Achieving long–term sustained drug delivery for electrospun

Science and Engineering, Donghua University, No. 2999 North ... College of Materials and Textile, Zhejiang Sci–Tech University, Hangzhou 310018, Chi...
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Achieving Long-Term Sustained Drug Delivery for Electrospun Biopolyester Nanofibrous Membranes by Introducing Cellulose Nanocrystals Miao Cheng,† Zongyi Qin,*,† Shuo Hu,† Shu Dong,† Zichu Ren,† and Houyong Yu*,‡ †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials and College of Material Science and Engineering, Donghua University, No. 2999 North Renmin Road, Songjiang, Shanghai 201620, China ‡ The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci−Tech University, Hangzhou 310018, China ABSTRACT: Electrospun nanofibrous membranes of poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) seems not to be ideal for biomedical applications because of their hydrophobicity, and high crystallinity, as well as weak mechanical properties. It is found that hydrophilic drug such as tetracycline hydrochloride (TH) generally is located on the hydrophobic surface of electrospun PHBV nanofibrous membranes, leading to fast drug release. Therefore, we used cellulose nanocrystals (CNCs) as rigid organic nanofillers for PHBV nanofibrous membranes to enhance their mechanical, thermal, and hydrophilic properties. The influences of the CNC contents on microstructures and properties of composite nanofibrous membranes were studied. It is found that at 6 wt % CNC content, the increase of tensile strength by 125%, Young’s modulus by 110%, and maximum decomposition temperature (Tmax) by 24.3 °C could be achieved, which could be contributed to strong hydrogen bonding between PHBV and CNCs. Moreover, with the introducing of the hydrophilic CNCs, the hydrophilicity of composite nanofibrous membranes was improved gradually. More importantly, good cytocompatibility, high drug loading and long-term sustained release property of composite nanofibrous membranes could be achieved. The maximum drug loading and drug loading efficiency were 25 and 98.8%, respectively, and more than 86% drug content was delivered within 540 h for the nanofibrous composite membranes with 6 wt % CNC content. KEYWORDS: electrospun nanofibrous membranes, composite nanofiber, PHBV, cellulose nanocrystals, sustained drug delivery

1. INTRODUCTION Nanoscale fibrous systems possess unique structure and properties including their large surface area to volume ratio, high porosity, as well as controllable crystal quality and degradation behaviors, and thus exhibit great potential in biomedical applications such as wound healing and tissue engineering.1−5 Electrospinning is one kind of most commonly approach for fabricating drug-loaded fibers, which have almost no impact on molecular structure and bioactivity of the loaded drugs during mild spinning process, and reduce in vitro drug burst release.1,2,4,6 It has been demonstrated that the unique structure of electrospun fibers can provide some distinct advantages in drug delivery applications, including high drug loading and encapsulation efficiency, suitable interactions of nanofibrous membranes with various cell cultures, ability to modulate release, process simplicity, and cost-effectiveness.7−11 Most importantly, the drug release process can be well modulated by controlling the composition of nanofibers as well as the microstructure of membranes, and moreover, the bioavailability of the drug-loaded in the nanofibers can be significantly enhanced. How to develop long-acting drug © XXXX American Chemical Society

formulations is still a great challenge for overcoming adherence and emerging drug resistance, meanwhile, the control of drug release can be beneficial to decrease the use of drugs and the frequency of drug administration and toxicity through improving the effectiveness of drugs.1,2,6 Polymer carriers used in electrospun fibers play an important role in designing suitable release of pharmaceutical dosage especially as delayed as well as modified dissolution. Furthermore, biodegradable polyesters are believed to be one kind of the most promising carriers, for example, polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic) acid (PLGA), and polycaprolactone (PCL).2,3,5 As one of the most important members of biodegradable thermoplastic polyester produced by bacteria, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer commonly composed of hydroxybutyrate (HB) units with less hydroxyvalerate (HV) units (0− 24%) has been evaluated as a biomedical material for in vitro Received: March 18, 2017 Accepted: May 30, 2017 Published: May 30, 2017 A

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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cellulose (MCC) was obtained from Shanghai Chemical Reagents (China). Hydrochloric acid (HCl), ammonia solution (NH3·H2O), acetone, methanol, chloroform, N,N-dimethylformamide (DMF), potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen (Na2HPO4·12H2O) were purchased from Guoyao Group Chemical Reagent CO., LTD (China). The human MG-63 cells (an osteosarcoma cell line), Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM), Cell Counting Kit-8, and ActinTracker Green were received from Shanghai Institute of Biochemistry and Cell Biology (China), Gibco (USA), Beyotime Institute of Biotechnology (China), and Biyuntian Biology Technology Co., Ltd. (China), respectively. Tetracycline hydrochloride (TH) was provided from Sigma-Aldrich (USA) without further purification. All other chemicals used were of analytical grade and obtained from Hangzhou Mike Chemical Agents Company (China). 2.2. Preparation. 2.2.1. Extraction of Cellulose Nanocrystals. The extraction of the CNCs was carried out by the previous reported method.19 Briefly, the MCC was first putted into the hydrothermal kettle filled with HCl aqueous solution. After the reaction at 110 °C for 3 h, the hydrothermal kettle was cooled to room temperature, and 3 M ammonia solution was added to neutralize the residual HCl in the as-prepared CNC suspension. Then the suspension was centrifuged at 12000 r min−1 for 20 min at 4 °C and washed with deionized water several times to remove the residual impurities. Finally, the obtained CNCs were dispersed into aqueous solution under sonication (Kunshan Ultrasonic Instruments Co., Ltd., China; 50 W, 40 Hz) for 5 min for forming a stable suspension. 2.2.2. Preparation of Electrospinning Solution. Composite Electrospinning Solution. PHBV powder was first dissolved in chloroform under constant stirring for at least 24 h. After the adding of DMF, the solution was continuously stirred for another 24 h. The final solution was composed of 0.1 g mL−1 PHBV in 9:1 (w/w) chloroform/DMF co-solvent. A solvent exchange approach was applied to disperse the CNCs into organic polymer solution for avoiding the aggregation of the CNCs during the preparation of PHBV nanocomposites. Briefly, the CNCs were first transferred from aqueous suspension to acetone, and then to DMF through several successive centrifugations, and the obtained sediments were fully redispersed under ultrasonic condition. Then 1−10 wt % CNCs suspensions were added into above-mentioned PHBV solution for preparing the electrospinning solution. It should be noted that for the CNC content of 10 wt %, the spinning solution needed to be heated before electrospinning. Drug-Loaded Composite Electrospinning Solution. Tetracycline hydrochloride (TH) as a model drug was used in this work. 0.05 g mL−1 TH in methanol was added into the composite solution (with 6 wt % CNCs). The obtained solutions were yellow but clear, illustrating that the addition of TH did not affect well dispersion of the CNCs in PHBV solution. The mass ratio of TH and PHBV/CNC composites was 5, 15, and 25 wt %. For comparison, 5 wt % TH was added into PHBV solution. 2.2.3. Electrospinning Process. The electrospinning parameters for fabricating (drug-loaded) neat PHBV and composite fibrous membranes were optimized. The applied voltage (GAMMA/ES50P10W, USA), temperature, tip-to-collector distance, and collecting time were 15 kV, 30 °C, 18 cm, and 6 h, respectively. A stainless steel needle with 0.7 mm diameter was used, and the feeding rate (KD scientific model 100, USA) was selected at 1 mL h−1. The as-prepared electrospun PHBV and composite nanofibrous membranes were dried at room temperature in a vacuum for 12 h. 2.3. Characterization. The shear viscosity and conductivity of electrospinning solution were determined by a rotating viscometer (Digital viscometer DV-79, NiRun), and a conductimeter (DDS-307A, Lei Ci, Shanghai), respectively. The morphologies of (drug-loaded) PHBV and composite nanofibrous membranes were observed on a field emission scanning electron microscopy (FE-SEM, HITACHI S-4800) at 5.0 kV. The microstructure was characterized on an attenuated total reflection Fourier-transform infrared spectrometer (ATR-FTIR, Nicolet 8700).

and in vivo studies especially in controlled drug delivery because of its proven biocompatibility, mechanical strength, and oxygen permeability as well as degradation product (dominantly (R)-3-hydroxybutyric acid) as a normal constituent of human blood.7,9−11 The mechanical properties are wellknown to have a great influence on the performance of nanoscale fibrous systems in tissue engineering application as reported in previous works.8,9 For example, the nanofibrous scaffolds must provide temporary mechanical support for the regenerating tissue, even possess good mechanical properties similar to the tissue to be repaired. However, electrospun PHBV nanofibrous membranes seem not to be ideal for biomedical applications in particular as tissue scaffolds and drug carriers. The weak orientation of polymer chains achieved at low stretching rate during electrospinning, which should induce low Young’s modulus and tensile strength as well as poor thermal stability of PHBV nanofibrous membranes.12−16 Furthermore, PHBV might not be a suitable carrier for hydrophilic drugs because of its lack of polar groups and high crystallinity, and a fast drug release behavior can be observed because most of drug molecules appeared on the surface of electrospun PHBV nanofibers.6 Meanwhile, the drug loading capacity of nanofibers is not high, and furthermore the loaded drug is released only around 30%.14 In addition, hydrophobic characteristics of PHBV nanofibrous membranes is not beneficial to living cell adhesion and mobility. To overcome these limits, controlling the hydrophobicity of nanofibers with organic nanofiller is an effective route to modulating the drug release rate by controlling water diffusion into extraordinary porous nanofibrous membranes.2 Recently, the use of cellulose nanocrystals (CNCs) as unique organic nanoreinforcer for enhancing the properties of biodegradable polymeric matrixes has been paid more and more attention because of their high strength with Young’s modulus of 150 GPa, good biodegradability and biocompatibility as well as abundant hydrophilic hydroxyl groups and nontoxicity.17−21 It has been demonstrated that great improvement in mechanical and thermal properties of PHBV matrix can be achieved at low loading levels.13,15,17,18,22 Furthermore, a series of electrospun nanofibrous membranes have been successfully fabricated for PHBV/CNC composite with various CNC contents. However, studies in the drug release behaviors for neat PHBV nanofibrous membrane are rarely available in the literature, let alone in CNC-reinforced PHBV nanofibrous membranes.6,14 In this work, we should propose a combination of biodegradable CNCs as organic reinforcing materials in PHBV nanofibers via electrospinning, and a series of PHBV/ CNC composite membranes with different CNC loadings would be fabricated. The morphology, microstructure, mechanical and thermal properties, crystallization behavior, hydrophilicity, and in vitro degradation behavior of composite nanofibrous membranes would also be investigated. Besides, to evaluate their potential application as biomedical materials, we would also investigate comparative cytocompatibility of neat PHBV and composite nanofibrous membrane with human MG63 cells. Moreover, the influence of different drug loadings on the morphology, hydrophilicity, and drug release behavior of composite nanofibrous membranes would be also evaluated.

2. EXPERIMENTAL SECTIONS 2.1. Materials. PHBV with Mn of 5.90 × 104, Mw of 1.58 × 105, and the molar ratio of HV of 2.57% was provided from Tianan Biological Material Co., Ltd. (China). Commercial microcrystalline B

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 1. FE-SEM images for neat PHBV (a) and composite nanofibrous membranes with the CNC contents of (b) 1, (c) 3, (d) 6, (e) 9, and (f) 10 wt %. Each spectrum was recorded with 64 scans and a resolution of 2 cm−1 at room temperature. Differential scanning calorimetry (DSC) measurements were performed on a differential scanning calorimeter (TA Q20) at a nitrogen flow rate of 40 mL min−1. The membrane prior to the experimental measurement was heated from room temperature to 200 °C at a rate of 20 °C min−1, and kept at 200 °C for 5 min for eliminating the previous heat history. Then the membrane was first cooled to −50 °C, and heated again to 200 °C at a rate of 10 °C min−1. Some thermal parameters including melt crystallization temperature (Tmc) and melting temperature (TM1, TM2) as well as melt crystallization enthalpies (ΔHmc) and melting enthalpies (ΔHm) were calculated from DSC curves. Thermal stability was evaluated on a Netzsch TG209 F1 thermogravimetric analyzer (TGA) instrument coupled with QMS. The membranes were heated from room temperature to 600 °C at a heating rate of 10 °C min−1 and nitrogen flow rate of 30 mL min−1. Tensile properties of (CNC-loaded) PHBV and composite nanofibrous membranes were determined on an electronic universal testing machine (Kexin WDW3020). The membrane was cut into specimens of 80 mm length, 20 mm width, and 50−60 μm thickness. The test was carried out by loading the tensile specimen at a constant tensile rate of 1 mm min−1 at 20 °C and 65% relative humidity, and ten replicates were measured for each membrane.

The contact angles were determined on a Dataphysics OCA40 contact angle analyzer at room temperature. According to pendant drop method, about 2 μL of aqueous droplet was dropped onto the membrane surface at a contact time of 5 s. The average value was obtained from 20 independent measurements at different sites of the membrane. The in vitro degradation tests were carried out at 37 °C for 40 days in 0.1 M phosphate buffer solutions (PBS) of pH 7.4 prepared by using KH2PO4 and Na2HPO4·12H2O. Membranes were cut into 30 × 30 mm2 strips with a thickness of 50−60 μm. The degradation behaviors of PHBV and the nanofibrous membranes were investigated by detecting weight loss of specimens, which were washed with the distilled water and dried to a constant weight at given time intervals at room temperature in a vacuum. Percent weight loss = (m0 − mt) × 100%, where m0 is the initial mass, mt is the mass after a given time of hydrolysis. The experiments about cell culture assay, morphology, and attachment were performed according to the previous reported method.22 The human MG-63 cells were maintained at 37 °C in an atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) which was consisted of fetal calf serum (FCS, 10%) and antibiotics (penicillin of 100 U mL−1 and streptomycin of 100 U mL−1). After about 1 min digestion by using 0.25% trypsin, MG-63 cells were redispersed into the medium. Then the suspended solution was diluted to the concentration of 1 × 105 cells mL−1, in which the C

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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1052 ± 55 nm with obviously narrowed distribution. Table 1 lists the viscosity and electrical conductivity for PHBV

numbers of MG-63 cells were determined by counting with a hemeacytometer. Before being placed into the 24-well cell culture plates followed by adding 1 mL cell suspension to each well, all the membranes were disinfected with 75% ethanol and washed with PBS buffer solution for three times. The growth of MG-63 cells on the membranes carries out for 2 h, and the culture medium with unattached cells was removed from the wells before the membranes were washed with PBS buffer. The cell attachment in different composites was examined with a Cell Count Kit-8 (CCK-8). The optical density (O.D) was tested using a microplate reader at a wavelength of 450 nm, and an average value was obtained according to the instruction of CCK-8 for three places of each membrane. The MG63 cells were identified with the assistance of Actin-Tracker Green through measuring the cytoplasmic area and consequently estimating cellular spreading. The fluorescence images were recorded on an Olympus IX71-22FL/PH fluorescence microscope. 2.4. Drug Release Behavior. Determination of Drug Loading and Drug Loading Efficiency. First, various contents of THs (0, 2, 4, 6, 8, 10, and 12 μg μL−1) were dissolved in PBS buffer solution, and the absorbance was measured at a wavelength of 360 nm on a TU1901 spectrophotometer (Beijing Purkinje General Instrument). The standard curve with a linear correlation (R2 = 0.9998) was obtained. Then, 100 mg of membrane was dissolved into 15 mL of chloroform, and consequently 50 mL of 0.1 M PBS was added into the solution for the extraction of TH. After the drug was completely dissolved in PBS under sonication and centrifugation at a speed of 12000 r min−1 for 20 min, the resultant TH solution was analyzed spectroscopically at a wavelength of 360 nm and then the drug weight could be determined from the absorbance data according to the standard curve. That is to say, the drug loading and drug loading efficiency were calculated, in which drug loading (%) = 100 × [weight of drug measured/weight of drug unloaded nanofibers], and drug loading efficiency (%) = 100 × [weight of drug measured/weight of drug added]. In Vitro Drug Release Behaviors of Drug-Loaded Nanofibrous Membranes. One hundred milligrams of membrane was enclosed in dialysis bag and then immersed into 20 mL of PBS at 37 °C under continuous stirring achieved with a magnetic stir bar (100 r min−1). 5.0 mL solution was taken out from the medium at certain time intervals, and after being spectroscopically analyzed, the solution was again poured into the medium. The change in the drug release was monitored as a function of time. The cumulative release percentage of drug-loaded membranes was calculated from the results for triplicate tests. To better compare the rate of drug release for neat PHBV and composite nanofibrous membranes with 6 wt % CNC content, we fitted in vitro drug release data by using a first-order model

ln(1 − M t /M∞) = kt

Table 1. Viscosity and Electrical Conductivity for PHBV Electrospinning Solution As a Function of the CNC Content sample neat PHBV PHBV/CNC

CNC content (%)

viscosity (Pa s)

conductivity (μS cm−1)

0 1 3 6 9 10

0.31 0.45 0.67 0.98 1.34 1.71

0.1 0.18 0.42 0.76 0.93 1.05

electrospinning solution as a function of the CNC content. It has been reported that the addition of rigid particles can improve the shear viscosity of polymeric electrospinning solutions, as a result, a significant increase occurred in diameter of composite nanofibers.14,23 When the CNC contents increased from 3 to 6 wt %, more uniform composite nanofibers can be observed as displayed in Figure 1, accompanying with the reduce in the average diameter from 748 ± 62 to 620 ± 33 nm. The decrease in diameter of the nanofibers could be observed by increasing the CNC content at a fixed PHBV fraction, or reducing PHBV fraction at a constant CNC loading, contributing to the weakening effects of the CNCs on the PHBV chain entanglement and inter- or intramolecular interactions. It has been reported that the CNCs extracted from the MCC through hydrothermal hydrolysis have high electronegativity.19 The ionic strength of electrospinning solution brought about by the negatively charged CNCs can increase the charge density in ejected jets as well as electrostatic forces. If the enhanced conductivity is over more than the effect of the increased viscosity on the nanofiber diameter, the elongation of the charged jet would be allowed to continue longer and then to form thinner fibers. In contrast, the fiber diameter increased.6,11 Therefore, the nanofiber diameter was increased to 712 ± 72 nm when the CNC content further increased to 9 wt %, meanwhile some protuberances appeared on the fiber surface and the size distribution was broadened. For the CNC content of 10 wt %, the electrospinning solution would be heated for decreasing its very high viscosity to ensure the electrospinning process being performed smoothly. Obviously, the nanofibers were annealed and connected together to form “annealed structure” as illustrated in Figure 1 because of the heating treatment of the electrospinning solution. 3.2. Chemical Structure. The FT-IR spectra for neat PHBV and composite nanofibrous membranes with various CNC contents are displayed in Figure 2. The PHBV nanofibrous membranes showed the characteristic bands at around 2875, 3000, and 2935 cm−1 attributing to −CH3 symmetric and asymmetric stretching and −CH2 antisymmetric stretching, and in the range of 1227−1478 and 826−979 cm−1 region to various aliphatic C−H vibrational bands of PHBV, respectively.11,23−25 The CNCs exhibited the characteristic bands in the 3650−3000 cm−1 region assigning to O−H stretching vibrations, at 1049 cm−1 to asymmetric C−O−C bond stretching from the pyranose ring, at 1160 cm−1 to C− O−C glycosidic linkage, in the range of 2800 and 3000 cm−1 to stretching vibrations of C−H of methylene groups.17,19,20,22 The characteristic bands of PHBV and CNCs were all observed

(1)

where Mt and M∞ are respectively the cumulative absolute amount of drug released at time t and at infinite time, k a kinetic constant and t the release time.

3. RESULTS AND DISCUSSION 3.1. Morphology. Figure 1 displays the FE-SEM images for neat PHBV and composite nanofibrous membranes with various CNC contents. It is necessary to point out that the electrospinning conditions had been optimized through adjusting the electrostatic spinning process by varying applied voltage, flow rate, collection distance, and the nature of the spinning solution including viscosity, surface tension, and solvent ratio to generate uniform nanofibers without beads. Note that the surfaces of the CNC-loaded fibers appeared to be similarly smooth and significantly improved fiber uniformity compared with those for neat PHBV nanofibers, and only slightly agglomeration of the CNCs in the composite nanofibers could be observed even the CNCs content exceed 10 wt %. The diameter of PHBV nanofibers is about 1025 ± 96 nm with wide size distribution. By adding 1 wt % CNCs, the average diameter of composite nanofibers increased slightly to D

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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obtained thermal parameters are presented in Table 2. A sharp crystallization peak appeared for all the membranes in the cooling scan from the DSC curves, indicating obvious heat release accompanying with the exothermic crystallization. As one of the most important thermal parameters, the Tmc of composite nanofibrous membranes except for 10 wt % CNCs shifted to high temperature once the CNCs were loaded into the nanofibers.14,17,22,27 The crystallization of PHBV can be accelerated in the presence of CNCs which could act as heterogeneous nucleating agent.15,18,22,28,29 With the increasing CNC contents, the Tmc increased gradually from 92.1 °C for neat PHBV nanofibers to 95.9 °C for composite nanofibrous membranes with 6 wt % CNC content, and then reduced to 67.9 °C at 10 wt % CNC content, in which heterogeneous nucleation effect became weaken obviously, leading to a significant reduction in the crystallization temperature of composite nanofibrous membranes.17,25,27,30 Moreover, the PHBV presented double melting peaks (TM1 and TM2) as shown in Figure 3, indicating a melting−recrystallization− melting behavior of PHBV. This result can be explained by the fact that more perfect crystals can be formed when those crystals with more defective or smaller size were melted during heating process and subsequently recrystallized.15,18,20,29,31 Furthermore, compared with those for neat PHBV, the melt peaks, TM1 and TM2, for composite nanofibrous membranes increased slightly from 145.2 to 158.5 °C and from 157.5 to 169.8 °C, respectively, indicating that the crystal perfection of PHBV is improved by the addition of CNCs.16,22,23,27 Strangely, triple melting peaks appeared for composite nanofibrous membranes with 10 wt % CNC content, in which TM1 was obviously lower than those for PHBV nanofibers and other composite nanofibrous membranes. These results suggest that there are more imperfect PHBV crystallites in composite nanofibrous membranes due to special annealed structure, which can get a support from the crystallinity (Xc) of composite nanofibrous membranes as listed in Table 2. The Xc increased gradually from 60.7% for neat PHBV to 70.1% for composite nanofibrous membranes with 9 wt % CNC content, but decreased to 68.8% when the CNC loading further increased to 10 wt %. It is well-known that semicrystalline and glassy polymers are generally believed to be promising carriers for achieving sustained drug release because the water diffusion into these materials is relatively slow.2 3.4. Thermal Stability. Figure 4a gives the TGA curves for neat PHBV and composite nanofibrous membranes at the heating rate of 10 °C min−1, and the obtained thermal parameters are presented in Table 2. It is found that all composite nanofibrous membranes exhibited an onset of degradation process and higher thermal stability than neat PHBV nanofibers.24,30 Along with the increase of the CNC loading, the Tmax continuously increased from 263.5 °C for neat PHBV nanofibrous membranes to the highest temperature of 287.8 °C for composite membranes at 6 wt % CNC content, and then reduced lightly to 275.2 °C at 10 wt % CNC content. The similar change trend was also found for T0, T5%, and Tf. Compared with those for neat PHBV membranes, the T0, T5%, Tmax, and Tf for composite nanofibrous membranes with the highest thermal stability improved by 25.3, 24.1, 24.3, and 26.0 °C, respectively, benefiting from strong hydrogen bonding between PHBV and CNCs.17,19,20 More clearly, the presence of hydrogen bonds should restrict the motion of the polymer segments, and further the formation of the six-membered ring

Figure 2. FT-IR spectra and carbonyl stretching region in the infrared spectra for PHBV and composite nanofibrous membranes.

in the infrared spectra of composite nanofibrous membranes. Furthermore, along with the increasing content of the CNCs, the intensity of the peak at 2883 cm−1 assigned to C−H stretching vibrations in cellulose increased, whereas the band intensity at 3445 cm−1 contributing to characteristic absorptions of the hydrogen-bonded hydroxyl groups appeared to first increase and then decrease. The highest band intensity was obtained at the 6 wt % CNCs, indicating that the strongest hydrogen bonding between CNCs and PHBV formed among all the composite nanofibers.17,19,20,22 The existence of hydrogen bonds between CNC and PHBV was also supported by comparing the shift in the carbonyl stretching frequency among neat PHBV and composite nanofibrous membranes. As illustrated in Figure 2, with the increase in CNC contents, the CO band shifted first from 1728 cm−1 for neat PHBV to 1722 cm−1 for composite nanofibrous membranes, and then to 1725 cm−1 when the CNC content reached to 10 wt %.16,22,26 This result shows that the hydrogen bonding between hydroxyl groups of CNCs and carbonyl groups of PHBV would provide protons for oxygen atom in the carbonyl group and reduce its polarity.17,22 It is worth mentioning that composite nanofibrous membranes from heated electrospinning solution could severely weaken hydrogen-bonding interactions. 3.3. Crystallization and Melting Behavior. Figure 3 presents the nonisothermal crystallization and melting curves for neat PHBV and composite nanofibrous membranes, and the

Figure 3. DSC traces for neat PHBV and composite nanofibrous membranes recorded during the first cooling and second heating scans at a rate of 10 °C min−1. E

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Table 2. Degree of Crystallinity and Thermal Parameters for Neat PHBV and Composite Nanofibrous Membranes

a

sample

Tmc (°C)

ΔHmc (J g−1)

TM1 (°C)

TM2 (°C)

ΔHm (J g−1)

Xc (%)a

T0 (°C)

T5% (°C)

Tmax (°C)

Tf (°C)

PHBV 1% 3% 6% 9% 10% CNC

92.1 93.4 95.0 95.9 95.1 67.9

64.4 68.3 71.8 75.2 73.5 73.1

145.2 148.1 152.2 158.5 158.3 140.5

157.5 161.7 162.9 169.8 168.5 165.8

89.3 90.5 92.9 95.8 93.5 90.9

60.7 62.3 65.3 69.6 70.1 68.8 88.6

247.3 250.8 252.3 272.6 268.5 253.4 336.1

232.0 238.5 244.6 256.1 254.6 245.2 297.0

263.5 265.2 269.0 287.8 286.8 275.2 363.9

271.5 274.1 276.1 297.5 297.9 295.2 420.9

Xc = ΔHm/[ΔH100%PHBV × (1−wCNC)], where ΔH100%PHBV = 146.6 J g−1.13,14

Figure 4. (a) TGA curves, (b) tensile strength and Young’s modulus, and (c) elongation to break as a function of the CNC contents for neat PHBV and composite nanofibrous membranes.

Figure 5. (a) Contact angles and (b) cell attachment and growth of MG-63 cells cultured on (6 wt % CNCs) neat PHBV and composite nanofibrous membranes.

fiber diameter and orientation, porous structure and strong interaction between the components in the composite fibers.16,25,29,31 Figure 4b, c give tensile strength and Young’s modulus, and elongation to break for neat PHBV and composite nanofibrous membranes with various CNC loadings. It is obvious that compared to neat PHBV membranes, a great improvement in both the tensile strength and Young’s modulus could be achieved for composite nanofibrous membranes with the incorporation of the CNCs. The tensile strength and Young’s modulus of composite nanofibrous membranes increased first with the increase of the CNC content, and reached to a maximum at 6 wt % CNC content, and then

ester in the initial step of PHBV degradation.17,19,20,22 In addition, hydrogen bonding could also be helpful to facilitate uniform dispersion of CNCs in PHBV solution by using a mixed solvent of chloroform and DMF.17 But at high CNC content, the aggregation of the CNCs and “annealed structure” of the nanofibers would reduce thermal stability of composite nanofibrous membranes.17,30 3.5. Mechanical Properties. Mechanical properties of electrospun nanofibrous membranes are important parameters in biomedical applications, especially being required to provide sufficient mechanical strength for supporting the growth and migration of cells, depending on the nature of polymeric matrix, F

DOI: 10.1021/acsbiomaterials.7b00169 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

3.7. In Vitro Degradation Behavior. Figure 6 presents the in vitro degradation behavior of neat PHBV and composite

decreased gradually, whereas the elongation at break showed a reverse trend.27,29,30 As already mentioned above, composite nanofibrous membranes became more uniform and finer at higher CNC content, and exhibited strong hydrogen bonding between PHBV and CNC and relatively perfect crystal structure, further leading to great enhancement in mechanical properties of nanofibrous membranes.17,20,23,24 Compared with those for neat PHBV membrane, the highest tensile strength and Young’s modulus for composite nanofibrous membrane at 6 wt % CNC content increased by about 125 and 110%; meanwhile the lowest elongation at break reduced to 3.8%. When the CNC content further increased, the CNCs agglomeration in the polymer matrix, wide distribution of fiber diameter, and defective crystals would weaken the mechanical strength of composite nanofibrous membranes.17,22,27,29 3.6. Hydrophilicity and Cytocompatibility. The hydrophilicity of biopolyester nanofibers is usually used to evaluate the biocompatibility and tissue compatibility of the material, and the improvement of the hydrophilicity of composite nanofibrous membranes can promote cell adhesion, proliferation and differentiation on the fiber surfaces.11,12,22 The hydrophilicities of neat PHBV and composites nanofibrous membranes were estimated by contact angle measurement as plotted in Figure 5a. The hydrophilicity of PHBV nanofibrous membrane did not only depend on the microstructure of electrospun nanofibers such as fiber diameter, fiber shape and functional groups on the nanofiber surface but also pore structure of electrospun nanofibrous membrane. It can be seen that the contact angle of PHBV nanofibrous membrane was about 138°. The hydrophobic property of nanofibrous membrane was mainly depended on its surface roughness which can induce multiple contacting points of water droplet with the membrane surface.13,14,31,32 It is found that with the increase in CNCs content, the hydrophilicity of PHBV composite nanofibrous membranes were gradually improved as displayed in Figure 5a. It is suggested that controlling of the hydrophilic properties for composite nanofibrous membranes could be achieved efficiently by simply adjusting the addition of the CNCs.18,22 The cytocompatibility of nanofibrous membranes plays an important role in the potential applications for biomaterials in tissue engineering, which can be evaluated by observing cellular growth and proliferation on membrane surfaces. Furthermore, the osteoblast cell line MG-63 has been commonly used to evaluate the biocompatibility of the material. Thus, the cell attachments for neat PHBV and composite nanofibrous membranes were examined according to the instruction of CCK-8 in this work. The fluorescent image was applied for qualitatively analyzing the attachment of MG-63 cells on neat PHBV and composite nanofibrous membranes. It has been reported that the random-oriented nanofibrous membrane is the most favorable scaffold for cell growth compared to other scaffolds.8,33 As shown in Figure 5b, compared with that for neat PHBV membranes, more larger amount of cells can be cultured on composite nanofibrous membranes, benefiting from the improved hydrophilicity of composite membranes and promote attachment of cells on the membrane surface.22 The O.D value increased from 0.27 for neat PHBV nanofibrous membrane to 0.38 for membrane by incorporation of CNCs, which strongly indicated that such composite nanofibrous membrane with excellent biocompatibility exhibited great potential as biomedical materials

Figure 6. Degradation behavior of neat PHBV and composite nanofibrous membranes.

nanofibrous membranes. It could be observed that the weight loss of all the membranes increased when the incubation time was prolonged, and further the incorporation of CNCs resulted in rapid degradation of composite nanofibrous membranes. More detailedly, the degradation rate of neat PHBV nanofibers was very slow, and only 12% weight loss of neat PHBV nanofibers occurred after 40 days. It is well-known that the crystallinity and hydrophilicity are important factors for the degradation of PHBV segment through random hydrolytic ester bond cleavage, that is to say, high crystallinity and hydrophobicity result in slow degradation of PHBV matrix.6,12,18,23 It is easy to understand that with the incorporation of hydrophilic CNCs, composite nanofibrous membranes exhibited a rapid degradation process, and about 35% weight loss of composite nanofibrous membranes with 10 wt % CNC content could be achieved after same 40 days as displayed in Figure 6. The enhanced surface hydrophilicity make the invasion of the degradation medium and water into amorphous regions of the nanofibers more easily, thus speeding up their degradation rate.8,12,18 In addition, relative small fiber diameter and large amount of pore structure are beneficial to increase the number of reactive sites during hydrolysis process, because the nanofibrous membrane can fully contact with the degradation medium. These results show that the degradation rate of composite nanofibrous membranes could be chosen by varying CNC content to satisfy the degradation requirements for biomedical materials. 3.8. Drug-Loading Capacity. The application of electrospun nanofibrous membranes in drug delivery has get more attention because of its high drug loading capacity and ability to enclose a variety of biomolecules. Design of controlled release drug delivery systems is expected to better improve drug effectiveness and reduce harmful side effect especially at high doses.1,2,6 TH as one kind of broad spectrum antibiotics has been abundantly utilized for many biomedical uses because of its multiple roles as a therapeutic and antibacterial reagent. The sustained control release of TH from a carrier, e.g., nanofibers may benefit faster regeneration of osteoblasts, fibroblasts and other types of cells.34,35 Furthermore, TH has lots of oxygencontaining functional groups, which can form strong hydrogenbonding with CNCs. It is possible that being accompanied by CNCs, TH could be incorporated into nanofibers to control G

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105 nm, and for composite nanofibers, the diameter reduced to about 412 ± 122 nm. It is suggested that the improvement in solution conductivity due to the introducing of TH (a hydrochloride salt with high charge density) would lead to relatively high elongation during the electrospinning process, and thus produced smaller nanofibers with narrow diameter distribution.6,14 However, when the drug loading was further increased to 15% or more, the nanofibers would stick together and form interconnection structure. It can be explained that the drug at low loading content could be well dispersed into PHBV matrix in the presence of the CNCs due to strong interaction between drug and CNCs, whereas redundant drugs (15 or 25%) did not enter fully into the PHBV nanofibers, resulting in the nanofibers being bonded together by external drug. Furthermore, large amount of TH attached on the nanofiber surface would cause burst drug release behavior. It has been demonstrated that polymer solubility as well as diameter and microstructure of the nanofibers played important roles in the drug release behaviors.1,14 Hydrophilic properties of electrospun drug-loaded nanofibers were further evaluated by using contact angle measurement as plotted in Figure 7. The contact angles for PHBV and composite nanofibrous membranes with 5 wt % drug loading were about 106° and 95°, respectively. Lower water contact angle, stronger hydrophilicity of the membranes.6 Thus, the drug-loaded nanofibrous membranes had higher hydrophilicity than those without drug loading. It is found that when the CNCs and TH with a large number of hydrophilic groups were simultaneously introduced into the nanofibrous membrane, their contact angle gradually decreased with the increase in the drugs as displayed in Figure 7, which strongly support the improvement on the hydrophilic properties. However, the nanofibers were adhered together because of the aggregation of redundant drugs on the nanofiber surface. 3.10. Drug Release Behavior. Figure 8 presents in vitro accumulated drug release behaviors and theoretical curves derived from the first-order model for the drug-loaded neat PHBV and composite nanofibrous membranes. It is found that the drug release processes for all the membranes were involved in an initial burst release, with a significant amount of drug released at a relative rapid rate within first several 10 h, and subsequently a sustained drugs release pattern as described in Figure 8a. The PHBV nanofibrous membranes delivered about

infections, for example, in applications related to skin and bones. Therefore, TH as a model drug was used in this work. The drug loading and drug loading efficiency are extremely important indicators to evaluate the feasibility of clinical application of the material processing technology.14 Here neat PHBV and composite nanofibrous membranes with 6 wt % CNC content were chosen to evaluate the feasibility as longterm sustained release drug delivery systems. Table 3 shows Table 3. Drug Loading and Drug Loading Efficiency of Drug-Loaded PHBV Nanofibers and Composite Nanofibrous Membranes with Different Drug Loadings sample PHBV PHBV/CNC (6 wt %)

mean drug loading (%)

experimental drug loading (%)

drug loading efficiency (%)

5 5 15 25

4.85 4.99 14.92 24.71

97.0 99.8 99.5 98.8

drug loading and drug loading efficiency of neat PHBV and composite nanofibrous membranes under optimized electrospinning condition. It could be seen that the drug loading efficiency of composite nanofibrous membrane exceeded 97%, benefiting from slight drug loss or denaturation under relatively mild electrospinning condition. Also, the drug loading efficiency of composite nanofibrous membranes was higher than neat PHBV nanofibers, which might be contributed to interaction between polar hydroxyl groups of CNCs and polar groups of TH drug. 3.9. Morphology and Hydrophilicity for Drug-Loaded Nanofibers. Figure 7 illustrates FE-SEM images of drugloaded PHBV nanofibers and composite nanofibrous membranes with different drug loadings. Note that neat PHBV and composite nanofibrous membrane with the 5% drug loading exhibited a smooth and bead-free appearance and no visible TH crystals attached on the nanofiber surface, but high porosities and small fiber diameter compared with those without drug loading. It indicates that the difference in the volatility between chloroform and DMF almost had no impact on the solidification of polymer and tetracycline hydrochloride from the solution because of the quick evaporation of the solvents during the electrospinning process.14 The average diameter of PHBV nanofibers with 5 wt % drug loading was about 705 ±

Figure 7. FE-SEM images and contact angles for drug-loaded PHBV nanofibers and composite nanofibrous membranes with different drug loading. H

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Figure 8. (a) Accumulated drug release and (b) theoretical curves derived from the first-order model for neat PHBV and composite nanofibrous membranes with 6 wt % CNC loading.

4. CONCLUSIONS Nanofibrous membranes based on biodegradable PHBV with various CNC contents were successfully produced via electrospinning technique. The resultant nanofiber morphology; microstructure; and mechanical, thermalm and hydrophilic properties of as-prepared nanofibrous membranes were investigated by SEM, FT-IR, XRD, TGA, and contact angle measurement. It is observed that composite nanofibers became more uniform and smaller in diameter along with the increasing of CNC loading. The contact angle of nanofibrous membrane decreased from 127° for neat PHBV to 87° for composite membranes at 6 wt % CNC content. Meanwhile, the tensile strength and Young’s modulus were enhanced by about 125% and 110%, meanwhile the T0, T5%, Tmax and Tf were increased by about 25.3, 24.1, 24.3, and 26.0 °C, respectively. These enhancements were attributed mainly to strong hydrogen bonding between PHBV and CNCs. Moreover, the biocompatibility of nanofibrous membranes in MG-63 cells got a significant improvement by introducing CNCs. More importantly, high drug loading efficiency of more than 98% and longterm sustained release behaviors of composite nanofibrous membranes were achieved because of the higher specific surface area and better hydrophilicity. It is observed that composite nanofibrous membrane with 5 wt % drug loading delivered more than 86% drug content within 540 h, whereas drugloaded PHBV nanofibrous membranes gave only 37.6% content over the same period. It is clear that composite nanofibrous membrane displayed a low initial burst release followed by a slower rate of release, exhibiting great potential applications especially in long-term sustained drug delivery system and tissue engineering scaffolds.

36.1% drug content after 50 h, but only 1.5% content when the time was prolonged to 490 h. Moreover, all the drug-loaded composite nanofibrous membranes delivered more than 86% drug content after 540 h, meanwhile PHBV nanofibrous membranes gave only about 37.6% content over the same period. The higher drug release within 50 h was mainly from the surface and amorphous region of nanofibrous membranes, whereas less drug release in the later period is mainly from crystalline region which could hinder the water diffusion into the polymer inner layers.6,14 As illustrated in Figure 8b, the first-order model is demonstrated to be very suitable for fitting the drug release profile, and the results were presented in Table 4. It is found that the 5% drug loading composite nanofibrous Table 4. Statistical Parameters Obtained from the Vitro Release Profiles through the First-Order Model sample

drug loading (%)

k

R2

neat PHBV PHBV/CNC (6 wt %)

5 5 15 25

−0.0653 −0.0094 −0.0157 −0.0425

0.998 0.984 0.990 0.985

membranes exhibit the minimum absolute value of the kinetic constant (k) as listed in Table 4, whereas the nanofibers without CNCs show a maximum absolute value. This result indicates that compared with neat PHBV nanofibers, CNCreinforced electrospun nanofibers exhibit a lower initial burst and slower release rate. These results show that the introducing of CNCs could efficiently overcome the drawback of PHBV nanofibrous membrane as the drug carrier, in which most of hydrophilic drug molecules only appeared on electrospun nanofiber surface, resulting in rapid drug released.6,14 Composite nanofibrous membranes exhibited smoother and well-regulated drug release with higher accumulated drug content at a high rate during the experiment period. These excellent abilities could be attributed to their smaller fiber diameter and improved hydrophilicity, benefiting water penetration into the inner regions to release more drug molecules from the nanofibers.1,14 In addition, with the increase in drug loadings, the release rate was significantly accelerated. It is believed that the unique structure and properties of such nanofibrous systems, having large specific surface area to volume ratio, high porosity, similar microarchitecture with the extracellular matrixm and the possibility of controlling their hydrophilicity, make them a promising formulation pathway to promote pharmaceutical research and development.1

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.Q.). Fax: +86 21 67792855. *E-mail: [email protected] (H.Y.). ORCID

Zongyi Qin: 0000-0002-6329-4005 Houyong Yu: 0000-0002-6543-5924 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by Key Basic Research Project of Science and Technology of Shanghai (15Q10622). Dr. Miao Cheng is thankful to the support from the Innovation I

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Research Funds for the Doctoral candidate of Donghua University (15D310606). Prof. Houyong Yu also acknowledges the support from the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University (LK1428).

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K

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