Materials Science and Engineering C 33 (2013) 461–465

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Electrospun polyvinyl alcohol/chitosan composite nanofibers involving Au nanoparticles and their in vitro release properties Eryun Yan a, Shan Fan a, Xunqi Li a, Cheng Wang b,⁎, Zhiyao Sun b, Liang Ni b, Deqing Zhang a a b

College of Materials Science and Engineering, Qiqihar University, Qiqihar 161006, PR China Key Laboratory of Functional Inorganic Material Chemistry (Heilongjiang University), Ministry of Education, PR China

a r t i c l e

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Article history: Received 24 May 2012 Received in revised form 9 August 2012 Accepted 17 September 2012 Available online 23 September 2012 Keywords: Composite Chitosan Nanoparticles Electrospinning

a b s t r a c t Au nanoparticles (Au NPs) containing polyvinyl alcohol (PVA)/chitosan (CS) composite nanofibers were successfully prepared by a simple and effective method called electrospinning. Au NPs were firstly synthesized under a mild condition with CS as the reducing agent and stabilizer, followed by being mixed with PVA solution and then the resulting fibers were fabricated. The research indicated that Au NPs were indeed doped into the as-prepared fibers and the composite fibers well preserved Au NPs' unique optical characteristics. Additionally, with the adjustment of the weight ratios between PVA and CS, the diameter distribution and the morphology of the nanofibers were largely changed. In vitro drug release experiments demonstrated that the drug release rate can be conveniently controlled by changing the crosslink time. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Recently, electrospinning technology has emerged as a versatile method to produce biomimetic non-woven mats comprising a large network of interconnected fibers and pores [1,2]. A large number of biomacromolecules, such as poly (ε-caprolactone) [3], poly (lactic acid) [4], poly (lactide-co-glycoside) [5], collagen [6] and gelatin [7] were successfully fabricated into non-woven mats by the electrospinning method. Especially, a series of electrospun nanofibers based on chitin and chitosan have gained great attention owing to their extensive biomedical applications in such as tissue engineering scaffolds, drug delivery, etc. [8]. As an inorganic functional material, Au NPs are of particular interest due to their unique optical and electronic characteristics as well as their excellent biocompatibility [9]. They have been exploited for a wide variety of applications, such as biological [10] and chemical sensing [11], surface-enhanced Raman scattering [12], photovoltaic cells [13], surface patterning [14], and the diagnosis and treatment of cancer [15]. If Au NPs can be integrated into CS nanofibers, it would largely expand the applications of CS in the field of biomedicine. CS is a copolymer of N-acetyl-D-glucosamine and D-glucosamine that is produced by alkaline deacetylation of chitin. It has been used as both a reducing agent and stabilizer to synthesize Ag NPs through γ [16] or UV irradiation [17]. Based on the above work, Cheng and co-authors prepared Ag NPs containing CS/gelation nanofibers in a mild condition [7], in which microcrystalline chitosan was utilized as the initiating material to fabricate Ag NPs. But the preparation procedure was relatively fussy. In the present study, HAuCl4 was reduced ⁎ Corresponding author. Tel.: +86 451 86608038. E-mail address: [email protected] (C. Wang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.014

by CS under heating in the presence of acrylic acid, which was responsible for the solubility of CS. The obtained Au NPs stabilized by CS was mixed with 10% PVA solution at different weight ratios, which was electrospun to produce Au NPs involving PVA/CS hybrid nanofibers. Although PVA/CS nanofibers with uniform structure were successfully fabricated in previous work and they were also used to load other functional materials [18,19], PVA/CS based nanofibers in the application of drug delivery have not been reported. Herein, we will explore the drug loading and release abilities of Au NPs involving PVA/CS hybrid nanofibers. Taking the diagnosis and treatment applications of Au NPs, the biocompatible, non-toxic, antibacterial and biogradable properties of chitosan, and the simple preparation process into account, we anticipate that the Au NPs loaded with CS/PVA hybrid nanofibers will have tremendous potential in advanced biomedical applications. 2. Materials and methods 2.1. Materials Chitosan (CS, degree of deacetylation 0.90, MW 200 kDa) provided by Nantong Shuanglin Biological Product Inc. was refined firstly. PVA (Dp = 1750) was supplied by Changchun Institute of Applied Chemistry Chinese Academy of Science (China). HAuCl4 glutaraldehyde (GA) and acrylic acid (AA) were used as received. 2.2. Preparation of Au NPs stabilized by chitosan 0.12 g of purified CS was dissolved in an aqueous AA solution (20 mL, 0.06 g AA). Then, 50 μL of 0.1 M HAuCl4 was added into the

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system. The mixture was heated at 70 °C until a red solution is generated. Au NPs stabilized by chitosan (Au/CS) were obtained. 2.3. Preparation of Au containing PVA/CS spinning solutions and electrospinning process 10% (w/t) PVA solution was mixed with the Au/CS suspension mentioned above at weight ratios of 100/0, 90/10, 80/20, 70/30, 60/40 and 50/50. The mixtures were stirred at room temperature for 48 h, which were used for electrospinning. The electrospinning process was done on home-made setup as described in our previous study [20]. The applied voltage is 18 kV and the tip-to-collector distance is 20 cm. 2.4. In vitro drug release experiments 10 mg of doxorubicin (DOX) was dispersed into 10 g of Au NPs containing PVA/CS solution with the weight ratio of 70/30, which was stirred for 24 h. The mixed solution was electrospun into nanofibers. The obtained DOX loaded hybrid nanofibers were taken off from the aluminum foil, and immersed into 1.25 wt.% glutaraldehyde (GA)/acetone mixture for 1, 2, 4 and 8 h, respectively. After immersion, the hybrid nanofibers were dried in the ventilation cabinet for 24 h. Then, the dried nanofibers were immersed into 2 mL pH 7.4 phosphate buffer solution (PBS) at 37 °C. At periodic interval, the release media was withdrawn and another 2 mL fresh PBS solution was added. The amount of doxorubicin was determined by measuring the adsorption at 470 nm (TU-1901 spectrometer, China) and using a calibration curve. 2.5. Characterization The morphology of nanofibers was observed using scanning electron microscopy (SEM, Hitachi S-4300), and the fiber samples were coated with gold prior to observation. The hybrid nanofibers were electrospun onto Cu network, which was used for transmission electron microscopy (TEM, Hitachi H-7650) investigation, using LaB6 radiation. The solutions in quartz glassware or the nanofibers electrospun on quartz plate were directly performed on a TU-1901 spectrometer for UV–vis spectra analysis. CS powder or the as-prepared nanofibers were mixed with dry KBr, triturated, and pressed into a tablet, which was used for the FT-IR spectra examination on a Magna 560 FT-IR spectrometer. 3. Results and discussion CS is a weak base, and the pKb value of the D-glucosamine residue is about 6.2–7.0. As a result, CS is insoluble at neutral and alkaline environments but soluble in acidic condition. Based on this, at first, we mixed CS and HAuCl4 together and attempted to make CS dissolve in this system and reduce HAuCl4 further. But, it failed. CS and HAuCl4 deposited simultaneously. We deduced that the acidity of solution was not enough to dissolve CS. Afterwards, a determined amount of AA was added into the system, and CS dissolved completely without any precipitation. Consequently, Au NPs stabilized by CS were successfully prepared and their TEM image was given in Fig. 2a. As can be seen, Au NPs were well dispersed in the solution. This solution was not electrospinnable since CS has changed into a polyelectrolyte in acidic conditions. It was reported that the repulsive forces between ionic groups on polyelectrolyte arise due to the fact that the application of a high electric field during electrospinning would restrict the formation of continuous fibers [21]. To solve this problem, another biodegradable polymer PVA was added into the system, and nanofibers with ideal morphology were obtained. To apply the hybrid nanofibers in biomedical field, as many Au NPs as possible would be introduced into the resulting fibers, thus the content of CS in the fibers was also increased. Fig. 1 showed the SEM images of Au containing PVA/CS composite nanofibers with

different PVA/CS weight ratios. As can be seen, the average diameter of pure PVA nanofibers (Fig. 1a) was 675 nm, and it was the largest in all the samples. The surface of the fibers was quite smooth. Conglutination among the fibers was observed obviously due to the slow evaporation of the solvent. When a little amount of CS-stabilized Au NPs was added into the PVA solution (Fig. 1b), the morphology of the fibers had no conspicuous change, except that the average size of the fibers decreased to 600 nm. With the ratios between PVA and CS reduced to 80/20 and 70/30, the average diameter of the fibers dropped to 350 and 260 nm, respectively. In addition, the conglutination disappeared and the cross section size of the fibers was quite uniform. It may be because that the existence of CS would improve the morphology and decrease the size of the fibers. In fact, the morphology of these hybrid nanofibers was basically same to the reported Ag/PVA/CS non-woven mats [18], but the diameter distributions of both were not completely consistent with each other, owing to the different molecule weights of PVA and CS used. When the ratio of PVA/CS was further decreased (Fig. 1e), spindle-like fibers appeared and the cross section size in one fiber became inhomogeneous. When the ratio was reduced to as low as 50/50, the average diameter of the fibers was only 95 nm. More spindle-like structures existed on the fibers. Besides, a striking feature observed was that, rupture on the fibers arose. According to Ref. [21], high content of CS went indeed against forming continuous fibers. The existence and distribution of Au NPs in the composite nanofibers were characterized by TEM, presented in Fig. 2. Fig. 2a illustrated the Au NPs dispersed in CS solution. These particles were spherical mostly in shape with a mean size of less than 10 nm and they dispersed well in the solution with no congregation. Fig. 2b gave the TEM image of Au containing CS/PVA nanofibers. It can be seen that Au NPs have exactly combined into the hybrid fibers, and they distributed well in the fibers. The presence of Au NPs in the as-prepared fibers was further approved by UV–vis spectra, shown in Fig. 3. The Au NPs stabilized by CS (Fig. 3a) displayed a characteristic absorption peak at approximately 525 nm, which was assigned to the surface plasmon resonance (SPR) band of Au NPs [9]. The standing PVA/CS nanofibers (Fig. 3g) had no absorption around 525 nm, thus they would not interfere with the examination of Au NPs. Fig. 3b–f presented the UV–vis spectra of Au NPs containing CS/PVA nanofibers. It can be seen that these hybrid nanofiber samples also exhibited the same SPR band to the Au NPs in CS solution, revealing that they well preserved the optical properties of the encapsulated Au NPs, and hence the hybrid fibers should also possess the functionalities originating from the Au NPs' unique optical characteristics. As the amount of Au NPs in the fibers increased, the intensity of SPR peak increased accordingly, implying that more Au NPs have been doped into the resulting fibers. Fig. 4 showed the FT-IR spectra of Au containing PVA/CS nanofiber mats blended in different weight ratios, pure PVA nanofibers and CS powder. The peaks at 3430, 2940, 1430 and 1090 cm −1 were attributed to the ν(O\H), νs(CH2), δ(CH\O\H) and ν(C\O) of pure PVA, and they were well preserved in the resulting hybrid fibers. The peaks at 1638 and 1560 cm −1 belonging to the amide band of CS both existed in the hybrid fibers. But, with the increase of the amount of CS in the fibers, the intensity of the peak at 1430 cm −1 was weakened to some extent, implying that hydrogen bond between CS and PVA chains formed. For the future clinical applications, as many Au NPs as possible would be incorporated into the resulting fibers. But when the weight ratio between PVA and CS was lower than 70/30, spindle-like fibers appeared and the cross section size in one fiber became inhomogeneous (Fig. 1e). Consequently, at last, PVA/CS nanofibers (weight ratio of 70/30) with relatively high amount of Au NPs and ideal morphology were chosen for the drug delivery study. To investigate the in vitro drug release behavior of the Au NPs containing PVA/CS hybrid nanofibers, an antitumor drug DOX was loaded into the fibers with an encapsulation efficiency of 100%. This

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Fig. 1. SEM images of Au containing PVA/CS composite nanofibers with different PVA/CS weight ratios: (a) 100/0, (b) 90/10, (c) 80/20, (d) 70/30, (e) 60/40 and (f) 50/50. All scale bar=2 μm.

high encapsulation efficiency that originated from that all the drugs in the solution was loaded into the resulting fibers with no DOX vaporized in the electrospinning process. In fact, in the initial drug release exploration, the DOX molecules in the as-prepared nanofibers

with different weight ratios between PVA and CS (not crosslinked) were all released within 1 h (data not given), therefore, the discrepancy in the size of nanofibers had no obvious effect on the drug release speed. Consequently, the purpose of sustaining drug release

Fig. 2. TEM images of Au NPs stabilized by CS (a) and Au NPs containing PVA/CS nanofibers (b).

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Fig. 5. Typical in vitro release profile of DOX loaded Au containing PVA/CS nanofibers (weight ratio of PVA/CS: 70/30) with different crosslink time at 37 °C and pH 7.4.

can't be obtained from the hybrid fibers (not crosslinked) and moderately crosslink of the as-prepared fibers was necessary. Herein, GA was used to selectively crosslink the hybrid nanofibers, and the drug release rate was largely slowed. Fig. 5 showed the DOX release profile (at 37 °C, pH 7.4) of the drug loaded nanofibers with different crosslink time. The percentage released was calculated based on the initial DOX amount in the hybrid nanofibers. For the nanofibers crosslinked for 1 h, an initial burst release of about 21% of the total loaded drug in the first hour was observed, and about 63% of the drug was released within 48 h. With the crosslink time extended to 2 h, a small release burst was also observed (24%), and the ultimate DOX amount released decreased to 47%. The release burst in the initial stage of test can be explained as follows: the swelling of the soluble polymers in physiological condition (pH = 7.4) was inevitable although part of the \NH2 on CS chains and DOX was crosslinked together, accordingly, DOX molecules near the surface of the hybrid fibers were diffused into the medium quickly. After that, the drug deep into the fibers was released gradually. For the hybrid nanofibers crosslinked for 4 h and 8 h, there was almost no burst release, and the release curves were mild all along within the test time. Finally, only 25% and 23% of the DOX were examined, indicating that most of the drugs were bound on the CS chains by GA and the swelling of polymers was effectively restrained. It can be concluded that the

DOX release rate can be effectively adjusted by changing the crosslink time for the nanofibers and the nanofibers crosslinked by GA were desirable carriers for anticancer drug delivery systems. In addition, besides the swelling of polymers, the effect of the morphology change of fibers on the drug release rate was also proposed. The SEM images of DOX loaded PVA/CS hybrid nanofibers involving Au NPs and the fibers crosslinked by GA were presented in Fig. 6. The as-prepared drug encapsulated nanofibers (Fig. 6a) were well distributed on the aluminum foil and some fibers were stuck together due to the reservation of solvent. After the nanofibers were crosslinked for 1 h, the fiber sample looks like a fishing net resulting from the conglutination of more fibers. With the further increase of crosslink time, the conglutination degree was larger and larger, at last, the fibers formed a film (Fig. 6e). For the same sample, the total surface area of fibers would largely exceed that of a film. Thus, compared with the film, the nanofibers were much more in favor of the release of drug. As a result, the DOX release rate of the nanofibers with short crosslink time was larger than the ones with long crosslink time. The results were in agreement with the DOX release profiles.

4. Conclusions PVA/CS nanofibers involving Au NPs with mean size less than 10 nm were successfully fabricated by the electrospinning method. With the increase of Au NPs, the average diameter of the hybrid nanofibers decreased from 675 nm to 95 nm. Moreover, the asprepared fibers well preserved Au NPs' unique optical characteristics. FT-IR results indicated that hydrogen bond between CS and PVA chains formed. These nanofibers held advantages in terms of facile, versatile synthesis, the unique optical and electronic characteristics from Au NPs, the high drug encapsulation efficiency and the controllable drug release rate provided by the crosslink with GA. Thus, the PVA/CS nanofibers involving Au NPs may serve as a good candidate for loading and releasing some functional nanomaterials and may also be extended to other advanced biomedical applications.

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The study has been supported by the NSFC (51143002, 51273056, 21072049, 21202091), CPDF (201104456), NSF of Heilongjiang (E201118, E201144), APAFHP (2010td03, 1251G070), IFF of Heilongjiang University (Hdtd2010-11), and Program for Young Teachers Scientific Research in Qiqihar University (2010k-M25).

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Fig. 6. SEM images of DOX loaded Au containing PVA/CS hybrid nanofibers (weight ratio of PVA/CS: 70/30) (a) and the fibers crosslinked by GA for 1 h (b), 2 h (c), 4 h (d) and 8 h (e). All scale bar=2 μm.

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