New Spanish Broom dressings based on Vitamin E and Lactobacillus plantarum for superficial skin wounds

Journal Pre-proof New Spanish Broom dressing based on vitamin E and Lactobacillus plantarum for superficial skin wounds Teresa Cerchiara, Barbara Giordani, Luz Maria Melgoza, Carola Parolin, Francesco Dalena, Angela Abruzzo, Federica Bigucci, Barbara Luppi, Beatrice Vitali PII:

S1773-2247(19)31075-5

DOI:

https://doi.org/10.1016/j.jddst.2020.101499

Reference:

JDDST 101499

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 24 July 2019 Revised Date:

20 December 2019

Accepted Date: 2 January 2020

Please cite this article as: T. Cerchiara, B. Giordani, L.M. Melgoza, C. Parolin, F. Dalena, A. Abruzzo, F. Bigucci, B. Luppi, B. Vitali, New Spanish Broom dressing based on vitamin E and Lactobacillus plantarum for superficial skin wounds, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2020.101499. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Spanish Broom wound dressing based on Vitamin E in association with Lactobacillus plantarum

Biological characterization

Release studies 40

Vitamin E released (%)

* 30 * * 20

10

0 0

O/W emulsion

5

10

15

20

25

Time (h) Spanish Broom wound dressing cotton wound dressing

Antioxidant activity Vitamin E

90

L. plantarum

+ Alginate

Freeze-drying

L. plantarum released (%)

80

*

70 60 50 40

*

30 *

20

*

10

*

*

0 0

5

10

15

20

25

Time (h) Spanish Broom wound dressing (PBS)

Spanish Broom wound dressing (SWF)

cotton wound dressing (PBS)

cotton wound dressing (SWF)

Antibacterial activity towards S. aureus (left) and P. aeruginosa (right)

1

New Spanish Broom dressing based on Vitamin E and Lactobacillus plantarum

2

for superficial skin wounds

3 4

Teresa Cerchiaraa*#, Barbara Giordania#, Luz Maria Melgozab, Carola Parolina, Francesco Dalenac,

5

Angela Abruzzoa, Federica Biguccia, Barbara Luppia, Beatrice Vitalia

6 7

a

Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Via

8

San Donato 19/2, 40127 Bologna, Italy

9

[email protected],

[email protected],

[email protected],

10

[email protected], [email protected], [email protected], [email protected]

11

b

12

Ciudad de México, México

13

[email protected]

14

c

15

15D, 87036 Arcavacata di Rende, CS, Italy

16

[email protected]

Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco,

Department of Chemistry and Chemical Technology, University of Calabria, Via P. Bucci, Cubo

17 18

#

Contributed equally to the work and should be considered joint first authors

19 20

*Corresponding author at: Department of Pharmacy and Biotechnology, University of Bologna, Via

21

San Donato 19/2, 40127 Bologna, Italy.

22

Telephone number: +39 051 2095615

23

E-mail address: [email protected] (T. Cerchiara)

24 25 26 27 28 29 30 31 32 33 34 1

35

Abstract

36 37

The focus of this work was to develop innovative dressings based on Spanish Broom gauzes loaded

38

with Vitamin E or Vitamin E- L. plantarum association for the treatment of skin wounds. Vitamin E

39

was selected for its well-known anti-oxidants properties and incorporated inside an emulsion

40

characterized by the presence of sodium alginate in the external phase. Two different techniques,

41

namely spray-drying and lyophilization, were then employed to set the Vitamin E emulsion on

42

Spanish Broom fibers. Sodium alginate was used as a coating polymer and Vitamin E was

43

successfully encapsulated, as confirmed by FT-IR spectroscopy. Scanning electron microscopy

44

revealed that emulsions obtained with both methods were well distributed on gauzes. In particular,

45

freeze-drying emulsion was able to provide the higher yield and entrapment efficiency. For this

46

reason, a freeze-drying emulsion enclosing a probiotic strain in addiction to Vitamin E was

47

prepared, in order to confer antibacterial properties to the final preparation. Indeed, Spanish Broom

48

dressing based on Vitamin E- Lactobacillus plantarum association revealed good antimicrobial

49

activities against pathogens that play a prominent role in skin wounds, such as Staphylococcus

50

aureus and Pseudomonas aeruginosa. Moreover, sustained release of both Vitamin E and L.

51

plantarum were guaranteed, as well as a good viability of L. plantarum during storage conditions.

52

In conclusion, Spanish Broom dressing loaded with the freeze-dried emulsion based on Vitamin E-

53

Lactobacillus plantarum could be a promising delivery system for the wound care.

54 55 56

Keywords: Wound dressing, Spanish Broom fibers, Vitamin E, Lactobacillus plantarum, sodium

57

alginate.

58 59 60 61 62 63 64 65 66 67 68 2

69

1. Introduction

70

Currently worldwide, delayed wound healing has a high social and economic impact. Consequently,

71

new strategies, based on alternative and innovative approaches to improve the healing of wounds,

72

are widely desirable in the care market [1-3]. Wound healing is a complicated process needful to

73

rapidly restore the integrity of skin barrier, limit dehydration and prevent the onset of infections. In

74

particular, four different phases happen in cascade: hemostasis occurs immediately after injury,

75

followed by the inflammatory phase, the formations of new vessels and finally the maturation of

76

new tissues. [2, 4].

77

Traditionally, cotton gauzes are widely employed as dressings in virtue of their favourable

78

properties, including absorbent capacity of wound exudates, low cost, easy use and fabrication.

79

Unfortunately, cotton cultivation requires an intensive use of pesticides and a large amount of water

80

leading to environmental problems. As alternative, we proposed new dressings based on Spanish

81

Broom fibers that share with cotton fibers some features, such as the high cellulose content, the

82

wide spread and the hydrophilic character [5]. Moreover, Spanish Broom is one of the most

83

common natural fibers that may be used in biocomposite materials, i.e. wound dressing. At the

84

present time, wound dressings based on cellulose fibers (i.e. cotton and linen) can be combined with

85

natural products like honey [6], propolis [7], Aloe vera [8], retynil palmitate [9, 10], hyaluronic acid

86

[11] and Vitamin E [12], that can considerably promote the healing process.

87

Especially, recent studies [12, 13] reported that Vitamin E can significantly contribute to healing of

88

wounds, through diverse mechanisms such as protection of biological membranes against lipid

89

peroxidation, anti-oxidant and anti-inflammatory activities [14-16].

90

Vitamin E is a group of lipophilic molecules enclosing eight different isoforms divided into

91

tocotrienols and tocopherols on the basis of their chemical structure. In particular, α-tocopherol is

92

the most widely present and potent form in the biological systems. In addition, α-tocopherol acetate

93

is widely used for topical treatments due to its stability. Considering the hydrophobicity and the

94

susceptibility to degradation of Vitamin E, a delivery system able to improve its solubility and

95

control the release is highly recommended. In the present study we encapsulated Vitamin E acetate

96

by means of two techniques, spray-dried and lyophilization, using sodium alginate as a coating

97

material.

98

Alginate is a polysaccharide of natural origin belonging to the family of linear, block copolymer

99

containing guluronic and mannuronic acids residues. It’s mainly presents in the cell wall of brown

100

seaweed, conferring high resistance and flexibility. Alginate is of particular interest for tissue repair

101

due to its abilities to keep the wound microenvironment protected from bacterial infections and

102

physiologically humid at the same time, facilitating in this way the healing process [17, 18]. 3

103 104

Recently, probiotics have been studied as a valid alternative approach for therapeutic uses,

105

including cure of skin diseases, i.e. atopic dermatitis [19], and acceleration of wound healing [20-

106

23].

107

To date only few studies have been reported showing the positive effects on cutaneous wound

108

healing of various probiotic bacteria, such as L. plantarum, L. brevis and L. fermentum [24, 25].

109

Topical application of lactobacilli can facilitate the recovery from skin injuries through different

110

actions, such as the reduction of phlogosis and the immunomodulation of immune system.

111

Furthermore, the production of antimicrobial metabolites by lactobacilli (i.e. lactic acid) protects the

112

wound from possible infections by opportunistic bacteria present on the skin.

113

This work aimed to develop and characterize innovative wound dressings using two drying

114

technological processes (spray-drying and freeze-drying) to obtain Spanish Broom fibers loaded

115

with Vitamin E and Vitamin E- L. plantarum association.

116

Process parameters (yield, Vitamin E entrapment efficiency and loading) and release behavior of

117

vitamin and lactobacilli were assessed, as well as the antibacterial activity of the final dressing.

118 119 120

2. Materials and methods

121 122

2.1 Materials

123

Spanish Broom dressing was provided by Prof. Giuseppe Chidichimo, from University of Calabria

124

(Arcavacata di Rende, CS, Italy). Cotton gauzes were purchased from a local drugstore. Vitamin E

125

acetate (alpha tocopherol acetate), sodium alginate and arabic gum were purchased from

126

Farmalabor (Canosa di Puglia, BT, Italy). Medium-chain triglycerides (TMC, Labrafac CC) were a

127

kind gift of Gattefossè (Saint-Priest, France). Tween 80 and Yeast Extract were supplied by Fluka

128

(Buchs, Switzerland).

129

Difco De Man, Rogosa e Sharpe (MRS) broth and GasPak EZ were obtained from Becton,

130

Dickinson and Co. (Sparks, USA). L-(+)-ascorbic acid, L-cysteine monohydrate hydrochloride and

131

Cetrimide broth were from Merck KGaA (Darmstadt, Germany). Mannitol salt was from Oxoid

132

(Basingstoke, UK).

133

S. aureus ATCC 29213, P. aeruginosa ATCC 10145 and L. plantarum ATCC 14917 were

134

purchased from American Tissue and Cell Culture Corp. (Virginia, USA).

135

Ethanol 96%, lactose, skimmed milk, bovine serum albumin (BSA) and all salts were from Sigma-

136

Aldrich (Milan, Italy). 4

137

Phosphate buffer (PBS: 6.65 mM Na2HPO4 × 12 H2O; 1.09 mM K2PO4; 0.14 M NaCl, pH 7.4) and

138

simulated wound fluid (SWF: 2% w/v BSA; 0.4 M NaCl; 0.05 M Trizma base; 0.02 M CaCl2, pH

139

7.4) were used for in vitro release studies.

140 141

2.2 Spanish Broom fibers for wound dressings

142

Spanish Broom fibers used to prepare wound dressings were extracted by patented DiCoDe process

143

as reported by Gabriele et al. [26] providing fibers with high cellulose content (91.7 ± 0.1%),

144

excellent mechanical properties (tenacity 35.9 ± 1.6 cN/tex, strain at break 5.8 ± 1.7%) and good

145

cytocompatibility [27]. Considering the increasing interest in finding new biomaterials for wound

146

concerns [28], the possible employment of Spanish Broom gauzes was investigated as an attractive

147

and ecologically sustainable alternative to traditional cotton wound dressing.

148 149

2.3 Preparation of Spanish Broom wound dressings loaded with Vitamin E

150 151

2.3.1 Preparation of Vitamin E emulsion

152

An oil in water emulsion containing arabic gum as emulsifier was prepared for the encapsulation of

153

Vitamin E. The lipid phase was produced by mixing Vitamin E (5 g) in TMC (1.5 g), added to the

154

aqueous phase containing arabic gum (2.5 g) and homogenized until its complete dispersion. The

155

emulsion was diluted with 86 g of an aqueous solution containing sodium alginate (0.3% w/v) and

156

subsequently processed by spray-drying or freeze-drying.

157 158

2.3.2. Preparation of Spanish Broom wound dressings loaded with Vitamin E spray-dried

159

microcapsules

160

Vitamin E microcapsules were obtained by processing the emulsion, prepared as reported in the

161

section 2.3.1, with a Mini Büchi spray dryer B-191 (Büchi Labortechnik AG, Flawil, Switzerland).

162

Preliminary studies were performed to determine the best drying conditions: inlet temperature 160

163

°C, outlet temperature 106 °C, air flow rate 700 NI/h, aspirator 100%, pump feed rate 25%. The

164

microcapsules were laid on Spanish Broom wound dressings (cut into rectangle 2×3 cm) applied at

165

the end of the cyclone in a way that doesn’t impair the entire spray-drying process. Cotton wound

166

dressings loaded with Vitamin E microcapsules were used as comparison. Spanish Broom and

167

cotton wound dressings loaded with Vitamin E microcapsules were stored at 4 °C.

168 169

2.3.3 Preparation of Spanish Broom wound dressings loaded with Vitamin E freeze-dried emulsion

5

170

250 µL of the emulsion, prepared as reported in the section 2.3.1, were spread on Spanish Broom

171

wound dressings (cut into rectangle 2×3 cm), frozen at −18 °C overnight, and finally lyophilized for

172

18 h (Christ Freeze Dryer ALPHA 1-2, Milan, Italy). Cotton wound dressings prepared with the

173

same method were used as comparison. The freeze-dried wound dressings were stored at 4 °C.

174 175

2.4 Preparation of Spanish Broom wound dressings loaded with Vitamin E and Lactobacillus

176

plantarum

177 178

2.4.1. Lactobacillus plantarum culture conditions

179

L. plantarum was grown in 500 mL of MRS broth containing L-cysteine (0.05% w/v) and incubated

180

at 37 °C for 48 h under anaerobically conditions with the addition of GasPak™ EZ.

181

L. plantarum suspension was centrifuged (5000 g for 10 min), recovered cells were washed with

182

sterile saline (NaCl 0.9% w/v) and suspended in the medium for lyophilization (~ 5×1011 CFU/mL),

183

consisting of skimmed milk (10% w/v), yeast extract (0.5% w/v), lactose (0.5% w/v) and ascorbic

184

acid (0.1% w/v).

185 186

2.4.2. Preparation of Spanish Broom wound dressings loaded with freeze-dried emulsion

187

containing Vitamin E and L. plantarum

188

The lipid phase of the emulsion was prepared by mixing Vitamin E (5 g) in TMC (1.5 g) and added

189

to the aqueous phase containing arabic gum (2.5 g) and L. plantarum suspended in freeze-drying

190

medium (5 g). The emulsion was diluted with 86 g of an aqueous solution of sodium alginate (0.3%

191

w/v) and 250 µL were deposited on Spanish Broom wound dressing (cut into rectangle 2×3 cm) and

192

lyophilized as reported in section 2.3.3. Cotton wound dressings prepared with the same method

193

were used as comparison. The freeze-dried wound dressings were stored at 4 °C.

194 195

2.5. Process yields and determination of Vitamin E loading and entrapment efficiency

196

The following equation was used to calculate the percent yields (Yield %) of spray-drying and

197

freeze-drying processes (Eq.1):

198

Yield % = final weight of formulation (microcapsules or freeze-dried emulsion) x 100/ overall

199

weight of all initial components (Eq.1)

200

For the determination of entrapment efficiency (EE %) (Eq. 2) and Vitamin E loading percentage

201

(VL %) (Eq. 3), exact amounts of microcapsules or freeze-dried emulsion were dissolved in 10 mL

202

of ethanol. Vitamin E was spectroscopically determined (UV-1601 Shimadzu, Milan, Italy) at 285

6

203

nm and quantified by using a standard curve set up in the concentration range 12-25 µg/mL

204

(R2=0.9991).

205

EE % = vitamin completely released from formulation x 100/ initial amount of vitamin (Eq. 2)

206

VL %= vitamin completely released from formulation x 100/ weight of formulation (Eq. 3).

207 208

2.6 Determination of L. plantarum loading capacity and survival during storage conditions

209

L. plantarum content inside emulsion was assessed before and after lyophilization to determine both

210

the loading and the impact of manufacturing procedures on probiotic cell survival, as previously

211

reported [29]. Briefly, an accurately weighed amount of freeze-dried powder was firstly incubated

212

in MRS for 1 h at 37 °C and then allowed to grow on MRS agar plates (agar 1.5% w/v) for 24 h at

213

37 °C under anaerobic conditions. Afterwards, colony forming units (CFU) were counted and L.

214

plantarum loading was expressed as number of CFU per gram of freeze-dried emulsion.

215

The same procedure was also applied for the determination of probiotic viability during 12 months

216

of storage at 4 °C.

217 218

2.7. Fourier Transform Infrared Spectroscopy (FT-IR)

219

Potassium bromide and sample (Vitamin E spray-dried microcapsules or freeze-dried emulsion)

220

were tritured together (weigh ratio 1:10) and KBr disks were obtained with a hydraulic press

221

applying a pressure of 100 tons for 5 min. Infrared spectra were recorded between 4000 and 650

222

cm-1 with a Jasco FT-IR 4100 spectrophotometer (Jasco Lecco, Italy).

223 224

2.8. Scanning Electron Microscopy (SEM)

225

The morphology and particle size of spray-dried microcapsules and the morphology of freeze-dried

226

emulsions deposited on Spanish Broom and cotton dressing were indagated by Scanning Electron

227

Microscopy (SEM). Samples were spread out on carbon tape and coated with a thin layer of gold

228

under argon atmosphere using a sputter module in a high vacuum evaporator. Samples were then

229

observed with LEO 420 (LEO Electron Microscopy Ltd, Cambridge, UK) at 15 kV under high

230

vacuum conditions.

231 232

2.9. In vitro Vitamin E and L. plantarum release studies

233

According to Taepaiboon et al. [30], Vitamin E release from wound dressing loaded with freeze-

234

dried emulsions was evaluated at 37 °C applying the total immersion method with some

235

modifications. Briefly, wound dressing samples (cut into rectangle 2×3 cm) were immersed in 10

236

mL of releasing medium, composed of PBS added to ethanol (30% v/v) and tween 80 (0.5% w/v) in 7

237

order to ensure sink conditions. At pre-established time intervals (1, 3, 5 and 24 h), the entire

238

amount of medium was collected and released vitamin was separated from freeze-dried emulsion by

239

centrifugation (12400 g, 20 min, 25 °C). Vitamin E content was quantified in the supernatant as

240

reported in section 2.5.

241

L. plantarum release studies were carried out at 37 °C by placing Spanish Broom and cotton wound

242

dressing in 10 mL of PBS or SWF to simulate wound exudate, as reported by Boateng et al. [31].

243

Aliquots were taken at predetermined time points (1, 3, 5, 7 and 24 h) and viable cells released over

244

time were determined through plate count method as described in section 2.6.

245 246

2.10 Water uptake ability of Spanish Broom wound dressings loaded with Vitamin E and L.

247

plantarum

248

In order to evaluate the capability of Spanish Broom and cotton wound dressings to absorb the

249

wound exudate, water uptake studies were performed in SWF following the method reported by

250

Bigucci et al. [32]. Wound dressings loaded with freeze-dried emulsion were placed on cellulose

251

nitrate membrane filter (diameter of 4.7 cm and pore size of 0.45 mm) soaked in SWF. The filter

252

was then positioned on the top of a sponge (7×5×2 cm), previously waterlogged in SWF and kept

253

hydrated by immersing it in the same fluid.

254

The increase in weight of the wound dressings was monitored for 5 h and water uptake percentage

255

(WU %) was defined as follows (Eq. 4):

256

WU %  = (WHWF – WF - WDW) × 100/ WDW (Eq. 4)

257

where WHWF is the weight of the hydrated wound dressing and the soaked cellulose filter, WF is the

258

weight of the soaked cellulose filter and WDW is the weight of the dry wound dressing.

259 260

2.11. Antibacterial activity of wound dressings loaded with Vitamin E and L. plantarum

261

L. plantarum formulated in Spanish Broom wound dressings was assessed for its ability to

262

counteract the growth of two pathogenic bacteria, Staphylococcus aureus and Pseudomonas

263

aeruginosa, commonly responsible for wound infections [33, 34]. The antibacterial activity of L.

264

plantarum was tested by means of agar overlay assay, as described in a previous work [27], with

265

slight modifications. Briefly, Spanish Broom wound dressings loaded with freeze-dried emulsion

266

(cut in disks of 1 cm in diameter) were placed on the surface of MRS agar plates and anaerobically

267

incubated at 37 °C. 100 µL of an overnight culture (108 CFU/mL) of S. aureus or P. aeruginosa

268

were then inoculated into 10 mL of mannitol salt or cetrimid soft agar (agar 0.7% w/v),

269

respectively, and poured on the plates were L. plantarum had been recovered. Plates were further

270

incubated for 24 h and the capability of L. plantarum formulated in Spanish Broom wound 8

271

dressings to exert an antimicrobial activity was assessed by determining the size of inhibition halo.

272

To assure that antibacterial activity of wound dressing loaded with Vitamin E and L. plantarum was

273

due to L. plantarum, the formulation loaded with only Vitamin E freeze-dried emulsion was also

274

tested.

275 276

2.12. Statistical analysis

277

All the experiments were performed in triplicate. Results were expressed as mean ± SD. Statistical

278

analysis was performed using ANOVA test. Differences were deemed significant for p < 0.05.

279 280

3. Results and discussion

281 282

3.1 Characterization of Vitamin E microcapsules and freeze-dried emulsion

283

Vitamin E microcapsules and freeze-dried emulsion were prepared by spray-drying and

284

lyophilization, respectively. Both formulations were obtained by oil in water emulsion, diluted with

285

an aqueous solution of sodium alginate (0.3% w/v). The use of this polymer is particularly

286

favorable because its capability to improve the recovery of wounds. Moreover, it is biocompatible,

287

easily available and not expensive [18]. Among drying processes, spray-drying is a fast and

288

economical, single step, drying method, which is widely used in pharmacy to produce a dry powder

289

from a liquid phase [35]. Moreover, this process plays a key role to have powder with good water

290

dispersibility [36]. The yield of spray-drying process was calculated considering both the

291

microcapsules recovered from the collection chamber and those entrapped in Spanish Broom or

292

cotton fibers (ca 25 mg of microcapsules loaded on gauze every 20 mL of nebulized emulsion).

293

However, the low yield (9.12 ± 2.01 %) of spray-drying process was due to high viscosity

294

(determined at 25 °C by using a falling ball viscometer, HAAKETM Falling Ball Viscometer Type

295

C, Thermo electron corporation, Karlsruhe, Germany) of sodium alginate emulsion (73.58 ± 0.84

296

mPa×sec), producing many microcapsules adherent to the wall of instrument’s drying chamber. So,

297

these microcapsules are not considered for the determination of encapsulation yield. In addition,

298

some of the microcapsules owing small size did not separate from the cyclone, others were trapped

299

in the filter and therefore not collected [37]. In this regard, Ceschan et al. [38] and Gallo et al. [39]

300

showed that the yield of the spray-drying process decreased with high viscosity of emulsion,

301

confirming our results. The yield results affected Vitamin E loading too. In fact, we observed a

302

significant inverse correlation between vitamin loading (64.52 ± 2.50 %) and yield due to the

303

difficulty to recover microcapsules from spray-dryer. Tan et al. reported similar results [40].

9

304

Moreover, also the low EE (9.09 ± 0.12 %) of Vitamin E in sodium alginate microcapsules was due

305

to the high viscosity of the emulsion.

306

Alternatively, lyophilization is a drying technique widely used to ameliorate the long-term stability

307

of several pharmaceutical products, like vaccines, proteins, and vitamins [41]. Although Vitamin E

308

loading obtained using both drying processes was considered statistically equal (65.35 ± 2.36 %),

309

freeze-drying provided better results in terms of yield and EE (100 %). It was due to the fact that no

310

mass loss occurred during the freeze-drying process. Taking into account these observations, we

311

concluded that freeze-drying was more effective at drying Vitamin E emulsion, so we select this

312

method for the next studies.

313 314

3.2 Fourier Transform Infrared Spectroscopy (FT-IR)

315

The FT-IR spectra of Vitamin E, spray-dried microcapsules and freeze-dried emulsion are reported

316

in Fig. 1. FT-IR spectrum of Vitamin E presented characteristic C=O stretching vibration at around

317

900–1078 cm−1, C-O vibration at 1209 cm−1, C=O stretching at 1759 cm−1, and C-H alkanes group

318

at 2927 cm−1. Absorption band of Vitamin E at 3400–3650 cm−1 was ascribed to the terminal

319

hydroxyl group and peak at 1078–1251 cm−1 was due to the C–O stretching [42]. The FT-IR spectra

320

of spray-dried microcapsules and freeze-dried emulsion showed similar peaks with Vitamin E

321

suggesting that there was interaction between Vitamin E and sodium alginate without any variations

322

in their structure. This finding was in agreement with Tarigan et al. [37].

323 324

3.3 Morphology of Spanish Broom wound dressings loaded with Vitamin E spray-dried

325

microcapsules or freeze-dried emulsion

326

The SEM micrographs showed that both spray-dried microcapsules and spray-dried microcapsules

327

deposited on Spanish Broom dressing (Fig. 2A-B) possessed spherical shape (mean particle size:

328

2.94 µm ± 1.12) with several indentations, that are typically formed during drying and cooling

329

processes as a consequence of the rapid shrinkage of nebulized particles [43]. Moreover,

330

microcapsules were easily deposited on the wound dressing preserving its fibrous structure. Similar

331

results were obtained with cotton dressing (data not shown).

332

Freeze-dried emulsion based on Vitamin E appeared uniformly distributed on Spanish Broom (Fig.

333

2C) and cotton fibers (data not shown). However, flakes with rough and porous structure were

334

observed on the surface of wound dressing, and they are attributed to the direct sublimation of ice

335

into water vapor during lyophilization [44].

336 337 10

338

3.4 Preparation of freeze-dried emulsion containing Vitamin E and L. plantarum

339

Nowadays, microbial contaminations of skin lesions are responsible for the high rates of morbidity

340

and mortality [45] and dressings with multifunctional properties such as antioxidant and

341

antibacterial activity enhancing wound healing process are highly required [46]. In this work, we

342

selected L. plantarum which could contribute to accelerate the wound healing process thanks to its

343

antibacterial activity against pathogens like P. aeruginosa and S. aureus that play a prominent role

344

in superficial skin and burn wound infections [47]. According to Lee et al. [48], the most largely

345

used preservation method for different probiotic species including L. plantarum is the freeze-drying.

346

So, in order to obtain a formulation with antioxidant and antibacterial properties, we prepared an

347

emulsion characterized by a lipid core based on Vitamin E and an aqueous phase (sodium alginate

348

0.3% w/v) which incorporates L. plantarum. The selected polymer, in addiction to promoting

349

wound healing [47], is also reported to be able to protect probiotic microorganisms during

350

manufacturing procedures and storage [49].

351

L. plantarum loading in freeze-dried powder was found to be 10.9 ± 0.1 log CFU g-1. Each gauze

352

was loaded with 26.13 ± 2.92 mg of freeze-dried emulsion, containing 2.3 ± 0.38 × 109 viable

353

probiotic cells, with no significant differences between Spanish Broom and cotton wound dressings.

354

Interestingly, the decrease in L. plantarum viability after freeze-drying procedure was very low (0.3

355

log units), supporting the choice of this productive technique.

356

Moreover, Vitamin E loading was not influenced by the presence of lactobacilli and it was the same

357

obtained for freeze-dried emulsion containing only with Vitamin E (section 3.1).

358 359

3.5 Morphology of Spanish Broom wound dressings loaded with freeze-dried emulsion of Vitamin E

360

and L. plantarum

361

SEM micrographs showed that freeze-dried emulsion based on the association of Vitamin E and L.

362

plantarum, as well as Vitamin E freeze-dried emulsion, were uniformly distributed on Spanish

363

Broom and cotton fibers (Fig. 3A-B). The presence of L. plantarum in the emulsion did not affect

364

morphology of freeze-dried emulsion, which maintained its irregular flake like structures.

365 366

3.6 In vitro Vitamin E and L. plantarum release studies

367

Fig. 4 shows in vitro Vitamin E release from freeze-dried emulsion based on the association

368

Vitamin E-L. plantarum on Spanish Broom and cotton wound dressing.

369

Both wound dressings provided a burst release of Vitamin E in the first 5 h followed by a sustained

370

release over 24 h. The results highlighted that the presence of sodium alginate in the emulsion and

371

the freeze-drying process affected the Vitamin E release for a longer period of 24 h, assuring the 11

372

antioxidant effect for several hours. This is a particularly important aspect considering that the first

373

12-48 h are decisive during wound healing process [50]. Moreover, the sustained release of Vitamin

374

E could increase patient compliance reducing wound dressing changes. Comparing the two wound

375

dressings, Vitamin E release from Spanish Broom wound dressing resulted slightly higher than that

376

from the cotton dressing (p < 0.05), and it was due to higher aqueous media absorption capacity of

377

Spanish Broom fibers with respect to cotton. This behaviour is probably due to the higher fineness

378

of Spanish Broom fibers [51].

379

A similar release profile was obtained when Spanish Broom and cotton wound dressings where

380

loaded with freeze-dried emulsion containing only vitamin (data not shown).

381

Moreover, both Spanish Broom and cotton wound dressings provided a sustained release of L.

382

plantarum over 24 h. L. plantarum release was higher in PBS than in SWF, probably due to the

383

different solubility of sodium alginate freeze-dried emulsion in these two environments. In

384

particular, after 7 h Spanish Broom wound dressing released 5.29 ± 0.57 × 108 and 2.76 ± 0.14 ×

385

108 viable probiotic cells when immersed in PBS or SWF, respectively.

386

Similarly, to Vitamin E release, Spanish Broom wound dressing allowed a higher release of L.

387

plantarum with respect to cotton wound dressing. According to this result, Spanish Broom dressing

388

with Vitamin E-L. plantarum freeze-dried emulsion was able to absorb a higher volume of SWF

389

(265.1 ± 1.5 %) than cotton dressing (252.4 ± 2.1 %). In conclusion, these results showed that both

390

wound dressings can be used to improve healing process reducing the frequency of dressing

391

changes, nurse time and travelling costs. A better release performance, however, was observed with

392

Spanish Broom fibers.

393 394

3.7 Survival of L. plantarum during storage

395

L. plantarum showed a good stability for at least 12 months (> 9 log CFU g-1), suggesting that

396

freeze-dried emulsion was able to maintain high bacterial viability for a prolonged period of time.

397 398

3.8. Antibacterial activity of freeze-dried emulsion loaded on Spanish Broom wound dressing

399

The biological activity of Spanish Broom wound dressing loaded with Vitamin E and L. plantarum

400

was evaluated towards S. aureus and P. aeruginosa, chosen as representative pathogens responsible

401

for skin infections. This wound dressing exhibited antibacterial activity, as demonstrated by the

402

diameters of the zones of growth inhibition: 2.90 ± 0.28 cm for S. aureus and 3.45 ± 0.21 cm for P.

403

aeruginosa. Since the Spanish Broom wound dressing loaded with Vitamin E freeze-dried emulsion

404

was not antibacterial, the biological activity can be attributed only to L. plantarum. These results

405

highlighted the multiplicity of functional properties of the novel Spanish Broom-based skin 12

406

dressing, combining the anti-oxidant activity of Vitamin E with the antibacterial activity of

407

lactobacilli, both crucial in the wound healing process.

408 409

Conclusions

410

In this work, innovative wound dressings based on Spanish Broom fibers with antioxidant and

411

antioxidant-antibacterial properties due to Vitamin E and the association of Vitamin E with L.

412

plantarum were successfully developed. Vitamin E, with and without L. plantarum, was dispersed

413

in a solution of sodium alginate (0.3 %) and the resulting emulsion was freeze-dried on the Spanish

414

Broom wound dressing. These formulations assured a sustained release of Vitamin E. The

415

formulation based on the association of Vitamin E and L. plantarum showed a good antibacterial

416

activity against Staphylococcus aureus and Pseudomonas aeruginosa and guaranteed a sustained

417

release of probiotic cells over 24 h. Compared to cotton dressing, Spanish Broom wound dressing

418

showed higher release of Vitamin E and L. plantarum, suggesting that the Spanish Broom is a

419

successful alternative to the cotton in wound care.

420 421

Conflict of interest

422

The authors report no conflicts of interest.

423 424

Acknowledgments: This work was supported by Fondazione Cassa di Risparmio di Imola [grant

425

no. 269.0316-2016.0260]. The authors are grateful to Martina Conti and Prof. G. Chidichimo for

426

their contribution to this work.

427 428 429 430 431 432 433 434

13

435

Figure captions

436

Fig. 1. FT-IR spectra of Vitamin E (A), spray-dried microcapsules (B) and freeze-dried emulsion

437

(C).

438

Fig. 2. SEM micrographs of spray-dried microcapsules (2000 X) (A), Spanish Broom wound

439

dressing loaded with spray-dried microcapsules (100 X) (B) and freeze-dried emulsion of Vitamin

440

E (200 X) (C).

441

Fig.3. SEM micrographs (50 X) of freeze-dried emulsion of Vitamin E and L. plantarum loaded on

442

(A) Spanish Broom and (B) cotton wound dressings.

443

Fig. 4. In vitro Vitamin E release in releasing medium (PBS with ethanol 30% v/v and tween 80

444

0.5% w/v) from freeze-dried emulsion loaded on Spanish Broom and cotton wound dressings (mean

445

± SD, n=3).

446

Fig. 5. In vitro L. plantarum release in PBS and SWF from freeze-dried emulsion loaded on Spanish

447

Broom and cotton wound dressings (mean ± SD, n=3).

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 14

469 470

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22 (2014) 25-28.

611 612

19

A

B

A

B

A

B

40

Vitamin E released (%)

* 30 * * 20

10

0 0

5

10

15 Time (h)

Spanish Broom wound dressing cotton wound dressing

20

25

90

L. plantarum released (%)

80

*

70 60 50 40

*

30 *

20

*

10

*

*

0 0

5

10

15

20

25

Time (h) Spanish Broom wound dressing (PBS)

Spanish Broom wound dressing (SWF)

cotton wound dressing (PBS)

cotton wound dressing (SWF)

L. plantarum viability (log CFU g-1) 12

11

10 9

8

7

6 0 2 4 6 8 Time (months) 10 12

T. C. and B.V. designed the study. B.G., L.M.M., C.P., C.P., F.D., A.A., F.B. and B.L. performed the experiments. All authors contributed in data interpretation and discussion. T.C., B.G., C.P., C.P. and B.V. contributed to manuscript generation. All authors have read and agreed to the revised version of the manuscript.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: