Electrospun Nanofibrous Mats Containing ... - ACS Publications

Biomacromolecules 2010, 11, 1633–1645

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Electrospun Nanofibrous Mats Containing Quaternized Chitosan and Polylactide with In Vitro Antitumor Activity against HeLa Cells Milena G. Ignatova,† Nevena E. Manolova,*,† Reneta A. Toshkova,§ Iliya B. Rashkov,*,† Elena G. Gardeva,§ Lilia S. Yossifova,§ and Marin T. Alexandrov§ Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St. bl. 103A, BG-1113 Sofia, Bulgaria, and Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Acad. G. Bonchev St. bl. 25, BG-1113 Sofia, Bulgaria Received March 16, 2010; Revised Manuscript Received April 25, 2010

Nanofibrous materials containing the antitumor drug doxorubicin hydrochloride (DOX) were easily prepared using a one-step method by electrospinning of DOX/poly(L-lactide-co-D,L-lactide) (coPLA) and DOX/quaternized chitosan (QCh)/coPLA solutions. The pristine and DOX-containing mats were characterized by ATR-FTIR and X-ray photoelectron spectroscopy (XPS). The release rate of DOX from the prepared fibers increased with the increase in DOX content. The DOX release process was diffusion-controlled. MTT cell viability studies revealed that incorporation of DOX and QCh in the nanofibrous mats led to a significant reduction in the HeLa cells viability. It was found, that the antitumor efficacy of the DOX-containing mats at 6 h was higher than that of the free DOX. SEM, TEM, and fluorescence microscopic observations confirmed that the antitumor effect of QCh-based and DOX-containing fibrous mats was mainly due to induction of apoptosis in the HeLa cells.

Introduction Doxorubicin hydrochloride (DOX) is one of the most employed antracycline antitumor drugs in clinical use nowadays. It acts against various tumors including cervical and breast tumors.1-3 However, when DOX is used at therapeutic concentrations, severe side effects are observed, such as cytotoxicity in normal tissue, pervasive cardiotoxic effects, and inherent multidrug resistance effect, which further limit its therapeutic efficacy. To overcome these disadvantages, various DOX delivery systems, such as polymer-drug conjugates, micro- and nanoparticles, liposomes, and micelles, have been developed.4-6 It has been reported that these systems have led to some improvement of DOX antitumor therapeutic efficacy and to a smaller extent of drug availability in normal tissues. Recently, the use of electrospun micro- and nanofibers as antitumor drug carriers has attracted a great deal of attention because it is a promising approach for the targeting delivery of the antitumor drugs at tumor tissue, especially in postoperative local chemotherapy. The drug release profile from these systems can be controlled by modulation of the nanofiber morphology, porosity, and composition. Biodegradable aliphatic polyesters are among the most preferred polymers as antitumor drug delivery carriers. Poly(L-lactide-co-D,L-lactide) (coPLA) is a biodegradable and biocompatible aliphatic polyester with relatively good mechanical properties and an appropriate degradation rate for most musculoskeletal applications. co-PLA-based nanofibrous materials are suitable for biomedical applications such as drug delivery systems, implants, and wound-dressing materials.7,8 Up to now, a few studies on the preparation of * Corresponding authors. Tel: +359 2 9793289. Fax: +359 2 8700309. E-mail: [email protected], [email protected]. † Institute of Polymers, Bulgarian Academy of Sciences. § Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences.

electrospun mats from (co)polymers of lactic-acid-containing antitumor drugs have been reported.9-14 In recent years, the natural polysaccharide chitosan and its derivatives have drawn a great attention as antitumor drug carriers.5,15,16 This is due to the set of advantageous properties of these polymers, for example, nontoxicity, biodegradability, biocompatibility, intrinsic antibacterial properties, and immunostimulating effect. Chitosan has shown good antitumor activity, which is mainly due to its polycationic nature.17 Quaternized derivatives of chitosan (QCh) are well-known for their high efficacy against bacteria and fungi18,19 and good in vitro antitumor activity.20 Recently, we have reported on the successful preparation of continuous QCh-containing micro- and nanofibers by one-step electrospinning of mixed QCh/poly(vinyl alcohol), QCh/poly(vinyl pyrrolidone), or QCh/coPLA solutions.7,8,21,22 It has been found that the fabricated QCh-containing micro- and nanofibrous materials show good antibacterial and antimycotic activity. To the best of our knowledge, until now, no data about in vitro antitumor activity of electrospun QCh-based fibrous materials on human cervical cancer cell line (HeLa) have been reported. The combination of the advantageous properties of the antitumor drug DOX and the biological properties of QCh derivatives is a promising strategy for the preparation of nanofibrous materials suitable for local tumor treatment. The present work aims at studying the possibility for the preparation of novel DOX-containing nanofibrous mats by onestep electrospinning of coPLA/DOX and QCh/coPLA/DOX solutions. The chemical composition and the morphology of the fibrous materials were characterized by ATR-FTIR spectroscopy, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). The in vitro release profile of DOX from the DOX-containing nanofibers was followed. An evaluation of the in vitro antitumor activity of the QCh-based and DOX-containing mats on human cervical cancer cell line (HeLa) using the MTT assay was also performed.

10.1021/bm100285n  2010 American Chemical Society Published on Web 05/14/2010

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Biomacromolecules, Vol. 11, No. 6, 2010

Scheme 1. Schematic Representation of the Structure of (a) the Antitumor Drug and (b,c) Polymers Used in the Present Study

Experimental Section Materials. Formaldehyde solution (∼36% in water) (Fluka), NaBH4 (Fluka), CH3I (Fluka), NaI (Fluka), glutaraldehyde solution (∼50% in water) (Fluka), and doxorubicin hydrochloride (DOX) (Scheme 1a) (Sigma-Aldrich) were of analytical grade of purity and were used as received. Prior to use, N-methyl-2-pyrrolidone (NMP) (Fluka) was freshly distilled under reduced pressure. Dimethylformamide (DMF) (Fluka) and dimethyl sulfoxide (DMSO) (Fluka) were dried with molecular sieves (4 Å) prior to use. Poly(L-lactide-co-D,L-lactide), Resomer LR j w/M j n ) 2.46) (molar ratio j w 911 000 g/mol, M 708 (Scheme 1c) (M L-lactide/D,L-lactide 69:31), was kindly donated by Boerhinger-Ingheleim Chemicals (Germany). Chitosan with a molecular weight of 380 000 g/mol (Aldrich) and degree of deacetylation 80% was used. Cell Culture. Human cervical cancer cell line (HeLa) was cultured in DMEM medium (Gibco, Austria) with 10% fetal bovine serum (Gibco, Austria), 100 U/mL penicillin and 0.1 mg/mL streptomycin in 75 cm3 tissue plastic flasks. Disposable items (75 mL tissue culture flask, filter system, well plates) were purchased from Orange Scientific, Belgium. Trypsin-EDTA, penicillin, and streptomycin were obtained from FlowLab, Australia. DMSO and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) powder were purchased from Sigma Aldrich, Germany. Cells were maintained in log growth phase at 37 °C in humidified air with 5% CO2. Preparation and Characterization of N,N,N-Trimethyl Chitosan Iodide. The quaternized chitosan derivative (QCh) (Scheme 1b), N,N,Ntrimethyl chitosan iodide, was prepared according to a known procedure.18 In brief, in the first step, N-methyl chitosan was prepared by reacting chitosan and formaldehyde via Schiff’s base intermediate and subsequent hydrogenation with NaBH4. The product was purified from unreacted aldehyde and inorganic products by Soxhlet extraction with ethanol and diethylether for 4 days. The obtained N-methyl chitosan was filtered out and vacuum dried at 40 °C for 2 days. Yield: 94%. The resulting N-methyl chitosan was quaternized using methyl iodide.

Ignatova et al. N,N,N-Trimethyl chitosan iodide was purified by two-fold precipitation in acetone and dried under reduced pressure at 40 °C. Yield: 96%. Fourier transform infrared (FT-IR) spectra of QCh (films) were recorded using a Bruker Vector 22 infrared spectrometer at room temperature on KBr pellets. 1H NMR spectra were taken on a Bruker spectrometer (400.13 MHz) at 333 K in D2O. IR: N,N,N-trimethyl chitosan iodide (film): 3417 (N-H stretching vibration), 2924, 2879 (C-H stretching vibration from N,N,N-trimethyl residues and from polysaccharide structure), 1654 (amide I), 1561 (amide II), 1470 (antisymmetric C-H deformation of the N,N,N-trimethyl group), 1376 (symmetric C-H deformation), and 1068 cm-1 (C-O-C stretching vibration). 1H NMR (D2O, δ): 2.07 (s, -NHCOCH3), 3.12 (s, -N+-(CH3)3I-), 3.38 (s, CH3-O-6), 3.51 (s, CH3-O-3), 3.84 (m, H2 and H6), 4.0 (m, H5), 4.18 (m, H3), 4.33 (m, H4), and 5.21 (m, H1). The quaternization degree of QCh was determined using two independent methods: 1H NMR spectroscopy and potentiometric titration of the iodide form with aqueous silver nitrate, using working silver electrode and reference calomel electrode. The quaternization degree was calculated from the intensity ratio of the signal at 3.12 ppm for -N+-(CH3)3I- to that of H-1. This value (70%) is in very good agreement with the quaternization degree determined potentiometrically (72%). The degree of methylation of -OH functions was determined from the intensity ratios of the signal of CH3-O at 3.51 and 3.38 ppm for OH at C-3 and C-6 position, respectively, and the H1 signal. The degree of methylation of H3 and H6 was 98%. Preparation of Pristine and DOX-Containing QCh/coPLA and coPLA Nanofibers. coPLA nanofibers were prepared by electrospinning of their solutions in dry DMF/DMSO (60/40 v/v) at polymer concentration of 5 wt %. DOX-containing coPLA mats were prepared by electrospinning of coPLA solutions containing DOX (3 and 6%) (in weight percent to coPLA content) in dry DMF/DMSO (60/40 v/v) at polymer concentration of 5 wt %. QCh/coPLA nanofibers at weight ratio of QCh/coPLA 30:70 were prepared by electrospinning of their mixed solutions in dry DMF/DMSO (60/40 v/v) at total polymer concentration of 5 wt %. DOX-containing QCh/coPLA mats were prepared by electrospinning of mixed QCh/coPLA solutions (weight ratio of QCh/coPLA 30:70) containing DOX (3 and 6%) (in weight percent to QCh/coPLA content) in dry DMF/DMSO (60/40 v/v) at total polymer concentration of 5 wt %. In typical, the mixed solutions were placed into a plastic syringe (5 mL) equipped with a conical nozzle connected to an electrode. The electrode was connected to a high voltage power supply capable of generating positive DC voltages from 10 to 40 kV. The rotating, grounded drum with a diameter of 45 mm was placed 18 cm from the needle tip, and the rotating speed was maintained at 1100 rpm. The spinning solution was delivered using a syringe pump at a controlled feed rate of 1.0 mL/h at a constant value of the applied voltage (25 kV). The electrospun fibrous mats were placed under vacuum overnight at 40 °C to remove any solvent residues. Prior to electrospinning, the dynamic viscosity of the spinning solutions was measured using a Brookfield LVT viscometer equipped with a small-sample thermostatted adapter, spindle and chamber SC418/13R at 25 ( 0.1 °C. The electrical resistance of the spinning solutions was measured in an electrolytic cell equipped with rectangular sheet platinum electrodes having a surface area of 0.6 cm2 and disposed at a distance of 2.0 cm. During the measurements, short electric pulses with opposite direction were applied to the Pt electrodes to avoid accumulation of ionic charge and polarization effects in the vicinity of electrode surface. This allowed solution resistance in the range of 20-2000 kΩ to be measured with an accuracy of (3%. Calibration of the electrolytic cell was performed using standard solution of KCl (conductivity 140.8 mS/m), and the cell constant (Kcell) was determined. The conductivity of the spinning solutions (σ, µS/cm) was calculated from the following equation

Electrospun Mats with In Vitro Antitumor Activity σ)

1 1 ) F KcellR

Biomacromolecules, Vol. 11, No. 6, 2010 (1)

where F is the specific resistance of the solution (µΩ · cm) and R is the electrical resistance of the solution (µΩ). Characterization of Pristine and DOX-Containing Electrospun Mats. The fibers collected onto aluminum plates were vacuum-covered with carbon and gold and examined in a SEM Philips 515. The average fiber diameter and the standard deviation were estimated in terms of the criteria for complex evaluation of electrospun mats reported elsewhere23 using the Image J software program24 by measuring at least 30 fibers from each SEM image. The electrospun pristine and DOX-containing mats were analyzed by XPS. The XPS measurements were carried out in the UHV chamber of an ESCALAB-MkII (VG Scientific) electron spectrometer using Mg KR excitation with a total instrumental resolution of ∼1 eV. Energy calibration was performed, taking the C1s line at 285 eV as a reference. Surface atomic concentrations were evaluated using Scofield’s ionization cross sections with no corrections for λ (the mean free path of photoelectrons) and the analyzer transmission function. The experimental values for element atomic percentage obtained from XPS analysis are the average of three independent measurements. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses were performed using an IRAffinity-1 spectrophotometer (Shimadzu, Kyoto, Japan) equipped with a MIRacle ATR (diamond crystal, depth of penetration of the IR beam into the sample is ∼2 µm) accessory (PIKE Technologies). The spectra were recorded from 4000 to 500 cm-1 with spectral resolution of 4 cm-1 using a DLATGS detector equipped with temperature controller. All spectra were corrected for H2O and CO2 using IRsolution internal software. All samples were dried under reduced pressure prior to analysis. Static contact angle measurements were performed using a Kru¨ss drop shape analysis system (model DSA 10-MK2) at 20 ( 0.2 °C. A sessile drop of deionized water controlled by a computer dosing system was placed onto the samples. Temporal images of the droplet were taken. The contact angles were calculated by computer analysis of the acquired images. The data are average from 15 measurements. To demonstrate the presence of DOX in the DOX-containing electrospun mats, they were analyzed by fluorescence microscopy (Leika DM 500B, Wetzlar, Germany). For comparison, the pristine coPLA and QCh/coPLA mats were also observed under fluorescence microscope. In vitro DOX Release from the DOX-Containing Nanofibrous Mats. A piece of DOX-containing nanofibrous mat (16 mg) was placed in a vial filled with 10 mL of PBS (pH 7.4). The vial was incubated at 37 °C in a thermostatted shaker and shaken horizontally at 300 rpm. At predetermined time intervals, aliquots were drawn off, and the amount of released DOX in the buffer solution was determined by a UV-vis spectrophotometer at the wavelength of 483.5 nm. The accumulative weight and relative percentage of the released DOX were calculated as a function of incubation time. All DOX release tests were performed in triplicate. The total content of DOX in the electrospun fibers was determined using the same detection procedure, as mentioned above, after three pieces from DOX-containing nanofibrous mats (16 mg) were degraded thoroughly in 20 mL of Tris-HCl buffer solution (pH 8.6) containing 100 µg/mL of proteinase K at 37 °C. The total content of DOX was determined as the average value of the three experiments. MTT Cytotoxicity Assay. HeLa cells were trypsinized by 0.25% Trypsin-EDTA and counted using a hemocytometer. Cells were transferred to a 96-well microtiter plate to ensure a concentration of 2 × 104 cells per well. After incubation overnight at 37 °C in humidified air with 5% CO2 to allow cells attachment, the medium was changed and HeLa cells were treated with various formulations of nanofibrous mats (coPLA, QCh/coPLA, DOX-containing coPLA, and DOXcontaining QCh/coPLA) with DOX (positive control) and were cultivated only in medium (negative control) for different time periods (6, 24, 48, and 72 h). In parallel with this experiment and to determine

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the cytotoxic effect of QCh, the cells were also incubated with QCh/ coPLA mats containing different concentrations of QCh (100, 1050, and 1900 µg/mL). The effect of different nanofibrous mats on cell viability was assessed by the MTT assay as referred by Mossmann.25 Cell number and viability were evaluated by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. Each variant of nanofibrous mats was assayed by five measurements. Upon culturing on mats, the HeLa cells were washed twice with PBS (pH 7.4) and further incubated with 100 µL of MTT working solution (Sigma Chemical) at 37 °C for 3 h; the supernatants were aspirated, and 100 µL of lysing solution (DMSO/ethanol 1:1) was added to each well to dissolve the resulting formazan. MTT assay reading was performed using ELISA plate reader (TECAN, SunriseTM, Gro¨dig/Sazburg, Austria). The percentage of cell growth inhibition was calculated as follows

cell viability (%) ) OD570 (experimental)/OD570 (control) × 100 Ultrastructural Cell Morphology. HeLa cells in logarithmic growth phase (2 × 105 cells/mL) were seeded on pristine and on DOXcontaining electrospun coPLA and QCh/coPLA mats in 24-well tissue culture plate and were incubated at 37 °C for 24 h in CO2 incubator. Untreated cells were used as negative control, and DOX-treated HeLa cells were used as positive control. After incubation for a predetermined time, the nanofibrous mats were prepared for SEM and TEM observations and for double staining with propidium iodide (PI) and acridine orange (AO). Scanning Electron Microscopy. The pristine and the DOXcontaining electrospun mats were washed twice with PBS (pH 7.4) immediately after the incubation period to remove unattached HeLa cells, then immersed into 2.5 vol % glutaraldehyde PBS solution and kept at 4 °C for 5 h for HeLa cell fixation. Then, the samples were again washed twice with PBS and dehydrated in an ethanol solution of varying concentration -30, 50, 70, and 90%, and finally, with absolute ethanol for about 10 min each. The samples were then dried in 100% hexamethyldisilazane (HMDS, Sigma-Aldrich) for 5 min and later dried in air after the removal of HMDS. The morphology of the HeLa cells after contact with the electrospun QCh/coPLA, coPLA and DOX-containing electrospun mats was observed by an SEM Jeol JSM5510 after vacuum gold coating (Jeol JFC-1200 fine coater). The microscope was operated at an accelerating voltage of 10 kV with a high emission current of 50 mA and a working distance of 12 mm. For comparison, the morphology of the HeLa cells that had been cultured on Termanox plastic disks without or in the presence of PBS solution of DOX (10 µg/mL) was also studied. Transmission Electron Microscopy (TEM). The pristine and DOXcontaining coPLA and QCh/coPLA mats with HeLa cells on their surface were fixed for 6 h in 4% glutaraldehyde in 0.2 Μ cacodylate buffer (pH 7.2) at 4 °C, post-fixed for 2 h in 2% OsO4, and dehydrated in graded alcohols and through propylene oxide, propylene oxide-resin embedded in Durcupan (Fluka). Pyramids with 4 mm2 surface were formed, and semithin sections (0.5 up to 1 µm thick) were prepared with a glass knife on a Reichert ultratome. The sections were mounted on glass slides, stained with 1% toluidine blue in 1% borax, and examined under a light microscope. Areas with attached HeLa cells on the mat surface were selected, and pyramids were additionally trimmed for ultratomy. Yellow-gold ultrathin sections (70-100 nm thick) were prepared and mounted on 400 mesh copper grids and stained with saturated solution of uranyl acetate followed by lead citrate according to the standard technique. All TEM examinations were carried out using an electron microscope JEOL 1200 EX at an accelerating voltage of 80 kV and an instrumental magnification of 2000-12 000×. Transmission electron micrographs were taken on Kodak EM film 4489, 61/2 × 9 cm. To evaluate the images, we scanned the negatives directly on a HP Scanjet 4890 scanner at 600 dpi resolution using the “SCAN FILM” option.

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Quantification of Apoptosis using Double Staining with PI and AO. Apoptotic nuclear morphology was assessed using PI and AO double staining according to standard procedures26 with the following modification: HeLa cells were grown on 13 mm diameter nanofibrous mats and examined under fluorescence microscope (Leika DM 500B, Wetzlar, Germany). In brief, HeLa cells were cultured on different formulations of nanofibrous mats (coPLA, QCh/coPLA, DOXcontaining coPLA, and DOX-containing QCh/coPLA) in 24-well plates in CO2 incubator, as described in the previous section. After 24 and 48 h of incubation, the nanofibrous mats were washed twice with phosphate buffer saline (PBS) for 10 min to remove culture media. Equal volumes of fluorescent dyes containing AO (10 µg/mL) and PI (10 µg/mL) were added to the mats. Fresh staining HeLa cells on nanofibrous mats were placed on a glass slide, covered by coverslip, and were observed within 30 min before the fluorescence color started to fade. The degree of apoptosis resulting from treatment was observed by double staining, where a bright-green nucleus with chromatin condensation as dense green areas (early apoptotic cells) and an orange nucleus showing condensation of chromatin (late apoptotic cells) indicated apoptotic cell death. Statistical Analysis. The data are given as the mean ( standard deviation (SD). Significance testing was performed using one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Values of *p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant.

Results and Discussion Electrospun DOX-Containing coPLA and QCh/coPLA Mats. In the present study, to obtain hybrid DOX-containing QCh/coPLA nanofibers an appropriate solvent system was chosen, consisting of DMF/DMSO (60:40 v/v), which allows the mixed solutions to be obtained and successfully electrospun. Preliminary experiments on the electrospinning conditions varying the DOX concentration in the range from 3 to 6 wt %, the AFS from 1.0 to 1.5 kV/cm, and the feeding rate from 0.8 to 1.2 mL/h were performed. These experiments enabled us to select the optimal process conditions for preparing continuous cylindrical defect-free QCh/coPLA/DOX nanofibers with relatively narrow diameter distribution (total polymer concentration 5 wt %, feeding rate 1.0 mL/h, and AFS 1.4 kV/cm). Under the same electrospinning conditions, defect-free and cylindrically shaped coPLA/DOX nanofibers were also obtained. The morphology of the fibers and their average diameters were characterized by SEM. As seen from the SEM micrographs (Figure 1a-d), the fiber surface was smooth and no DOX crystals were detected. Moreover, on adding DOX to QCh/ coPLA solutions as well as with increasing its concentration from 3 to 6 wt %, the average fiber diameter decreased: for example, from 590, to 470, to 310 nm in the case of QCh/ coPLA mat, QCh/coPLA mat containing 3 wt % DOX, and QCh/coPLA mat containing 6 wt % DOX, respectively. A similar tendency was also observed for the system coPLA/ DOX (Table 1). This may be explained by the increase in the conductivity of coPLA and QCh/coPLA solutions and by slightly lower solution viscosity of coPLA and QCh/coPLA on adding the low-molecular-weight organic salt DOX (Table 1). Similar behavior has been observed in other systems21,27-29 and is explained by the higher charge density on the surface of ejected jet during electrospinning, thus imposing higher elongation forces to the jet. A narrowing of the fiber diameter distribution was also observed (Figure 1a-d). ATR-FTIR spectrum of the coPLA/DOX mats (Figure 2b) showed the absorption characteristic bands of both coPLA (Figure 2a) (1751 cm-1: νCdO; 1088 cm-1: νC-O-C) and DOX constituents (1624 and 1578 cm-1 characteristic for νCdO from

Ignatova et al.

the anthraquinone ring). In the spectrum of the QCh/coPLA characteristic bands at 1651 and 1548 cm-1, respectively, for amide I and amide II from polysaccharide structure of QCh, with the exception of the bands of coPLA, were observed (Figure 2c). In the ATR-FTIR spectrum of the QCh/coPLA/ DOX mats new bands were observed at 1624 cm-1 and at 1578 cm-1 and assigned to CdO stretching vibrations from the anthraquinone ring of the DOX included in the mats (Figure 2d). The observation of the nanofibers by fluorescence microscopy revealed that coPLA/DOX and QCh/coPLA/DOX nanofibers showed fluorescence distributed uniformly along the fibers, which is attributed to the incorporated DOX (Figure 3b,c,e,f). In contrast, in the case of pristine nanofibers, no fluorescence was detected (Figure 3a,d). The composition of the surface layer of the mats was analyzed by XPS (Figure 4). XPS analysis of the coPLA mats revealed the presence of C1s (285 eV) and O1s (532.5 eV) (Figure 4a,b). The high-resolution signal of the C1s consists of peaks at 285.0 (-C-H or -C-C-), 286.9 (-C-O), and 288.9 eV (-O-CdO) (Figure 4a). The theoretically calculated ratio of the peak areas that correspond to the respective carbon atoms is [C-C/C-H]/ [C-O]/[O-CdO] 33.3/33.3/33.3, whereas the experimentally determined one is 46.2/27.2/26.6. This indicates that the surface layer of the coPLA mats is enriched with ca. 12.9% in carbon atoms participating in C-H/C-C bonds. The O1s peak was deconvoluted into two peaks at 533.3 and 531.9 eV, assigned to -C-O and -O-CdO, respectively (Figure 4b). Moreover, the atomic percentages (54.7% C and 45.3% O) experimentally determined from the XPS peaks are close to the theoretical values calculated from the chemical composition of pristine coPLA mats (52.9% C and 47.1% O). A confirmation of the successful incorporation of DOX in the surface layer of the coPLA mats was obtained from the performed XPS analyses. The appearance of the N1s peak in the spectrum of the DOX-containing coPLA mats at 400.5 eV assigned to -NH3+ of DOX component was observed (Figure 4e). In addition, the spectrum showed the appearance of Cl2p peak at 198.0 eV attributed to the DOX incorporated in the mats (Figure 4f). Considerable differences were observed in the C1s and O1s spectra of the DOX-containing coPLA mats as well (Figure 4 c,d). The C1s detailed spectrum is deconvoluted to three peaks at 285 eV for -C-H- and -C-C- of coPLA component and for -C-H-, -C-C-, and -C-N- of DOX component, at 286.9 eV for -C-O- of coPLA component and for -C-OH, -C-OCH3 and -C-O- of DOX component, and at 288.9 eV for -O-CdO of coPLA component and for -C-CdO and -O-C-O- of DOX component (Figure 4c). The theoretically calculated ratio of the peak areas [C-H/C-C/ C-N]/[C-O/C-OH/C-OCH3]/[O-CdO/C-CdO/ O-C-O] was 35.0/32.7/32.3, whereas the experimentally determined one is 46.9/24.9/28.2; that is, the peak assigned to the carbon atoms participating in C-H/C-C bonds was characterized by the largest area (Figure 4c). Therefore, it can be assumed that the mat surface is enriched with methyl groups of low binding energy. In the expanded O1s spectrum, the appearance of a new peak with a low intensity at 532.4 eV assigned to C-OH and -C-OCH3 of DOX component was observed (Figure 4d). In the case of QCh/coPLA mats the constitutive atoms were detected by XPS (Figure 4g-i): carbon (C1s) at 285.0 eV, oxygen (O1s) at 532.3 eV, nitrogen (N1s) at 399.6 and 402.4 eV and iodine (I3d) at 618.0 and 629.5 eV. Moreover, the C1s, N1s and I3d spectral regions were analyzed by peak reconstruction.

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Figure 1. SEM micrographs and fiber diameter distribution of electrospun mats of (a) coPLA, (b) coPLA/DOX (6 wt % DOX), (c) QCh/coPLA, and (d) QCh/coPLA/DOX (6 wt % DOX).

The detailed N1s spectrum showed two components at 399.6 eV characteristic of -N-CdO and -NH2 groups and at 402.4 eV typical of ammonium group (-N+(CH3)3) from QCh (Figure 4 h). The presence of N1s and I3d peaks (at 618.0 eV (I3d5/2) and at 629.5 eV (I3d3/2)) (Figure 4i) proved the presence of QCh in the surface layer of the QCh/coPLA mat. Considerable differences were observed in the C1s spectra of the QCh/coPLA nanofibers, as well. The C1s peak (Figure 4g) was deconvoluted into four contributions at 285.0 eV assigned to -C-H or -C-C- of coPLA and of QCh components and also to -C-NH2 of QCh component, at 287.0 eV for -C-O, -C-OH, -C-OCH3, and -C-N-CdO of QCh component

and to -C-O of coPLA component, at 288.0 eV to -O-C-Oand -N-CdO of QCh component and at 289.2 eV for -O-CdO of coPLA component. The theoretical ratio of the peaks corresponding to the respective carbon atoms in the high resolution C1s spectrum of QCh/coPLA mat is [C-H/C-C/ C-NH 2 ]/[C-O/C-OH/C-OCH 3 /C-N-CdO]/[O-C-O/ N-CdO]/[O-CdO] 40.4/29.0/7.3/23.3. The experimentally determined one is 44.8/28.8/6.1/20.3. Therefore, in the case of QCh/coPLA mat the surface composition of the mats does not significantly differ from that of the feed. The experimentally determined atomic percentages of the elements (53.1% C, 37.6%

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Table 1. Dynamic Viscosity (η) and Conductivity (σ) of Spinning j ) and Standard Deviation Solutions, Average Fiber Diameter (d (SD) of Electrospun Fibers, and Contact Angle of Water of Electrospun Mats electrospun mats

η (cP)

σ (µS/cm)

j d (nm)

SD

contact angle of water (deg)

coPLA coPLA/DOX (3 wt % DOX) coPLA/DOX (6 wt % DOX) QCh/coPLA QCh/coPLA/DOX (3 wt % DOX) QCh/coPLA/DOX (6 wt % DOX)

5300 4900

27 109

830 640

115 112

128 ( 3.9 152 ( 3.3

4900

184

580

90

149 ( 4.2

1450 1390

956 963

590 470

80 80

50 ( 8.4 43 ( 3.4

1380

1018

310

54

42 ( 5.2

O, 1.3% N, 8.0% I) are close to the theoretically calculated ones (49.9% C, 39.7% O, 1.4% N, 9.0% I). Consistently, XPS analysis showed all peaks expected for QCh/ coPLA/DOX mat: C1s (285 eV), O1s (532.3 eV), N1s (399.5 and 402.0 eV), I3d (618.0 and 629.4 eV), and Cl2p (198.3 eV). The detailed spectrum in the C1s region (Figure 4j) showed four peaks: peak at 285 eV, which was assigned to -C-H or -C-C- of coPLA an d of QCh and DOX components and also to -C-NH2 of QCh and DOX components, peak at 286.8 eV for -C-O, -C-OH, and -C-OCH3 of QCh and DOX components, for -C-O of coPLA component, and for -C-N-CdO of QCh component, peak at 288.3 eV for -O-C-O- and -N-CdO

of QCh component and for -O-C-O- of DOX component, and peak at 289.2 eV for -O-CdO of coPLA component and for -C-CdO of DOX component. Moreover, the experimentally determined ratio of the peak areas that corresponded to the respective carbon atoms in the detailed C1s spectrum of a QCh/coPLA/DOX mat, [C-C/C-H/C-N]/[C-O/C-OH/ C-OCH 3 /C-N-CdO]/[O-C-O/N-CdO]/[O-CdO/ C-CdO] 42.2/28.8/6.7/22.3, was close to the theoretically calculated one (41.8/28.7/6.9/22.6). In the N1s region, two components were identified at 399.5 eV, which was assigned to -N-CdO and -NH2 groups of QCh component, and at 402.0 eV assigned to ammonium group (-N+(CH3)3) of QCh component and to -NH3+ groups of DOX component (Figure 4k). The appearance of the Cl2p peak at 198.3 eV confirmed the successful incorporation of DOX in the QCh/coPLA nanofibrous mats (Figure 4m). The hydrophilic/hydrophobic characteristics of the nanofibrous mats can influence the initial adhesion of the cells and their proliferation to a higher extent.30 For that reason, water contact angles of the prepared electrospun mats were determined. The water contact angles of electrospun mats are mostly influenced by the morphology, average diameter, and surface chemical composition of the fibers.31,32 CoPLA mats and, in particular, coPLA/DOX mats exhibit very high values of water contact angle (Table 1), which could be accounted for by the combined effects of the surface enrichment of the fibers with methyl groups of low binding energy and the surface roughness

Figure 2. ATR-FTIR spectra of electrospun mats of (a) coPLA, (b) coPLA/DOX (6 wt % DOX), (c) QCh/coPLA, and (d) QCh/coPLA/DOX (6 wt % DOX).

Figure 3. Fluorescence micrographs of mats of (a) coPLA, (b) coPLA/DOX (3 wt % DOX), (c) coPLA/DOX (6 wt % DOX), (d) QCh/coPLA, (e) QCh/coPLA/DOX (3 wt % DOX), and (f) QCh/coPLA/DOX (6 wt % DOX). Magnification ×40.



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Figure 4. XPS peak fittings for electrospun mats of: coPLA, (a) C1s and (b) O1s; DOX-containing coPLA (6 wt % DOX), (c) C1s, (d) O1s, (e) N1s, and (f) Cl2p; QCh/coPLA, (g) C1s, (h) N1s, and (i) I3d; and DOX-containing QCh/coPLA (6 wt % DOX), (j) C1s, (k) N1s, (l) I3d, and (m) Cl2p.

of the electrospun mat itself.33 The contact angles of water on QCh/coPLA and QCh/coPLA/DOX agree with an increased hydrophilicity, which is attributed to the presence of QCh in the fibers. The incorporation of 3-6 wt % DOX in the fibers slightly changes the water contact angle values. The observed hydrophilization of the mat surface is in accordance with the above-mentioned surface composition of the electrospun QCh/ coPLA and QCh/coPLA/DOX mats. It may be considered that in this case the chemical composition is the determinant factor for the determined water contact angle values. In Vitro DOX Release Profile from DOX-Containing coPLA and QCh/coPLA Nanofibrous Mats. In vitro release profiles of DOX from 3 and 6 wt % DOX-containing coPLA and QCh/coPLA nanofibers in PBS (pH 7.4) as a function of time, respectively, were studied (Figure 5). Similar DOX release behavior was detected for nanofibrous DOX-containing QCh/ coPLA and for coPLA mats. An initial fast release for 20 min in the case of QCh/coPLA/DOX mats and for 36 min in the case of coPLA/DOX mats was observed. Diffusion of DOX

nearby the surface layer of fibers could contribute to the initial burst release. However, initial burst release increased with increasing DOX content in the fibers. For example, the release percentages were about 44.5 and 63.8 at the 20th min in the cases of QCh/coPLA mats containing 3 and 6 wt % DOX, respectively. As seen from Figure 5a, the DOX from QCh/ coPLA/DOX nanofibers was released much faster than DOX from coPLA/DOX nanofibers. This result might be attributed to the presence of QCh in the nanofibers, known for its remarkable hydrophilicity and water affinity, which can enable water molecules to diffuse into the fiber interior, resulting in an increased exchange between the DOX molecules and the solution. We replotted the release fraction of DOX against the square root of time to assess the release mechanism of DOX from DOX-containing nanofibers (Figure 5b). For the first 20 and 36 min, in the case of QCh/coPLA/DOX and coPLA/DOX nanofibers, respectively, that is, the first stage in the DOX release profile, DOX diffuses out of the fibers at a rather high rate. As

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Figure 5. Release profiles of DOX from DOX-containing electrospun mats: (a) QCh/coPLA (4,2) and coPLA (0,9) in PBS (pH 7.4) at 37 °C. DOX content in the nanofibers: 6.0 (4,0) and 3.0 wt % (2,9). (b) Curves in part (a) replotted against square root of time.

it is seen from Figure 5b, an approximately linear relationship between Mt/M∞ and t1/2 for the first stage of DOX release was observed. This indicates that the DOX release process is diffusion-controlled in this stage. As it is shown in Figure 5b, the DOX release profiles contain in addition two more stages. For the second stage, a linear relationship between Mt/M∞ and t1/2 was also detected. In the case of the second stage, the slope of the straight lines was less than that for the first stage. This indicates that in the second stage the release rate was slower probably because of the fact that DOX distributed in the inner layer of the fiber matrix traversed a longer path and hence it needed a longer time to be released. In the third stage, the release rate further decreased. This might be explained with the decrease in the total DOX content in the inner layer of the fibers with the increase in the release time. As mentioned above, on increasing the DOX content in the nanofibrous mats, the release rate of DOX increased (Figure 5). For the QCh/coPLA/DOX nanofibers, after release time of 30 h, about 58.6% and 72.3% DOX were released from the 3 and 6 wt % DOX-containing fibers, respectively. About 12.5% and 13.1% of the DOX release were detected for 30 h for 3 and 6 wt % DOX-containing coPLA, respectively. According to the data reported for other systems,11,34,35 the present results on the DOX release behavior and for the concentration dependence of DOX release demonstrated that DOX was dispersed throughout the entire coPLA and QCh/coPLA nanofibers; that is, the DOX release nanofiber system is a matrix-type one. Therefore, the prepared DOX-containing nanofibrous mats showed an initial burst release of DOX and then provide a sustained DOX release. To inhibit the tumor cell growth for tumor treatment, a certain initial burst drug release is preferable to achieve sufficient initial dosage of the antitumor drug. For the cancer cells that survive the initial stage of the DOX release, the sustained DOX release is necessary to prevent their further proliferation. Therefore, the performance of these nanofibrous mats makes them promising candidates for tumor treatment. The next step of our studies has been devoted to the in vitro assessment of the cytotoxic activity of the nanofibrous mats. MTT Cytotoxicity Assay. The viability of HeLa cells, cultured for different periods of time on the pristine and DOX-containing nanofibrous mats, was evaluated by MTT cytotoxicity assay. The antineoplastic drug DOX was used as a positive control in this experiment. As shown in Figure 6a, the effect on HeLa cell viability is less pronounced when cultured on coPLA mat. Low cytotoxicity of poly(D,L-lactide) has been shown by other authors32 on human skin fibroblasts

and is explained by the high hydrophobicity of the nanofibrous poly(D,L-lactide) mat (water contact angle 136.2 ( 3.4°, a value close to that obtained for coPLA mats, 128 ( 3.9°). For the same time period, a significant reduction in the HeLa cell viability was observed when cultured on DOX-containing mats (the viability of HeLa cells in the presence of coPLA/ DOX and QCh/coPLA/DOX mats was reduced to 64.42 ( 15.4% and 61.13 ( 17.6%, respectively) (Figure 6a). On the sixth hour of incubation, the DOX-containing mats showed a higher cytotoxicity than the free DOX (Figure 6a). At the 24th, 48th, and 72nd h of incubation, the DOX-containing mats displayed a high cytotoxicity to the HeLa cells, similar to that of the free DOX (Figure 6b-d). For the sake of comparison, the QCh/coPLA mat (concentration of QCh-100 µg/mL) was tested as control. As seen in Figure 6, the QCh/coPLA mat showed a significant inhibition of HeLa cell growth. The strongest effect was observed at the 48th hour of incubation. (HeLa cell viability was reduced to 61.13 ( 17.00; 46.25 ( 5.13; 31.16 ( 5.74; and 43.90 ( 3.52% at the 6th, 24th, 48th, and 72nd h, respectively.) The observation that the nanofibrous QCh/coPLA mat displays a significant antitumor potential is of particular interest. The effect of various concentrations of QCh in the QCh/ coPLA nanofibrous mat on the viability of HeLa cells was examined as well. It was found that with increasing the concentration of QCh in the nanofibrous mats of QCh/coPLA, HeLa cell viability decreased (Figure 7). The growth inhibition in HeLa cells was best manifested at the 72nd hour of cultivation. Approximately 43.90 ( 3.52, 21.21 ( 5.11, and 17.87 ( 0.99% of the HeLa cells remained viable after incubation for 72 h in the presence of QCh/coPLA nanofibrous mats with concentrations of QCh 100, 1050, and 1900 µg/mL, respectively (Figure 7). The results have shown that the QCh/coPLA mats exert a significant cytotoxic effect on HeLa cells, which depends on the duration of the incubation period and the QCh concentration. QCh/coPLA/DOX mats inhibit the proliferation of the human cervical tumor cell line HeLa in vitro to a greater extent than the conventionally applied chemotherapeutic DOX (for the 6th hour of incubation). These results most probably are due to the synergistic action of QCh and DOX. To determine whether the antiproliferative activity of QChbased nanofibrous mats on HeLa cells is effectuated via an induction of apoptosis, SEM and TEM were applied. These methods were used because the morphological description of apoptosis using electron microscopy remains one of the best methods for determining apoptosis and distinguishing it from



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Figure 6. Cell viability of HeLa cell line tested by MTT method after (a) 6, (b) 24, (c) 48, and (d) 72 h for incubation with different formulations: control, untreated HeLa cells (1); coPLA mat (2); QCh/coPLA mat (3); DOX-containing QCh/coPLA mat (4); DOX-containing coPLA mat (5); and free DOX (10 µg/mL) (6). The total DOX content was 10 µg/mL; concentration of coPLA, 233 µg/mL; concentration of QCh, 100 µg/mL in the DOX-containing, coPLA-containing, and QCh-containing formulations, respectively. ***p < 0.001.

Figure 7. Cell viability of HeLa cell line tested by MTT method after 6, 24, 48, and 72 h of incubation in the presence of QCh/coPLA mats with different QCh concentrations: 100, 1050, and 1900 µg/mL, and of free DOX (10 µg/mL). ***p < 0.001.

necrosis.36 The method of double staining with fluorescent dyes (PI and AO) was applied to establish the condensation of nuclear chromatin in apoptotic cells. Effects of Pristine and DOX-Containing coPLA and QCh/ coPLA Mats on Human Cervical Cancer Cells HeLa using Scanning Electron Microscopy. The morphological changes and growth characteristics of HeLa cells grown on the pristine and DOX-containing mats were analyzed by means of SEM. As it can be seen from the SEM analyses, the control HeLa cells show numerous microvilli on their surface and extending

lamellipodia (Figure 8a). The HeLa cells grown for 6 h on coPLA nanofibrous mats showed shrinkage and blebbing of cell surface thus exhibiting morphological features of early apoptosis (Figure 8c). SEM micrographs of HeLa cells grown on QCh/ coPLA nanofibrous mats showed narrowing of cells, cell membrane blebbing, holes, and cytoplasmic extrusions (Figure 8e). The specific surface cell changes characteristic of the late apoptosis including cell shrinkage, cell membrane blebs, or apoptotic bodies, microvilli disappearance or reduction (blunt microvillus), and cytoplasmic extrusions and pore formation on the cell surface were observed in HeLa cells grown on coPLA/ DOX and QCh/coPLA/DOX mats (Figure 8d,f). Similar findings concerning the morphological changes of HeLa cells undergoing apoptosis have been also reported in other studies.37 For comparison, the HeLa cells grown for 6 h in a DOX-containing medium (10 µg/mL) showed cell shrinkage, blebbings on the cell surface, and shortening of the lamellipodia (Figure 8b). The SEM images clearly demonstrate that QCh/coPLA, coPLA/ DOX, and QCh/coPLA/DOX mats have led to a considerably larger number of morphological changes in the HeLa cells, and their cytotoxic effect is more pronounced than that of the DOXcontaining medium to HeLa cells. These results are in agreement with the results from the MTT assay. Effects of Pristine and DOX-Containing coPLA and QCh/ coPLA Mats on Human Cervical Cancer HeLa Cells using Transmission Electron Microscopy. The presented TEM images (Figure 9) show the ultrastructural changes within the HeLa cells grown on DOX-containing nanofibrous mats (the sixth hour of incubation). The ultrastructural features of untreated HeLa cells are presented in Figure 9a, where well distinguishable nuclear and cytoplasm organelles and membranous structures as well as

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Figure 8. SEM micrographs of surface ultrastructural characteristics of HeLa cells after 6 h of incubation. (a) untreated HeLa cells, (b) HeLa cells incubated with DOX and incubated with mats of (c) coPLA, (d) DOX-containing coPLA, (e) QCh/coPLA mats, and (f) DOX-containing QCh/coPLA mats. DOX content 10 µg/mL; L: lamellipodia extensions, B: blebs, E: cytoplasmic extrusions, H: holes; magnification: × 800 (a-f).

Figure 9. Electron micrograph of HeLa cells at the sixth hour following the seeding of the cells. (a) untreated, (b) grown on coPLA mat, (c) grown on QCh/coPLA mat, (d) grown on a DOX-containing coPLA mat, and (e) grown on a DOX-containing QCh/coPLA mat. Uranyl acetatelead citrate staining. Bar ) 2 µm.

many pseudopodia arising from the plasma membrane can be observed. When grown on coPLA mat, pycnotic cells with homogeneously condensed chromatin, hypergranularity of the cytoplasm, and decomposed plasma membrane are observed (Figure 9b). As shown in Figure 9c, the cells incubated on the QCh/coPLA mats are with a broken plasma membrane. Chromatin fragmentation, cytoplasm hypergranularity, and many vacuoles at the cell periphery could be also detected. Cell shrinkage, chromatin condensation, cloudy nuclear membranes as well as picnotic or disintegrated cytoplasm were a usual finding in the HeLa cells incubated on coPLA/DOX mats (Figure 9d). Nearly all HeLa cells incubated on QCh/coPLA/ DOX mats were in advanced decomposition: picnotic nuclei,

disintegrated cytoplasm, many membrane bound vesicles and vacuoles, mitochondria swelling, and disrupted organelles (Figure 9e). Qualitative assessment of the severity of morphological changes in the HeLa cells was performed based on the losses and destruction of the cells. It was shown that the cell damages were less severe in the cells incubated solely on a coPLA mat, followed by the cells incubated on a QCh/coPLA mat and a DOX-containing coPLA mat and were most pronounced in cells incubated on a DOX-containing QCh/coPLA mat. Morphologically, it can be presumed that the destruction of the cells in these cases has taken place following the apoptotic pathway. This conclusion is based on the features including retraction



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Figure 10. Fluorescence micrographs of AO and PI double-stained human cervical cancer cells HeLa lines incubated for 24 h. (a) untreated cells; after incubation with (b) QCh/coPLA mat, (c) free DOX, and (d) QCh/coPLA/DOX mat; bar ) 20 µm.

Figure 11. Percentages of apoptotic cells after incubation on pristine and DOX-containing mats for (a) 24 and (b) 48 h. Cells were cultured in DMEM and maintained at 37 °C and 5% CO2. DOX content was 10 µg/mL. Data are expressed as mean ( SD percent apoptotic cell and representative of 10 experiments. ***p < 0.001.

and fragmentation of the nuclear chromatin as well as the cytoplasmic damages. The results from the MTT assay and data from the ultrastructural cell morphology are complementary. To get more insight into the mechanism of mat-induced cytotoxicity in HeLa human cervical cancer cells, we have studied the type of cell death (apoptosis and/or necrosis). Fluorescence Using PI and AO Double Staining for Quantification of Apoptosis. To determine whether the inhibitory activity of the pristine and DOX-containing nanofibrous mats is associated with induction of apoptosis, the fluorescence test with double staining with PI and AO was applied. Morphological alterations of HeLa cells cultivated for 24 or 48 h on different nanofibrous mats were analyzed (Figure 10). For the sake of comparison, HeLa cells cultivated in the presence of free DOX (in concentration 10 µg/mL) were used. Stained control, untreated HeLa cells, are with homogeneous pale green nuclei and bright green nucleoli (Figure 10a). Perinuclear accumulations of orange granules were also found. In contrast, when cells were cultured on QCh/coPLA, coPLA/DOX, and QCh/coPLA/DOX mats, cell rounding, retraction of the pseudopods, blebbing of the cellular membrane, reduction of the cellular and nuclear volume (picnosis), and fragmentation of

the nucleus (karyorrhexis) occurred (Figure 10b-d), which are typical morphological features of apoptosis. At the 24th hour of incubation on a QCh/coPLA mat, a marked discoloration of a great part of the cells and a presence of cells in an early stage of apoptosis were observed (condensation of nuclear chromatin) (Figure 10b). The free DOX and the QCh/coPLA/DOX mat induce apoptosis in the majority of HeLa cells (Figure 10c and 10d). HeLa cells after incubation on QCh/ coPLA/DOX mat showed distinct morphological changes (rounding-up of the cell, retraction of pseudopodes, plasma membrane blebbing, and nuclear margination) corresponding to typical late apoptosis. The percentage of apoptotic HeLa cells, incubated in the presence of free DOX, was 92.69 ( 10.27%, whereas apoptotic HeLa cells, cultured on coPLA and QCh/coPLA mats, were 35.52 ( 13.18 and 54.50 ( 8.71%, respectively (Figure 11a). For instance, all HeLa cells cultured on QCh/coPLA/DOX and on coPLA/DOX were 100% apoptotic. Therefore, DOXcontaining nanofibrous mats exhibit a strong antitumor effect, comparable to that of free DOX. With increasing the time of incubation to 48 h, the percentage of the apoptotic cells incubated on coPLA and QCh/coPLA nanofibrous mats increases (42.48 ( 7.07 and 69.25 ( 12.74% for incubation on

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coPLA and QCh/coPLA, respectively) (Figure 11b). At the 48th hour of incubation, QCh/coPLA/DOX and coPLA/DOX mats and the free DOX induce apoptosis in all HeLa cells. To the best of our knowledge, there are no data available in the literature on the antitumor effect of QCh-containing nanofibrous materials on the human cervical cancer HeLa cell line. The data regarding the mechanism of antitumor action of chitosan and its derivatives are contradictory. It is considered that as polycations, the surface charge of chitosan and its derivatives is one of the major factors affecting their cytotoxic activity.17,20,38 Huang et al.20 suggest that quarternized derivatives of chitosan, having positively charged amino groups, interact electrostatically with the negatively charged membranes of the tumor cells and achieve their cytotoxic effect by membrane disintegration, causing death of the tumor cells by induction of necrosis, and not of apoptosis. Another presumed mechanism of the cytotoxic action of chitosan is the induction of apoptosis in the cells. Apoptosis is a mechanism of programmed cell death, which plays a major role as a protective mechanism against carcinogenesis by elimination of genetically damaged cells or cells that give rise to neoplasia.39-42 In developing of chemotherapeutic and chemo-prophylactic strategies for the control of malignant diseases, chemotherapeutics whose main mechanism of action is associated with inducing of a cell death through apoptosis are preferred. During the past decade, a predominant statement is that the cytotoxic effect of chitosan is due to the induction of apoptosis [Pae et al. for HL-60 cell line,43 Huang et al. for three human tumor cell lines, HeLa, Hep3B, and SW480 (mitochondrial apoptosis),20 Xu et al. for hepatocellular tumor cells (SMMC-7721),44 Lin et al. for human gastric tumor cells (AGS)45]. We assume that the significant antitumor effect of the nanofibrous materials containing both QCh and DOX is due to the superposition of two effects. The electrostatic interactions between the positively charged tertiary amino groups of QCh and the negatively charged areas of the tumor cell membrane probably lead to destruction of the structure of the cell membrane, thus facilitating the penetration of DOX into the cells and inducing apoptotic effect at the level of DNA46 or at the level of mitochondria.47 Therefore, QCh- and DOXcontaining nanofibrous mats could be of interest as a means for local delivery and release of antitumor drugs directly at the tumor site.

Conclusions One-step preparation of DOX-containing nanofibrous materials was achieved by electrospinning. The DOX-containing coPLA and QCh/coPLA fibers exhibited high cytotoxicity against HeLa cells, close to that of free DOX. Moreover, QCh/ coPLA mats showed an inhibitory effect on HeLa cells that is concentration- and time-dependent. The high antitumor activity renders these types of nanofibrous materials promising candidates for the treatment of cervical tumor, which remains a critical public health problem.48 Our results on the antitumor activity of the QCh/coPLA and QCh/coPLA/DOX fibrous materials are consistent with the hypothesis that the induction of apoptosis is one of the major mechanisms of their antitumor action. Acknowledgment. Financial support from the Bulgarian National Science Fund (grant DO-02-164/2008) is gratefully acknowledged.

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