meat-peptone bullion purchased from the NationalCenter of Infectious and Parasitic Diseases (NCIPD, Bulgaria).

Synthesis and Characterization of PLA-b-PDMAEMA DiblockCopolymers. Poly(D-lactide)-b-poly(N,N-dimethylamino-2-ethyl meth-acrylate) (PDLA-b-PDMAEMA) and poly(L-lactide)-b-poly(N,N-di-methylamino-2-ethyl methacrylate) (PLLA-b-PDMAEMA) block co-polymers were synthesized using a three-step procedure described indetail elsewhere.19 The first stage consists of controlled ring-openingpolymerization of D-lactide or L-lactide initiated by aluminum triiso-propoxide (Al(OiPr)3) to form R-isopropyloxy, ω-hydroxy polylactide(PLA-OH), followed by quantitative conversion of PLA-OH intoR-isopropyloxy, ω-bromoisobutyratepolylactide macroinitiator (PLA-Br), and atom transfer radical polymerization (ATRP) of DMAEMAinitiated by PLA-Br. The polymerization was performed in THF at 60°C in the presence of a CuBr/HMTETA catalytic system under nitrogen.The composition and macromolecular characteristics of the diblockcopolymers were determined by 1H NMR and SEC. The number-average molar mass of the diblock copolymers was calculated usingthe relative intensity of the R-amino methylene protons of DMAEMAresidue from repeating units at δ ) 2.3 ppm and the methine protonsof PLA at δ ) 5.15 ppm knowing Mj n of the macroinitiator.

High-molecular-weight poly(D-lactide) (HMPDLA, Mj n ) 75 000g/mol and Mj w/Mj n ) 1.58) was synthesized using ring-openingpolymerization of D-lactide initiated by aluminum triisopropoxide at amonomer/initiator molar ratio [LA]0/[Al] ) 1667. The optical purityof the synthesized PDLA-OH and PLLA-OH was determinedmeasuring the specific optical rotation [R] of polymers in chloroform

solution at a concentration of 1 g/dl and 20 °C using a digitalPolarimeter OA AA5 (Optical Activity, U.K.) at a wavelength of589 nm.

Preparation of Fibrous Mats by Electrospinning. HMPLLA fiberswere prepared by electrospinning of its solution in CHCl3 (concentra-tion: 7 wt %). “sc” PLLA-b-PDMAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLA fibers were prepared by electrospinning of theirsolutions in CHCl3 at a constant total concentration: 15 wt % [PL(D)LAblock/PD(L)LA] ) 1/1 (mol/mol)]. Solutions were placed in a syringe(2 mL) equipped with a cylindrical metal nozzle with an inner diameterof 0.1 mm and a wall thickness of 0.05 mm. The nozzle was connectedto an electrode via an alligator clip. The electrode was connected to ahigh-voltage power supply generating positive DC voltage up to 30kV. The electrospun mats were collected onto a rotating drum (1000rpm). The spinning solution was delivered at a controlled feed rate of0.5 mL/h at an applied voltage of 15 kV and a distance between thetip of the conical nozzle and the collector of 12 cm. The electrospunsamples were placed under vacuum for 72 h to remove any solventresidues.

Characterization of the Electrospun Mats. The morphology ofthe electrospun mats was observed by SEM. For that purpose, the matswere vacuum-coated with carbon and gold and analyzed using a PhilipsSEM 515. The fiber morphology was evaluated in terms of the criteriafor complex evaluation of electrospun mats reported elsewhere27 usingImage J software28 by measuring at least 20 fibers/pore from each SEMimage.

Differential scanning calorimetry (DSC) measurements were carriedout with a DSC Q50 apparatus from T.A. Instruments under nitrogenflow (heating rate of 10 °C/min).

XRD morphological analyses were performed on a Siemens D5000diffractometer using Cu KR radiation (wavelength: 1.5418 Å) at roomtemperature. The samples were step-scanned from 10 to 25° in 2θ withsteps of 0.02°with fixed counting time of 4 s (40 kV, 30 mA).

For the detection of the tertiary amino groups in the fiber structurethe mat of PLLA or PLA/copolymer were immersed in 10 mL of 0.02wt % aqueous solution of fluorescein for 7 h and then washed severaltimes with distilled water.

The surface chemical composition of the fibers was determinedquantitatively by XPS using VG ESCALAB 220iXL spectrometer withmonochromatic Al KR X-ray source (hν ) 1486.6 eV) of 250 W.

Behavior of the HMPLLA, “sc” PLLA-b-PDMAEMA/HMP-DLA, and “sc” PDLA-b-PDMAEMA/HMPLLA Mats on Contactwith Blood. Platelet, Erythrocyte, and Thrombocyte Adhesion on theSurface of the HMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA, and“sc” PDLA-b-PDMAEMA/HMPLLA Mats. Platelet, erythrocyte, andthrombocyte adhesion was monitored by direct SEM observation ofthe blood cells adhered to the surfaces of mats contacting with wholecitrated human blood. Each mat was immersed in human whole citratedblood for 1 h. After that, the samples were carefully washed with salineto remove the nonadhered blood cells. The adhered blood cells werefixed on the mat surface by immersion of the mat into 2.5 wt %glutaraldehyde solution in saline at room temperature for 30 min. Then,the samples were carefully washed three times with saline and, priorto freeze-drying, washed with distilled water to remove NaCl. Beforethe SEM observations, the specimens were vacuum-coated with carbonand gold.

Blood Cell Counting. A hematological analyzer (Sysmex-SF 3000,Japan) was used for blood cell counting. The investigations were carriedout using fresh venous whole blood from healthy volunteers collectedinto EDTA tubes. Fibrous mats were incubated in whole humanblood at 25 °C for 30 min. The interaction of the fibrous mats withblood was evaluated by counting the white blood cells, red blood cells,and platelets.

Behavior of the HMPLLA, “sc” PLLA-b-PDMAEMA/HMP-DLA, and “sc” PDLA-b-PDMAEMA/HMPLLA Mats on Contactwith Pathogenic Microorganisms S. aureus and E. coli. Evaluationof the interaction of fibrous HMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA, and “sc” PDLA-b-PDMAEMA/HMPLLA mats with S.

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aureus and E. coli was performed by direct SEM observation of theadhered S. aureus and E. coli cells onto the surface of the mats. Matshad been placed in contact with a cell culture of S. aureus (pH ∼5.5)and E. coli (pH ∼7) for 24 h (2 × 107 cells/ml). Then, the sampleswere washed three times with phosphate-buffered saline (PBS, pH 7.4)for the removal of nonadhered bacteria. The adhered bacteria on thesurface of the mats were fixed by immersion of the mats in 2.5 wt %glutaraldehyde solution in PBS at 4 °C for 5 h. Then, the samples werewashed again carefully with PBS and distilled water and freeze-dried.After that, the samples were coated with gold, and the bacterialmorphology was observed by Jeol JSM-5510.

Statistical Analysis. Blood counting data were presented as mean( standard deviation (S.D.). The statistical significance of the dataobtained was analyzed by one-way ANOVA. GraphPad Prism 5 wasutilized to conduct statistical analysis. Probability values of p < 0.05were interpreted as denoting statistical significance.

Results and Discussion

In the present study, diblock copolymers based on PLA andPDMAEMA blocks (PLA-b-PDMAEMA) were used. Theywere synthesized in a controlled manner using a three-stepprocedure recently described in detail.19 Schematic representa-tion of the structure of the synthesized diblock copolymers isgiven in Scheme 1, and their macromolecular characteristicsare listed in Table 1.

The macromolecular characteristics of the high-molecular-weight PLLA (HMPLLA) and PDLA (HMPDLA) used aspartners for the copolymers to obtain stereocomplex (“sc”) fibersby electrospinning are listed as well in Table 1. The specificoptical rotation [R] of the PDLA and PLLA homopolymers,which were used as macroinitiators for ATRP of DMAEMA,was +145.5 and -146.5°, respectively, attesting that enantio-meric polylactides with high optical purity were obtained.

Preparation of Fibrous Materials by Electrospinning ofSolutions Containing PD(L)LA-b-PDMAEMA. Preliminaryexperiments on preparation of PLLA-b-PDMAEMA/PDLA-b-PDMAEMA fibers were performed by electrospinning ofsolutions at total polymer concentration 25 wt % and molar ratio[PDLA block]/[PLLA block] ) 1/1. The experiments resultedin obtaining spherical microparticles with mean diameter of thetwo most abundant populations 13.5 ( 2 and 2.9 ( 1.2 µm,respectively; that is, the process performed under these condi-

tions was electrospraying rather than electrospinning. This resultmay be attributed to the low molecular weight of the usedcopolymers (Table 1), and it is in good agreement with ourprevious results on electrospinning of block copolymers con-taining PCL and PDMAEMA block.10

The replacement of one of the PLA-b-PDMAEMA copoly-mers with high-molecular-weight homopolymer PLA (HPLA)rendered feasible the obtaining of fibers from mixed PLLA-b-PDMAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLAsolutions by electrospinning. It should be noted that recentlywe have shown stereocomplex formation on casting of mixedsolutions of polylactides having significant differences in theirmolecular weight, such as HPLA and PLA-b-PDMAEMAcopolymers.19 In addition, a suitable approach for the preparationof fibers containing block copolymers of PCL and PDMAEMAby using high-molecular-weight homopolymer partners has beenpreviously reported.10 In the present study, the molar ratiobetween the repeating units of the PLA block in the PLA-b-PDMAEMA copolymers and in HPLA was 1/1, and the totalpolymer concentration was 15 wt %. SEM micrographs of theobtained fibers are shown in Figure 1. For the sake ofcomparison, HMPLLA fibers are shown as well. The HMPLLAfibers were defect-free with mean fiber diameter 1800 ( 500nm. Pores with mean pore length and width 380 ( 110 and160 ( 50 nm, respectively, were observed along the fiber length.The fibers obtained by electrospinning of PLLA-b-PDMAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLA were defect-free as well. However, pores were not detected along theirlength. A similar result was obtained in the case of electrospin-ning of mixed solutions of high-molecular-weight PCL and itscopolymer with PDMAEMA.10

Fibers with smooth surface were obtained in the presence ofPDMAEMA blocks. The mean diameters of PLLA-b-PD-MAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLA fi-bers were 1400 ( 450 and 1700 ( 600 nm, respectively.Presumably, the higher values of the mean diameter for PDLA-b-PDMAEMA/HMPLLA fibers may be attributed to the highermolecular weight of HMPLLA (Mj n 99 000) as compared withthat of HMPDLA (Mj n 75 000).

Stereocomplex Formation in the “sc” PLLA-b-PDMAEMA/HMPDLA and “sc” PDLA-b-PDMAEMA/HMPLLA Fibers.PLA stereocomplex formation leads to significant changes inthermal behavior of the polymers. The obtained mats werestudied by DSC and XRD to prove the stereocomplex formationbetween the PLA block of the copolymer and the HPLAhomopolymer in the fibers obtained by electrospinning ofpartners PLLA-b-PDMAEMA/HMPDLA and PDLA-b-PD-MAEMA/HMPLLA. DSC curves of as-spun PLLA-b-PD-MAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLA areshown in Figure 2. For the sake of comparison, the DSC curveof as-spun HMPLLA fibers is shown as well.

The shape of the DSC thermogram of HMPLLA fibers isvery similar to that reported for electrospun PLLA fibers.16

Glass-transition temperature, cold crystallization, and meltingtemperature peaks are clearly observed. The presence of coldcrystallization temperature (Tcc) attests that the fibers containcrystallizable free amorphous regions. This is attributed to thefast evaporation of the solvent during the electrospinning processthat hampers the complete crystallization. Additionally, thecrystallization is further hampered by the PDMAEMA segmentin PLA-b-PDMAEMA copolymers. The Tcc of “sc” PLA-b-PDMAEMA/HMPLA fibers appears at lower temperature thanthat for HMPLLA fibers (∼80 °C, compared with 95 °C forHMPLLA).

Scheme 1. Schematic Representation of the Structure ofPLA-b-PDMAEMA Diblock Copolymers

Table 1. Macromolecular Characteristics of the Studied HMPLLA(Fluka), HMPDLA, PLLA-b-PDMAEMA, and PDLA-b-PDMAEMA(Co)polymers

(co)polymer

PDMAEMAa

Mj n PLA block Mj w/Mj nFw [%] DP Mj n

HMPLLA 99 000b 1.5b

HMPDLA 70 000c 1.58d

PLLA-b-PDMAEMA 36 38 6000 10 500c 1.36d

PDLA-b-PDMAEMA 35 35 5500 10 350c 1.46d

a Weight fraction (Fw), degree of polymerization (DP), and number-average molecular mass (Mj n, g/mol) of PDMAEMA blocks, as determinedby 1H NMR spectroscopy in CDCl3. b According to Fluka specification.c Number-average molecular weight (Mj n, g/mol) of PLA blocks, asdetermined by SEC in THF (+2 wt % Et3N) using polystyrene standards.d Polydispersity index of the (co)polymers, as determined by SEC in THF(+2 wt % Et3N) using polystyrene standards.

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It has been reported that stereocomplex formation results ina significant increase in the Tm values of the used materials.11,29,30

Recently, “sc” PLA-b-PDMAEMA/HMPLA films were pro-duced, and it was shown that the presence of the PDMAEMA

block in the structure of copolymers did not hamper thestereocomplex formation, and the Tm was 212 °C.19 Similarlyto these findings, in the present study, it was found that “sc”PLLA-b-PDMAEMA/HMPDLA and “sc” PDLA-b-PDMAEMA/

Figure 1. SEM micrographs of fibers prepared by electrospinning of (A) HMPLLA (polymer concentration: 7 wt %), (B) PLLA-b-PDMAEMA/HMPDLA (15 wt %), and (C) PDLA-b-PDMAEMA/HMPLLA (15 wt %); molar ratio [PL(D)LA block/PD(L)LA] ) 1/1; solvent: CHCl3; feed rate: 0.5mL/h; applied voltage: 15 kV; distance between the capillary and collector: 12 cm; magnification: ×1000 and ×10 000.

Figure 2. DSC curves of as-spun fibrous mats of HMPLLA, PLLA-b-PDMAEMA/HMPDLA, and PDLA-b-PDMAEMA/HMPLLA. Heating rate of10 °C/min under N2 flow. First heating run.

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HMPLLA fibers showed higher melting temperature (Tm ≈ 220°C) than HMPDLA or HMPLLA fibers (Tm around 177 °C). Itshould be noted that in films as well as in fibers, crystallizationof PDLA or PLLA enantiomers is not observed (no trace ofmelting at ca. 175 °C or lower). In addition, it was found thatthe enthalpy of melting (∆Hm) for “sc” PLA-b-PDMAEMA/HMPLA fibers was ∼60 J/g. This value is close to the previouslyfound one (55 J/g) for “sc” PDLA-b-PDMAEMA/PLLA-b-PDMAEMA stereocomplex films. This value is lower than ∆Hm

for the stereocomplex of the PLA homopolymers (∆Hm ) 118J/g) and is attributed to the presence of PDMAEMA segmentsthat disturb the structural organization of the PLA segmentsinto highly crystalline domains.19

XRD analyses were performed to determine the crystallinestructure of the HMPLLA, “sc” PLLA-b-PDMAEMA/HMP-DLA, and “sc” PDLA-b-PDMAEMA/HMPLLA fibers. Figure3A shows the recorded XRD patterns. In the XRD pattern of

as-spun HMPLLA fibers, only a wide halo that begins at 2θaround 22.8° is observed. This result is in agreement with datareported for PLLA electrospun from different solvents (chlo-roform/acetone, methylene chloride/dimethylformamide, andchloroform or 1,1,1,3,3,3-hexafluoro-2-propanol), where nocrystalline peak is detected.3,4,31 There is only one reportshowing weak diffraction at 2θ around 17° detected in elec-trospun PLLA fibers;16 however, this finding is still notexplained.

Crystalline peaks could not be detected for the as-spun “sc”PLA-b-PDMAEMA/HMPLA fibers. This result is in agreementwith literature data for PLLA/PDLA stereocomplex fibers17 andis attributed to fast unfolding of the polymer chains caused bythe electrical field and to fast solvent evaporation as well.

Annealing of the PLA fibers allows the crystallization of thepolymer and the appearance of characteristic peaks for crystal-line structures in XRD patterns. In the present study, the samples

Figure 3. XRD patterns of HMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA and “sc” PDLA-b-PDMAEMA/HMPLLA fibers: (A) as spun and (B) annealedat 100 °C for 8 h.

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were annealed at 100 °C for 8 h. The recorded patterns arepresented in Figure 3B. Annealing of HMPLLA fibers led tothe appearance of clearly observed peaks at 2θ ) 16.2 and 18.5°,which are attributed to the R form of PLLA crystallized in apseudo-orthorhombic unit cell of dimensions a ) 0.595 nm andc ) 2.78 nm, which contains two 103 helices.16,32 Diffractionpeaks at 2θ ) 12, 21, and 24° were detected for the annealed“sc” PLA-b-PDMAEMA/HPLA fibers. These 2θ values are ingood agreement with the data reported for PLA stereocomplexformation that crystallized in triclinic unit cell of dimensions a) 0.916 nm, b ) 0.916 nm, c ) 0.870 nm, R ) 109.2°, and γ) 109.8°, in which L-lactide and D-lactide segments are packedparallel taking a 31 helical conformation.16,32

The absence of diffraction peaks for the R form of PLA at2θ ) 16.2 and 18.5° is an indication of the absence ofhomocrystallites of HMPLA or PLA block from the copolymerin the annealed samples. This result is in agreement with thedata obtained from the DSC.

Surface Chemical Composition of “sc” PLLA-b-PD-MAEMA/HMPDLA and “sc” PDLA-b-PDMAEMA/HM-PLLA Fibers. We demonstrated the presence of PDMAEMAsegments in the “sc” PLA-b-PDMAEMA/HMPLA fibers bystaining HMPLLA and “sc” PLA-b-PDMAEMA/HMPLA matswith negatively charged dye fluorescein. Photographs of fibrousmats after their 7 h stay in dye solution are shown in Figure 4.Before immersion, the color of all fibrous mats was white.Changes in the color of the mats were observed after stay inthe aqueous fluorescein solution, followed by washing with

distilled water. The HMPLLA fibrous mat did not change itscolor because of the lack of functional groups able to interactwith the dye. Because of the presence of PDMAEMA in the“sc” PLA-b-PDMAEMA/HPLA mats, the contact of the novelmaterials with fluorescein led to their intense orange color. Thisis an indication that ionic interactions had taken place betweennegatively charged carboxyl anions of the dye and the positivelycharged (protonated) tertiary amino groups of PDMAEMA.

Studies on the surface chemical composition of the preparedfibers were performed by XPS. The analyses were performedat a detection limit of ca. 0.1 wt % (according to themanufacturer) and probe depth (ca. 1-3 nm). Only the expectedelements were detected in the samples. In the spectrum (notpresented) of HMPLLA fibers, peaks for C1s at 288.3 eV andfor O1s at 534.2 eV were observed. In the spectrum of “sc”PLLA-b-PDMAEMA/HMPDLA and “sc” PDLA-b-PDMAEMA/HMPLLA fibers, besides the peaks for carbon and oxygen, apeak for N1s at 400 eV was observed. This peak was attributedto the tertiary amino groups of the PDMAEMA block and wasused for quantitative analysis. The theoretically calculatednitrogen content in PLA-b-PDMAEMA copolymer was 4.2%,whereas in “sc” PLA-b-PDMAEMA/HPLA fibers, it was∼1.5%. Experimentally determined values for the nitrogencontent on the surface of the “sc” PLA-b-PDMAEMA/HMPLAfibers were twice higher than the theoretical one. These valuesimply that most probably the surface of the “sc” PLA-b-PDMAEMA/HMPLA fibers is enriched in PLA-b-PDMAEMAblock copolymer. This finding is in agreement with our previousstudies, demonstrating that by electrospinning of solutionscontaining diblock copolymer with PDMAEMA block, fibrousmats from PCL with surface enriched in tertiary amino groupsof PDMAEMA units could be obtained.10 Because of thespecific biological properties of PDMAEMA, these materialsraise great interest.

Behavior of the Prepared Fibrous HMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA, and “sc” PDLA-b-PDMAEMA/HMPLLA Materials in Contact with Blood and PathogenicMicroorganisms. In contrast with PLA, which is inert to bloodcells, PDMAEMA possesses hemostatic and antibacterialactivity.20-25 These properties of PDMAEMA are explained byelectrostatic interactions between the positively charged tertiaryamino groups and the negatively charged moieties on the surfaceof the blood cells and pathogenic microorganisms. Knowingthat the surface of the fibrous “sc” PLA-b-PDMAEMA/HMPLAmats is enriched in tertiary amino groups of PDMAEMA, weevaluated the behavior of the prepared novel fibrous mats incontact with whole blood and pathogenic microorganisms.

The behavior of the fibrous mats placed in contact with bloodwas studied by blood cell counting and by SEM. Blood cellcounting allows quantitative evaluation of the changes that occur

Figure 5. Dependence of the blood cell counts on the type of thefibrous mat after a 30 min stay in whole blood. All values are anestimate of the standard uncertainty expressed as the mean ( S.D.(n ) 3), **: p < 0.01; ***: p < 0.001.

Figure 4. Photographs of fibrous mats of (A) HMPLLA, (B) “sc” PLLA-b-PDMAEMA/HMPDLA, and (C) “sc” PDLA-b-PDMAEMA/HMPLLA aftera 7 h stay in aqueous solution of fluorescein.

156 Biomacromolecules, Vol. 11, No. 1, 2010 Spasova et al.

in the blood cell counts after contact of the polymers with wholehuman blood.21 Fibrous mats were incubated for 30 min at 37°C in fresh human blood from healthy volunteers. The depen-dence of the blood cell counts (white blood cells, red bloodcells, and platelets) on the nature of the fibrous mat after 30min of incubation in blood is presented in Figure 5. As seen, inthe case of the HMPLLA mat, there is almost no reduction inthe blood cell counts. The insignificant reduction of the numberof blood cells is most probably due to their mechanical retentioninto the highly porous 3D structure of the mat.

In contrast, fibrous “sc” PDLA-b-PDMAEMA/HMPLLA and“sc” PLLA-b-PDMAEMA/HMPDLA mats caused a consider-able decrease in red blood cell counts and the number ofthrombocytes, indicating significant hemostatic activity of theprepared materials. All of the mats preserved their integrity afterstay in whole blood. In contrast with HMPLA mats, the “sc”

PLA-b-PDMAEMA/HMPLA mats were intensely red (Figure6). The obtained result is in good agreement with the dataobtained by cell counting, indicating a decrease in red bloodcell counts and the number of thrombocytes.

Fibrous mats placed in contact with blood were observed bySEM (Figure 7). SEM analysis showed the presence of singleerythrocytes with nonaltered shape on the surface of theHMPLLA fibrous mat (Figures 8A), indicating that this matdoes not exhibit hemostatic activity. The presence of such singleerythrocytes is most probably due to mechanical retention ofthe cells in the highly porous structure of the fibrous mat. Incontrast, in the case of fibrous PLLA-b-PDMAEMA/HMPDLA(Figures 7B) and PDLA-b-PDMAEMA/HMPLLA (Figures 7C)mats, a significant number of adhered red blood cells as wellas thrombus formation is observed. The red blood cells areaggregated, agglutinated, and highly deformed. The observed

Figure 6. Photographs of fibrous mats of (A) HMPLLA, (B) “sc” PLLA-b-PDMAEMA/HMPDLA, and (C) “sc” PDLA-b-PDMAEMA/HMPLLA aftera 1 h stay in human whole blood.

Figure 7. SEM micrographs of fibrous mats after contact with whole human blood (1 h): (A) HMPLLA, (B) “sc” PLLA-b-PDMAEMA/HMPDLA,and (C) “sc” PDLA-b-PDMAEMA/HMPLLA.

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significant number of red blood cells adhered onto the stereo-complex mats is in good agreement with the intensive redcoloring of these mats after a 1 h stay in whole blood (Figure6) as well as with results obtained by cell counting (Figure 5).The obtained results indicate that enriching the surface of thestereocomplex fibers in tertiary amino groups of PDMAEMAimparts hemostatic properties to the novel fibrous materials.

To evaluate the adhesion of pathogenic microorganisms S.aureus and E. coli onto the surface of the prepared novel fibrousmaterials, HMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA, and“sc” PDLA-b-PDMAEMA/HMPLLA mats were incubated for24 h in a culture medium containing S. aureus or E. coli. Then,the cells were fixed with glutaraldehyde solution in PBS, washedwith distilled water, and freeze-dried. SEM micrographs ofHMPLLA, “sc” PLLA-b-PDMAEMA/HMPDLA, and “sc”PDLA-b-PDMAEMA/HMPLLA that were in contact with S.aureus are shown in Figure 8.

A significant number of adhered S. aureus cells can beobserved on the surface of the HMPLLA mat (more than 40cells/100 µm2) and E. coli (more than 50 cells/100 µm2, notshown here). The morphology of the adhered cells was notaltered, indicating that HMPLLA fibers are a favorable substratefor growth of pathogenic microorganisms, and a bacterial biofilmis formed on them. This result is in good agreement with resultsobtained by some of us by studying the possibility of preparingnovel fibrous materials from HMPLLA and natural polymerchitosan.7 As seen from the presented SEM micrographs, thepreparation of “sc” fibers containing PDMAEMA led to asignificant decrease in the number of adhered cells to ca. 1 cell/100 µm2 and 2 cells/100 µm2 for PLLA-b-PDMAEMA/

HMPDLA and PDLA-b-PDMAEMA/HMPLLA mats placed incontact with S. aureus and 2 cell/100 µm2 and 4 cells/100 µm2

for PLLA-b-PDMAEMA/HMPDLA and PDLA-b-PDMAEMA/HMPLLA mats placed in contact with E. coli, respectively. Thereduced number of adhered cells on the surface of the matscontaining PDMAEMA is a result of the antibacterial propertiesof PDMAEMA, which is a contact biocide and leads toinhibition of bacterial growth. The obtained results indicate thatenrichment of the surface of the stereocomplex fibers in tertiaryamino groups of PDMAEMA impart hemostatic as well asantibacterial properties to the novel fibrous materials.

Conclusions

The present study is the first one showing a possibility forone-step preparation of PLA stereocomplex fibrous materialshaving surface enriched in tertiary amino groups of PD-MAEMA. This was achieved by electrospinning of solutioncontaining high-molecular-weight P(D- or L-)LA and P(L- orD)LA-b-PDMAEMA block copolymers. The enrichment of thesurface in tertiary amino groups from the PDMAEMA impartshemostatic and antibacterial properties to the stereocomplexmaterials. The obtained results showed potentialities for theapplication of these novel materials as wound healing materialsas well as devices contacting with pathogenic microorganisms.

Acknowledgment. The authors thank the bilateral cooperationbetween the Bulgarian Academy of Sciences and CommissariatGeneral aux Relations Internationales (CGRI, Brussels) and theNational Science Fund of Bulgaria (grant no. DO-02-82/2008)

Figure 8. SEM micrographs of fibrous mats after 24 h contact with S. aureus: (A) HMPLLA mat, (B) “sc” PLLA-b-PDMAEMA/HMPDLA mat, and(C) “sc” PDLA-b-PDMAEMA/HMPLLA mat; magnification: ×1000.

158 Biomacromolecules, Vol. 11, No. 1, 2010 Spasova et al.

for the financial support. R.M. and P.D. are very grateful to“Region Wallonne” and European Community (FEDER, FSE)for general support in the frame of “Objectif 1-Hainaut: MateriaNova” as well as to the Belgian Federal Science Policy Officeof (SSTC-PAI 5/3).

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