Quaternized chitosan-coated nanofibrous materials containing gossypol: Preparation by electrospinning, characterization and antiproliferative activity towards HeLa cells

International Journal of Pharmaceutics 436 (2012) 10–24

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Quaternized chitosan-coated nanofibrous materials containing gossypol: Preparation by electrospinning, characterization and antiproliferative activity towards HeLa cells Milena Ignatova a , Nevena Manolova a,∗ , Reneta Toshkova b , Iliya Rashkov a,∗ , Elena Gardeva b , Liliya Yossifova b , Marin Alexandrov b a b

Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St., bl. 103A, BG-1113 Sofia, Bulgaria Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev St., bl. 25, BG-1113 Sofia, Bulgaria

a r t i c l e

i n f o

Article history: Received 3 March 2012 Received in revised form 12 June 2012 Accepted 13 June 2012 Available online 21 June 2012 Keywords: Electrospinning Gossypol Quaternized chitosan Polylactide Antitumor activity Apoptosis

a b s t r a c t Nanofibrous polylactide-based materials loaded with a natural polyphenolic compound gossypol (GOS) with antitumor properties were prepared by electrospinning. The nanofibrous materials were coated with a thin film of crosslinked quaternized chitosan (QCh). GOS incorporated in the nanofibrous mats was in the amorphous state. GOS release was diffusion-controlled and its in vitro release profiles depended on the mat composition. The nanofibrous materials exhibited high cytotoxicity towards HeLa tumor cells. Interestingly, it was particularly pronounced in the case of fibrous materials, which contain both QCh and GOS. The observed strong antiproliferative effect of the nanofibrous mats was mainly due to induction of cell apoptosis. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The conventional drugs used in cancer treatment (alkylating agents, antimetabolites, topoisomerase inhibitors and antimicrotubule agents) block DNA synthesis and cell division. Despite their effectiveness, these agents exhibit low selectivity (they destroy both cancer cells and normal mitotic cells). Their administration is accompanied by severe side effects: myelosuppression, cardio-, hepato- and nephrotoxicity, etc. The targeted and controlled delivery of drugs to tumor tissues is crucial for the enhancement of their therapeutic efficacy and the minimization of their side effects. The development of highly effective polymer systems for targeted delivery of antitumor drugs has been the subject of a number of studies (Allen, 2002; Duncan, 2003). The investigations on the biological activity of compounds with a potentially preventive or therapeutic potential for pre-cancerous and cancerous lesions, secondary tumor formation and metastases are of particular interest. Chitosan is a natural polysaccharide that is usually prepared by N-deacetylation of chitin. It is composed of ␤(1→4)-linked

∗ Corresponding authors. Tel.: +359 2 9793289; fax: +359 2 8700309. E-mail addresses: [email protected] (N. Manolova), [email protected] (I. Rashkov). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.06.035

2-acetamido-2-deoxy-␤-d-glucopyranose and 2-amino-2-deoxy␤-d-glucopyranose residues. Chitosan is a non-toxic, biocompatible, biodegradable and non-immunogenic polymer with inherent antitumor and immunomodulating properties (Aranaz et al., 2009; Bshena et al., 2011; Kato et al., 2005). Chitosan and its derivatives have been extensively used in the development of polymer systems for targeted drug delivery (Kim et al., 2008; Park et al., 2010; Ta et al., 2008). It has been reported that quaternized chitosan derivatives (QCh) exhibit higher antimicrobial and antimycotic activity as compared to chitosan (Jia et al., 2001; Kim et al., 1997, 2003), as well as good in vitro antitumor activity (Huang et al., 2006). Previously, some of us have reported on the preparation of QChcontaining nanofibers by electrospinning of QCh in the presence of poly(vinyl alcohol), poly(vinyl pyrrolidone) or poly(l-lactideco-d,l-lactide) (coPLA) (Ignatova et al., 2006, 2007, 2009; Paneva et al., 2009). It has been demonstrated that the incorporation of QCh in the electrospun fibers imparts them high antibacterial and antimycotic activity. In addition, the possibility for preparation of electrospun nanofibers based on poly(l-lactide) coated with a film of chitosan or N-carboxyethylchitosan, which possess antibacterial and hemostatic properties has been shown (Spasova et al., 2008; Yancheva et al., 2010). The natural polyphenolic compound GOS has been reported to display antiviral, antimalarial and antitumor activities (Ligueros

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

et al., 1997). It disturbs different cell processes such as energy metabolism and other mitochondrial functions in tumor cells (Oliver et al., 2005). It has been found that GOS manifests antiproliferative and antimetastatic properties towards several types of tumor cells, such as leukemic cells, carcinoma cells isolated from the colon or prostate and glioma cells (Macoska et al., 2008; Meng et al., 2008). Clinical studies have revealed that it inhibits metastasis formation in adrenocortical carcinoma, malignant glioma and breast carcinoma (Oliver et al., 2005). Blocking of the cell cycle and cell death has been observed in the case of cells placed in contact with GOS and a number of studies have assumed that cell death in this case takes place by induction of apoptosis. It has been demonstrated that GOS inhibits DNA synthesis and causes DNA fragmentation in tumor cells in vitro (Oliver et al., 2005). Another important advantage of GOS is that it induces apoptosis in drugresistant cancer cells (Oliver et al., 2005). The micro- and nanofibrous polymer systems for drug delivery evoke interest because of a number of advantages of these materials: high specific surface area, possibilities for sustained drug release and for reduction of the side effects of the drugs and for enhancement of their therapeutic effect. Polylactide is suitable as a matrix for drug delivery, since it is a biodegradable and biocompatible aliphatic polyester with relatively good mechanical properties. Recently, we have reported on nanofibrous materials containing doxorubicin that manifest good antitumor activity towards the HeLa human cervical cancer cell line (Ignatova et al., 2010) and MCF-7 human breast cancer cell line (Ignatova et al., 2011) in vitro. These materials composed of blended coPLA and QCh have been prepared by electrospinning of mixed solutions. Implants prepared thereof exhibit good antitumor activity in vitro and in vivo towards myeloid Graffi tumor, as well (Toshkova et al., 2010). The present contribution aims at the preparation and characterization of nanofibrous materials which combine the beneficial properties of GOS with those of QCh. GOS-containing fibers were prepared from coPLA or coPLA/poly(ethylene glycol) (PEG) by electrospinning. A different approach was used to introduce QCh in the system – the nanofibrous materials were coated with a thin film of crosslinked QCh. The in vitro release profiles of GOS from the mats and their cytotoxicity towards human HeLa tumor cells were studied. The cell death was assessed by staining assays.

2. Experimental 2.1. Materials N-butyraldehyde (Fluka), NaBH4 (Fluka), CH3 I (Fluka), NaI (Fluka), genipin (Challenge Bioproducts Co., Ltd.) and GOS (Molekula, Dorset, UK) 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) ˚ prior to use. Poly(l-lactidewere dried using molecular sieves (4 A) co-d,l-lactide) Resomer® LR 708 (M w 911,000 g mol−1 , M w /M n = 2.46) – (l-lactide: d,l-lactide molar ratio = 69:31) was kindly donated by Boerhinger-Ingelheim Chemicals Inc. (Germany). Chitosan with a molecular weight of 380,000 g mol−1 (Aldrich) and a deacetylation degree of 80% was used. For culturing of the HeLa tumor cell line Dulbecco Modified Eagle’s Medium (DMEM) (Sigma–Aldrich, Germany), enriched with fetal calf serum (FCS) (Gibco, Austria) and containing antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, LONZA) was used. Trypsin–EDTA was supplied by FlowLab, Australia. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide (EtBr) and acridine orange (AO) were purchased from Sigma–Aldrich, Germany. The disposable consumables

11

Scheme 1. Quaternized chitosan (QCh) chain sequence.

(75 cm3 tissue culture containers, a filtration system, tissue culture plates) were supplied by Orange Scientific, Belgium. 2.2. N-Butyl-N,N-dimethyl chitosan iodide synthesis and characterization A quaternized chitosan derivative (QCh) (Scheme 1) – N-butylN,N-dimethyl chitosan iodide, was prepared according to a known procedure (Kim et al., 1997). Briefly, in the first step N-butyl chitosan was prepared by reacting chitosan and n-butyraldehyde 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-butyl chitosan was filtered out and vacuum-dried at 40 ◦ C for 2 days. Yield – 92%. The resulting N-butyl chitosan was quaternized using methyl iodide. N-butyl-N,N-dimethyl chitosan iodide was purified by two-fold precipitation in acetone and dried under reduced pressure at 40 ◦ C. Yield – 90%. The quaternization degree of QCh was determined from the 1 H NMR data and by potentiometric titration of the iodide form with an aqueous silver nitrate solution, using a working silver electrode and a reference calomel electrode. The quaternization degree was calculated from the intensity ratio of the signal at 3.39 ppm for CH2 N+ (CH3 )2 I− to the signals at ı 3.64–4.54 ppm for H-2, H-3, H-4, H-5, H-6, H-6 (6H). This value (82%) is in agreement with the quaternization degree determined potentiometrically (80%). The degree of methylation of the OH functions was determined from the intensity ratios of the signals of CH3 O at 3.51 and 3.42 ppm, for OH at C-3 and C-6 positions, respectively, and the H-2, H-3, H-4, H5, H-6, H-6 (6H) signals at ı 3.64–4.54. The degree of methylation of H-3 and H-6 was 13 and 88%, respectively. 2.3. Preparation of pristine and GOS-containing coPLA and coPLA/PEG nanofibrous mats CoPLA nanofibers were prepared by electrospinning of coPLA solution in dry DMF/DMSO (60/40, v/v; 5 wt.% coPLA). CoPLA/PEG nanofibers with a coPLA/PEG weight ratio of 70/30 were obtained by electrospinning of their mixed solutions in dry DMF/DMSO (60/40, v/v) (total polymer concentration – 5 wt.%). GOS-containing coPLA or coPLA/PEG mats were prepared by electrospinning of coPLA/GOS or coPLA/PEG/GOS mixed solutions (3 and 10% in weight percent to coPLA or coPLA/PEG content) in dry DMF/DMSO (60/40, v/v) at a total polymer concentration of 5 wt.%. The mixed solutions were placed in 5 mL syringes equipped with 20G needles. The positive lead of a direct current (DC) high voltage power supply was connected to the needles via alligator clips. The electrospinning solutions were delivered at a controlled flow rate (1.0 mL/h), at a constant value of the applied voltage (25 kV) and constant tip-tocollector distance (20 cm). The fibers were collected directly onto an aluminum foil fixed to a rotating grounded drum (with a diameter of 45 mm) at a rotating speed of 1200 rpm. In this way, uniform electrospun fibrous mats with size of 16 cm × 19 cm were prepared. The electrospun fibrous mats were placed under vacuum overnight

12

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

at 40 ◦ C to remove any solvent residues. The GOS-containing fibers are further denoted as coPLA/GOS and coPLA/PEG/GOS. Prior to electrospinning, the dynamic viscosity of the spinning solutions was measured using a Brookfield LVT viscometer equipped with a small-sample thermostated adapter, a spindle and SC4-18/13R chamber, at 25 ± 0.1 ◦ C. The electrical resistance of the spinning solutions was measured in an electrolytic cell equipped with rectangular sheet platinum electrodes according to a previously described procedure (Ignatova et al., 2010, 2006). The conductivity of the spinning solutions (, ␮S/cm) was calculated from the following equation: =

1 1 =  Kcell · R

(1)

where  is the specific resistance of the solution (␮ cm), R is the electrical resistance of the solution (␮), and Kcell is the constant of the cell. 2.4. Preparation of crosslinked QCh coating on the mats The next step comprised first coating of the fibrous mats with a thin film of QCh, and then crosslinking of the film. The mats were threefold soaked in a 0.1 wt.% water/ethanol (3/1, v/v) solution of QCh for 30 min, then withdrawn and dried to a constant weight. In order to impart stability in aqueous medium to the QCh coating, it was crosslinked with genipin, similarly to a previously described method (Zhang et al., 2010). A 5 wt.% solution of genipin was prepared in ethanol and then 20 mL of genipin solution were placed in a desiccator. The QCh-coated mats were kept for 48 h in a desiccator saturated with vapors of genipin solution in ethanol at 30 ◦ C and then dried in a vacuum oven at room temperature overnight. Our preliminary experiments have shown that this period is sufficient for the formation of a water-insoluble QCh coating. After crosslinking the QCh-coated mats were washed several times with a mixture of water/ethanol (1/1, v/v) to remove any non-reacted genipin and were dried in a vacuum oven to constant weight and then observed by SEM. The coating of crosslinked QCh is further denoted as crQCh-coat. 2.5. Characterization of the electrospun mats The morphology of the mats was evaluated by SEM. For this purpose, the mats were vacuum-coated with gold in a Jeol JFC-1200 fine coater and examined by a Jeol JSM-5510 scanning electron microscope. The average fiber diameter and the fiber morphology were estimated in terms of the criteria for complex evaluation of electrospun mats reported elsewhere (Spasova et al., 2006) using the Image J software program (Rasband, 2006) by measuring at least 30 fibers from each SEM image. Attenuated total reflection Fourier-transform infrared (ATRFTIR) spectroscopic analyses were performed using an IRAffinity-1 spectrophotometer (Shimadzu Co., Kyoto, Japan) equipped with a MIRacleTM ATR (diamond crystal, depth of penetration of the IR beam into the sample – about 2 ␮m) accessory (PIKE Technologies, USA). The spectra were recorded from 4000 to 500 cm−1 with a resolution of 4 cm−1 using a DLATGS detector equipped with a temperature controller. All spectra were corrected for H2 O and CO2 using IRsolution internal software. Static contact angle measurements were performed using a Krüss drop shape analysis system (DSA 10-MK2 model) at 20 ± 0.2 ◦ C. A drop of deionized water (10 ␮L) 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 20 measurements on different areas of the surface. The 1 H

NMR spectra of QCh were recorded using a Bruker Avance II+ 600 spectrometer at 353 K in D2 O. The surface chemical composition of the mats was determined quantitatively by X-ray photoelectron spectroscopy (XPS). The XPS measurements were carried out in the ultrahigh-vacuum (UHV) chamber of an ESCALAB-MkII (VG Scientific) electron spectrometer using Mg K␣ excitation with a total instrumental resolution of ∼1 eV. Energy calibration was performed, taking the C1 s line at 285 eV as a reference. The surface atomic concentrations were evaluated using Scofield’s ionization cross-sections with no corrections for  (the mean free path of photoelectrons) and the analyzer transmission function. The experimental values for the element atomic percentages obtained from the XPS analysis are the average of three independent measurements. The high-resolution spectra were dissected by means of special deconvolution software package. The crystalline structure of the nanofibrous mats was evaluated by X-ray diffraction analysis (XRD). XRD spectra were recorded at room temperature using a computer-controlled D8 Bruker Advance powder diffractometer with filtered Cu K␣ radiation. Data were collected in the 2 range from 5◦ to 50◦ with a step of 0.02◦ and counting time of 1 s/step. In order to demonstrate the presence of GOS in the non-coated and QCh-coated coPLA/GOS and coPLA/PEG/GOS mats, they were analysed by fluorescence microscopy (Leika DM 500B, Wetzlar, Germany). 2.6. In vitro GOS release study The release experiments were carried out in vitro as follows. A piece of GOS-containing nanofibrous mat (16 mg) was placed in a vial filled with 80 mL of PBS (pH 7.4). The vial was incubated at 37 ◦ C in a thermostated shaker and shaken horizontally at 250 rpm. At predetermined time intervals, aliquots were withdrawn and the same amount of fresh PBS was added back to maintain a constant condition before placing the vial back in the shaker. The amount of GOS released in the buffer solution was determined using a UV–vis spectrophotometer at a wavelength of 375 nm. The cumulative weight and relative percentage of the released GOS were calculated as a function of incubation time. All GOS release tests were performed in triplicate. To determine the total content of GOS in the electrospun fibers, three pieces of GOS-containing nanofibrous mats of known weight were dissolved in 10 mL of DMFA/DMSO mixed solvent = 60/40 (v/v). The amount of GOS was determined by a UV–vis spectrophotometer at a wavelength of 377 nm, as the mean of three experiments. It was found that the amount of incorporated GOS was equal to that present in the spinning solution. 2.7. Cell culturing A permanent HeLa human cancer line (cervical carcinoma) was used in the experiments. The culturing of HeLa cells was performed as previously described (Ignatova et al., 2010). 2.8. Cell proliferation assay HeLa tumor cells were trypsinized using 0.25% Trypsin–EDTA and counted using a hemocytometer. The cells were seeded in a 96-well microtiter plate at a concentration of 2 × 104 cells/well. After overnight incubation at 37 ◦ C in a humid atmosphere containing 5% CO2 required for cell attachment, the culture medium was replaced and the cells were treated with different types of nanofibrous mats (coPLA; coPLA/PEG; coPLA/GOS; coPLA/PEG/GOS; coPLA/crQCh-coat; coPLA/PEG/crQCh-coat; coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat), with GOS (positive control) and cultured only in nutritive medium (negative control) for different periods of time (6, 24, 48 and 72 h). The amount of GOS was 30 ␮g/g

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

polymer and the amount of QCh (crosslinked) was 186 ␮g/g electrospun polymer in the GOS-containing nanofibrous mats, and those coated with a film of QCh (crosslinked), respectively. 66.6 ␮g of GOS-containing nanofibrous mats, and 79 ␮g of nanofibrous mats coated with a film of QCh (crosslinked) were placed in contact with 100 ␮L of MTT solution. The concentration of free GOS was 20 ␮g/mL. The effect of the various nanofibrous mats on the viability of the tumor cells (HeLa) was assessed by a MTT assay according to a method described by Mossmann (1983). Each variant of the nanofibrous mats was assayed by five measurements. After culturing on mats, the HeLa cells were washed twice with PBS (pH 7.4), after which 100 ␮L of MTT solution were added to each well and the cells were incubated at 37 ◦ C for 3 h; the supernatants were aspirated and 100 ␮L of the lysing solution (DMSO:ethanol = 1:1) were added to each well in order to dissolve the obtained formazan. The results from the MTT assay were read using an ELISA plate reader (TECAN, SunriseTM, Grödig/Salzburg, Austria). The absorbance of the dissolved formazan was measured spectrophotometrically at a wavelength of 540 nm, ref. 620 nm. The percentage of cell growth inhibition was calculated as follows: Cell viability (%) =

OD570 (experimental) × 100 OD570 (control)

2.9. Determination of the inhibitory concentration (IC50 ) of GOS and QCh For the performance of the in vitro experiments with HeLa tumor cells GOS was initially dissolved in 70% ethanol to achieve a concentration of the stock solution of 2 mg/mL and further dissolved in DMEM culture medium, enriched with 10% FCS, until concentrations of 10, 20, 40, 80 and 100 ␮g/mL were attained. In order to determine the IC50 of GOS and QCh against HeLa tumor cells they were cultured in a 96-well microtiter tissue culture plate at a concentration of 2 × 104 cells/well. After overnight incubation at 37 ◦ C in a humid atmosphere containing 5% CO2 the culture medium was replaced and the cells were treated with different concentrations of GOS (10–100 mg/mL) or QCh (10–550 mg/mL). Non-treated HeLa cells cultured in DMEM containing 10% FCS served as a negative control. The cell viability was assessed after 24-h incubation under the above-mentioned conditions by a standard MTT assay according to the method of Mossmann (1983). 2.10. Study of the effect of the electrospun mats on HeLa cells using SEM The pristine coPLA mats, the coPLA/GOS, coPLA/crQCh-coat and coPLA/GOS/crQCh-coat mats were sterilized by UV-light for half an hour. Then they were placed in 24-well plates, seeded with HeLa cells at a concentration of 2 × 105 cells/mL and incubated at 37 ◦ C for 24 h in a CO2 incubator. The 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. After fixation, the samples were rinsed twice with PBS and dehydrated with graded concentrations of ethanol – 30, 50, 70 and 90%, and finally, with absolute ethanol. The samples were treated with 100% hexamethyldisilazane (HMDS, Sigma–Aldrich, USA) for 5 min and then air-dried after the removal of HMDS. The morphology of the HeLa cells after contact with the mats was observed by a Jeol JSM5510 scanning electron microscope after coating with gold (Jeol JFC-1200 fine coater). For the sake of comparison, the morphology

13

of HeLa cells cultured on Termanox® plastic disks in the absence or presence of PBS solution of GOS (20 ␮g/mL) was also studied. 2.11. Studying apoptosis by staining methods The morphology of the apoptotic nuclei was assessed by double staining with AO and EtBr similarly to standard procedures (Wahab et al., 2009). In brief, the cells were seeded on the various nanofibrous mats (coPLA, coPLA/GOS, coPLA/crQCh-coat and coPLA/GOS/crQCh-coat mats) in 24-well tissue culture plates, in a CO2 incubator, as described in the previous section. After 24-h incubation, the nanofibrous mats were washed twice with PBS for 10 min to remove the culture medium. Equivalent amounts of fluorescent dyes – AO (10 ␮g/mL) and EtBr (10 ␮g/mL) were added to the mats. Freshly stained HeLa cells attached to the nanofibrous mats were mounted on a slide, covered with a cover glass and observed by a fluorescence microscope (Leica DM 5000B, Wetzlar, Germany) within 30 min, before the fluorescence had started to fade away. Pappenheim’s staining method (WHO, 2006) was also used for studying cell morphology. HeLa tumor cells were cultured in the presence of the different types of mats: (coPLA, coPLA/GOS, coPLA/crQCh-coat and coPLA/GOS/crQCh-coat mats) and GOS (20 ␮g/mL) as positive control. For the sake of comparison, HeLa cells were cultured on Termanox® plastic disks at a concentration of 2 × 105 cells/well in a 24-well tissue culture plate. After 24-h incubation under standard conditions in a CO2 incubator the mats were removed, the incubation medium was replaced and the cells were examined using an inverted microscope (Olympus CK-40). Subsequently, the Termanox® plastic disks with HeLa cells were rinsed with PBS, air-dried at room temperature, stained by the Pappenheim’s method and examined microscopically (Leica DM 5000B; Leica Microsystems, Wetzlar, Germany). 2.12. 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 post hoc test.

3. Results and discussion 3.1. Preparation and characterization of pristine, GOS-containing and QCh-coated mats The incorporation of coPLA and PEG in materials designed for biomedical purposes attracts attention, because coPLA and PEG are polymers of low toxicity. The copolymers of lactic acid are biodegradable; PEG of low molecular weight is easily eliminated via the kidneys. Fibrous materials were successfully prepared by electrospinning coPLA or coPLA/PEG (coPLA:PEG = 70:30, w/w) solutions in a mixed DMFA/DMSO (60:40, v/v) solvent, total polymer concentration – 5 wt.%. The average diameter of the coPLA/PEG nanofibers (350 ± 50 nm) was approximately twice as small as that of the coPLA nanofibers (760 ± 110 nm) at constant AFS and constant total polymer concentration (Table 1). The observed effect may be explained by the decrease in the solution viscosity on adding a lower-molecular-weight polymer (PEG) to the spinning solution. Continuous cylindrical and defect-free coPLA/GOS and coPLA/PEG/GOS nanofibers were obtained under the same electrospinning conditions (Fig. 1). The surface of the fibers was smooth and no GOS crystals were detected on it. As evident from the SEM micrographs (Fig. 1A–D), all the nanofibers are partially

14

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

Fig. 1. SEM micrographs of electrospun mats of: coPLA/GOS (3 wt.% GOS) (A), coPLA/PEG/GOS (3 wt.% GOS) (B), coPLA/GOX (10 wt.% GOS) (C) and coPLA/PEG/GOX (10 wt.% GOS) (D); magnification 2000×. Total polymer concentration – 5 wt.% (DMF/DMSO = 60/40, v/v), AFS 1.25 kV/cm and feed rate of 1.0 mL/h.

Fig. 2. SEM micrographs of mats of: coPLA/crQCh-coat (A), coPLA/PEG/crQCh-coat (B), coPLA/GOS (3 wt.% GOS)/crQCh-coat (C), coPLA/PEG/GOS (3 wt.% GOS)/crQCh-coat (D), coPLA/GOS (10 wt.% GOS)/crQCh-coat (E), and coPLA/PEG/GOS (10 wt.% GOS)/crQCh-coat (F).

aligned in the case when the electrospinning of solutions of these polymers has been performed using a rotating collector. The coating of the fibers with QCh does not result in the formation of any continuous film between them and the mats preserve their porous structure to a great extent (Fig. 2).

Table 1 GOS content, solution dynamic viscosity (), conductivity (), average fiber diameter ¯ of electrospun fibers; SD – standard deviation. (d) Electrospun mats

GOS content (%)a

 (cP)

 (␮S/cm)

d¯ (nm)

SD

coPLA coPLA/GOS coPLA/GOS coPLA/PEG coPLA/PEG/GOS coPLA/PEG/GOS

– 3 10 – 3 10

5300 4945 4240 660 620 570

27.0 27.0 27.1 30.0 30.2 30.2

760 680 600 380 350 340

110 110 75 70 60 50

a

GOS content in weight percent to coPLA or coPLA/PEG.

Fluorescence micrographs of the mats are presented in Fig. 3. Fluorescence was observed only in the case of GOS-containing mats. It is well known that crystallinity has an impact on the drug release profile and for that reason XRD spectra of the nanofibrous mats of coPLA, coPLA/GOS, coPLA/PEG and coPLA/PEG/GOS were taken (Fig. 4). In the XRD spectrum of the pristine coPLA mat only an amorphous halo was observed. In the case of coPLA/PEG the appearance of low-intensity peaks characteristic of PEG (2 = 19.2◦ and 23.4◦ ) was detected and no peaks corresponding to coPLA crystal phase were observed. This result indicates that only crystallization of PEG occurs during electrospinning. The crystallization of coPLA is impeded which is in accordance with the data reported for similar systems (Spasova et al., 2010; Zhou et al., 2006a,b). GOS incorporated in the nanofibrous mats was also in the amorphous state (Fig. 4B and D). The obtained results are consistent with the data obtained for other drug-containing nanofibrous materials (Natu et al., 2010; Wei et al., 2010; Xu et al., 2005).

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

15

Fig. 3. Fluorescence micrographs of mats of: (A) coPLA, (B) coPLA/GOS (10 wt.% GOS), (C) coPLA/PEG, (D) coPLA/PEG/GOS (10 wt.% GOS), (E) coPLA/crQCh-coat, (F) coPLA/GOS/crQCh-coat, (G) coPLA/PEG/crQCh-coat, and (H) coPLA/PEG/GOS/crQCh-coat. Bar = 100 ␮m.

The GOS-containing nanofibrous mats and those coated with a thin film of QCh (crosslinked) were characterized by ATR-FTIR spectroscopy (Fig. 5) and by XPS analysis (Figs. 6 and 7). In the ATR-FTIR spectrum of coPLA/GOS mats in addition to the bands characteristic of coPLA (1751 cm−1 – C O ; 1086 cm−1 – C O C ) new bands appeared at 1622 and 1574 cm−1 characteristic for the C O vibrations and C C vibrations from the naphthalene ring of GOS incorporated in the mats (Fig. 5B). In the spectra of the coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats (not shown in the figure) new characteristic bands were observed at 1651 and 1558 cm−1 , assigned to amide I and amide II of the polysaccharide structure of QCh, respectively (Fig. 5C), as well as at 3366 cm−1 assigned to NH-stretching vibrations (not shown in the figure). In Fig. 6A the high resolution XPS spectrum of the C1s region of the coPLA mat is presented. This region was deconvoluted into three peaks: at 285.0 eV for C H or C C , at 286.9 eV for C O and at 288.9 eV for O C O. The expanded O1s spectrum of coPLA (Fig. 6B) is consisted of two peaks, assigned to C O at 533.3 eV and to O C O at 531.9 eV. The experimentally

determined elemental composition (54.7% C and 45.3% O) was close to the theoretically calculated values (52.9% C and 47.1% O). The theoretical ratio of the peaks corresponding to the respective carbon atoms in the high resolution C1s spectrum is [C C/C H]/[C O]/[O C O] = 33.3/33.3/33.3. The experimentally determined one is 46.2/27.2/26.6. Therefore, in the case of the coPLA mat the surface of the mat is enriched with methyl groups of low binding energy and this result is in conformity with data obtained by other authors and with our previous results (Cui et al., 2008; Ignatova et al., 2010; Yancheva et al., 2010), as well as with the observed high hydrophobization of the mat surface. The

Fig. 4. XRD spectra of mats of: coPLA (A), coPLA/GOS (B), coPLA/PEG (C) and coPLA/PEG/GOS (D).

Fig. 5. ATR-FTIR spectra of mats of: (A) coPLA, (B) coPLA/GOS and (C) coPLA/GOS/crQCh-coat.

16

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

C1s

O1s

294 292 290 288 286 284 282 280 278 276

538

Binding energy (eV)

536

C1s

534

532

A

526

294 292 290 288 286 284 282 280

Binding energy, eV

C

O1s

C1s

540 538 536 534 532 530 528 526

292

290

288

286

284

282

Binding energy, eV

Binding energy, eV

D

E

N1s

280

538

536

404

402

400

398

396

528

Binding energy, eV

J

526

C1s

630

620

610

Binding energy, eV

H

530

528

F

I I3d

N1s

532

530

Binding energy, eV

O1s

534

532

294 292 290 288 286 284 282 280 278

640

G

536

534

Binding energy, eV

I3d

Binding energy, eV

538

528

B

O1s

406

530

Binding energy (eV)

526

408

406

404

402

400 398 396 394

Binding energy, eV

K

640

630

620

610

600

Binding energy, eV

L

Fig. 6. XPS peak fittings for electrospun mats of: coPLA – C1s (A) and O1s (B); coPLA/GOS – C1s (C) and O1s (D); coPLA/crQCh-coat – C1s (E), O1s (F), N1s (G) and I3d (H); and coPLA/GOS/crQCh-coat – C1s (I), O1s (J), N1s (K) and I3d (L).

experimentally determined value of the water contact angle is 128.0 ± 3.9◦ . The high resolution C1s spectrum of the GOS-containing coPLA (Fig. 6C) shows three peaks – a peak at 285.0 eV, assigned to C H or C C from coPLA and GOS, a peak at 287.7 eV corresponding to C O from coPLA and C OH from GOS and a peak at 289.7 eV assigned to O C O from coPLA and to C(H) O from

GOS. In the expanded O1s spectrum a new peak of low intensity appears at 533.5 eV, assigned to C OH from GOS (Fig. 6D). The theoretically calculated ratio of the peak areas corresponding to the respective carbon atoms from the C1s spectrum should be [C C/C H]/[C O/C OH]/[O C O/C(H) O] = 37.3/32/30.7. experimentally determined ratio of the areas The corresponding to the respective carbon atoms

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

C1s

O1s

C1s

294 292 290 288 286 284 282 280

538

536

Binding energy, eV

534

536

534

532

530

528

292

290

288

402

400

398

396

640

O1s

286

284

282

280

538

532

630

620

610

600

532

530

528

526

528

294 292 290 288 286 284 282 280 278

Binding energy, eV

I

I3d

N1s

Binding energy, eV

530

F

H

O1s

534

534

Binding energy, eV

C1s

G

536

536

Binding energy, eV

Binding energy, eV

284 282 280

C

I3d

404

288 286

Binding energy, eV

E

N1s

538

294 292 290

Binding energy, eV

D

540

528

C1s

Binding energy, eV

406

530

B

O1s

538

532

Binding energy, eV

A

540

17

410 408 406 404 402 400 398 396 394

Binding energy, eV

J

K

640

630

620

610

600

Binding energy, eV

L

Fig. 7. XPS peak fittings for electrospun mats of: coPLA/PEG – C1s (A) and O1s (B); coPLA/PEG/GOS – C1s (C) and O1s (D); coPLA/PEG/crQCh-coat – C1s (E), O1s (F), N1s (G) and I3d (H); and coPLA/PEG/GOS/crQCh-coat – C1s (I), O1s (J), N1s (K) and I3d (L).

[C C/C H]/[C O/C OH]/[O C O/C(H) O] is 75.8/12.1/12.1. This indicates that the surface layer of the coPLA/GOS mat is enriched with ca. 38.5% carbon atoms participating in C H/C C bonds. The performed water contact angle measurements of the mats indicated that the incorporation of GOS in the coPLA mats

does not alter significantly their hydrophobicity. The incorporation of GOS in the mats results in a slight increase in the water contact angle value. The experimentally determined values of the water contact angles of coPLA/GOS (3 wt.%) and coPLA/GOS (10 wt.%) are close – 130.5 ± 3.5◦ and 131.0 ± 9.7◦ , respectively.

18

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

In the XPS spectrum of the coPLA/PEG mat the C1s (285 eV) and O1s (532.5 eV) peaks were observed (Fig. 7A and B). The C1s signal is composed of three components – a peak at 285 eV assigned to C H or C C from coPLA and PEG, a peak at 286.6 eV corresponding to C O from coPLA and PEG, and a peak at 289.6 eV assigned to O C O from coPLA. The O1s expanded spectrum showed two components at 532.5 eV assigned to C O from coPLA and at 534 eV for C O from coPLA and PEG (Fig. 7B). The experimentally determined atomic percentages of the elements (56.8% C and 43.2% O) were in accordance with the theoretically calculated ones (55.1% C and 44.9% O). The theoretically calculated ratio of the areas of the peaks corresponding to the respective carbon atoms is [C C/C H]/[C O]/[O C O] = 38.3/38.3/23.4, whereas the experimentally determined one is 39.3/48.6/12.2; thus, the peak assigned to carbon atoms participating in C O bonds is characterized by the largest area. This result is consistent with the observed hydrophilization of the mat surface. In the water contact angle determination experiments the water drop is rapidly absorbed by the nanofibrous mat. The high resolution XPS spectra for C1s and O1s detection (Fig. 7C and D) confirmed the successful incorporation of GOS in the surface of coPLA/PEG mats. In the expanded C1s spectra of the same mats three peaks were identified – at 285 eV for C H or C C from coPLA, PEG and GOS, at 287.0 eV for C O from coPLA and PEG as well as to C OH from GOS and at 289.1 eV for O C O from coPLA and C(H) O from GOS (Fig. 7C). In the detailed O1s spectrum the appearance of a new peak at 533.1 eV assigned to C OH from GOS was registered (Fig. 7D). The theoretical ratio of the areas of the peaks corresponding to the respective carbon atoms in the high resolution C1s spectrum of the GOS-containing coPLA/PEG mat should be [C C/C H]/[C O/C OH]/[O–C O/C(H) O] = 41.8/36.5/21.7. The experimentally determined ratio is 48.0/25.8/26.2. Thus, the surface of the GOS-containing coPLA/PEG mat is enriched with carbon atoms engaged in C H/C C and O C O/C(H) O bonds. These results are consistent with the results obtained from the performed water contact angle measurements of the mat. The incorporation of GOS in the coPLA/PEG mat leads to an increase in the water contact angle (30.9 ± 3.0◦ ) and the obtained mat remains hydrophilic. In the high resolution C1s spectra of the QCh (crosslinked)coated coPLA, coPLA/PEG, coPLA/GOS and coPLA/PEG/GOS mats as compared to those of the pristine mats a new peak was observed at 288.3 eV corresponding to O C O and N C O from QCh (Figs. 6E and I, 7E and I). In the expanded O1s spectra of the same mats new peaks were identified: at 532.5 eV, assigned to C OCH3 from QCh and at 530.8 eV, assigned to N C O from QCh (Figs. 6F and J, 7F and J). The appearance of a N1s peak composed of two components – at 399.5 eV, assigned to N C O and NH2 groups of QCh and at 402.0 eV corresponding to the ammonium group ( N+ (CH3 )3 ) from QCh (Figs. 6G and K, 7G and K) and I3d peaks [I3d5/2 at 618.0 eV and I3d3/2 at 630.0 eV, Figs. 6H and L, 7H and L] additionally confirmed the successful coating of the coPLA, coPLA/PEG, coPLA/GOS and coPLA/PEG/GOS fibers with a film of QCh. The atomic percentages of the nitrogen and iodine atoms of the coPLA/crQCh-coat and coPLA/GOS/crQCh-coat mats obtained from the XPS spectra are close to those of the coPLA/PEG/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats. The experimentally determined values of the water contact angles are: 65.4 ± 9.1 for coPLA/crQChcoat, and 66.3 ± 10.5 and 66.7 ± 11.1 for the coPLA/GOS/crQCh-coat mats containing 3 and 10 wt.% GOS, respectively. These values are consistent with the increased hydrophilicity of the mat surface, which is attributed to the presence of QCh (crosslinked) film on the fibers. The formation of a coating of crosslinked QCh on the surface of coPLA/PEG and coPLA/PEG/GOS mats leads to an increase in the

water contact angle and the measured water contact angle values of coPLA/PEG/crQCh-coat, coPLA/PEG/GOS (3 wt.%)/crQCh-coat and coPLA/PEG/GOS (10 wt.%)/crQCh-coat are 65.0 ± 17.3◦ , 67.5 ± 7.8◦ and 67.8 ± 7.2◦ , respectively. 3.2. In vitro GOS release studies The release profiles of GOS from pristine and QCh (crosslinked)coated mats containing 3 and 10 wt.% GOS are shown in Fig. 8. The release studies were performed at 37 ◦ C using a PBS buffer of pH 7.4. The release curves of GOS for coPLA/GOS, coPLA/PEG/GOS, coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats had similar characteristics: an initial rapid release was observed followed by a second stage of gradual release. It was found that the amount of GOS initially released increased on increasing the GOS content in the coPLA/GOS and coPLA/PEG/GOS mats (Fig. 8A). Around 9.9% and 11.7% of the loaded amount was released initially from coPLA/GOS mats containing 3 and 10 wt.% GOS. In the case of coPLA/PEG mats the amount of rapidly released GOS was considerably higher than that in the case of coPLA mats. Around 39.7% and 49.0% of GOS release were detected in the initial 20 min for coPLA/PEG mats containing 3 and 10 wt.% GOS, respectively (Fig. 8A). The observed faster release of GOS from coPLA/PEG/GOS mats as compared to coPLA/GOS mats during the first stage is most probably due to the hydrophilicity of the PEG-containing mats. As seen in Fig. 8B, GOS incorporated in the coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats is released more slowly during the first stage (for 60 min in the case of coPLA/crQCh-coat mats and 85 min in the case of coPLA/PEG/crQCh-coat mats) as compared to the non-coated coPLA/GOS and coPLA/PEG/GOS mats, the released amount being 9.4 and 54.0%, respectively. This is most probably due to the formation of a film of crosslinked QCh on the surface of the fibers, which serves as an additional barrier through which GOS needs to diffuse. The total amount of GOS released from the coPLA/GOS mats during incubation time of 420 min was 12.7% and 15.2%, respectively, for nanofibers containing 3 and 10 wt.% GOS. The amount of released GOS after 420 min from the coPLA/PEG/GOS mats, containing 3 and 10 wt.% GOS was 44.2% and 60.8%, respectively. In the case of coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats, containing 10 wt.% GOS, the total amount of released GOS was 13.3% and 58.9% for 420 min, respectively. From the plot of the released fraction of GOS (Mt /M∞ ) versus the square root of time (t1/2 ) (Fig. 8C and D) it is evident that the relationship is linear for the first stage of GOS release thus indicating a diffusion-controlled process. The obtained results confirm the successful preparation of QChcoated and non-coated nanofibrous materials, in which the natural compound GOS has been incorporated. Since GOS possesses antitumor activity, our study was next focused on the evaluation of the antitumor activity of the obtained nanofibrous materials towards HeLa human tumor cells in vitro. 3.3. Cytotoxicity of GOS-containing and QCh (crosslinked)-coated nanofibrous mats towards HeLa cancer cells First, the cytotoxic effect of GOS and QCh towards HeLa human cervical cancer cells was assessed by the MTT assay. GOS did not display any antiproliferative effect after 6 h of incubation (the result is not represented graphically). As seen from Fig. 9A, after 24 h and 48 h of incubation GOS inhibited the proliferation of HeLa tumor cells to a considerable extent and this effect became more clearly expressed at higher GOS concentration. The IC50 value for GOS was calculated to be 19.7 ␮g/mL after 24 h and 19.3 ␮g/mL – after 48 h of incubation. In contrast to GOS, QCh manifested an antiproliferative effect as early as 3 h after incubation (Fig. 9B).

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

A

70

B

70 60

Cumulative drug release (%)

Cumulative drug release (%)

60 50 40 coPLA/PEG/GOS (10 wt %) coPLA/PEG/GOS (3 wt %) coPLA/GOS (10 wt %) coPLA/GOS (3 wt %)

30 20

50 40 coPLA/PEG/GOS (10 wt%)/crQCh-coat

30

coPLA/GOS (10 wt%)/crQCh-coat

20

10

10

0 0

0

40 80 120 160 200 240 280 320 360 400 440

0

40 80 120 160 200 240 280 320 360 400 440

Time (min)

C

Time (min)

D

0.7

0.7 0.6

Drug release fraction (M t/M∝)

0.6

Drug release fraction (M t/M∝)

19

0.5 0.4 coPLA/PEG/GOS (10 wt%) coPLA/PEG/GOS (3 wt%) coPLA/GOS (10 wt%) coPLA/GOS (3 wt%)

0.3 0.2

0.4 0.3 coPLA/PEG/GOS(10 wt%)/crQCh-coat coPLA/GOS (10 wt%)/crQCh-coat

0.2 0.1

0.1 0.0

0.5

0

4

8

12

16

20

0.0

24

0

4

Square root of time (min 1/2 )

8

12

16

20

24

Square root of time (min 1/2 )

Fig. 8. Release profiles of GOS from electrospun mats in PBS (pH 7.4) at 37 ◦ C: (A) coPLA and coPLA/PEG and (B) coPLA/crQCh-coat and coPLA/PEG/crQCh-coat. GOS content in the nanofibers: 10.0 (, , 䊉, ) and 3.0 wt.% (, ). Curves in (A and B) replotted against square root of time.

A

B

100

80

Cell viability (%)

Cell viability (%)

80

60

40

20

0

100

24 h 48 h

0 10 20 30 40 50 60 70 80 90 100

Concentration ( μg/ml)

60

40 3h 6h 24h 48h 72h

20

0

0

10 0

200

30 0

40 0

50 0

600

Concentration ( μg/ml)

Fig. 9. Effect of GOS (A) and of QCh (B) on the viability of the HeLa cells incubated in the presence of an increasing concentration of the tested compounds in vitro. The data are expressed as mean ± SD.

20

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

Fig. 10. Effect of the nanofibrous mats on HeLa tumor cells after 6 (A), 24 (B), 48 (C) and 72 (D) h of incubation. 1 – coPLA; 2 – coPLA/PEG (70:30, w/w); 3 – coPLA/GOS; 4 – coPLA/PEG/GOS; 5 – coPLA/crQCh-coat; 6 – coPLA/PEG/crQCh-coat; 7 – coPLA/GOS/crQCh-coat; 8 – coPLA/PEG/GOS/crQCh-coat; con – control, HeLa cells; GOS (20 ␮g/mL). **p < 0.01, ***p < 0.001.

After a 3-h exposure the HeLa cell viability was decreased, statistically significant values being recorded at concentrations equal to and higher than 200 ␮g/mL. After 6-h and 24-h exposure the percentage of viable treated tumor cells was 60–65%, whereas a strong antiproliferative effect was observed after 48 and 72 h of incubation at all used concentrations of QCh, and the percentage of the viable cells was reduced to 20%. The obtained results are in accordance with the previously reported by us concentration-dependent antiproliferative effect of QCh (Ignatova et al., 2010). In Fig. 10 the antiproliferative effect of the obtained nanofibrous mats towards HeLa cells displayed between 6 and 72 h of incubation is presented. GOS (20 ␮g/mL) was used as a positive control and HeLa cells were used as a negative control. As seen in Fig. 10A, after 6 h of incubation the percentage of the viable HeLa cells treated with free GOS was reduced insignificantly as compared to the non-treated control. The coPLA and coPLA/PEG mats also did not display any statistically significant antiproliferative activity – the viable HeLa cells were 90.1 ± 7.2% and 82.4 ± 11.5%, respectively. In contrast to them, the coPLA/GOS and coPLA/PEG/GOS mats manifested high antiproliferative activity towards HeLa cells – the cell viability was decreased to 44.0 ± 9.4% and 52.1 ± 11.3% for coPLA/GOS and coPLA/PEG/GOS, respectively. It is noteworthy that after coating with a film of crosslinked QCh the nanofibrous mats exhibited higher antiproliferative activity as compared to the non-coated mats. The percentage of viable HeLa cells was 29.5 ± 10.4%; 32.9 ± 9.1%; 31.3 ± 7.7% and 29.9 ± 6.2% for coPLA/crQCh-coat, coPLA/PEG/crQCh-coat, coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats, respectively. After 24-h incubation (Fig. 10B) the GOS-containing mats and those coated with a film of crosslinked QCh displayed high cytotoxicity. The percentage of viable HeLa cells after contact with these mats was reduced to: 27.8 ± 7.3% for coPLA/GOS, 31.6 ± 4.0% for coPLA/PEG/GOS, 29.0 ± 10.4% for coPLA/crQCh-coat and 26.2 ± 6.3% for coPLA/PEG/crQCh-coat mats, respectively. The

strongest decrease in the proliferative activity of HeLa cells was observed in the case of mats containing a combination of GOS and QCh. For coPLA/GOS/crQCh-coat and coPLA/PEG/GOS/crQCh-coat mats the cell viability dropped down to 20.0 ± 3.2% and 22.8 ± 2.5%. It should be noted that free GOS (positive control) reduced much less the cell viability – to 50.7 ± 6.5%. After 48 h (Fig. 10C) and 72 h (Fig. 10D) of incubation, the inhibition of the growth of HeLa cells in the presence of all studied nanofibrous mats was close to that observed after 24-h incubation. The most pronounced antiproliferative effect was displayed by mats containing both GOS and QCh. This is probably due to synergistic action of QCh and GOS. A similar synergistic antiproliferative effect was observed in in vitro and in vivo tests performed with HeLa, MCF-7 and Graffi tumor cells upon contact with nanofibrous mats containing QCh and doxorubicin (Ignatova et al., 2011, 2010; Toshkova et al., 2010). 3.4. Study of the changes in the morphology of HeLa human cervical cancer cells after contact with GOS-containing and QCh (crosslinked)-coated nanofibrous mats After 6-h exposure the control HeLa cells were flat-shaped and possessed numerous microvilli on their surface and extended lamellipodia (Fig. 11A). After 6 h of culturing of HeLa cells in the presence of free GOS, cells with normal morphology were observed, as well as a small number of round-shaped cells and blebbings on the cell surface (Fig. 11B). In contrast to them, in the case of HeLa cells cultured on coPLA mats a change in the cell morphology from flat to round shape was observed, which was accompanied by shrinkage and blebbing of cell surface (Fig. 11C). These alterations of the cell surface characteristic of early apoptosis indicate that the coPLA mats display slight cytotoxicity towards HeLa cells. When HeLa cells were grown on GOS-containing mats, cell narrowing, cell membrane blebbing, holes and cytoplasmic extrusions were observed

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

21

Fig. 11. SEM micrographs of surface ultrastructural characteristics of HeLa cells after 6 h incubation. (A) Untreated HeLa cells, (B) HeLa cells incubated with GOS and HeLa cells incubated with mats of: (C) coPLA, (D) coPLA/GOS, (E) coPLA/crQCh-coat, and (F) coPLA/GOS/crQCh-coat. GOS content 20 ␮g/mL; L – lamellipodia extensions, B – blebs, H – holes, P – pores; magnification: 800×.

(Fig. 11D). Specific changes in the cell surface, characteristic of late apoptosis were observed for HeLa cells cultured on coPLA/crQChcoat and coPLA/GOS/crQCh-coat mats: appearance of pores on the cell surface, cell shrinkage, cell membrane blebs or apoptotic bodies, disappearance or shortening of the microvilli and cytoplasmic extrusions (Fig. 11E and F). In the case of coPLA/crQCh-coat mats lysis of some of the cells was observed, as well. The obtained results are consistent with the data acquired from the MTT assay. The observed alterations in the morphology of the HeLa cells indicate that the coPLA/crQCh-coat, coPLA/GOS/crQCh-coat and coPLA/GOS mats exhibit higher cytotoxicity towards HeLa cells as compared to free GOS. 3.5. Analysis of cell death by staining methods Apoptosis is characterized by internucleosomal DNA fragmentation and morphological changes such as chromatin condensation, cell shrinkage, membrane blebbing and disintegration of the cell into apoptotic bodies. These morphological changes in apoptotic cells are clearly distinguishable at the light microscopic level,

as well. In order to identify the morphological changes in damaged tumor cells the method of intravital double staining with fluorescent dyes (AO and EtBr) (Fig. 12) was applied in order to discriminate between different stages of viability of the cells – viable, early apoptotic, late apoptotic and necrotic cells. The morphological changes occurring in HeLa tumor cells after 24-h contact with the nanofibrous mats – coPLA, coPLA/GOS, coPLA/crQCh-coat and coPLA/GOS/crQCh-coat, were analysed. Untreated HeLa cells (negative control) are characterized by a normal morphological structure – pale green nuclei and bright yellow-green nucleoli, accumulation of orange granules in the perinuclear region. In contrast to them, the contact with the nanofibrous mats results in a decrease in the number of HeLa cells and in the occurrence of cell destruction. Pale green, yelloworange, orange-red coloured dead cells, the majority of which have the morphological characteristics of early or late apoptosis (cell rounding-up, cell shrinkage, condensation and aggregation of chromatin, DNA fragmentation, apoptotic bodies) are observed. A distinct cytotoxic effect is observed in the case of coPLA/GOS mats (Fig. 12D) and coPLA/crQCh-coat mats (Fig. 12E). A significant

22

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

Fig. 12. Fluorescence micrographs of AO and EtBr double-stained HeLa tumor cells incubated for 24 h in the presence of nanofibrous mats. (A) Untreated HeLa cells and cells after incubation with: (B) free GOS, (C) coPLA mat, (D) coPLA/GOS mat, (E) coPLA/crQCh-coat mat, and (F) coPLA/GOS/crQCh-coat mat. Bar = 20 ␮m.

number of cells have yellow-orange to red coloured nuclei and cytoplasm. These changes are considerably greater in the case of HeLa cells cultured on coPLA/GOS/crQCh-coat mats (Fig. 12F). Apoptotic morphological changes have been observed in HEp-2 laryngeal tumor cells after 6-h treatment with different GOS concentrations (Konac et al., 2005); in HT-29 human colorectal tumor cells (Zhang et al., 2003); in myelogenous leukemic cells K-562 (Ergun et al., 2004). Our results indicate significant morphological changes in HeLa cells exposed to GOS-containing mats and mats coated with QCh (crosslinked), as well as QCh (crosslinked)-coated mats, containing GOS and are in accordance with the data from the MTT assay. The morphological changes in HeLa tumor cells occurring after 24-h contact with the nanofibrous mats – coPLA, coPLA/GOS, coPLA/crQCh-coat and coPLA/GOS/crQCh-coat, were analysed after staining according to the Pappenheim’s method, as well. The untreated HeLa cells have normal morphology, whereas the treated cells are round-shaped, characterized by a loss of adhesion and intercellular contacts, with condensed and aggregated chromatin in the form of dense compact masses and apoptotic bodies. In the case of the coPLA/crQCh-coat (Fig. 13E), coPLA/GOS (Fig. 13D) and

coPLA/GOS/crQCh-coat (Fig. 13F) a significant decrease in the cell count was also observed. The observed changes in the morphology of the HeLa tumor cells occurring after contact with the GOS-containing nanofibrous mats are in accordance with the morphological changes in three human breast cancer cell lines treated with GOS, described by other authors (Gilbert et al., 1995). No data were found in the literature concerning the antitumor effect of electrospun nanofibrous materials containing GOS towards HeLa human cervical cancer cells. In our previous studies we have demonstrated that nanofibrous materials containing QCh and doxorubicin display good cytotoxicity towards HeLa and MCF-7 tumor cells in vitro (Ignatova et al., 2011, 2010) and good antitumor activity towards a solid myeloid tumor initially induced by the Graffi virus in vivo (Toshkova et al., 2010). The data regarding the mechanism according to which chitosan and its derivatives exhibit their antitumor activity are controversial. It is known that chitosan which contains positively charged amino groups interacts electrostatically with the negatively charged biological membranes of tumor cells. This induces changes in the cell permeability and inhibits the cell growth and division (Koyano et al.,

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

23

Fig. 13. Light micrographs of HeLa tumor cells cultured for 24 h on glass slips in the presence of nanofibrous mats, stained by the Pappenheim’s method: (A) untreated HeLa cells, and cells after incubation with: (B) free GOS, (C) coPLA mat, (D) coPLA/GOS mat, (E) coPLA/crQCh-coat mat, and (F) coPLA/GOS/crQCh-coat mat.

1998). A similar mechanism has been proposed by Huang et al. (2006) for the antitumor activity of quaternized chitosan derivatives. Murata et al. (1989) have made the assumption that chitosan directly inhibits the proliferation of tumor cells and induces apoptosis. GOS is a natural polyphenolic compound which displays antiproliferative and proapoptotic properties towards various tumor cells in vitro and towards experimental tumors in vivo (Konac et al., 2005; Le Blanc et al., 2002; Wu et al., 1989; Ye et al., 2010). It may be assumed that the observed strong cytotoxic effect of GOS-containing nanofibrous mats coated with QCh (crosslinked) on HeLa tumor cells is due to combining of the effects of QCh and the natural polyphenol GOS. The thin QCh film coating possesses positively charged tertiary amino groups which can interact electrostatically with negatively charged segments of the tumor cell membrane and therefore induce changes in the cell membrane permeability. This facilitates the penetration of GOS in the tumor cell as well as its binding at the site of contact to proteins from the Bcl-2 family (overexpressed in tumor cells and situated on the outer mitochondrial membrane) or to proapoptotic proteins (Bak). Most probably Bcl-2 inhibition or Bak activation occurs or a combination of the two effects simultaneously (Oliver et al., 2005).

4. Conclusions GOS-containing nanofibrous materials based on coPLA or coPLA/PEG were prepared by elecrospinning and subsequently coated with a thin film of QCh. The nanofibrous mats containing GOS (both non-coated and QCh-coated) displayed higher antiproliferative activity towards HeLa tumor cells as compared to free GOS. Furthermore, the cytotoxicity of the nanofibrous mats containing a combination of GOS and QCh was considerably higher as compared to that of GOS-containing mats or QCh-coated mats alone. This fact was attributed to the synergistic action of QCh and GOS. The induction of apoptosis is one of the major mechanisms of the antitumor activity of the prepared nanofibrous materials, which is verified by the performed fluorescence microscopy analyses and scanning electron microscopy observations. These results indicate that the obtained nanofibrous materials are promising candidates as systems for local delivery of antitumor drugs in the treatment of cervical tumors. Acknowledgment Financial support from the Bulgarian National Science Fund (Grant DO-02-164/2008) is gratefully acknowledged.

24

M. Ignatova et al. / International Journal of Pharmaceutics 436 (2012) 10–24

References Allen, T.M., 2002. Ligand-targeted therapeutics in anticancer therapy. Nat. Rev. Cancer 2, 750–763. ˜ I., Miralles, B., Acosta, N., Galed, G., Heras, Aranaz, I., Mengíbar, M., Harris, R., Panos, Á., 2009. Characterization of chitin and chitosan. Curr. Chem. Biol. 3, 203–230. Bshena, O., Heunis, T.D.J., Dicks, L.M.T., Klumperman, B., 2011. Antimicrobial fibers: therapeutic possibilities and recent advances. Future Med. Chem. 3, 1821–1847. Cui, W., Li, X., Zhou, S., Weng, J., 2008. Degradation patterns and surface wettability of electrospun fibrous mats. Polym. Degrad. Stabil. 93, 731–738. Duncan, R., 2003. The dawning era of polymer therapeutic. Nat. Rev. Drug Discov. 2, 347–360. Ergun, M.A., Konac, E., Erbas, D., Ekmekci, A., 2004. Apoptosis and nitric oxide release induced by thalidomide, gossypol and dexamethasone in cultured human chronic myelogenous leukemic K-562 cells. Cell Biol. Int. 28, 237–242. Gilbert, N.E., O’Reilly, J.E., Chang, C.J., Lin, Y.C., Brueggemeier, R.W., 1995. Antiproliferative activity of gossypol and gossypolone on human breast cancer cells. Life Sci. 57, 61–67. Huang, R., Mendis, E., Rajapakse, N., Kim, S.-K., 2006. Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS). Life Sci. 78, 2399–2408. Ignatova, M., Manolova, N., Markova, N., Rashkov, I., 2009. Electrospun non-woven nanofibrous hybrid mats based in chitosan and PLA for wound-dressing applications. Macromol. Biosci. 9, 102–111. Ignatova, M., Manolova, N., Rashkov, I., 2007. Novel antibacterial fibers of quaternized chitosan and poly(vinyl pyrrolidone) prepared by electrospinning. Eur. Polym. J. 43, 1112–1122. Ignatova, M., Manolova, N., Toshkova, R., Rashkov, I., Gardeva, E., Yossifova, L., Alexandrov, M., 2010. Electrospun nanofibrous mats containing quaternized chitosan and polylactide with in vitro antitumor activity against HeLa cells. Biomacromolecules 11, 1633–1645. Ignatova, M., Starbova, K., Markova, N., Manolova, N., Rashkov, I., 2006. Electrospun nano-fibre mats with antibacterial properties from quaternised chitosan and poly(vinyl alcohol). Carbohydr. Res. 341, 2098–2107. Ignatova, M., Yossifova, L., Gardeva, E., Manolova, N., Toshkova, R., Rashkov, I., Alexandrov, M., 2011. Antiproliferative activity of nanofibers containing quaternized chitosan and/or doxorubicin against MCF-7 human breast carcinoma cell line by apoptosis. J. Bioact. Compat. Polym. 26, 539–551. Jia, Z., Shen, D., Xu, W., 2001. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydr. Res. 333, 1–6. Kato, Y., Onishi, H., Machida, Y., 2005. Contribution of chitosan and its derivatives to cancer chemotherapy. In Vivo 19, 301–310. Kim, C.H., Choi, J.W., Chun, H.J., Choi, K.S., 1997. Synthesis of chitosan derivatives with quaternary ammonium salt and their antibacterial activity. Polym. Bull. 38, 387–393. Kim, J.H., Kim, Y.S., Park, K., Lee, S., Nam, H.Y., Min, K.H., Jo, H.G., Park, J.H., Choi, K., Jeong, S.Y., Park, R.W., Kim, I.S., Kim, K., Kwon, I.C., 2008. Antitumor efficacy of cisplatin-loaded glycol chitosan nanoparticles in tumor-bearing mice. J. Control. Release 127, 41–49. Kim, J.Y., Lee, J.K., Lee, T.S., Park, W.H., 2003. Synthesis of chitooligosaccharide derivative with quaternary ammonium group and its antimicrobial activity against Streptococcus mutans. Int. J. Biol. Macromol. 32, 23–27. Konac, E., Ekmekci, A., Yurtcu, E., Ergun, M.A., 2005. An in vitro study of cytotoxic effects of gossypol on human epidermoid larynx carcinoma cell line (Hep-2). Exp. Oncol. 27, 81–83. Koyano, T., Minoura, N., Nagura, M., Kobayashi, K., 1998. Attachment and growth of cultured fibroblast cells on PVA/chitosan-blended hydrogels. J. Biomed. Mater. Res. 39, 486–490. Le Blanc, M., Russo, J., Kudelka, A.P., Smith, J.A., 2002. An in vitro study of inhibitory activity of gossypol, a cottonseed extract, in human carcinoma cell lines. Pharmacol. Res. 46, 551–555. Ligueros, M., Jeoung, D., Tang, B., Hochhauser, D., Reidenberg, M.M., Sonenberg, M., 1997. Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells. Br. J. Cancer 76, 21–28. Macoska, J.A., Adsule, S., Tantivejkul, K., Wang, S., Pienta, K.J., Lee, C.T., 2008. (−)Gossypol promotes the apoptosis of bladder cancer cells in vitro. Pharmacol. Res. 58, 323–331. Meng, Y., Tang, W., Dai, Y., Wu, X., Liu, M., Ji, Q., Ji, M., Pienta, K., Lawrence, T., Xu, L., 2008. Natural BH3 mimetic (−)-gossypol chemosensitizes human prostate

cancer via Bcl-xL inhibition accompanied by increase of Puma and Noxa. Mol. Cancer Ther. 7, 2192–2202. Mossmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Murata, J., Saiki, I., Nishimura, S., Nishi, N., Tokura, S., Azuma, I., 1989. Inhibitory effect of chitin heparinoids on the lung metastasis of B16–B16 melanoma. Jpn. J. Cancer Res. 80, 866–872. Natu, M.V., de Sousa, H.C., Gil, M.H., 2010. Effects of drug solubility, state and loading on controlled release in bicomponent electrospun fibers. Int. J. Pharm. 397, 50–58. Oliver, C.L., Miranda, M.B., Shangary, S., Land, S., Wang, S., Johnson, D.E., 2005. (−)Gossypol acts directly on the mitochondria to overcome Bcl-2- and Bcl-X(L)mediated apoptosis resistance. Mol. Cancer Ther. 4, 23–31. Paneva, D., Ignatova, M., Manolova, N., Rashkov, I., 2009. Novel chitosan-containing micro- and nanofibrous materials by electrospinning: preparation and biomedical application. In: Chang, W.N. (Ed.), Nanofibers: Fabrication, Performance, and Applications. Nova Science Publishers, New York, pp. 73–151 (Chapter 3). Park, J.H., Saravanakumar, G., Kim, K., Kwon, I.C., 2010. Targeted delivery of low molecular drugs using chitosan and its derivatives. Adv. Drug Deliv. Rev. 62, 28–41. Rasband, W.S., 1997–2006. ImageJ. U.S. National Institute of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/. Spasova, M., Manolova, N., Paneva, D., Mincheva, R., Dubois Ph Rashkov, I., Maximova, V., Danchev, D., 2010. Polylactide stereocomplex-based electrospun materials possessing surface with antibacterial and haemostatic properties. Biomacromolecules 11, 151–159. Spasova, M., Mincheva, R., Paneva, D., Manolova, N., Rashkov, I., 2006. Perspectives on: criteria for complex evaluation of the morphology and alignment of electrospun polymer nanofibers. J. Bioact. Compat. Polym. 21, 465–479. Spasova, M., Paneva, D., Manolova, N., Radenkov, P., Rashkov, I., 2008. Electrospun chitosan-coated fibers of poly(l-lactide) and poly(l-lactide)/poly(ethylene glycol): preparation and characterization. Macromol. Biosci. 8, 153–162. Ta, H.T., Dass, C.R., Dunstan, D.E., 2008. Injectable chitosan hydrogels for localised cancer therapy. J. Control. Release 126, 205–216. Toshkova, R., Manolova, N., Gardeva, E., Ignatova, M., Yossifova, L., Rashkov, I., Alexandrov, M., 2010. Antitumor efficacy of electrospun nanofibrous implants containing quaternized chitosan and doxorubicin against Graffi myeloid tumor. Int. J. Pharm. 400, 221–233. Wahab, S.I.A., Abdul, A.B., Alzubairi, A.S., Elhassan, M.M., Mohan, S., 2009. In vitro ultramorphological assessment of apoptosis induced by zerumbone on (HeLa). J. Biomed. Biotechnol. 2009, 769568. Wei, A., Wang, J., Wang, X., Wei, Q., Ge, M., Hou, D., 2010. Preparation and characterization of the electrospun nanofibers loaded with clarithromycin. J. Appl. Polym. Sci. 118, 346–352. WHO, 2006. Guidelines on Standard Operating Procedures for Haematology, http://searo.who.int/en/Section10/Section17/Section53/Section480 1732.htm. Wu, Y.W., Chik, C.L., Knazek, R.A., 1989. An in vitro and in vivo study of antitumor effects of gossypol on human SW-13 adrenocortical carcinoma. Cancer Res. 49, 3754–3758. Xu, X., Yang, L., Xu, X., Wang, X., Chen, X., Liang, Q., Zeng, J., Jing, X., 2005. Ultrafine medicated fibers electrospun from W/O emulsions. J. Control. Release 108, 33–42. Yancheva, E., Paneva, D., Manolova, N., Mincheva, R., Danchev, D., Dubois, P.H., Rashkov, I., 2010. Tuning of the surface biological behavior of poly(llactide)-based electrospun materials by polyelectrolyte complex formation. Biomacromolecules 11, 521–532. Ye, W., Chang, H.L., Wang, L.S., Huang, Y.W., Shu, S., Sugimoto, Y., Dowd, M.K., Wan, P.J., Lin, Y.C., 2010. Induction of apoptosis by (−)-gossypol-enriched cottonseed oil in human breast cancer cells. Int. J. Mol. Med. 26, 113–119. Zhang, K., Qian, Y., Wang, H., Fan, L., Huang, C., Yin, A., Mo, X.J., 2010. Genipin-crosslinked silk fibroin/hydroxybutyl chitosan nanofibrous scaffolds for tissue-engineering application. J. Biomed. Mater. Res. Part A 95, 870–881. Zhang, M., Liu, H., Guo, R., Ling, Y., Wu, X., Li, B., Roller, P.P., Wang, S., Yang, D., 2003. Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells. Biochem. Pharmacol. 66, 93–103. Zhou, H., Green, T.B., Joo, Y.L., 2006a. The thermal effects on electrospinning of polylactic acid melts. Polymer 47, 7497–7505. Zhou, H., Kim, K.W., Giannelis, E.P., Joo, Y.L., 2006b. Polymeric nanofibers. In: Reneker, D.H., Fong, H. (Eds.), ACS Symposium Series, 918. American Chemical Society, Washington, DC, pp. 217–230.