soy protein isolated nanofibers and their release characteristics for biomedical applications

Therefore, our aims were to (i) fabricate nano ﬁbers using blended SA/SPI aqueous solutions with vancomycin simultaneously incor- porated via the electrospinning technique; (ii) investig[r]

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Original Article

release characteristics for biomedical applications

a Division of Polymer Materials Technology, Faculty of Agricultural Product Innovation Technology, Srinakharinwirot University, Nakhon-Nayok, Thailand

b Laboratory of Organic Synthesis, Chulabhorn Research Institute, Bangkok, Thailand

c Division of Biotechnology and Agricultural Products, Faculty of Agricultural Product Innovation Technology, Srinakharinwirot University, Nakhon-Nayok,

Thailand

d Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand

a r t i c l e i n f o

Article history:

Received 28 February 2017

Received in revised form

19 May 2017

Accepted 21 May 2017

Available online xxx

Keywords:

Electrospinning

Nanoﬁbers

Alginate

Soy protein isolated

Release characteristics

Biomedical applications

a b s t r a c t

Natural polymer-based nanofibers with functions of loading and releasing bioactive cues or drugs have recently gained interest for biomedical applications Nanotopography and large surface area to volume ratio of hydrophilic polymerfibers promote their use as carriers of hydrophilic drugs Here, sodium alginate (SA) and soy protein isolated (SPI) blendedfibers encapsulated with vancomycin were fabricated via electrospinning with the assistance of poly(ethylene oxide) (PEO) Morphological results showed submicron-sized, smooth and uniform as-spun SA/PEO/SPIfibers with an average diameter of 200 nm Beads on the fiber mats were formed with increasing SPI content in the blending system Optimal polymer composition of the electrospinning solution was determined as 5.6/2.4/2 SA/PEO/SPI Polymer blends were maintained after ionic crosslinking, as indicated by the FTIR result Investigation of release characteristic of vancomycin-loaded SA/PEO/SPI electrospun fibers exhibited initial burst release fol-lowed by a controlled release after 2 days of immersion in a phosphate buffered saline The release rate of SA/PEO/SPIfibers was significantly slower than that of SA/PEO fibers, and drug-loaded fibers inhibited bacterial growth against Staphylococcus aureus after 24 h of incubation Non-toxicity and biocompati-bility of thefibers were confirmed by an indirect cytotoxicity test using human dermal fibroblasts Re-sults suggested that the vancomycin-loaded SA/PEO/SPI blendedfibers were a promising nanomaterial for use in biomedicalfields such as scaffolds for tissue engineering and drug delivery systems

© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

1 Introduction

Electrospinning has recently gained interest as a potential

technique for fabricating polymericﬁbers with diameters ranging

between micrometers and nanometers due to ease of use,

cost-effectiveness and adaptability[1,2] Polymericﬁbers exhibit a

va-riety of advantages including interconnected porous structure,

large surface area to volume and the ability to encapsulate

bio-logical cues such as antibiotics, anticancer agents, proteins and

growth factors[2e4] Therefore, polymericﬁbers are widely used

as carriers of active biomolecules in applications of wound healing, biosensors, drug delivery and tissue engineering[3,5]

Sodium alginate (SA) is a naturally occurring algal anionic polysaccharide with excellent biocompatibility, low toxicity, non-immunogenicity and low cost [6] Electrospun SA fibers have been investigated for use as cell-growth scaffolds for bone, cartilage and nerve tissue engineering, and as drug carriers for bacterial inhibition[5,7,8] However, individual SA in water-based solution cannot be electrospun due to its insufficient chain entanglements [9] Thus, electrospinning of SA aqueous solutions is often co-blended either with poly(vinyl alcohol) (PVA) or with poly(-ethylene oxide) (PEO), leading to uniform and smooth fibers [5,9e11] Besides, SA lacks cell recognition and is, therefore, inap-propriate for use in the field of biomedicine To overcome this limitation, blending with protein-based polymers such as collagen,

* Corresponding author.

E-mail address: patcharakamon@g.swu.ac.th (P Nooeaid).

Peer review under responsibility of Vietnam National University, Hanoi.

Contents lists available atScienceDirect Journal of Science: Advanced Materials and Devices

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d

http://dx.doi.org/10.1016/j.jsamd.2017.05.010

2468-2179/© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

Journal of Science: Advanced Materials and Devices xxx (2017) 1e8

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gelatin, elastin and silkﬁbroin is the most common modiﬁcation

method used to achieve more protein-binding sites and

subse-quently enhance cell adhesion[1,7,8,12e16] Protein-based

poly-mers derived from plants, such as soy protein isolated from

soybeans, are alternatives to synthetic and animal-derived

protein-based polymers because of their abundant renewable resource,

non-toxicity, biodegradability and cost-effectiveness [17e20]

In-vitro biocompatibility of soy protein isolated (SPI) has been proved

with mouseﬁbroblasts and human mesenchymal stem cells[17] In

addition, SPI from non-animal origin has low immunogenicity, long

storage time and stability, with medicinal properties that accelerate

wound healing and tissue regeneration[21] Accordingly, SPI

pre-sents attractive features for controlled release, wound healing and

tissue engineering Previous studies determined that SA/SPI blends

could be fabricated into many forms including denseﬁlms, porous

foams, hydrogels and ﬁbers Olami et al [21] reported SA/SPI

blended foams fabricated by freeze-drying with chemical

cross-linking, and in-vitro investigation of these porous foams suggested

that SA/SPI blends promoted cell inﬁltration and stability Silva

et al.[20]developed SA/SPI hydrogels with bioactive glass for bone

regeneration, while Wang et al.[18]reported the successful

fabri-cation of SA/SPIﬁbers by viscous spinning into a coagulating bath

However, the diameters of these ﬁbers were much larger than

nanoﬁbers prepared by electrospinning technique Electrospun

nanoﬁbers are composed of highly interconnected porous

struc-tures with a large surface area as desirable feastruc-tures for biomedical

applications[22,23]

Several forms of SA/SPI blends have been investigated in

regenerative biomedicine[15,19,24e26]; however, electrospinning

of blended SA/SPI aqueous solutions has not been explored

Therefore, our aims were to (i) fabricate nanoﬁbers using blended

SA/SPI aqueous solutions with vancomycin simultaneously

incor-porated via the electrospinning technique; (ii) investigate the effect

of SPI contents in the blends on morphology, chemical composition

and in-vitro release characteristics in addition to antibacterial

properties; and (iii) evaluate the potential of the fabricatedﬁbers

for biomedical applications using an indirect cytotoxicity test

against human dermalﬁbroblasts (HDFs)

2 Experimental

2.1 Materials

Sodium alginate (SA) (alginic acid sodium salt from brown algae,

suitable for immobilization of microorganisms, molecular weight

100,000e200,000 g/mol) and poly(ethylene oxide) (PEO;

molecu-lar weight 106g/mol), sodium hydroxide (NaOH), calcium chloride

dihydrate (CaCl2$2H2O), vancomycin antibiotic, phosphate buffered

saline (PBS tablets) and 3-(4,

5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from

Sigma-eAldrich (St Louis, MO, USA) Soy protein isolated (SPI) containing

99% protein was purchased from Now Sports (Bloomingdale, IL,

USA) Dulbecco's modiﬁed Eagle's medium (DMEM),

penicillin-streptomycin solution, and Antibiotic-Antimycotic solution were

purchased from Gibco (Grand Island, NY, USA) Fetal bovine serum

(FBS) was purchased from Hyclone, USA

2.2 Electrospinning of alginate/soy protein isolated (SA/SPI) blends

The starting solutions were 3 wt% SA and 3 wt% PEO Both SA

and PEO were dissolved separately in deionized water at room

temperature under vigorous stirring until homogeneous solutions

were obtained PEO was mixed with SA because the alginate needs

a co-blending polymer that acts as a carrier from the needle tip to

the sample collector during the electrospinning process An SA/PEO

blended solution at a weight ratio of 7/3 was chosen based on preliminary tests (results not shown) The polymer mixture was stirred to obtain a homogeneous solution Then, SPI was dissolved

in deionized water at a concentration of 3 wt% at 90C and ho-mogenized at 5000 rpm for 10 min After that, the mixture was stirred continuously at 90 C for 30 min The SPI solution was adjusted to a pH at around 9 by addition of 1 M NaOH to avoid aggregation and precipitation of soybean protein [27] Finally, different contents of 3 wt% SPI solution were added into the pre-vious SA/PEO (7/3) solution The compositions of polymer blended solutions are listed inTable 1 Each composition of SA/PEO/SPI was loaded into a 10 ml glass syringefitted with a stainless steel blunt needle (22-gauge) High voltage of 15 kV was applied to the solu-tion forcing the polymer from the syringe onto the collector with a needle-collector distance of 15 cm The volumetricflow rate of the electrospinning solution was set at 0.5 ml/h Electrospinning con-ditions were chosen based on the preliminary testing The resulting fibers were then crosslinked by spraying an excess amount of 0.5 M CaCl2 crosslinking agent on the meshes and dried at room tem-perature for 24 h

Prior to fabrication of vancomycin-loadedﬁbers, vancomycin at

a concentration of 0.1 wt% of polymer was added into the blended solution of optimal polymer composition The mixture was stirred until the drug was completely dissolved in the polymer solution Electrospinning of vancomycin-loaded SA/PEO/SPI solutions was performed under similar condition to SA/PEO/SPI solutions without drug loading

2.3 Characterization 2.3.1 Morphological observation Morphology of electrospun SA/SPIfibers was observed under a scanning electron microscope (SEM, Quanta 250 microscope, Japan) Thefibers were gold-coated using a sputtering device (Jeol, JFC-1200) prior to SEM observation Averagefiber diameter was determined from the SEM images using ImageJ software from a hundred randomly selectedfibers

2.3.2 Fourier transform infrared spectroscopy Chemical composition of SA/SPI ﬁber mats was investigated using attenuated total reﬂection Fourier transform infrared spec-troscopy (ATR-FTIR; Nicolet170-SX, Thermo Nicolet Ltd., USA) Spectra were recorded between wavenumbers 4000 to 600 cm1at

32 scans with a resolution of 2 cm1at room temperature 2.3.3 In-vitro drug release study

Vancomycin-loaded SA/SPIﬁber mats with thickness of around

100mm were cut into circular disks with a diameter of 12 mm Each ﬁber disk was immersed in 15 ml of phosphate buffer saline solution (PBS, pH 7.4) Experiments were performed at 37C with agitation at

90 rpm in an orbit shaker (Labtech, LSI-3016A, Korea) At each im-mersion time, ranging from 0 to 48 h, 1 ml of PBS solution was withdrawn and used to determine the amount of drug released

Table 1 Electrospinning solution compositions.

SA/PEO/SPI ﬁbers Amount of polymer content (wt%)

R Wongkanya et al / Journal of Science: Advanced Materials and Devices xxx (2017) 1e8

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Simultaneously, an equivalent volume of fresh PBS solution was

replaced to maintain the sink condition The amount of vancomycin

released at each time point was determined by a UV-vis

spectro-photometer (GENESYS 10S, Thermo) at a wavelength of 280 nm The

absorbance detection of the drug was converted to drug

concen-tration according to the calibration curve of vancomycin in PBS

so-lution Results were calculated from Eq.(1)and were presented as %

cumulative release as a function of immersion time (h)

Ctotal 100 (1)

where Ctis the cumulative amount of drug release at time t and

Ctotalis the total drug release after theﬁber disks are completely

decomposed Four replicates were analyzed for each sample

Re-sults were presented as the mean± standard deviation (n ¼ 4)

2.3.4 Release kinetics study

Release kinetics of vancomycin from SA/PEO/SPI ﬁbers were

determined using the Ritger-Peppas model which is suitable for

describing drug release from polymeric systems[28,29] To

esti-mate the release mechanism, an initial 60% of the antibiotic release

data wasﬁtted to Eq.(2)as this equation is only typically applicable

to up to 60% of early-stage release[28,29]

where Mt/M∞is the fraction of drug release at time t, K is the rate

constant and n is the release exponent which characterizes the

release mechanism The n value was determined by the slope of the

plot of logarithm Mt/M∞as a function of the logarithm of time

2.3.5 Antibacterial determination

The antibacterial activity of the vancomycin-loaded SA/PEO/SPI

ﬁbers was investigated using the disk diffusion method of the US

Clinical and Laboratory Standards Institute against two pathogenic

bacteria (Gram-positive and negative)[30] Staphylococcus aureus

(S aureus) and Escherichia coli (E coli) were chosen due to their

frequent involvement in infections[31] The strains were grown

overnight on agar plates at 37C prior to use Then, bacteria

sus-pension at a density 106CFU/ml was spread over an agar plate, and

specimens (thickness 100mm) were cut as circular disks with a

diameter of 12 mm SA/PEOﬁber mats without vancomycin were

used as the control Sample disks were pre-treated under UV light

for 30 min prior to testing for released drug-induced bacterial

in-hibition Each specimen was adhered to the agar plate and

incu-bated at 37C for 24 h After that, the diameters of the inhibition

zones around the sample disks were determined Four replicates

were tested for each sample

2.3.6 In-vitro cytotoxicity evaluation

Indirect cytotoxicity evaluation of SA/PEO/SPIﬁbers with and

without vancomycin loading was performed in accordance with

ISO10993-5 standard test method[32,33] Brieﬂy, ﬁber samples

(circular disks of 12 mm diameter and around 100mm thickness)

were sterilized under UV radiation for 30 min and then immersed

in cell culture medium (DMEM supplemented with 10% v/v FBS and

antibiotics) in a 96-well tissue culture polystyrene plate (TCPS) The

ﬁber samples were incubated for 1 and 3 days to produce the

sample extraction Three extraction ratios of the extraction medium

(i.e., 0.5, 5, and 10 mg/ml) were investigated Human dermal

ﬁ-broblasts (HDFs) were cultured separately in wells of TCPS at

15,000 cells/well in the culture medium for 16 h to allow cell

attachment onto the well surface After that, the medium was

replaced with an extraction medium and HDFs were incubated for another 24 h MTT assay was used to determine the viability of the treated cells [34] After treatment, medium in the wells was removed and new medium containing 0.5 mg/ml MTT was added After 2 h of incubation, the formazan crystals formed by cellular dehydrogenase were solubilized by adding DMSO to the wells The absorbance was determined at 550 nm and subtracted with the absorbance at 650 nm The absorbance of formazan formed by the control cells was taken as 100% cell viability Three replicates were investigated for each sample

2.4 Statistical analysis Data were reported as mean± standard derivation (SD) One-way ANOVA was used to compare the means of different data sets, and signiﬁcance was accepted when the p-value was less than 0.05

3 Results and discussion 3.1 Electrospinning and morphology of electrospunfibers Successful electrospinning of SA/PEO/SPI blended fibers was achieved and non-woven morphology of as-spunfibers is shown in Fig 1 Electrospun SA fibers were previously manufactured with assistance of a co-blending polymer such as polyKtn(ethylene ox-ide) (PEO) and poly(vinyl alcohol) (PVA)[5,34] SA is rigid and its chain conformation is extended in aqueous solution; therefore, SA solution lacks chain entanglements and has relatively low spinn-ability[5,10,11,34] Consequently, PEO with properties of biocom-patibility, biodegradability and non-toxicity was used to improve the spinnability of SA aqueous solutions As a result, at the optimal electrospinning conditions of 15 kV high voltage, 15 cm needle tip-collector distance and 0.5 ml/h feed rate, SA/PEO solution at a weight ratio of 7/3 was conveniently electrospun and non-woven fibers were achieved as shown in Fig 1(a) The SA/PEO fibers were produced in the nano-sized range of 60e600 nm with mean fiber diameter of 200 nm (Fig 1(a0)) Previous electrospun alginate-basedfibers recorded mean diameter of SA fibers in the range of 250e300 nm [5,7,10,34,35] Lee et al [10] reported successful electrospinning of alginate with co-blending of PVA with weight ratio of SA/PVA at 1/20, while Li et al.[5]fabricated electrospun SA/ PVAfibers forming few beads using SA/PVA solution at a ratio of 4/6

by weight These studies, both determined that increased alginate content showed a negative impact on spinnability and obtained ﬁber morphology In contrast, our results indicated that electro-spinning of SA aqueous solutions could be satisfactorily performed with addition of PEO (molecular weight 106 g/mol) at the maximum weight ratio of 7/3 SA/PEO Therefore, enhanced chain entanglements of high molecular weight PEO dominated the electro-spinnability of alginate solutions

Furthermore, we extended our study to SPI blends, since SPI is a promising protein in tissue engineering [20] and a possible co-blending candidate to improve the performance of polymer ma-trix like alginate Following this approach, electrospinning of SA/SPI blended solutions in the presence of PEO was carried out As-spun SA/PEO/SPI nanofibers with several blending ratios (Table 1) are shown inFig 1(bef) Compared to SA/PEO fibers inFig 1(a), SA/ PEO/SPI (6.3/2.7/1 and 5.6/2.4/2)fibers (10e20 wt% SPI) did not show significant difference in morphology and mean fiber size, as shown inFig 1(b, b0, c and c0) Diameters of SA/PEO/SPIfibers at weight ratio of 6.3/2.7/1 were in the range of 100e300 nm and SA/ PEO/SPI (5.6/2.4/2)fibers showed fiber diameters in the range of 200e600 nm In addition, beads tended to form on the fibers with increasing SPI content in the blends, probably because the addition

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of SPI minimized the chain entanglements of the SA/PEO blending

system In detail, when SPI content was increased to 30 wt% (SA/

PEO/SPI 4.9/2.1/3), an increased degree of beading on theﬁbers was

observed (Fig 1(d)) and the size distribution of the ﬁbers

(300 nme2mm) (Fig 1(d0)) increased Thisﬁnding was consistent

with the non-uniformity ofﬁber formation during electrospinning

Especially, in the case of SA/PEO/SPI 4.2/1.8/4, large quantities of

beads occurred along the ﬁber mesh (Fig 1(e)), while

electro-spinning of SA/PEO/SPI 3.5/1.5/5 solution was not possible as only

electrospraying took place, resulting in aggregation of

microparti-cles as conﬁrmed by the SEM image inFig 1(f)

Based on the morphological results, it was suggested that

increasing SPI content hindered the electrospinning of SA-based

solutions Subsequently, formation of ﬁbers was not well

ach-ieved with SPI above 30 wt% in the electrospinning solutions This

may be attributed to insufﬁcient chain entanglements of PEO

car-rier in the solution systems Since the amount of SPI (30e50 wt%) in

these compositions was larger than their PEO content (15e21 wt%)

as detailed inTable 1, the PEO could not dominate the spinnability

This phenomenon was in agreement with previous studies that

determined PEO as a crucial factor, which favored the

electro-spinnability of alginate aqueous solution [34] Our results

indi-cated that the SPI content could be maximized at 20 wt% to the SA/

PEO solution (SA/PEO/SPI formulation of 5.6/2.4/2) to facilitate

formation of uniform and smooth nanoﬁbers

3.2 Crosslinking of nanoﬁber mats

On the basis of spinnability andﬁber morphology, SA/PEO/SPI

5.6/2.4/2 (56 wt% SA, 24 wt% PEO and 20 wt% SPI;Table 1)ﬁbers

were used as a carrier of vancomycin antibiotic drug They were

comparatively investigated with SA/PEOfibers without the pres-ence of SPI (70 wt% SA and 30 wt% PEO;Table 1) Vancomycin-loaded nanofibers in both types were fabricated using blending electrospinning, as blending is a simple method of incorporating a drug into electrospun polymericfibers

Thefibrous structure did not change in morphology after drug loading, as confirmed byFig 2(a, a0) compared toFig 1(a) for SA/ PEOfibers, andFig 2(c, c0) compared toFig 1(c) for SA/PEO/SPI fibers This result could be suggested by homogeneous dissolution

of the water-soluble drug into hydrophilic polymers Thefiber mats were then crosslinked using an agent containing calcium ions to improve water resistance By ionic crosslinking, thefibers in both cases swelled while their surface remained smooth, as observed in Fig 2(b, b0and d, d0) Thefibers fused at junctions, forming more intersected regions, especially in the case of SA/PEO/SPI fibers (Fig 2 (d, d0)) A morphological change of SA-based fibers after crosslinking was also noted in a previous study of SA/PVAfibers [36] The SA/PVAfibers fused, flattened and their macrostructure was damaged[32,36] This might be caused by high swelling of hydrophilic polymers in aqueous solutions Nevertheless, cross-linked mats withfibrous structure were still visible in our study 3.3 Chemical composition of crosslinked electrospunfibers FTIR analysis was performed on SA/PEO and SA/PEO/SPI electrospun fibers after ionic crosslinking and compared to as-received SA and SPI to identify characteristic absorption peaks corresponding to the chemical structures (Fig 3) In the spectrum of neat SA, the main characteristic peaks of SA appeared as a broad peak at 3700e3000 cm1, and at 1595 and 1408 cm1, which were attributed to eOH stretching vibration, and antisymmetric and

Fig 1 SEM images and ﬁber diameter histograms of electrospun SA/PEO/SPI ﬁbers at weight ratios of (a, a 0 ) 7/3/0, (b, b 0 ) 6.3/2.7/1, (c, c 0 ) 5.6/2.4/2, (d, d 0 ) 4.9/2.1/3, (e, e 0 ) 4.2/1.8/4 and (f, f 0 ) 3.5/1.5/5.

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symmetric eCOOe stretching vibration, respectively[18,20] In the

spectrum of SA/PEO/SPI 7/3/0ﬁbers, in addition to the

character-istic peaks of pure SA at 1610 and 1408 cm1, a sharp absorption

band at 1100 cm1appeared which was related to the CeOeC

ab-sorption complex of PEO[37] Moreover, the intensity of the

ab-sorption band at 2884 cm1in the spectrum of SA/PEOﬁbers was

higher compared to the spectrum of neat SA and resulted from

eCH2 bending vibration in the presence of PEO When SPI was

blended with SA/PEO, a sharp absorption band appeared at

3277 cm1which was assigned to stretching vibrations of the eNH

group[18,20,38] The absorption band of the carboxylate group at

1595 cm1shifted to a higher wavenumber at 1618 cm1, while a

new peak existed at 1525 cm1, indicating amide I (eC]O) and

amide II (eNH) of SPI, respectively Similar to the spectrum of neat

SPI, amide I and II absorption bands were found at 1633 and

1526 cm1, respectively [18,38] In addition, the characteristic

peaks of PEO still appeared in the spectrum of SA/PEO/SPI 5.6/2.4/2

ﬁbers, including the peak of CeOeC absorption complex at

1084 cm1 and the peak of eCH2 bending at 2883 cm1 This

conﬁrmed the occurrence of SA/PEO/SPI blends and the remains of

the three polymers after crosslinking

3.4 In-vitro drug release and release kinetics Release behaviors of vancomycin from the nanofibers are pre-sented inFig 4(a) Van-SA/PEO/SPI 7/3/0fibers showed a gradual release of vancomycin along the investigation time Rapid initial burst release of drug was observed after thefirst 4 h of immersion reaching around 33% Initial burst release is a typical characteristic

of electrospun blended polymeric fibers since blended electro-spinning usually leads to the distribution of drug on thefiber sur-face[4] Later, the drug release continuously increased for 2 days of immersion In the case of Van-SA/PEO/SPI 5.6/2.4/2fibers, an initial burst release was followed by sustained release The initial burst release was significantly minimized compared to the fibers without SPI, indicated by the release trends before the intersected point of both curves at 18 h Drug release of Van-SA/PEO/SPI 5.6/2.4/2fibers was approximately 4% at the samefirst stage of 4 h, followed by about 69% of drug release within 18 h of immersion Finally, release

of vancomycin maintained at around 85% after 30 h of immersion This result indicated that SA/PEO/SPI blended ﬁbers showed reduced initial burst release and provided a more controlled release, while SA/PEOﬁbers promoted drug release in a conven-tional manner

Subsequently, the release profiles were extended to the study of release kinetics to investigate the release mechanism of vanco-mycin from thefibers Release profiles inFig 4(a) werefitted with the Ritger and Peppas model which is suitable for swellable drug delivery systems[28] Values of kinetic exponent (n) indicating the release mechanism and regression coefficient (R2) were reported in Fig 4(b) In general, the n value depends on sample geometry such

as thin films, cylinders and spheres [20,39] Electrospun fiber meshes could be regarded as a thinfilm[20,39] When n< 0.5, the drug release mechanism is completely controlled by Fickian diffu-sion A value of n> 1.0 means that the release mechanism is cor-responding to case II transport If 0.5 n 1.0, this indicates an overlapping of the two previous means of transport [39e42] In Fig 4(b), the n value of SA/PEOfibers without the presence of SPI was 0.54, implying a release mechanism overlapping Fickian and case II diffusion, whereas the SA/PEO/SPI blendedfibers showed an

n value of 1.78, corresponding to case II transport mechanism Thus, the release of SA/PEOﬁbers was controlled by penetration of the

Fig 2 SEM images of Van-SA/PEO/SPI 7/3/0 fibers including (a, a 0 ) before crosslinking and (b, b0) after crosslinking with different magnifications, and SEM images of Van-SA/PEO/ SPI 5.6/2.47/2 fibers including (c, c 0 ) before crosslinking and (d, d0) after crosslinking with different magnifications.

Fig 3 FTIR spectra of crosslinked SA/PEO/SPI 5.6/2.4/2 ﬁbers compared to the spectra

of neat SA, SPI and SA/PEO/SPI 7/3/0 ﬁbers.

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aqueous medium, swelling and polymer chain disentanglement

and relaxation[4,41], while the release mechanism of SA/PEO/SPI

ﬁbers was controlled by the rate of polymer chain relaxation like a

polymer dissolution, which was found as a typical release

mecha-nism of drug-loaded polysaccharide-based matrix[4,41] This

dif-ference in release mechanisms may be caused by morphological

changes of SA/PEO/SPIﬁbers after crosslinking leading to entangled

and overlappedﬁbers Consequently, most of the ﬁbers lost their

shape and their surface area changed, while the cylindrical shape of

the SA/PEOﬁbers was maintained This might be the reason for the

release kinetic change since the n value is inﬂuenced by the

ge-ometry of devices Moreover, sample dimensions, i.e.,ﬁber size, are

believed to be important factors affecting the n value and

subse-quently drug release mechanisms In our investigation, the mean

ﬁber diameters and ﬁber distribution of both types did not show

signiﬁcant difference Therefore, the factor of ﬁber size might not

provide the impact in this case Other studies on drug-loaded

polymericﬁbers have suggested that the release properties of the

ﬁbers could be tailored by changing the polymer used[43,44] Also,

blending one polymer matrix with another altered release kinetics

from the kinetics of pure polymer matrix[43] In addition, it was

suggested that the morphology ofﬁbers plays an important role in

the release kinetics [31,43] Similarly, we determined ﬁber

morphology as a dominant factor affecting release properties

3.5 Antibacterial activity evaluation

The antibacterial property of vancomycin-loaded SA/PEO/SPI

ﬁbers against S aureus (Gram-positive) and E coli (Gram-negative)

was tested following the disk diffusion method Disks of

electro-spun SA/PEOﬁbers without loading vancomycin served as control

Bacterial inhibition zones around the drug-loadedﬁber disks are

shown inFig 5 After 24 h of incubation, both Van-SA/PEO/SPI (7/3/

0) and Van-SA/PEO/SPI (5.6/2.4/2) ﬁbers inhibited the growth of

S aureus, showing inhibition zones of 22.8± 0.5 and 21 ± 1 mm,

respectively, while no inhibition zone was observed around the

control disk as expected The antibacterial activity of Van-SA/PEO/

SPI (7/3/0)ﬁbers was slightly higher than that of Van-SA/PEO/SPI

(5.6/2.4/2)ﬁbers in contact with S aureus This result related to

the faster rate of initial drug release by Van-SA/PEO/SPI (7/3/0)

ﬁ-bers On the contrary, none of the drug-loaded ﬁber disks

pre-vented the growth of Gram-negative E coli as expected, because

vancomycin is only effective against Gram-positive bacteria This confirmed that the released drug was responsible for inhibiting bacterial growth, not thefiber materials used Thus, the SA/PEO/SPI fibers acted as an effective device, offering functions for loading and releasing drugs against bacterial growth

3.6 Indirect cytotoxicity evaluation

To confirm the potential of electrospun SA/PEO/SPI fibers as a biomedical device, non-cytotoxicity and biocompatibility of the drug-free and drug-loadedfibers were investigated using in-vitro indirect cytotoxicity evaluation Following the ISO10993-5 standard test method for biological evaluation of medical devices, human dermalfibroblasts (HDFs) were cultured with the extraction media

of thefibers[32,33,45] As shown inFig 6, the non-toxicity and biocompatibility of thefibers were indicated by cell viability after exposure to the extraction media (0.5, 5 and 10 mg/ml) InFig 6(a), both drug-free SA/PEO/SPI 7/3/0 and 5.6/2.4/2 fibers at all

Fig 4 (a) In-vitro drug release of fibers fabricated from vancomycin-loaded SA/PEO/SPI blends after immersion in the PBS solution at pH 7.4 and 37 C () compared to that of vancomycin-loaded SA/PEO fibers (:) and (b) linear fits of Ritger and Peppas model for vancomycin release from fabricated fibers showing fitting parameters, including kinetic exponent (n) and regression coefficient (R 2 ).

Fig 5 Antibacterial activity of representative samples of vancomycin-loaded Van-SA/ PEO/SPI (7/3/0) and Van-SA/PEO/SPI (5.6/2.4/2) fibers against Staphylococcus aureus (S aureus) and Escherichia coli (E coli) in comparison with the activity of unloaded SA/ PEO fibers as negative control The result reports diameter (mm) of inhibition zone occurred around the fiber disks in contact with bacteria for 24 h.

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concentrations of extraction media were not toxic to the cells,

indicated by cell viability in the range of 91e113% These viability

values agreed with a previous study[46,47].Fig 6(b) showed that

vancomycin loading led to reduction of cell viability compared to

control (100%), signiﬁcantly in the case of the highest extraction

concentration (5e10 mg/ml) of SA/PEO/SPI 7/3/0 ﬁbers after 1 and 3

days extraction The viability of cells was found in the range of

44e76% as shown inFig 6(b) This suggested that antibiotic loading

at high extraction concentration had a negative effect on cell

viability In contrast, vancomycin-loadedﬁbers at the lowest

con-centration (0.5 mg/ml) appeared to be non-toxic to cells showing

high viability of 91e92% Thus, the result of cell viability conﬁrmed

that both drug-free and drug-loaded fabricatedﬁbers at 0.5 mg/ml

of extraction media were not toxic to HDFs and compatible with the

cells In addition to the drug releasing function, SA/PEO/SPI blended

ﬁbers would be a useful candidate for tissue engineering

applica-tions as scaffolds based on polymer nanoﬁbers to support cell

ac-tivity and subsequently promote speciﬁc tissue regeneration

4 Conclusion

Electrospinning was used to fabricate nano-sizedﬁbers based

on alginate, soy protein isolated and poly(ethylene oxide) blended

aqueous solutions with vancomycin antibacterial drug loading

Aqueous solutions of vancomycin-loaded SA/PEO/SPI (5.6/2.4/2)

were successfully electrospun to produce uniform ﬁbers with

diameter range of 60e600 nm Composition of polymer blends was

believed to strongly affectﬁber morphology and consequently drug

release behavior The SA/PEO/SPIﬁbers provided a slower release of

vancomycin in the initial stage, followed by constant release over a

longer time compared to SA/PEOﬁbers According to the Ritger and

Peppas model, SA/PEO/SPIﬁbers followed the release mechanisms

of polymer chain relaxation In addition, theﬁbers provided

anti-bacterial activity against Gram-positive S aureus related to the

released vancomycin dose Finally, the result of in-vitro cytotoxicity

of theﬁbers tested with HDFs conﬁrmed their non-cytotoxicity and

biocompatibility As a result, the electrospun SA/PEO/SPI ﬁbers

could act as a biomedical device offering several advantages

including drug encapsulation and controlled release, antibacterial

activity and compatibility with cells suitable for biomedical

applications

Acknowledgements Financial support from Srinakharinwirot University with the grant number 744/2558 matching with funding from Faculty

of Agricultural Product Technology and Innovation, Srinakhar-inwirot University with grant number 517/2558 was gratefully acknowledged

References

[1] K.Y Lee, L Jeong, Y.O Kang, S.J Lee, W.H Park, Electrospinning of poly-saccharides for regenerative medicine, Adv Drug Deliv Rev 61 (2009) 1020e1032

[2] T.J Sill, H.A Von Recum, Electrospinning: applications in drug delivery and tissue engineering, Biomaterials 29 (2008) 1989e2006

[3] S Agarwal, J.H Wendorff, A Greiner, Use of electrospinning technique for biomedical applications, Polymer 49 (2008) 5603e5621 (Guildf)

[4] A.K Gaharwar, S.M Mihaila, A.A Kulkarni, A Patel, A Di Luca, R.L Reis, M.E Gomes, C Van Blitterswijk, L Moroni, A Khademhosseini, Amphiphilic beads as depots for sustained drug release integrated into ﬁbrillar scaffolds,

J Control Release 187 (2014) 66e73 [5] W Li, X Li, Y Chen, X Li, H Deng, T Wang, R Huang, G Fan, Poly(vinyl alcohol)/sodium alginate/layered silicate based nanoﬁbrous mats for bacterial inhibition, Carbohydr Polym 92 (2013) 2232e2238

[6] P Nooeaid, J.A Roether, E Weber, D.W Schubert, A.R Boccaccini, Technolo-gies for multilayered scaffolds suitable for interface tissue engineering, Adv Eng Mater 16 (2013) 319e327

[7] S.I Jeong, M.D Krebs, C.A Bonino, S.A Khan, E Alsberg, Electrospun alginate nanoﬁbers with controlled cell adhesion for tissue engineering, Macromol Biosci 10 (2010) 934e943

[8] S.I Jeong, D Ph, M.D Krebs, C.A Bonino, J.E Samorezov, S.A Khan, E Alsberg, Electrospun chitosanealginate nanoﬁbers with in situ polyelectrolyte complexation for use as tissue engineering scaffolds, Tissue Eng Regen Med.

17 (2011) 59e70 [9] H Nie, A He, J Zheng, S Xu, J Li, C.C Han, Effects of chain conformation and entanglement on the electrospinning of pure alginate, Biomacromolecules 9 (2008) 1362e1365

[10] Y.J Lee, D.S Shin, O.W Kwon, W.H Park, H.G Choi, S.S Han, S.K Noh, W.S Lyoo, Preparation of atactic poly (vinyl alcohol)/sodium alginate blend nanowebs by electrospinning, J Appl Pol 106 (2007) 1337e1342 [11] S Saﬁ, M Morshed, S.A.H Ravandi, M Ghiaci, Study of electrospinning of sodium alginate, blended solutions of sodium alginate/poly (vinyl alcohol) and sodium alginate/poly (ethylene oxide), J Appl Polym Sci 104 (2007) 3245e3255

[12] N Sultana, M Wang, PHBV/PLLA-based composite scaffolds fabricated using

an emulsion freezing/freeze-drying technique for bone tissue engineering: surface modiﬁcation and in vitro biological evaluation, Biofabrication 4 (2012), 015003

[13] N Bhattarai, M Zhang, Controlled synthesis and structural stability of alginate-based nanoﬁbers, Nanotechnology 18 (2007), 455601

Fig 6 Indirect cytotoxicity of (a) electrospun drug-free fibers and (b) Van-SA/PEO/SPI fibers based on viability of human dermal fibroblasts (HDFs) after exposure to the 1 and 3 days fiber extraction media for 24 h Cells that cultured in fresh culture medium in the absence of fiber extraction were used as the control Data are reported as mean ± SD (n ¼ 3) * indicates significant difference of fiber sample from control (*p < 0.05).

Trang 8

[14] S Moon, R.J Farris, Electrospinning of heated gelatin-sodium alginate-water

solutions, Polym Eng Sci (2009) 1616e1620

[15] J Chang, Y.-H Lee, M Wu, M.-C Yang, C Chien, Preparation of electrospun

alginate ﬁbers with chitosan sheath, Carbohydr Polym 87 (2012) 2357e2361

[16] J.-J Chang, Y.-H Lee, M.-H Wu, M.-C Yang, C.-T Chien, Electrospun

anti-adhesion barrier made of chitosan alginate for reducing peritoneal

adhe-sions, Carbohydr Polym 88 (2012) 1304e1312

[17] K.B Chien, R.N Shah, Novel soy protein scaffolds for tissue regeneration:

material characterization and interaction with human mesenchymal stem

cells, Acta Biomater 8 (2012) 694e703

[18] Q Wang, Y Du, X Hu, J Yang, L Fan, T Feng, Preparation of alginate/soy

protein isolate blend ﬁbers through a novel coagulating bath, J Appl Polym.

Sci 101 (2006) 425e431

[19] X Xu, L Jiang, Z Zhou, X Wu, Y Wang, Preparation and properties of

elec-trospun soy protein isolate/polyethylene oxide nanoﬁber membranes, ACS

Appl Mater Interfaces 4 (2012) 4331e4337

[20] R Silva, B Bulut, J.A Roether, J Kaschta, D.W Schubert, A.R Boccaccini,

Sonochemical processing and characterization of composite materials based

on soy protein and alginate containing micron-sized bioactive glass particles,

J Mol Struct 1073 (2014) 87e96

[21] Z.M Olami, Microstructure and in vitro cellular response to novel soy

protein-based porous structures for tissue regeneration applications, J Biomater Appl.

30 (2016) 1004e1015

[22] W Cui, X Zhu, Y Yang, X Li, Y Jin, Evaluation of electrospun ﬁbrous scaffolds

of poly(dl-lactide) and poly(ethylene glycol) for skin tissue engineering,

Mater Sci Eng C 29 (2009) 1869e1876

[23] N.M Neves, R Campos, A Pedro, J Cunha, F Macedo, R.L Reis, Patterning of

polymer nanoﬁber meshes by electrospinning for biomedical applications, Int.

J Nanomed 2 (2007) 433e448

[24] D Fang, Y Liu, S Jiang, J Nie, G Ma, Effect of intermolecular interaction on

electrospinning of sodium alginate, Carbohydr Polym 85 (2011) 276e279

[25] G Kim, K Park, Alginate-nanoﬁbers fabricated by an electrohydrodynamic

process, Polym Eng Sci (2009) 2242e2248

[26] K Tarun, N Gobi, Calcium alginate/PVA blended nano ﬁbre matrix for wound

dressing, Indian J Fibre Text Res 37 (2012) 127e132

[27] Y.J Yuan, O.D Velev, K Chen, B.E Campbell, E.W Kaler, A.M Lenhoff, Effect of

pH and Ca2þ-induced associations of soybean proteins, J Agric Food Chem 50

(2002) 4953e4958

[28] P.L Ritger, N.A Peppas, A simple equation for description of solute release I.

Fickian and non-ﬁckian release from non-swellable devices in the form of

slabs, spheres, cylinders or discs, J Control Release 5 (1987) 23e36

[29] P.C.S Dash, P.N Murthy, L Nath, Kinetic modeling on drug release from controlled

drug delivery systems, Acta Pol Pharm e Drug Res 67 (2010) 217e223

[30] H Olami, I Berdicevsky, M Zilberman, Novel soy protein blend scaffolds loaded

with antibiotics: drug release proﬁle d bacterial inhibition effects, (2015) 23e31.

[31] A Shukla, J.C Fang, S Puranam, P.T Hammond, Release of vancomycin from

multilayer coated absorbent gelatin sponges, J Control Release 157 (2012)

64e71

[32] G Ma, D Fang, Y Liu, X Zhu, J Nie, Electrospun sodium

alginate/poly(-ethylene oxide) coreeshell nanoﬁbers scaffolds potential for tissue

engi-neering applications, Carbohydr Polym 87 (2012) 737e743

[33] K Sombatmankhong, N Sanchavanakit, P Pavasant, P Supaphol, Bone scaf-folds from electrospun ﬁber mats of hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and their blend, Polymer 48 (2007) 1419e1427 (Guildf)

[34] P Jantaree, K Lirdprapamongkol, W Kaewsri, C Thongsornkleeb,

K Choowongkomon, K Atjanasuppat, S Ruchirawat, J Svasti, Homodimers of vanillin and apocynin Ddecrease the metastatic potential of human cancer cells by inhibiting the FAK/PI3K/Akt signaling pathway, J Agric Food Chem.

65 (2017) 2299e2306 [35] C.D Saquing, C Tang, B Monian, C.A Bonino, J.L Manasco, E Alsberg, S.A Khan, Alginate-polyethylene oxide blend nanoﬁbers and the role of the carrier polymer in electrospinning, Ind Eng Chem Res 52 (2013) 8692e8704 [36] T Pan, W Song, X Cao, Y Wang, 3D bioplotting of gelatin/alginate scaffolds for tissue engineering: inﬂuence of crosslinking degree and pore architecture

on physicochemical properties, J Mater Sci Technol (2015) 1e12 [37] S.A Stone, P Gosavi, T.J Athauda, R.R Ozer, In situ citric acid crosslinking of alginate/polyvinyl alcohol electrospun nanoﬁbers, Mater Lett 112 (2013) 32e35

[38] S.V.G Nista, J Bettini, L.H.I Mei, Coaxial nanoﬁbers of chitosan-alginate-PEO polycomplex obtained by electrospinning, Carbohydr Polym 127 (2015) 222e228

[39] S.S Silva, R.L Reis, Morphology and miscibility of chitosan/soy protein blended membranes 70 (2007) 25e31

[40] W Pon-On, N Charoenphandhu, J Teerapornpuntakit, J Thongbunchoo,

N Krishnamra, I.M Tang, Mechanical properties, biological activity and pro-tein controlled release by poly(vinyl alcohol)-bioglass/chitosan-collagen composite scaffolds: a bone tissue engineering applications, Mater Sci Eng.

C 38 (2014) 63e72 [41] Z Xie, G Buschle-diller, Electrospun poly (D, L -lactide ) ﬁbers for drug de-livery: the inﬂuence of cosolvent and the mechanism of drug release, J Appl Polym Sci 115 (2010) 1e8

[42] H Yu, Y Jia, C Yao, Y Lu, PCL/PEG core/sheath ﬁbers with controlled drug release rate fabricated on the basis of a novel combined technique, Int J Pharm 469 (2014) 17e22

[43] K Kosmidis, E Rinaki, P Argyrakis, P Macheras, Analysis of Case II drug transport with radial and axial release from cylinders, Int J Pharm 254 (2003) 183e188

[44] K Garg, G.L Bowlin, Electrospinning jets and nanoﬁbrous structures, Bio-microﬂuidics 5 (2011) 13403

[45] G Chen, J Guo, J Nie, G Ma, Preparation, characterization, and application of PEO/HA core shell nanofibers based on electric field induced phase separation during electrospinning, Polymer 83 (2016) 12e19 (United Kingdom) [46] O Suwantonga, U Ruktanonchai, P Supaphol, Electrospun cellulose acetate fiber mats containing asiaticoside or Centella asiatica crude extract and the release characteristics of asiaticoside, Polymer 49 (2008) 4239e4247 [47] R Ke, W Yi, S Tao, Y Wen, Z Hongyu, Electrospun PCL/gelatin composite nanofiber structures for effective guided bone regeneration membranes, Mater Sci Eng C 78 (2017) 324e332

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