Accepted Manuscript Title: Electrospinning polyvinylidene fluoride fibrous membranes containing anti-bacterial drugs used as wound dressing Author: Ting He Jingnan Wang Peilin Huang Baozhen Zeng Haihong Li Qingyun Cao Shiying Zhang Zhuo Luo David Y.B. Deng Hongwu Zhang Wuyi Zhou PII: DOI: Reference:
S0927-7765(15)00240-4 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.04.026 COLSUB 7030
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-12-2014 30-3-2015 12-4-2015
Please cite this article as: T. He, J. Wang, P. Huang, B. Zeng, H. Li, Q. Cao, S. Zhang, Z. Luo, D.Y.B. Deng, H. Zhang, W. Zhou, Electrospinning polyvinylidene fluoride fibrous membranes containing anti-bacterial drugs used as wound dressing, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Drug-loaded PVDF fibrous membranes were fabricated by an electrospinning process.
was good for handling and replacement.
cr
5
ip t
The PVDF fibrous membranes owned excellent mechanical properties which
us
The drug-loaded fibrous membranes showed a suitable release behavior for enrofloxacin (Enro) drugs.
an
The drug-loaded PVDF fibrous membranes were biocompatible and displayed excellent antibacterial activity.
Ac ce p
te
d
M
10
1
Page 1 of 36
A novel fibrous wound-dressing with a slow - drug release behavior，high mechanical strength, excellent anti-bacterial activity and wound healing effect was fabricated by an electrospinning process, which might be a promising
ip t
dressing in wound healing applications.
Ac ce p
te
d
M
an
us
cr
5
2
Page 2 of 36
Electrospinning polyvinylidene fluoride fibrous membranes containing anti-bacterial drugs used as wound dressing Ting He1, 4ǂ, Jingnan Wang2ǂ, Peilin Huang1, Baozhen Zeng5, Haihong Li1,
Zhang5* and Wuyi Zhou1*
cr
5
ip t
Qingyun Cao3, Shiying Zhang4, Zhuo Luo4, David Y.B. Deng2*, Hongwu
us
1. Institute of Biomaterial, Department of Applied Chemistry, College of Science, South China Agricultural University, Guangzhou 510642, China;
an
2. Research Center of Translational Medicine, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China;
3. College of Animal Science, South China Agricultural University, Guangzhou
M
10
d
510642, China;
te
4. Key Laboratory of Application Technology of Environmental Photocatalysis of Hunan Province, Changsha University, Changsha 410022, China
15
Ac ce p
5. Department of Anatomy, Guangdong Provincial Key laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangzhou 510515, China. ǂ
Both authors contribute equally to this work.
Abstract: The aim of this study was to synthesis drug-loaded fibrous membrane scaffolds for potential applications as wound dressing. Polyvinylidene fluoride 20
(PVDF) fibrous membranes were loaded with enrofloxacin (Enro) drugs by using an electrospinning process, and their mechanical strength, drug release profile and anti-bacterial properties were evaluated. Enro drug-loaded PVDF
3
Page 3 of 36
membranes exhibited good elasticity, flexibility and excellent mechanical strength. The electrospinning Enro/PVDF membranes showed a burst drug release in the initial 12 hrs, followed by sustained release for the next 3 days,
The drug-loaded PVDF fibrous membranes displayed excellent anti-bacterial
cr
5
ip t
which was an essential property for antibiotic drugs applied for wound healing.
us
activity towards Escherichia coli and Staphylococcus aureus. The results suggest that electrospinning PVDF membrane scaffolds loaded with drugs can
an
be used as wound dressing.
Key words: Electrospinning, Enro/PVDF fibrous membranes, drug release,
M
wound dressing
15
20
Ac ce p
te
d
10
* Corresponding authors information. E-mail
address:
[email protected]
(WY.
Zhou)
and
[email protected] ( HW. Zhang) 4
Page 4 of 36
1. Introduction Electrospinning is a straightforward, versatile and cost-effective method for manufacturing fibrous membranes with fibres of diameters ranging from several
advantages over the conventional dry or wet spinning methods. These include
cr
5
ip t
nanometres to a few micrometres [1], [2], [3]. Electrospinning has several
the ease of use, greater control over the fibre diameter and versatility for
us
spinning a wide variety of polymers [4]. In electrospinning process, an external
an
electric field is applied to a polymer solution to fabricate nano/micro fibres [5]. In the recent years, electrospun fibres have been the subject of extensive studies due to their applications in drug delivery, tissue engineering and wound healing
M
10
[6], [7], [8]. Electrospun fibrous membranes exhibit favourable properties such
d
as high surface area, high length/diameter ratio, tunable surface morphology,
te
flexible surface functionality and superior mechanical performance that promote
15
Ac ce p
cell adhesion and proliferation [9], [10], [11], [12]. When used as delivery vehicles in wound healing, the drug-loaded fibrous membranes could minimise systemic absorption and side effects of drugs and improve the local drug concentration at specific sites to promote therapeutic efficacy [13], [14], [15], [16].
Because of their biodegradability and biocompatibility, Polylactic acid (PLA), 20
poly(lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL) and Chitosan (CA) have been used as wound dressing [17], [18], [19]. However, these polymers generally had poor mechanical properties, which limited their
5
Page 5 of 36
application. The materials used as wound dressing should possess good mechanical properties such as tensile strength, flexibility and elasticity, which are required for handling and replacement of the dressing. Polyvinylidene
polymer [20]. PVDF fibrous membranes possess excellent physical properties
cr
5
ip t
fluoride (PVDF) is a stable, non-degradable, biocompatible and non-toxic
such as high tensile strength, good impact strength, wear resistance and
us
hydrophobicity that enable their application as wound dressing scaffolds [21].
cell activities in vitro and in vivo [22].
Enrofloxacin (Enro) is a fluoroquinolone antibiotic widely used in wound
M
10
an
Additionally, the piezoelectric property of PVDF fibrous membranes enhances
healing because of its strong bactericidal activity, broad bactericidal effect and
d
lower minimum inhibitory concentration [23]. The drug is released rapidly
te
during inflammation at early stages of the wound healing process [24].
15
Ac ce p
In the present study, by exploiting the advantages of electrospinning, we attempted to develop a simple method for fabricating drug-loaded PVDF fibrous membranes. Enro was absorbed onto PVDF nanofibres by eletrospinning. The surface morphology, mechanical properties, hydrophilicity, physical form and components of fibrous membranes were investigated. Analysis of the in vitro release of Enro from the Enro/PVDF fibrous membrane into PBS solution (pH = 20
7.4) showed that the sustained release of the drug followed Fickian diffusion process. The fibrous membranes showed excellent anti-bacterial activities against Escherichia coli and Staphylococcus aureus. Results of the in vitro cell
6
Page 6 of 36
proliferation and adhesion assays revealed high biocompatibility of Enro-loaded PVDF fibrous membranes. The results suggested that electrospinning is a promising method for the fabrication of drug-loaded membranes as wound
2. Experiment
cr
5
ip t
dressing.
2.1. Materials
us
Polyvinylidene fluoride (PVDF) was supplied by Arkema (Kynar 761, France)
an
Enrofloxacin was purchased from Huihua Animal Health Products & Co., Ltd. (Guangdong, China). Human dermal fibroblasts (HDF) used for this study were purchased from American Type Culture Collection, USA and human adipose
M
10
derived stem cells. N, N-dimethylacetamide (DMAC) and acetone (ACE) as the
d
solvent was supported by Fuyu Chemical Industry & Co., Ltd. (Tianjin, China).
te
Phosphate buffer saline (PBS) was prepared as the solvent for drug delivery in
15
Ac ce p
vitro. All of the chemicals and reagents are analytical grade. 2.2. Preparation of spinning solutions Four different concentration of spinning solutions of PVDF were prepared by dissolving 0.8 g, 1.0 g, 1.2 g and 1.4 g of PVDF powder in 10 ml of DMAC/ACE (1:1, v/v). Before electrospinning, the solutions were degassed by sonicating for 15 min in a KQ-100 ultrasonicator (Kunshan Ultrasonic 20
Instrument Co., Ltd., China). Four different concentrations of Enro/PVDF spinning solutions were prepared by first dissolving PVDF in 10 ml of DMAC/ACE (1:1, v/v) followed by the
7
Page 7 of 36
addition of 0.13 g, 0.19 g, 0.25 g or 0.33 g of Enro in the PVDF casting solution. The corresponding percentage composition of the drug in the fibres was 10%, 14%, 18% and 22% (w/w), respectively. All solutions were degassed before
2.3. Electrospinning process
cr
5
ip t
electrospinning.
The spinning solutions were fed into a 10-ml syringe connected to a 22-gauge
us
blunted stainless steel needle. The flow rate of spinning solutions were
an
controlled by a DT-200 precision syringe pump (Dalian Dingtong Technology Co., Ltd., China) and the flow was set to 0.50 ml/h. The needle was attached to a DE-100 high-voltage power supply (Dalian Dingtong Technology Co., Ltd.,
M
10
China) with a maximum voltage of 50 kV. A grounded aluminium foil was used
d
as the collector. The distance between the needle and the collector was 15 cm
te
and the applied voltage was set at 15 kV. The spinning room was maintained at
15
Ac ce p
25 °C. The details of the electrospinning process is shown in Fig. 1. The fibrous membranes were dried at 50 °C for 24 h in a GZX-9070MBE oven (Boxun Industry & Commerce Co., Ltd., China) to remove residual organic solvents and moisture. The drug-loaded fibrous membranes in which the percentage composition of Enro was 10%, 14%, 18% and 22% were denoted as E1, E2, E3 and E4, respectively. The pure PVDF fibrous membranes were denoted as E0. 20
2.4. Material characterization The surface morphology of the electrospun fibrous membranes was examined under a scanning electron microscope (SEM, Hitachi S-4800). The diameter
8
Page 8 of 36
distributions of the nanofibres were determined by image analysis using nano-measure for >200 points from SEM images. The hydrophilicity of the fibrous membranes was tested using a contact angle meter by measuring the
fibrous membranes. Wide-angle X-ray diffraction (XRD) analyses were
cr
5
ip t
contact angle between an ultrapure water droplet (10 μl) and the surface of the
performed using a Bruker D8 Advance (Cu Kα irradiation radiation)
us
diffractometer to evaluate the physical state of the drug in the fibrous
an
membranes. Fourier transform infrared spectroscopy (FTIR) was used to determine the structure and stability of the drug in the fibrous membranes through a Nicolet-Avatar 360 FTIR spectrometer (Nicolet Instrument
M
10
Corporation). The mechanical properties of the fibrous membranes (3.00 cm in
d
length, 1.00 cm in width and 0.40 mm in thickness) were assessed by using an
te
UTM-4202 electronic universal testing machine (Sansi Zongheng Technology &
15
Ac ce p
Co., Ltd.) at a strain rate of 10 mm/min. 2.5. Drug release in vitro
To evaluate the drug release profile of the fibrous membranes, 10–15 mg of Enro/PVDF fibrous membranes were transferred to sealed dialysis bags to ensure no nanofibres leak; 2 ml of 0.01-M PBS (7.4 and 9.0) were added to the bags, and they were placed in 500 ml of conical flasks containing 498 ml of 20
0.01-M PBS at two pH values (7.4 and 9.0). The contents of the flask were stirred with a magnetic stirrer at 37 °C and 100 rpm. At regular intervals, 3 ml of solution was taken from the flask and its absorbance at 271 nm was measured
9
Page 9 of 36
using an UV-Vis spectrophotometer (UV-2550, Shimadzu). After measuring the absorbance, the solution was returned to the flask to ensure constant volume of
by using the following equation and plotted against time. Accumulati ve Drug Release =
Drug Concentrat ion × V × 100 % Total Entrapped Drug
(1)
cr
5
ip t
the release solution. The cumulative percentage of Enro released was calculated
us
2.6. Cell culture
The cytocompatibility of the fibrous membranes, including the pure PVDF
an
and the 18% Enro/PVDF samples, were determined using the human dermal
10
M
fibroblast (HDF) as a cell model. First, HDFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (NBS, Sijiqing, China), supplemented with
d
10% foetal bovine serum (FBS) and 1% anti-biotic and anti-mycotic solutions
te
(Sangon, China) in 25-cm2 cell culture flasks at 37 °C in a humidified
Ac ce p
atmosphere of 5% CO2, and the culture medium was changed every 2 days. The fibrous membranes were cut into circular pieces 14 mm in diameter and placed 15
in 24-well plates. The samples were disinfected by soaking in ethanol for 1 h, washed three times with PBS and soaked in PBS for 1 h to remove the residual ethanol. Before seeding the cells, fibrous membranes were soaked overnight in the cell culture medium to promote cell attachment. HDF cells were then seeded on the fibrous membranes and empty tissue culture plates at a density of 1 × 104
20
cells/well. 2.7. Cell proliferation and morphology assay The number of viable HDFs on different fibrous membranes was determined
1
Page 10 of 36
after 1, 3, 5 and 7 days of culture. HDFs were separated from the fibrous membranes by trypsinisation and counted using a Beckman Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Inc., California, USA). The results were
surface. After 1, 3, 5, or 7 days of culture, the morphology of HDFs on fibrous
cr
5
ip t
expressed as the mean number of cells adhering per cubic centimetre of the
membranes was observed under an SEM. The fibrous membranes with adhering
us
cells were washed with PBS, fixed in 2.5% glutaraldehyde solution for 6 h,
an
dehydrated in a graded series of ethanol (30%–100%) and finally dried on a
10
ion sputter and then under an SEM. 2.8. Anti-bacterial assessment
M
critical point dryer. Thus the samples obtained were coated with gold using an
d
The anti-bacterial activity of the Enro/PVDF fibrous membrane against E.
te
coli and S. aureus was determined by the disk diffusion method. E. coli and S.
15
Ac ce p
aureus were incubated in Luria–Bertani broth overnight at 37 °C and then diluted with sterile water to 1 × 107 cells/ml. A 40-μl aliquot of the diluted bacterial suspension was plated by the spread plate method on nutrient agar in duplicate. The fibrous membrane was cut into discs 6 mm in diameter and >3.5 mg in weight and placed on the surface of nutrient agar. The cultures were incubated for 24 h at 37 °C. Following this, the zones of inhibition around the 20
discs were measured. 2.9. Wound healing test
1
Page 11 of 36
Male Sprague-Dawley (SD) rats weighing 240-280g were used in this study. After anesthetization, the neck area of the dorsal of each rat was shaved and cleaned with 70% ethanol. Two wounds were created on the neck area of each
an equal size (1.5 cm × 1.5 cm) of pure PVDF membrane, 18% Enro/PVDF
cr
5
ip t
rat using scalpel (wound area of 0.8 cm2). The wounds were treated by placing
membrane and gauze dressing (n=6) over it. The wound dressing were fixed by
us
medical proof fabric to ensure without removing throughout the study period.
an
The area of the wound was measured every 3 days using the planimetry method until the wound completely healed. The percentage of wound closure is defined as Eq [32], [33].
M
10
d
W ound c losure (%) = (1 -
An ) × 100 Ao
(2)
te
where A0 is the initial wound area and An is the wound area after n days.
15
Ac ce p
3. Results and discussion
3.1. The morphology of different pure PVDF and Enro/PVDF fibrous membranes.
The concentration of the polymer is one of key factors for successful preparation of polymer nanofibres. The concentration of the polymer should be suitable such that it should enable fabrication without beads while maintaining a 20
nanoscale diameter of the fibres. This optimal concentration differs with the polymer and can be adjusted according to the molecular weight and the type of solvent. We selected DMAC and ACE as the solvents because Enro is highly
1
Page 12 of 36
soluble in them. The SEM micrographs and diameter distribution graphs of the pure PVDF nanofibres fabricated using 8%–14% solutions of the polymer are shown in Fig. 2. When PVDF nanofibres were prepared by using polymer
were commonly observed. Higher concentrations of the PVDF polymer
cr
5
ip t
solutions with concentration
S0927-7765(15)00240-4 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.04.026 COLSUB 7030
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-12-2014 30-3-2015 12-4-2015
Please cite this article as: T. He, J. Wang, P. Huang, B. Zeng, H. Li, Q. Cao, S. Zhang, Z. Luo, D.Y.B. Deng, H. Zhang, W. Zhou, Electrospinning polyvinylidene fluoride fibrous membranes containing anti-bacterial drugs used as wound dressing, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Drug-loaded PVDF fibrous membranes were fabricated by an electrospinning process.
was good for handling and replacement.
cr
5
ip t
The PVDF fibrous membranes owned excellent mechanical properties which
us
The drug-loaded fibrous membranes showed a suitable release behavior for enrofloxacin (Enro) drugs.
an
The drug-loaded PVDF fibrous membranes were biocompatible and displayed excellent antibacterial activity.
Ac ce p
te
d
M
10
1
Page 1 of 36
A novel fibrous wound-dressing with a slow - drug release behavior，high mechanical strength, excellent anti-bacterial activity and wound healing effect was fabricated by an electrospinning process, which might be a promising
ip t
dressing in wound healing applications.
Ac ce p
te
d
M
an
us
cr
5
2
Page 2 of 36
Electrospinning polyvinylidene fluoride fibrous membranes containing anti-bacterial drugs used as wound dressing Ting He1, 4ǂ, Jingnan Wang2ǂ, Peilin Huang1, Baozhen Zeng5, Haihong Li1,
Zhang5* and Wuyi Zhou1*
cr
5
ip t
Qingyun Cao3, Shiying Zhang4, Zhuo Luo4, David Y.B. Deng2*, Hongwu
us
1. Institute of Biomaterial, Department of Applied Chemistry, College of Science, South China Agricultural University, Guangzhou 510642, China;
an
2. Research Center of Translational Medicine, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China;
3. College of Animal Science, South China Agricultural University, Guangzhou
M
10
d
510642, China;
te
4. Key Laboratory of Application Technology of Environmental Photocatalysis of Hunan Province, Changsha University, Changsha 410022, China
15
Ac ce p
5. Department of Anatomy, Guangdong Provincial Key laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangzhou 510515, China. ǂ
Both authors contribute equally to this work.
Abstract: The aim of this study was to synthesis drug-loaded fibrous membrane scaffolds for potential applications as wound dressing. Polyvinylidene fluoride 20
(PVDF) fibrous membranes were loaded with enrofloxacin (Enro) drugs by using an electrospinning process, and their mechanical strength, drug release profile and anti-bacterial properties were evaluated. Enro drug-loaded PVDF
3
Page 3 of 36
membranes exhibited good elasticity, flexibility and excellent mechanical strength. The electrospinning Enro/PVDF membranes showed a burst drug release in the initial 12 hrs, followed by sustained release for the next 3 days,
The drug-loaded PVDF fibrous membranes displayed excellent anti-bacterial
cr
5
ip t
which was an essential property for antibiotic drugs applied for wound healing.
us
activity towards Escherichia coli and Staphylococcus aureus. The results suggest that electrospinning PVDF membrane scaffolds loaded with drugs can
an
be used as wound dressing.
Key words: Electrospinning, Enro/PVDF fibrous membranes, drug release,
M
wound dressing
15
20
Ac ce p
te
d
10
* Corresponding authors information. E-mail
address:
[email protected]
(WY.
Zhou)
and
[email protected] ( HW. Zhang) 4
Page 4 of 36
1. Introduction Electrospinning is a straightforward, versatile and cost-effective method for manufacturing fibrous membranes with fibres of diameters ranging from several
advantages over the conventional dry or wet spinning methods. These include
cr
5
ip t
nanometres to a few micrometres [1], [2], [3]. Electrospinning has several
the ease of use, greater control over the fibre diameter and versatility for
us
spinning a wide variety of polymers [4]. In electrospinning process, an external
an
electric field is applied to a polymer solution to fabricate nano/micro fibres [5]. In the recent years, electrospun fibres have been the subject of extensive studies due to their applications in drug delivery, tissue engineering and wound healing
M
10
[6], [7], [8]. Electrospun fibrous membranes exhibit favourable properties such
d
as high surface area, high length/diameter ratio, tunable surface morphology,
te
flexible surface functionality and superior mechanical performance that promote
15
Ac ce p
cell adhesion and proliferation [9], [10], [11], [12]. When used as delivery vehicles in wound healing, the drug-loaded fibrous membranes could minimise systemic absorption and side effects of drugs and improve the local drug concentration at specific sites to promote therapeutic efficacy [13], [14], [15], [16].
Because of their biodegradability and biocompatibility, Polylactic acid (PLA), 20
poly(lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL) and Chitosan (CA) have been used as wound dressing [17], [18], [19]. However, these polymers generally had poor mechanical properties, which limited their
5
Page 5 of 36
application. The materials used as wound dressing should possess good mechanical properties such as tensile strength, flexibility and elasticity, which are required for handling and replacement of the dressing. Polyvinylidene
polymer [20]. PVDF fibrous membranes possess excellent physical properties
cr
5
ip t
fluoride (PVDF) is a stable, non-degradable, biocompatible and non-toxic
such as high tensile strength, good impact strength, wear resistance and
us
hydrophobicity that enable their application as wound dressing scaffolds [21].
cell activities in vitro and in vivo [22].
Enrofloxacin (Enro) is a fluoroquinolone antibiotic widely used in wound
M
10
an
Additionally, the piezoelectric property of PVDF fibrous membranes enhances
healing because of its strong bactericidal activity, broad bactericidal effect and
d
lower minimum inhibitory concentration [23]. The drug is released rapidly
te
during inflammation at early stages of the wound healing process [24].
15
Ac ce p
In the present study, by exploiting the advantages of electrospinning, we attempted to develop a simple method for fabricating drug-loaded PVDF fibrous membranes. Enro was absorbed onto PVDF nanofibres by eletrospinning. The surface morphology, mechanical properties, hydrophilicity, physical form and components of fibrous membranes were investigated. Analysis of the in vitro release of Enro from the Enro/PVDF fibrous membrane into PBS solution (pH = 20
7.4) showed that the sustained release of the drug followed Fickian diffusion process. The fibrous membranes showed excellent anti-bacterial activities against Escherichia coli and Staphylococcus aureus. Results of the in vitro cell
6
Page 6 of 36
proliferation and adhesion assays revealed high biocompatibility of Enro-loaded PVDF fibrous membranes. The results suggested that electrospinning is a promising method for the fabrication of drug-loaded membranes as wound
2. Experiment
cr
5
ip t
dressing.
2.1. Materials
us
Polyvinylidene fluoride (PVDF) was supplied by Arkema (Kynar 761, France)
an
Enrofloxacin was purchased from Huihua Animal Health Products & Co., Ltd. (Guangdong, China). Human dermal fibroblasts (HDF) used for this study were purchased from American Type Culture Collection, USA and human adipose
M
10
derived stem cells. N, N-dimethylacetamide (DMAC) and acetone (ACE) as the
d
solvent was supported by Fuyu Chemical Industry & Co., Ltd. (Tianjin, China).
te
Phosphate buffer saline (PBS) was prepared as the solvent for drug delivery in
15
Ac ce p
vitro. All of the chemicals and reagents are analytical grade. 2.2. Preparation of spinning solutions Four different concentration of spinning solutions of PVDF were prepared by dissolving 0.8 g, 1.0 g, 1.2 g and 1.4 g of PVDF powder in 10 ml of DMAC/ACE (1:1, v/v). Before electrospinning, the solutions were degassed by sonicating for 15 min in a KQ-100 ultrasonicator (Kunshan Ultrasonic 20
Instrument Co., Ltd., China). Four different concentrations of Enro/PVDF spinning solutions were prepared by first dissolving PVDF in 10 ml of DMAC/ACE (1:1, v/v) followed by the
7
Page 7 of 36
addition of 0.13 g, 0.19 g, 0.25 g or 0.33 g of Enro in the PVDF casting solution. The corresponding percentage composition of the drug in the fibres was 10%, 14%, 18% and 22% (w/w), respectively. All solutions were degassed before
2.3. Electrospinning process
cr
5
ip t
electrospinning.
The spinning solutions were fed into a 10-ml syringe connected to a 22-gauge
us
blunted stainless steel needle. The flow rate of spinning solutions were
an
controlled by a DT-200 precision syringe pump (Dalian Dingtong Technology Co., Ltd., China) and the flow was set to 0.50 ml/h. The needle was attached to a DE-100 high-voltage power supply (Dalian Dingtong Technology Co., Ltd.,
M
10
China) with a maximum voltage of 50 kV. A grounded aluminium foil was used
d
as the collector. The distance between the needle and the collector was 15 cm
te
and the applied voltage was set at 15 kV. The spinning room was maintained at
15
Ac ce p
25 °C. The details of the electrospinning process is shown in Fig. 1. The fibrous membranes were dried at 50 °C for 24 h in a GZX-9070MBE oven (Boxun Industry & Commerce Co., Ltd., China) to remove residual organic solvents and moisture. The drug-loaded fibrous membranes in which the percentage composition of Enro was 10%, 14%, 18% and 22% were denoted as E1, E2, E3 and E4, respectively. The pure PVDF fibrous membranes were denoted as E0. 20
2.4. Material characterization The surface morphology of the electrospun fibrous membranes was examined under a scanning electron microscope (SEM, Hitachi S-4800). The diameter
8
Page 8 of 36
distributions of the nanofibres were determined by image analysis using nano-measure for >200 points from SEM images. The hydrophilicity of the fibrous membranes was tested using a contact angle meter by measuring the
fibrous membranes. Wide-angle X-ray diffraction (XRD) analyses were
cr
5
ip t
contact angle between an ultrapure water droplet (10 μl) and the surface of the
performed using a Bruker D8 Advance (Cu Kα irradiation radiation)
us
diffractometer to evaluate the physical state of the drug in the fibrous
an
membranes. Fourier transform infrared spectroscopy (FTIR) was used to determine the structure and stability of the drug in the fibrous membranes through a Nicolet-Avatar 360 FTIR spectrometer (Nicolet Instrument
M
10
Corporation). The mechanical properties of the fibrous membranes (3.00 cm in
d
length, 1.00 cm in width and 0.40 mm in thickness) were assessed by using an
te
UTM-4202 electronic universal testing machine (Sansi Zongheng Technology &
15
Ac ce p
Co., Ltd.) at a strain rate of 10 mm/min. 2.5. Drug release in vitro
To evaluate the drug release profile of the fibrous membranes, 10–15 mg of Enro/PVDF fibrous membranes were transferred to sealed dialysis bags to ensure no nanofibres leak; 2 ml of 0.01-M PBS (7.4 and 9.0) were added to the bags, and they were placed in 500 ml of conical flasks containing 498 ml of 20
0.01-M PBS at two pH values (7.4 and 9.0). The contents of the flask were stirred with a magnetic stirrer at 37 °C and 100 rpm. At regular intervals, 3 ml of solution was taken from the flask and its absorbance at 271 nm was measured
9
Page 9 of 36
using an UV-Vis spectrophotometer (UV-2550, Shimadzu). After measuring the absorbance, the solution was returned to the flask to ensure constant volume of
by using the following equation and plotted against time. Accumulati ve Drug Release =
Drug Concentrat ion × V × 100 % Total Entrapped Drug
(1)
cr
5
ip t
the release solution. The cumulative percentage of Enro released was calculated
us
2.6. Cell culture
The cytocompatibility of the fibrous membranes, including the pure PVDF
an
and the 18% Enro/PVDF samples, were determined using the human dermal
10
M
fibroblast (HDF) as a cell model. First, HDFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (NBS, Sijiqing, China), supplemented with
d
10% foetal bovine serum (FBS) and 1% anti-biotic and anti-mycotic solutions
te
(Sangon, China) in 25-cm2 cell culture flasks at 37 °C in a humidified
Ac ce p
atmosphere of 5% CO2, and the culture medium was changed every 2 days. The fibrous membranes were cut into circular pieces 14 mm in diameter and placed 15
in 24-well plates. The samples were disinfected by soaking in ethanol for 1 h, washed three times with PBS and soaked in PBS for 1 h to remove the residual ethanol. Before seeding the cells, fibrous membranes were soaked overnight in the cell culture medium to promote cell attachment. HDF cells were then seeded on the fibrous membranes and empty tissue culture plates at a density of 1 × 104
20
cells/well. 2.7. Cell proliferation and morphology assay The number of viable HDFs on different fibrous membranes was determined
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after 1, 3, 5 and 7 days of culture. HDFs were separated from the fibrous membranes by trypsinisation and counted using a Beckman Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Inc., California, USA). The results were
surface. After 1, 3, 5, or 7 days of culture, the morphology of HDFs on fibrous
cr
5
ip t
expressed as the mean number of cells adhering per cubic centimetre of the
membranes was observed under an SEM. The fibrous membranes with adhering
us
cells were washed with PBS, fixed in 2.5% glutaraldehyde solution for 6 h,
an
dehydrated in a graded series of ethanol (30%–100%) and finally dried on a
10
ion sputter and then under an SEM. 2.8. Anti-bacterial assessment
M
critical point dryer. Thus the samples obtained were coated with gold using an
d
The anti-bacterial activity of the Enro/PVDF fibrous membrane against E.
te
coli and S. aureus was determined by the disk diffusion method. E. coli and S.
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Ac ce p
aureus were incubated in Luria–Bertani broth overnight at 37 °C and then diluted with sterile water to 1 × 107 cells/ml. A 40-μl aliquot of the diluted bacterial suspension was plated by the spread plate method on nutrient agar in duplicate. The fibrous membrane was cut into discs 6 mm in diameter and >3.5 mg in weight and placed on the surface of nutrient agar. The cultures were incubated for 24 h at 37 °C. Following this, the zones of inhibition around the 20
discs were measured. 2.9. Wound healing test
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Male Sprague-Dawley (SD) rats weighing 240-280g were used in this study. After anesthetization, the neck area of the dorsal of each rat was shaved and cleaned with 70% ethanol. Two wounds were created on the neck area of each
an equal size (1.5 cm × 1.5 cm) of pure PVDF membrane, 18% Enro/PVDF
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ip t
rat using scalpel (wound area of 0.8 cm2). The wounds were treated by placing
membrane and gauze dressing (n=6) over it. The wound dressing were fixed by
us
medical proof fabric to ensure without removing throughout the study period.
an
The area of the wound was measured every 3 days using the planimetry method until the wound completely healed. The percentage of wound closure is defined as Eq [32], [33].
M
10
d
W ound c losure (%) = (1 -
An ) × 100 Ao
(2)
te
where A0 is the initial wound area and An is the wound area after n days.
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Ac ce p
3. Results and discussion
3.1. The morphology of different pure PVDF and Enro/PVDF fibrous membranes.
The concentration of the polymer is one of key factors for successful preparation of polymer nanofibres. The concentration of the polymer should be suitable such that it should enable fabrication without beads while maintaining a 20
nanoscale diameter of the fibres. This optimal concentration differs with the polymer and can be adjusted according to the molecular weight and the type of solvent. We selected DMAC and ACE as the solvents because Enro is highly
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soluble in them. The SEM micrographs and diameter distribution graphs of the pure PVDF nanofibres fabricated using 8%–14% solutions of the polymer are shown in Fig. 2. When PVDF nanofibres were prepared by using polymer
were commonly observed. Higher concentrations of the PVDF polymer
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solutions with concentration
