Drug-Loaded Halloysite Nanotube-Reinforced Electrospun Alginate

Subscriber access provided by University of South Dakota

Biological and Medical Applications of Materials and Interfaces

DRUG-LOADED HALLOYSITE NANOTUBES REINFORCED ELECTROSPUN ALGINATE-BASED NANOFIBROUS SCAFFOLDS WITH SUSTAINED ANTIMICROBIAL PROTECTION Rangika Thilan De Silva, Ranga K Dissanayake, M. M. M. G. Prasanga Gayanath Mantilaka, Sanjeewa Wijesinghe, Shehan Shalinda Kaleel, Thejani Nisansala Premachandra, Laksiri Weerasinghe, Gehan A J Amaratunge, and K.M. Nalin De Silva ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11013 • Publication Date (Web): 16 Sep 2018 Downloaded from http://pubs.acs.org on September 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

1

Drug-loaded Halloysite Nanotubes Reinforced

2

Electrospun Alginate-based Nanofibrous Scaffolds

3

with Sustained Antimicrobial Protection

4

Rangika Thilan De Silva†, Ranga K. Dissanayake†, M. M. M. G. Prasanga Gayanath Mantilaka†,

5

W. P. Sanjeewa Lakmal Wijesinghe†, Shehan Shalinda Kaleel†, Thejani Nisansala

6

Premachandra‡, Laksiri Weerasinghe†, Gehan A. J. Amaratunga†ǁ, K. M. Nalin de Silva*†§

7

†

8

Mahenwatte, Pitipana, Homagama, Sri Lanka.

9

‡

Sri Lanka Institute of Nanotechnology (SLINTEC), Nanotechnology and Science Park,

Department of Veterinary Pathobiology, Faculty of Veterinary Medicine, University of

10

Peradeniya, Peradeniya, Sri Lanka

11

§

12

ǁ

13

Thomson Avenue, Cambridge CB3 0FA, UK.

14

KEYWORDS: halloysite nanotubes; electrospinning; artificial scaffolds; mechanical properties;

15

sustained antimicrobial protection

Department of Chemistry, University of Colombo, Colombo 3, Sri Lanka.

Electrical Engineering Division, Department of Engineering, University of Cambridge, 9 J. J.

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

16

ABSTRACT. Halloysite nanotubes (HNT) reinforced alginate-based nanofibrous scaffolds were

17

successfully fabricated by electrospinning to mimic the natural extracellular matrix (ECM)

18

structure which is beneficial for tissue regeneration. An antiseptic drug, cephalexin (CEF) loaded

19

HNT, was incorporated into the alginate-based matrix in order to obtain sustained antimicrobial

20

protection and robust mechanical properties, key criteria for tissue engineering applications.

21

Electron microscopic imaging and drug release studies revealed that the CEF had penetrated into

22

the lumen space of HNT and also deposited on the outer walls, with a total loading capacity of 30

23

wt%. Moreover, the diameter of alginate-based nanofibers of the scaffolds ranged from 40-522 nm

24

with well-aligned HNT, resulting in superior mechanical properties. For instance, the addition of

25

5 (w/w %) HNT improved the tensile strength (σ) and elastic modulus by three-fold and two-fold,

26

respectively, compared to those of the alginate-based scaffolds without HNT. The fabricated

27

scaffolds exhibited remarkable antimicrobial properties against both gram-negative and gram-

28

positive bacteria, while the cytotoxicity studies confirmed the non-toxicity of the fabricated

29

scaffolds. Drug-release kinetics showed that CEF inside HNT diffuses within 24 hours and

30

diffusion of the drug is delayed by 7 days once CEF loaded HNT are incorporated into the alginate-

31

based nanofibers. These fabricated alginate-based electrospun scaffolds with enhanced mechanical

32

properties and sustained antimicrobial protection hold great potential to be used as artificial ECM

33

scaffolds for tissue engineering applications.

34 35 36 37


2

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38

1. INTRODUCTION

39

In the recent past, biopolymeric nanofibrous scaffolds have become a suitable substitute for

40

artificial scaffolds in tissue engineering applications due to their ability to mimic the spatial-

41

porous-structured morphology of extracellular matrices (ECM) which can be found in native

42

tissues and organs of the human body. These biopolymeric nanofibrous scaffolds facilitate cell

43

growth and proliferation. With the development of such nanofibrous scaffolds with desired

44

physical properties such as mechanical strength, biocompatibility and degradability, conventional

45

membrane-removal surgeries may no longer be required 1. Moreover, these artificial scaffolds

46

should have sufficient prolonged antimicrobial protection to inhibit secondary infections in order

47

to prevent implant failures 2. Compared to many artificial ECM scaffold-fabricating techniques

48

available such as wet-spinning 3, freeze-drying 4, template-based solution casting 5 and 3D printing

49

6

, electrospinning is one of the most versatile and robust techniques.

50

Electrospun scaffolds take a three-dimensional nanofibrous structure with nano/micro-scaled

51

interfibre porosity, which resemble the natural ECM structure that help to promote cell adhesion

52

and proliferation, and permit sufficient gases to exchange. Up to date, different biopolymers such

53

as alginate 7, poly(lactic acid) 8, chitosan 9, polyvinyl alcohol 10, polycaprolactone 11, poly(ethylene

54

oxide) 12 have been used to fabricate nanofibrous scaffolds. In particular, alginate has been deemed

55

to be a promising biopolymer for artificial scaffolds due to its high biocompatibility,

56

biodegradability and relatively low cost for mass production

57

strength limit its use in tissue engineering applications. Artificial scaffolds should be mechanically

58

robust and strong enough to withstand surgical procedures. Therefore attention needs to be directed

59

to reinforcing alginate-based fibres/scaffolds. Although certain studies have been carried out to

60

investigate the cell-adhesion

14

, scaffold degradation

15

13

, but its inadequate mechanical

and alteration of fibre dimensions

16

in


3


Page 4 of 36

61

alginate-based electrospun scaffolds, only a limited number of studies have been carried out on the

62

reinforcement of alginate-based electrospun scaffolds. To-date, range of micro/nano-fillers such

63

as hydroxyapatite (HA) 17-18, chitin whiskers 19, ZnO 20 and Ag nanoparticles 21 have been used to

64

reinforce electrospun alginate nanofibrous scaffolds. Apart from the mechanical inadequacy,

65

alginate based-scaffolds are also not resistant to bacterial infections which is a major drawback for

66

its use in artificial ECM scaffolds. Regenerative tissue scaffolds should prevent secondary

67

infections in order for cells to adhere and proliferate during the required time period. One way to

68

overcome this issue is by incorporating an antimicrobial drug. However, incorporation of drugs

69

lead to burst release and may not provide sustained antimicrobial protection. Here, halloysite

70

nanotubes (HNT) are used as multifunctional filler to mechanically reinforce and act as a carrier

71

of an antimicrobial drug in order to achieve the desired characteristics of artificial ECM scaffolds.

72

HNT are naturally occurring, tubular shaped aluminoslicate ((Al2Si2O5(OH)4.nH2O) nano-clay

73

type, with an external diameter of 20-200 nm, internal diameter of 10-70 nm and length of 50-

74

5000 nm

75

biopolymers including alginate 24-25. Liu et al. fabricated alginate/HNT scaffolds by a freeze-dying

76

method with a well interconnected pore structure (porosity of 100 – 200 μm), and obtained

77

enhanced thermo-mechanical properties 26. HNT have also been used to reinforce other forms of

78

alginate composites such as beads and hydrogels 27-28. In addition to the reinforcement, the lumen

79

space of HNT have been used to encapsulate different types of organic drugs, metal oxides (Au,

80

etc.), biocides, essential oils, natural compounds (curcumin, etc.) and inhibitors for various

81

nanocomposite related engineering applications 29-32. Xue et al. encapsulated metronidazole inside

82

the hollow space of HNT and incorporated drug-loaded HNT in poly(caprolactone)/gelatin

83

electrospun nanofibers 2. These scaffolds exhibited sustained antimicrobial protection due the

22-23

. HNT have been proven as a successful reinforcing nano-filler for a range of


4

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


84

control release of the drug. Similarly, HNT reinforced poly(lactic acid) electrospun nanofibrous

85

scaffolds with gentamicin (as a third phase) has been fabricated for bone regeneration applications

86

33

. pH sensitive HNT nanocomposite hydrogels have also been used for colon cancer drug delivery

87

34

. Sustain release of drugs may also be achieved by synthesizing alginate-based nanohydrogels

88

within the lumen space of HNT 35. Supramolecular functionalization or covalent modification of

89

HNT have also been used for drug loading, and controlled and sustained drug release applications

90

36-37

.

91

In this work, an antiseptic drug, cephalexin, was loaded into HNT (CEF-HNT) and CEF-HNT is

92

incorporated into alginate-based nanofibrous scaffolds to obtain sustained antimicrobial protection

93

while enhancing mechanical properties simultaneously. Alginate-based nanofibrous scaffolds with

94

varied HNT loadings (0 – 10 (w/w %)) were fabricated by electrospinning and their characteristics

95

were systematically investigated. The CEF loading in HNT is confirmed with electron micrographs

96

and drug-release kinetics. Morphological, mechanical, antimicrobial and thermal properties of the

97

fabricated alginate-based nanocomposite scaffolds are also evaluated. Furthermore, the sustained

98

protection of the fabricated scaffolds are confirmed with drug-release studies. These electrospun

99

alginate-based scaffolds with CEF-HNT exhibit significant potential to be used as artificial

100

scaffolds and as a substitute for ECM.

101

2. EXPERIMENTAL SECTION

102

Materials: Nano-filler carrier vessel, halloysite nanotubes (HNT) and the antiseptic compound,

103

cephalexin monohydrate (CEF) were purchased from Sigma-Aldrich and Aurobindo Pharma-

104

India, respectively. Electrospun polymer matrices, sodium alginate powder and poly(vinyl

105

alcohol) (PVA) (with a Mw of 89,000), and the cross-linker, glutaraldehyde used in this study were


5


Page 6 of 36

106

purchased from Sisco Research Labs Pvt. Ltd. and Glochem Ltd. Muller Hinton agar (Hardy,

107

USA) and phosphate buffer saline (Sigma, GmbH) were also used for microbiology studies.

108

Fabrication of drug-loaded HNT incorporated alginate-based electrospun scaffolds: The

109

antimicrobial drug, CEF was incorporated into the lumen space of HNT using a standard vacuum-

110

evacuation process. 2 g of HNT and CEF (maintaining a 1:1 ratio) were dispersed in 30 mL of

111

distilled water for 1 h at 600 rpm followed by ultrasonication for 15 min. The flask containing the

112

resultant CEF-HNT suspension was evacuated using a vacuum pump for 10 min until a slight

113

fizzing of the suspension was observed (as a removal of entrapped air). After the fizzing stopped,

114

the suspension was kept uninterruptedly for 10 min to reach equilibrium and the entire vacuum-

115

evacuation cycle was repeated thrice to promote CEF inclusion into the lumen space of HNT.

116

Afterwards, the suspension was centrifuged and rinsed twice using distilled water to remove the

117

excess CEF. UV absorbance of the supernatant of the rinsed solution of CEF-HNT was measured

118

at 260 nm and the encapsulation percentage was calculated based on the standard calibration curve

119

of CEF.

120

Nanofibrous scaffolds were fabricated by electrospinning an alginate-based polymer solution

121

comprising a secondary polymer PVA. 10 (w/w %) of secondary polymer solution was prepared

122

by dissolving PVA in distilled water at 80 °C with continuous stirring for 1-2 h. Afterwards, 2 %

123

(w/v) alginate solution was prepared by dissolving alginate in distilled water and corresponding

124

amounts of drug-loaded HNT (2.5, 5, 7.5 and 10 (w/w %)) were incorporated and stirred for 1h.

125

The resultant alginate-HNT and PVA solutions were mixed in 3:2 weight ratios for 2 h under

126

vigorous stirring followed by ultrasonication for 15 min (at an amplitude of 80 Hz). It should be

127

noted that the corresponding amounts of drug-loaded HNT were calculated based on the total

128

weight of the final alginate/PVA polymer solution. The resultant solution was electrospun in a


6

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


129

horizontal setup with a flow rate of 8-10 μL/min, voltage of 25-28 kV, needle to collector distance

130

of 10 cm and needle diameter of 0.5 mm (electrospinning parameters (for selected compositions)

131

are given in Table 1). Fabricated alginate-based scaffolds were chemically cross-linked using

132

glutaraldehyde vapor for 24 h in order to improve the water insolubility. A control sample of

133

alginate-based scaffolds without HNT were also fabricated according to the aforementioned

134

method for comparison purposes.

135

Table 1. The optimized values of the operating parameters for spinnability and brief highlights of

136

the morphology of the fibers in the scaffolds Fibre

Viscosity (P); flow

composition

rate (μL/min);

Observed morphology

voltage (kV) Alginate/HNT 0

%

65-70; 10; 26

Diameter of 80-400 nm; randomly oriented and

(w/w)

continuous; wavy and smooth surface; beads-free; inter-fibre porosity of 1.1-6.7 μm

(control) Alginate/HNT

70-80; 10-12; 26

5 % (w/w)

Diameter: 40-484 nm; randomly oriented and continuous; ultrafine, wavy and smooth surface; beads-free; inter-fibre porosity of 0.96-7.2 μm

Alginate/HNT 10 % (w/w)

70-80; 10-12; 26

Diameter: 63-522 nm; randomly oriented and continuous; wavy and smooth surface; noticeable sized beads; inter-fibre porosity of 0.43-6.5 μm

137


7


Page 8 of 36

138

Characterization of drug-loaded HNT and alginate/HNT scaffolds:

139

Morphological analysis: Morphology of the drug-loaded HNT as well the fabricated nanofibrous

140

scaffolds were evaluated using field-emission scanning electron microscope (FE-SEM) (Hitachi

141

SU6600) and transmission electron microscope (TEM) (Jeol 2100). SEM samples were gold

142

sputtered to prevent electrostatic charging during the observation. Energy-dispersive X-ray (EDX)

143

spectroscopy studies were carried out to confirm the antiseptic compound impregnation onto the

144

inner and outer walls of HNT with a scanning rate of 192000 CPS for 4.5 min.

145

Chemical properties: Fourier transform infrared (FTIR) spectroscopic studies were carried out to

146

confirm the CEF attachment on to the inner and outer walls of HNT as well as to evaluate the

147

chemical compositions of alginate/HNT scaffolds. All spectra were obtained within 500 – 4000

148

cm-1 with 32 scans per measurement at 0.4 cm-1 resolution.

149

Physical properties: Tensile testing was carried out to evaluate the mechanical properties such as

150

tensile strength (σ), elastic modulus (E) and elongation at break (ɛ) of the electrospun alginate/HNT

151

scaffolds, using an Instron Tensile Testing rig. All samples were tested according to ASTM D882-

152

02 with a sample size of 40 x 10 mm at a strain rate of 5 mm/min. Statistical significance of the

153

means of the respective σ, E and ɛ of tested samples was evaluated using one-way analysis of

154

variance (ANOVA), complemented with the Tukey Post-Hoc test, using commercial software

155

(OriginPro 8).

156

The thermal stability of the electrospun scaffolds were determined by thermo gravimetric analysis

157

(TGA) (STD Q600) from 25 to 1000 ˚C at a heating rate of 10 ˚C/min in nitrogen medium.


8

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


158

In vitro antibacterial study: The antibacterial activity of drug-loaded scaffolds were tested against

159

two Gram positive (Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (Clinical

160

isolate)) and two Gram negative bacteria (Pseudomonas aeruginosa (ATCC 9027) and

161

Escherichia coli (ATCC 35218)) using disc diffusion method. The cell suspensions of the test

162

microorganisms were prepared using 24 h old fresh culture and the turbidity was compared with

163

0.5 McFarland standards. A volume 5 mL of each cell suspension was dispensed onto the surface

164

of dried Mueller Hinton Agar (MHA powder (Hardy, USA ), 38.0 g in 1000 mL of distilled water)

165

dishes, distributed all over the surface and the excess suspension removed.

166

The alginate-based scaffolds with 5, 7.5 and 10 (w/w %) of CEF-HNT were cut in to 6 mm circular

167

disc and placed on the prepared MHA medium. CEF soaked antimicrobial disc (20 µg/disc) was

168

used as the positive control and scaffold without cephalexin was used the negative control. After

169

the incubation period, the plates were checked for the inhibition zones and the diameters of those

170

were measured. The procedure adopted was according that specified by the National Committee

171

for Clinical Laboratory Standards (NCCLS).

172

Drug release studies: The release profile of CEF was analysed for the CEF-HNT composite and

173

the cross-linked alginate-based scaffold with 7.5 (w/w %) of CEF-HNT in phosphate buffer saline

174

(PBS). An amount of 100 mg of CEF-HNT was added to 100 mL of PBS at 30 oC with 200 rpm

175

stirring. At predetermined time points, 1 mL of the sample was taken and CEF concentration was

176

determined by absorbance measurement at 260 nm (Shimadzu UV-3600 UV-Vis-NIR

177

spectrophotometer) with respect to the standard curve. After the desired period, the mixture was

178

sonicated for 2h at room temperature in order to get the loading capacity of CEF. Same procedure

179

was followed for the final scaffold with some modifications (50 mg of scaffold, 5 mL of PBS and

180

100 µL of withdrawing amount).


9


Page 10 of 36

181

Evaluation of In vitro Cytotoxicity of alginate/HNT scaffolds: The alginate-based scaffolds with

182

5 (w/w %) HNT were sterilized using gamma irradiation (Co 60, 25 KGy). Mouse L929 cell line

183

(ATCC) was used to determine the cytotoxicity of the test material. L929 cells were cultured and

184

the evaluation was carried out under sterile conditions using a class 2 safety cabinet (Herasafe,

185

Kendro Laboratory products, Germany).

186

Sterilised 20 mg of scaffolds and controls were incubated with 3 mL Complete Dulbecco’s

187

modified Eagale’s medium (DMEM + 10% FCS) (DMEM, USA), separately on a rotary mixture.

188

Extracts were prepared by eluting test samples in complete medium at days 1, 2 and 3. After that,

189

a concentration of 1.0×105 cells/ml was seeded in a 96-well culture plate (density of 1.0×104

190

cells/well in 100 µl medium) and allowed to reach 90% of cell confluence. Then original medium

191

was replaced with 100 µl of elucidates, separately and were incubated at 37 ºC in humidified air

192

with 5% CO2 for 24 hours. Medium was removed and substituted with 10 μl of MTT diluted in the

193

culture medium (DMEM) and incubated at 37 ºC for 4 hours. Finally, absorbance was measured

194

by Dynatech MR 700 micro plate reader at a test wavelength of 570 nm with the reference

195

wavelength of 630 nm. RPMI 1640 supplemented with 10% FBS was used as negative controls

196

whereas ethanol was used as the positive controls. The percent viability is calculated using

197

equation 1. Viability index of the cells is determined using difference between absorbance at 570

198

nm (A570) and 630 nm (A630).

199

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =

(𝐴570 − 𝐴630) 𝑠𝑎𝑚𝑝𝑙𝑒 (A570 − A630) control

× 100%

− 01

200


10

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


201

3. RESULTS AND DISCUSSION

202

Morphological properties: Micrographs of the drug-loaded HNT are illustrated in Figure 1.

203

Typically HNT have an internal diameter of 10 – 70 nm and external diameter of 20 – 200 nm with

204

a length of 50 – 5000 nm, depending on the clay deposit

205

CEF to be deposited on the outer and inner walls of HNT. CEF deposition on the outer surfaces of

206

HNT were confirmed by EDX mapping (Figure 1 (b)), where traces of sulphur (a key element of

207

CEF) can be seen on the surfaces of HNT. The hydroxyl groups of HNT which are available on

208

the defected outer walls and edges of HNT, are very likely to chemically interact with N-H and O-

209

H groups of CEF via non-covalent interactions such as hydrogen bond and electrostatic

210

interactions. Moreover, TEM imaging showed the encapsulation of CEF within the lumen space

211

of HNT (Figure 1 (C), lumen space is partially loaded) as well as CEF deposition on the outer

212

walls of HNT (Figure 1 (d)). These results suggested that the antiseptic drug is grafted on the outer

213

surface of HNT and also partially entrapped within the hollow tubular space. This was further

214

confirmed by the drug release studies (Vide infra).

22-23

. The drug-loading process enabled

215


11


Page 12 of 36

216 217

a

b

218 219 220 221 222 223

c

d Loaded space

Unloaded lumen space

224 225 226 227

Figure. 1. (a) SEM image of CEF loaded HNT and (b) its EDX mapping (red denotes to sulphur

228

element). (d) and (c) TEM images demonstrating the CEF deposition in HNT

229


12

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


230

Figure 2 shows the morphology of electrospun alginate-based nanofibrous scaffolds. As prepared

231

nanofibrous scaffolds yield e a white flexible substrate with a thickness ranging from 20 - 100 μm

232

(Figure S1, in supplementary data). Electrospun alginate-based fibres with low HNT loadings are

233

uniform, continuous and randomly oriented, and the morphology has does not differ from the

234

control sample (alginate-based scaffolds without HNT) (Figure 2 (a) vs (b, C)). Although the

235

addition of low loadings of HNT (2.5 - 5 (w/w %)) did not change the morphology of the alginate-

236

based nanofibrous scaffolds, high HNT loadings (10 (w/w %)) led to formation of conical shaped

237

beads within the fibres (Figure 2 (d)). The bead formation could be due to the increased polymer

238

solution viscosity with the addition of HNT during the synthesis process. Additionally the high

239

loadings of HNT create unfavourable electrospinning conditions as localized charge accumulates

240

in the driven jet (at tailor cone) and leads to an inhomogeneous electric field. The interfibre

241

porosity of the alginate-based nanocomposite scaffolds ranges from 0.43 – 7.2 μm (Table 1) and

242

is well below an order-of-magnitude of the porosities of fibreblast cells (around 20 μm), which is

243

as desired for better cell adhesion and proliferation. Furthermore, this interfibre

244

porosity/nanofibrous structure of the electrospun scaffolds act as a barrier and would help to

245

prevent the ingrowth of fibroblasts into the defected tissues 2. The diameter of electrospun alginate-

246

based fibres (control sample) yielded to be 80-400 nm while the alginate-fibres with 5 (w/w %)

247

and 10 (w/w %) of HNT were 40-484 nm and 63-522 nm, respectively. These electrospun alginate-

248

based fibres resemble the collagen fibrils of tissues ranges from 30 to 350 nm 38. Moreover, the

249

diameter of the collagens fibrils of acelular dermal matrix (ADM) (used as ECM scaffolds), ranges

250

from 56 - 61 nm

251

nanofibrous scaffolds are well within the required morphological parameters for artificial ECM

252

scaffolds.

39

. These findings suggest that the electrospun alginate-based nanocomposite


13


Page 14 of 36

253

Impregnation of HNT within the electrospun alginate-based nanofibers is illustrated in Figure 3.

254

HNT exhibits a fairly good uniaxial alignment along the alginate nanofibers. This was further

255

confirmed with TEM-EDX mapping as illustrated in FigureS2 (in supplementary data). The

256

dispersion of 5 (w/w %) HNT within the alginate matrix is fairly good and minimum amount of

257

agglomerations are detected. However, HNT aggregates are likely to form at high filler loadings

258

(7 and 10 (w/w %)).

259 260

a

b

c

d

261 262 263 264 265 266 267 268 269

Figure 2. Morphology of electrospun alginate-based scaffolds with (a) 0, (b, C) 5 and (d) 10 (w/w

270

%) of HNT

271


14

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


272 273

Alginate nanofibre HNT

274 275 276 277 278

Figure 3. TEM of electrospun alginate-based nanofibrous scaffolds with 5 (w/w %) of HNTs

279

Chemical properties: As illustrated in FTIR spectra (Figure 4), characteristic peaks of alginate

280

such as hydroxyl groups at 3300 cm-1, asymmetric carboxyl at 1600 cm-1, symmetric carboxyl at

281

1400 cm-1 and carbonyl functional groups at 1015 cm-1 can be found in the spectra of alginate-

282

based electrospun scaffolds 19. O-H stretching at 3300 cm-1, C-H stretching of alkyl groups at 2933

283

cm-1, C=O and C-O stretching of acetate groups at 1730 cm-1 and C-C stretching at 1090 cm-1 34

284

can be attributed to the functional groups of PVA (scaffolds contain alginate to PVA weight ratio

285

of 3:2). Major functional groups of HNT such as O–H stretching of inner-surface hydroxyl groups

286

(3696 cm-1 and 3625 cm-1), Si–O in-plane stretching (1011 cm-1) and O–H deformation of inner

287

hydroxyl groups (911 cm-1) can be seen in the FTIR spectra of Alg/HNT 10 scaffolds. All the key

288

functional groups of CEF (aromatic ring stretch at 1455 cm-1, carboxylate C=O stretching at 1596

289

cm-1, amide C=O stretch at 1690 cm-1, β-lactam C=O stretch at 1758 cm-1 and N-H at 2614 cm-1)

290

could not be detected on the composite scaffolds due the overlap of those low intense peaks with

291

the dominant peaks of alginate and PVA. Presence of surfaces grafted CEF on inner and outer wall

292

of HNT was confirmed due to the appearance of the β-lactam C=O peak of CEF (stretch at 1758


15


Page 16 of 36

293

cm-1) in the FTIR spectra of CEF-HNT (Figure S3, in supplementary data). The proposed CEF-

294

HNT interaction model is illustrated in Figure 5. Hydroxyl groups present in the cavity of HNTs

295

will interact with the drug molecules via H-bond interactions to improve the drug-loading and it

296

was further confirmed with the shifts and intensity changes in the hydroxyl groups of CEF-HNT

297

(Figure S3, in supplementary data. CEF peaks at 3436 cm-1 and 1596 cm-1 had shifted to 3442 cm-

298

1

299

deprotonated and the overall charge of the drug molecule is negative. Hence there could be an

300

electrostatic interaction with HNT’s positive surfaces. The same non-covalent interaction model

301

will account for the extended drug release of cephalexin from nanotube (Figure 5). Moreover, the

302

presence of characteristic peaks of the polymer blend confirms that the addition of HNT did not

303

affect the structural integrity of the nanofinrous scaffolds.

and 1629 cm-1 of CEF-HNT, respectively). At pH 7.4, carboxylic moiety of cephalexin will be

304 305 306 307 308 309 310 311 312 313 314 315 316

Figure 4. FTIR spectrum of alginate, PVA, HNT and alginate-based scaffolds

317


16

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


318 319

Figure 5. Proposed mode of drug binding with HNT

320 321

Tensile properties: Typical stress-strain curves of the electrospun alginate nanocomposite

322

scaffolds are illustrated in Figure 6(a). Tensile stress (σ) and elastic modulus (E) of the

323

nanocomposite scaffolds derived from the stress-strain curves are shown in Figure 6(b).

324

Statistical analysis (ANOVA together with Turkeys Post-Hoc) revealed that there is significant

325

difference of the mean σ among the (i) Alg/HNT 0 vs Alg/HNT 2.5 and 5, and (ii) Alg/HNT 2.5

326

and 5 vs Alg/HNT 7.5 and 10. Incorporation of low HNT concentrations such as 2.5 and 5 (w/w

327

%) into alginate fibres enhanced the mechanical properties significantly. For instance, σ

328

improved by three-fold with the addition of 2.5 (w/w %) and 5 (w/w %) of HNT compared to

329

that of the control sample (σ = 1 ± 0.2 MPa). These improvements could be due to the better

330

interfacial interaction between the polymer matrix and HNT (hydroxyl groups of HNTs are

331

likely to interact with the C-H functional groups of alginate and PVA) which can result in

332

effective stress transfer from the matrix to filler. Further, the alignment of HNT along the fibre

333

direction (at low loadings) could bare the longitudinally induced stress more effectively. The

334

addition of high HNT loadings (7.5 and 10 (w/w %)) led to inferior σ due to the possibilities of

335

HNT aggregation as well as bead formation along the fibres (Figure 2 (d)). HNT aggregates and


17


Page 18 of 36

336

beads can act as stress concentrated areas, and material will be easily ruptured under applied

337

tensile force. E of the nanofibrous scaffolds exhibits a similar trend as σ, where the optimum E

338

yielded at 2.5 and 5 (w/w %) HNT concentrations, and decreased thereafter (Figure 6(b)). The

339

stiffness of the material increased as a result of the reinforcing effect of HNT. The mechanical

340

properties of the composite scaffolds are summarized in Table S1, in supplementary data.

341

Fabricated alginate scaffolds with 2.5 and 5 (w/w %) of HNT had tensile strength of 3.8 ± 0.3

342

MPa and σ = 3.4 ± 0.3 MPa, respectively. These improved parameters are well above the guided

343

tissue regeneration (GTR) requirements of artificial scaffolds (σ should be more than 2-3 MPa) 2.

344


18

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364

Figure 6. (a) Selected stress-strain curves of alginate composite scaffolds. (b) Tensile strength and

365

elastic modulus of alginate-based scaffolds at varied HNT concentrations

366 367


19


Page 20 of 36

368

Antibacterial activities of the electrospun scaffolds: The antibacterial activities of the scaffolds

369

are given in the Table 2, Figure 7 and Figure S4 (in supplementary data). Most promising activities

370

were shown against tested Gram positive strains (S. aureus and S. epidermidis). All drug loaded

371

testing materials showed significant antibacterial activity, which is comparable to the inhibitory

372

activity of the positive control. Therefore, the cross linking and composite formation did not affect

373

the anti-bacterial activity of the loaded drug and its diffusion pattern. Alginate-based scaffolds

374

with 10 (w/w %) of CEF-HNT showed the highest activity against all tested bacteria strains.

375

Nevertheless, there is no significant difference between 5, 7.5 and 10 (w/w %) CEF-HNT loadings.

376

Table 2. Antimicrobial activity of CEF-HNT incorporated alginate-based scaffolds (mm). Alg/CEF-HNT

Alg/CEF-

Alg/CEF-

Positive

Negative

5

HNT 7.5

HNT 10

control

control

S. aureus

17.0 ± 0.4

24.1 ± 1.4

26.1 ± 1.7

28.4 ± 1.6

-

S. epidermidis

21.3 ± 1.1

26.3 ± 0.9

25.0 ± 1.1

27.1 ± 0.4

-

P. aeruginosa

15.6 ± 0.8

19.8 ± 0.5

20.3 ± 0.7

24.5 ± 0.8

-

E. coli

15.0 ± 0.7

18.6 ± 1.3

19.6 ± 0.9

22.2 ± 1.3

-

S. epidermidis

E. coli

377 378

S. aureus

P. aeruginosa

379 380 381 382 383

Figure 7. Antimicrobial activity of alginate-based scaffolds with 10 (w/w %) CEF-HNT


20

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


384

Drug-release studies: The release profiles of CEF-HNT and alginate-based scaffolds with CEF-

385

HNT HNT-alginate scaffold are presented in Figure 8. The release of CEF is mainly due to the

386

passive diffusion. The trend of the graphs also fit with the typical diffusion of the small molecule.

387

The total entrapped CEF content within the lumen space of HNT from measurement is found as

388

30 wt %. This indicates that CEF molecules are not only encapsulated inside the nanotube (the

389

lumen capacity is 10 vol %), but also grafted on the outside of HNT. This is further evident from

390

the drug release profile, where the first release phase (up to 5 h) is ascribed to the rapid release of

391

surface grafted drug molecules and the second phase is attributed to the delayed release (up to 24

392

h) of the entrapped drug molecules within the lumen space of HNT. Drug-release kinetics revealed

393

that CEF inside HNT diffuses within 24 hours and the diffusion rate of CEF gets delayed by 7 days

394

when CEF loaded HNT is incorporated into the alginate-based nanofibrous scaffolds, exhibiting a

395

sustained release profile. The release percentage of the drug from CEF-HNT after 24 h was about

396

95%. The drug loaded electrospun scaffold showed a sustain release profile as a result of cross-

397

linking the polymer matrix of the nanofiber. The total released percentage of the drug after 8 h and

398

7 days was 76% and 89%, respectively. The release pattern is appropriate for the development of

399

artificial ECM scaffolds, since the initial rapid release would help to eradicate the growth of

400

bacteria and sustained release will prevent any further infection.

401 402 403 404 405


21


Page 22 of 36

406 407 408 409 410 411 412 413 414 415 416 417

Figure 8. CEF release profiles of (A) CEF loaded HNT, (B) alginate-based scaffolds with 7.5

418

(w/w %) of CEF-HNT and (C): percentage release of the drug from both pure HNT and the

419

scaffold

420

Cytotoxicity of the electrospun scaffolds: Cytotoxicity of the alginate-based scaffolds with 5

421

(w/w %) HNT was evaluated. Absorption values of MTT test elutions and those of both negative

422

and positive controls are shown in Figure 9 (a). According to these results, the test samples do

423

not release any toxic substance which would bring harmful effects to the cells and hence they do


22

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


424

not affect cellular metabolism. These results are comparable to the negative control and do not

425

show any significant difference in the responses within three days of incubation (P< 0.01).

426

Moreover, the results of the test samples showed a significant difference to that of the positive

427

toxic control. Hence, the evaluations of the response of L929 cells to the prepared scaffolds

428

confirm the non-cytotoxicity of the material 2, 40-41.

429

Viability index of the cells is determined using the difference between absorbance at 570 nm and

430

630 nm. The percent viability is calculated using equation 1. According to the calculated values

431

for three days (Figure 9 (b)), all the percentage values are higher than the 100 % viability.

432

Therefore, toxicity level of the samples is “0”. This toxicity level is identified by considering

433

standard percentage viability range 42-44. According to the absorption cytotoxicity results of

434

prepared scaffolds, we can conclude that the scaffold is nontoxic to the human body.

435

Morphological images of the cells in samples are shown in Figure 10, which are taken from the

436

inverted optical microscope. There is no observable morphology change between control and

437

tested samples (day 3 samples) due to the non-toxicity to the cells.

438

Apart from cytotoxicity, cell adhesion and proliferation are also key factors in developing

439

artificial ECM. Shalumon et al. demonstrated that the alginate and PVA blends-based

440

electrospun nanofibrous membranes support cell adhesion and proliferation 20. For instance,

441

alginate-based electrospun scaffolds well adhered L929 cells in 48 hours and showed good

442

spreading after 96 hours. Further supporting evidence on the cell adhesion and proliferation on

443

alginate-based electrospun nanofibrous scaffolds can be found elsewhere 20, 45. As it is well

444

proven that the alginate/PVA electrospun scaffolds support cell adhesion, it can be postulated

445

that the scaffolds developed in this study are comparable to that of the other reported alginate-

446

based electrospun scaffolds. Jiajia et al. had investigated the cell adhesion on extract substrates


23


447

of the electrospun HNT microfiber composite membranes using optical microscope which

448

demonstrated the cell adhesion and healthy-growth morphologies while having HNT 2. Their

449

micrographs are very similar to the micrographs of this study (Figure 10b) because of the

450

healthy-growth and adhesion of the L929 cells.

451 452 453 454 455 456 457

0.700

Absorbance at 570 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

a

0.600 0.500 0.400

0.300 0.200 0.100 0.000

Day 1 Sample

Day 2 Negative

458

Day 3 Positive

b

459 460 461 462 463 464

Figure 9. (a) Graphs of absorption values of scaffold, negative control and positive control which

465

were obtained from MTT assay. (b) Percent cell viability of tested scaffolds


24

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


466 467 468 469 470 471 472 473 474

Figure 10. Morphologies of L929 cells (a) day 3 negative control and (b) day 3 scaffold

475 476

Thermal characteristics: TGA curves of alginate-based scaffolds are illustrated in Figure 11. The

477

initial weight loss (10% of mass loss) of all alginate-based scaffolds has been occurred due to the

478

elimination of surface bound water molecules of nanofibers at temperature below 100 ˚C. The

479

mass loss due to the pyrolysis of alginate and PVA components of fibres initiates at 215 ˚C. C-H

480

bonds and C-O-C glycoside bonds of the backbone structure of alginate thermally decompose at

481

around 250 ˚C as a result of dehydration of saccharide chains 46. The addition of HNT enhances

482

the thermal stability of the scaffolds. For instance, at 400 ˚C, alginate-based scaffolds and their

483

composites with 5 (w/w %) of HNT showed 78% and 67% of mass loss, respectively. Moreover,

484

the temperatures at maximum mass loss rate of alginate-based scaffolds had shifted from 275 ˚C

485

to 285 ˚C with the addition of 5 (w/w % HNT). The thermal degradation rate had also reduced in

486

the composites (1.01 %/ ˚C) compared to that of the pure alginate-based scaffolds (1.14 %/ ˚C).

487

That signifies the thermal stability of the nanofibrous scaffolds, which has been increased due to

488

the presence of HNT and their high thermal stability.

489


25


Page 26 of 36

490 491 492 493 494 495 496 497 498 499 500

Figure 11. TGA (solid) and DTGA (dotted) curves of alginate-based scaffolds

501 502

4. CONCLUSIONS

503

Drug-loaded HNT-incorporated alginate-based nanocomposite fibrous scaffolds were successfully

504

fabricated by electrospinning. Alginate-based fibres ranged from 40 to 522 nm with an inter-fibre

505

porosity of 0.43-7.2 μm. Low HNT compositions (2.5 – 5 (w/w %)) yielded beads-free nanofibers

506

and a noticeable amount of beads was observed in the scaffolds with high HNT compositions (10

507

(w/w %)). An antiseptic drug, CEF, was loaded within the lumen space as well as the outer walls

508

of HNT using a vacuum-evacuation method. The CEF loading inside the HNT was confirmed

509

using SEM-EDX and TEM, and the UV-vis based drug release study revealed that the total CEF

510

loading capacity within the HNT was 30 wt%. It was further evident that HNT had dispersed well

511

within the alginate-based matrix with good uniaxial alignment, which resulted in significantly


26

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


512

enhanced mechanical properties. For instance, the addition of 5 (w/w %) HNT improved the tensile

513

strength and elastic modulus by three-fold and two-fold, respectively, compared to those of the

514

alginate-based scaffolds without HNT. Alginate-based scaffolds with CEF-loaded HNT exhibited

515

superior antimicrobial properties against S. aureus, S. epidermidis, P. aeruginosa and E. coli with

516

inhibition zones ranging from 15 to 28 mm. Drug-release kinetics revealed that CEF inside HNT

517

diffuses within 24 hours and the diffusion rate of CEF gets delayed by 7 days when CEF loaded

518

HNT is incorporated into the alginate-based nanofibrous scaffolds, exhibiting a sustained release

519

profile. For instance, the total released percentage of the drug after 8 hours and 7 days was 76%

520

and 89%, respectively. This release profile would assist in eradicating the initial bacteria growth

521

and the sustained release would prevent any further infection. The cytotoxicity and cell viability

522

studies confirmed that the fabricated scaffolds are non-toxic to cells and human body. The

523

incorporation of HNT also improved the thermal stability of the scaffolds. These electrospun

524

alginate-based nanofibrous scaffolds with drug-loaded HNT satisfy the key criteria of artificial

525

ECM such as inter-fibre porosity (< 20 μm), mechanical robustness (σ >3 MPa), sustained

526

antimicrobial protection and non-toxicity, and hence, hold great potential to be used as an artificial

527

scaffold for tissue regeneration applications

528 529

AUTHOR INFORMATION

530

Corresponding Author

531

* E-mail: [email protected], [email protected], Phone no: +94 11 465 0531, Fax: +94 11

532

465 0532.

533


27


Page 28 of 36

534

Author Contributions

535

The manuscript was written through contributions of all authors. All authors have given approval

536

to the final version of the manuscript.

537

Supporting Information

538

Supplementary document contains figures and plots related to morphological, physical and

539

chemical properties of the fabricated nanofibrous scaffolds.

540

Acknowledgments

541

Authors would like to thank Ms. Malini Damayanthi for her tremendous support in TEM

542

imaging. The cytotoxicity studies were supported by Prof. R.P.V.J. Rajapakse, Head,

543

Department of Veterinary Pathobiology, Faculty of Veterinary Medicine, University of

544

Peradeniya, Peradeniya, 20400, Sri Lanka.

545

Abbreviations

546

HNT, Halloysite nanotubes; ECM, extracellular matrix; CEF, cephalexin; PVA, poly(vinyl

547

alcohol).

548

REFERENCES

549

(1) Retzepi, M.; Donos, N. Guided Bone Regeneration: Biological Principle and Therapeutic

550

Applications. Clinical oral implants research 2010, 21 (6), 567-576.

551

(2) Xue, J.; Niu, Y.; Gong, M.; Shi, R.; Chen, D.; Zhang, L.; Lvov, Y. Electrospun Microfiber

552

Membranes Embedded with Drug-Loaded Clay Nanotubes for Sustained Antimicrobial

553

Protection. ACS Nano 2015, 9 (2), 1600-1612, DOI: 10.1021/nn506255e.


28

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


554

(3) Cheng, J.; Jun, Y.; Qin, J.; Lee, S.-H. Electrospinning Versus Microfluidic Spinning of

555

Functional Fibers for Biomedical Applications. Biomaterials 2017, 114 (Supplement C), 121-

556

143, DOI: https://doi.org/10.1016/j.biomaterials.2016.10.040.

557

(4) Reys, L. L.; Silva, S. S.; Pirraco, R. P.; Marques, A. P.; Mano, J. F.; Silva, T. H.; Reis, R. L.

558

Influence of Freezing Temperature and Deacetylation Degree on the Performance of Freeze-

559

dried Chitosan Scaffolds towards Cartilage Tissue Engineering. European Polymer Journal

560

2017, 95, 232-240.

561

(5) Huling, J.; Ko, I. K.; Atala, A.; Yoo, J. J. Fabrication of Biomimetic Vascular Scaffolds for

562

3D tissue Constructs Using Vascular Corrosion Casts. Acta biomaterialia 2016, 32, 190-197.

563

(6) Mondschein, R. J.; Kanitkar, A.; Williams, C. B.; Verbridge, S. S.; Long, T. E. Polymer

564

Structure-property Requirements for Stereolithographic 3D Printing of Soft Tissue Engineering

565

Scaffolds. Biomaterials 2017, 140 (Supplement C), 170-188, DOI:

566

https://doi.org/10.1016/j.biomaterials.2017.06.005.

567

(7) De Silva, R.; Mantilaka, M.; Goh, K.; Ratnayake, S.; Amaratunga, G.; de Silva, K.

568

Magnesium Oxide Nanoparticles Reinforced Electrospun Alginate-Based Nanofibrous Scaffolds

569

with Improved Physical Properties. International Journal of Biomaterials 2017, 2017.

570

(8) Binan, L.; Tendey, C.; De Crescenzo, G.; El Ayoubi, R.; Ajji, A.; Jolicoeur, M.

571

Differentiation of Neuronal Stem Cells into Motor Neurons Using Electrospun Poly-L-lactic

572

Acid/Gelatin Scaffold. Biomaterials 2014, 35 (2), 664-674.

573

(9) Kohsari, I.; Shariatinia, Z.; Pourmortazavi, S. M. Antibacterial Electrospun Chitosan–

574

polyethylene Oxide Nanocomposite Mats Containing Bioactive Silver Nanoparticles.

575

Carbohydrate polymers 2016, 140, 287-298.


29


Page 30 of 36

576

(10) Merkle, V. M.; Tran, P. L.; Hutchinson, M.; Ammann, K. R.; DeCook, K.; Wu, X.; Slepian,

577

M. J. Core–shell PVA/Gelatin Electrospun Nanofibers Promote Human Umbilical Vein

578

Endothelial Cell and Smooth Muscle Cell Proliferation and Migration. Acta biomaterialia 2015,

579

27, 77-87.

580

(11) Rajzer, I.; Menaszek, E.; Castano, O. Electrospun Polymer Scaffolds Modified with Drugs

581

for Tissue Engineering. Materials Science and Engineering: C 2017, 77 (Supplement C), 493-

582

499, DOI: https://doi.org/10.1016/j.msec.2017.03.306.

583

(12) Loiola, L. M.; Tornello, P. R. C.; Abraham, G. A.; Felisberti, M. I. Amphiphilic Electrospun

584

Scaffolds of PLLA–PEO–PPO Block Copolymers: Preparation, Characterization and Drug-

585

release Behaviour. RSC Advances 2017, 7 (1), 161-172.

586

(13) Venkatesan, J.; Bhatnagar, I.; Manivasagan, P.; Kang, K.-H.; Kim, S.-K. Alginate

587

Composites for Bone Tissue Engineering: A Review. International Journal of Biological

588

Macromolecules 2015, 72 (Supplement C), 269-281, DOI:

589

https://doi.org/10.1016/j.ijbiomac.2014.07.008.

590

(14) Jeong, S. I.; Krebs, M. D.; Bonino, C. A.; Khan, S. A.; Alsberg, E. Electrospun Alginate

591

Nanofibers with Controlled Cell Adhesion for Tissue Engineeringa. Macromolecular bioscience

592

2010, 10 (8), 934-943.

593

(15) Bouhadir, K. H.; Lee, K. Y.; Alsberg, E.; Damm, K. L.; Anderson, K. W.; Mooney, D. J.

594

Degradation of Partially Oxidized Alginate and its Potential Application for Tissue Engineering.

595

Biotechnology progress 2001, 17 (5), 945-950.

596

(16) Lu, J.-W.; Zhu, Y.-L.; Guo, Z.-X.; Hu, P.; Yu, J. Electrospinning of Sodium Alginate with

597

Poly(ethylene oxide). Polymer 2006, 47 (23), 8026-8031, DOI:

598

http://dx.doi.org/10.1016/j.polymer.2006.09.027.


30

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


599

(17) Chae, T.; Yang, H.; Leung, V.; Ko, F.; Troczynski, T. Novel Biomimetic

600

Hydroxyapatite/Alginate Nanocomposite Fibrous Scaffolds for Bone Tissue Regeneration.

601

Journal of Materials Science: Materials in Medicine 2013, 24 (8), 1885-1894, DOI:

602

10.1007/s10856-013-4957-7.

603

(18) Ito, Y.; Hasuda, H.; Kamitakahara, M.; Ohtsuki, C.; Tanihara, M.; Kang, I.-K.; Kwon, O. H.

604

A composite of Hydroxyapatite with Electrospun Biodegradable Nanofibers as a Tissue

605

Engineering Material. Journal of Bioscience and Bioengineering 2005, 100 (1), 43-49, DOI:

606

http://dx.doi.org/10.1263/jbb.100.43.

607

(19) Watthanaphanit, A.; Supaphol, P.; Tamura, H.; Tokura, S.; Rujiravanit, R. Fabrication,

608

Structure, and Properties of Chitin Whisker‐reinforced Alginate Nanocomposite Fibers. Journal

609

of Applied Polymer Science 2008, 110 (2), 890-899.

610

(20) Shalumon, K. T.; Anulekha, K. H.; Nair, S. V.; Nair, S. V.; Chennazhi, K. P.; Jayakumar, R.

611

Sodium Alginate/Poly(vinyl alcohol)/Nano ZnO Composite Nanofibers for Antibacterial Wound

612

Dressings. International Journal of Biological Macromolecules 2011, 49 (3), 247-254, DOI:

613

http://dx.doi.org/10.1016/j.ijbiomac.2011.04.005.

614

(21) Lee, Y. J.; Lyoo, W. S. Preparation and Characterization of High‐molecular‐weight atactic

615

Poly(vinyl alcohol)/Sodium Alginate/Silver Nanocomposite by Electrospinning. Journal of

616

Polymer Science Part B: Polymer Physics 2009, 47 (19), 1916-1926.

617

(22) Pasbakhsh, P.; De Silva, R.; Vahedi, V.; Jock Churchman, G. Halloysite Nanotubes:

618

Prospects and Challenges of their Use as Additives and Carriers–A Focused Review. Clay

619

Minerals 2016, 51 (3), 479-487.


31


Page 32 of 36

620

(23) Churchman, G. J.; Pasbakhsh, P.; Lowe, D. J.; Theng, B. Unique but Diverse: Some

621

Observations on the Formation, Structure and Morphology of Halloysite. Clay Minerals 2016, 51

622

(3), 395-416.

623

(24) Liu, M.; Jia, Z.; Jia, D.; Zhou, C. Recent advance in Research on Halloysite Nanotubes-

624

polymer Nanocomposite. Progress in polymer science 2014, 39 (8), 1498-1525.

625

(25) Kumar, A.; Rao, K. M.; Han, S. S. Synthesis of Mechanically Stiff and Bioactive Hybrid

626

Hydrogels for Bone Tissue Engineering Applications. Chemical Engineering Journal 2017, 317,

627

119-131, DOI: https://doi.org/10.1016/j.cej.2017.02.065.

628

(26) Liu, M.; Dai, L.; Shi, H.; Xiong, S.; Zhou, C. In vitro Evaluation of Alginate/Halloysite

629

Nanotube Composite Scaffolds for Tissue Engineering. Materials Science and Engineering: C

630

2015, 49 (Supplement C), 700-712, DOI: https://doi.org/10.1016/j.msec.2015.01.037.

631

(27) Chiew, C. S. C.; Yeoh, H. K.; Pasbakhsh, P.; Krishnaiah, K.; Poh, P. E.; Tey, B. T.; Chan,

632

E. S. Halloysite/Alginate Nanocomposite Beads: Kinetics, Equilibrium and Mechanism for Lead

633

Adsorption. Applied Clay Science 2016, 119 (Part 2), 301-310, DOI:

634

https://doi.org/10.1016/j.clay.2015.10.032.

635

(28) Chiew, C. S. C.; Poh, P. E.; Pasbakhsh, P.; Tey, B. T.; Yeoh, H. K.; Chan, E. S.

636

Physicochemical Characterization of Halloysite/Alginate Bionanocomposite Hydrogel. Applied

637

Clay Science 2014, 101 (Supplement C), 444-454, DOI:

638

https://doi.org/10.1016/j.clay.2014.09.007.

639

(29) Zahidah, K. A.; Kakooei, S.; Ismail, M. C.; Bothi Raja, P. Halloysite Nanotubes as

640

Nanocontainer for Smart Coating Application: A Review. Progress in Organic Coatings 2017,

641

111 (Supplement C), 175-185, DOI: https://doi.org/10.1016/j.porgcoat.2017.05.018.


32

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


642

(30) Makaremi, M.; Pasbakhsh, P.; Cavallaro, G.; Lazzara, G.; Aw, Y. K.; Lee, S. M.; Milioto, S.

643

Effect of Morphology and Size of Halloysite Nanotubes on Functional Pectin

644

Bionanocomposites for Food Packaging Applications. ACS Applied Materials & Interfaces 2017,

645

9 (20), 17476-17488, DOI: 10.1021/acsami.7b04297.

646

(31) Biddeci, G.; Cavallaro, G.; Di Blasi, F.; Lazzara, G.; Massaro, M.; Milioto, S.; Parisi, F.;

647

Riela, S.; Spinelli, G. Halloysite Nanotubes Loaded with Peppermint Essential Oil as Filler for

648

Functional Biopolymer Film. Carbohydrate Polymers 2016, 152, 548-557, DOI:

649

https://doi.org/10.1016/j.carbpol.2016.07.041.

650

(32) Rao, K. M.; Kumar, A.; Suneetha, M.; Han, S. S. pH and Near-infrared Active; Chitosan-

651

coated Halloysite Nanotubes Loaded with Curcumin-Au Hybrid Nanoparticles for Cancer Drug

652

Delivery. International Journal of Biological Macromolecules 2018, 112, 119-125, DOI:

653

https://doi.org/10.1016/j.ijbiomac.2018.01.163.

654

(33) Pierchala, M. K.; Makaremi, M.; Tan, H. L.; Pushpamalar, J.; Muniyandy, S.; Solouk, A.;

655

Lee, S. M.; Pasbakhsh, P. Nanotubes in nanofibers: Antibacterial Multilayered Polylactic

656

Acid/Halloysite/Gentamicin Membranes for Bone Regeneration Application. Applied Clay

657

Science, DOI: https://doi.org/10.1016/j.clay.2017.12.016.

658

(34) Rao, K. M.; Nagappan, S.; Seo, D. J.; Ha, C.-S. pH Sensitive Halloysite-sodium

659

hyaluronate/poly(hydroxyethyl methacrylate) Nanocomposites for Colon Cancer Drug Delivery.

660

Applied Clay Science 2014, 97-98, 33-42, DOI: https://doi.org/10.1016/j.clay.2014.06.002.

661

(35) Cavallaro, G.; Lazzara, G.; Milioto, S.; Parisi, F.; Evtugyn, V.; Rozhina, E.; Fakhrullin, R.

662

Nanohydrogel Formation within the Halloysite Lumen for Triggered and Sustained Release. ACS

663

Applied Materials & Interfaces 2018, 10 (9), 8265-8273, DOI: 10.1021/acsami.7b19361.


33


Page 34 of 36

664

(36) Rizzo, C.; Arrigo, R.; D'Anna, F.; Di Blasi, F.; Dintcheva, N.; Lazzara, G.; Parisi, F.; Riela,

665

S.; Spinelli, G.; Massaro, M. Hybrid Supramolecular Gels of Fmoc-F/Halloysite Nanotubes:

666

Systems for Sustained Release of Camptothecin. Journal of Materials Chemistry B 2017, 5 (17),

667

3217-3229.

668

(37) Massaro, M.; Cavallaro, G.; Colletti, C. G.; Lazzara, G.; Milioto, S.; Noto, R.; Riela, S.

669

Chemical Modification of Halloysite Nanotubes for Controlled Loading and Release. Journal of

670

Materials Chemistry B 2018, 6 (21), 3415-3433.

671

(38) Cen, L.; Liu, W.; Cui, L.; Zhang, W.; Cao, Y. Collagen Tissue Engineering: Development

672

of Novel Biomaterials and Applications. Pediatric research 2008, 63 (5), 492-496.

673

(39) Wells, H. C.; Sizeland, K. H.; Kayed, H. R.; Kirby, N.; Hawley, A.; Mudie, S. T.;

674

Haverkamp, R. G. Poisson's Ratio of Collagen Fibrils Measured by Small Angle X-ray

675

Scattering of Strained Bovine Pericardium. Journal of Applied Physics 2015, 117 (4), 044701.

676

(40) Heng, B. C.; Das, G. K.; Zhao, X.; Ma, L.-L.; Tan, T. T.-Y.; Ng, K. W.; Loo, J. S.-C.

677

Comparative Cytotoxicity Evaluation of Lanthanide Nanomaterials on Mouse and Human Cell

678

Lines with Metabolic and DNA-quantification Assays. Biointerphases 2010, 5 (3), FA88-FA97.

679

(41) Liu, H.; Chen, J.; Jiang, J.; Giesy, J. P.; Yu, H.; Wang, X. Cytotoxicity of HC Orange NO. 1

680

to L929 Fibroblast Cells. Environmental toxicology and pharmacology 2008, 26 (3), 309-314.

681

(42) Wijesinghe, W.; Mantilaka, M.; Senarathna, K. C.; Herath, H.; Premachandra, T.;

682

Ranasinghe, C.; Rajapakse, R.; Rajapakse, R.; Edirisinghe, M.; Mahalingam, S. Preparation of

683

Bone-implants by Coating Hydroxyapatite Nanoparticles on Self-formed Titanium Dioxide

684

Thin-layers on Titanium Metal Surfaces. Materials Science and Engineering: C 2016, 63, 172-

685

184.


34

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


686

(43) Janković, A.; Eraković, S.; Vukašinović-Sekulić, M.; Mišković-Stanković, V.; Park, S. J.;

687

Rhee, K. Y. Graphene-based Antibacterial Composite Coatings Electrodeposited on Titanium for

688

Biomedical Applications. Progress in organic Coatings 2015, 83, 1-10.

689

(44) Mumjitha, M.; Raj, V. Fabrication of TiO2–SiO2 Bioceramic Coatings on Ti Alloy and its

690

Synergetic Effect on Biocompatibility and Corrosion Resistance. Journal of the mechanical

691

behavior of biomedical materials 2015, 46, 205-221.

692

(45) Yang, J. M.; Yang, J. H.; Tsou, S. C.; Ding, C. H.; Hsu, C. C.; Yang, K. C.; Yang, C. C.;

693

Chen, K. S.; Chen, S. W.; Wang, J. S. Cell Proliferation on PVA/Sodium Alginate and

694

PVA/poly(γ-glutamic acid) Electrospun Fiber. Materials Science and Engineering: C 2016, 66,

695

170-177, DOI: https://doi.org/10.1016/j.msec.2016.04.068.

696

(46) Kumar, A.; Lee, Y.; Kim, D.; Rao, K. M.; Kim, J.; Park, S.; Haider, A.; Lee, D. H.; Han, S.

697

S. Effect of Crosslinking Functionality on Microstructure, Mechanical Properties, and in vitro

698

Cytocompatibility of Cellulose Nanocrystals Reinforced Poly(vinyl alcohol)/Sodium Alginate

699

Hybrid Scaffolds. International Journal of Biological Macromolecules 2017, 95, 962-973, DOI:

700

http://dx.doi.org/10.1016/j.ijbiomac.2016.10.085.

701 702


35


703

Page 36 of 36

TOC

704


36

Drug-Loaded Halloysite Nanotube-Reinforced Electrospun Alginate

Recommend Documents