HAN Feng(韓 峰)，CUI Cheng-jun(崔呈俊)，CHEN Meng-xia(陳夢(mèng)霞)，HE Chuang-long(何創(chuàng)龍)，MO Xiu-mei(莫秀梅)，WANG Hong-sheng(王紅聲)

1 College of Chemistry，Chemical Engineering and Biotechnology，Donghua University，Shanghai 201620，China

2 Key Laboratory of Textile Science and Technology，Ministry of Education，Donghua University，Shanghai 201620，China

3 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials，Donghua University，Shanghai 201620，China

Introduction

Ethosome carriers invented by Touitou et al.［1］，are a modified form of liposomes that contain a relatively high concentration of ethanol.It is reported that ethosomes are more efficient at delivering topical agents to the skin，in terms of quantity and depth，than either liposomes or hydroalcoholic solutions［2］.Recently，it has been reported that drug-loaded liposome fixed on scaffolds can release drug in a much more smooth way［3］，which implys ethosome-loaded scaffolds may be powerful on transdermal drug delivery systems.

Silk fibroin (SF)，a main component of silkworm silk，has unique properties including good biocompatibility，good oxygen and water vapor permeability，and low inflammatory response，which makes it an excellent biomaterial used widely in biomedical area［4-5］.Moreover，SF is favoured by skin with diverse properties such as maintainning aqueous environment for skin［6］.Recently，electrospun SF nanofibers have been widely applied in tissue engineering， wound healing， and drug delivery［7-8］.

However，the electrospinning process frequently involves toxic solvents which is unfriendly to environment and may do harm to cells.In our previous work［9-10］，we successfully produced vitamin-loaded SF nanofibrous matrices through a green process of aqueous electrospinning.Here，we aim to fabricate a novel ethosome-loaded SF nanofibrous mat based on the similar eco-friendly method.To enhance the eletrospinnability of the SF aqueous solution and improve the mechanical properties of eletrospun product，polyethylene oxide(PEO)was selected for blending based on its aqueous solubility and known biocompatibility［11-13］.As ethosome has powerful capacity of delivery various drugs into skin tissue， the ethosome-loaded SF/PEO nanofibrous scaffolds can possess promising application in transdermal drug delivery systems.

1 Experimental

1.1 Materials

Cocoons of Bombyx mori silkworm were kindly supplied by Jiaxing Silk Co.(China).Phosphatidyl choline，cholesterol，and PEO (with an average molecular weight of 600 000)were purchased from Sigma-Aldrich (USA).

1.2 Preparation of regenerated SF

The preparation of the regenerated SF was done as described in our previous research［9-10］.Briefly，Bombyx mori silkworm was degummed using the sodium carbonate (0.02 mol/L Na2CO3)in boiling water for three times，about 30 min each，to remove the glue-like sericin proteins.The degummed SF was dried in hot air oven(45℃)after six rinses in distilled water，then dissolved in a ternary solvent system of CaCl2/H2O/CH3CH2OH solution(1∶8∶2 in molar ratio)to obtain a clear solution.Following that the solution was dialyzed against distilled water using cellulose tube (molecular weight cutoff 14 000，Sigma-Aldrich)at room temperature for 3 d.Then the SF solution was filtered and lyophilized to obtain the regenerated SF sponges.

1.3 Preparation and characterization of cationic ethosome

Ethosomes were prepared according to the thin-film hydration method.Phosphatidyl choline， cholesterol， and octadecylamine (90 ∶15 ∶5 in mass ratio)were dissolved in methanol in a 250 mL round-bottomed flask.The mixture was evaporated in a rotary evaporator，and solvent traces were removed under vacuum overnight.Hydration of the film was performed in two steps.First，5 mL of a mixture of water and alcohol (7∶3 in volume ratio)was added to the flask and the concentrated dispersion was mechanically shaken for 1 h at room temperature.Then，a second 5 mL aliquot of water-alcohol was added and the dispersion shaken for another hour.The obtained suspensions were then sonicated until it turned to clear opalescent dispersion.The polydispersity index (PI)，representing the distribution of particle size，was determined with a Zetasizer 2000(Malvern Instruments).For transmission electron microscopy (TEM)，samples were placed on a copper mesh coated with carbon membrane，then observed under H-800 electron microscopy.

1.4 Preparation of the ethosome-loaded SF nanofibrous mats

The ethosomes were incorporated into SF nanofibers via blend electrospinning.SF and PEO were dissolved in distilled water to make SF/PEO solution and different volume (0，0.5，1.0，2.0，and 3.0 mL respectively)of the prepared ethosome solution (20 g/L)was added into the SF/PEO solution (with a final concentration of 160 g/L and 10 g/L respectively for SF and PEO)and stirred gently at room temperature to form homogenous blends with different final concentrations of ethosome (0，1.25%，2.5%，5.0%，and 7.5% by weight based on the content of SF)for subsequent electrospinning.Then，the solution was filled into a 2.5 mL plastic syringe capped with 7-gauge blunt needles (inner diameter is 0.21 mm).A voltage of 20 kV using a high voltage power supply(BGG6-358，BMEI Co.，Ltd.，China)was applied across the needle and ground aluminum foil collector，which was placed at a distance of 20 cm between the needle and the collector.A flow rate of 0.3 mL/h was adopted for all electrospinning process.

1.5 Characterization of the ethosome-loaded SF/PEO nanofibrous mats

The morphology of the ethosome-loaded SF/PEO composite nanofibers was observed with a scanning electronic microscope (SEM)(JSM-5600，Japan)at an accelerated voltage of 10 kV.The mean fiber widths were determined by an image analysis software (Image-J， National Institutes of Health，USA)from selecting 80 fibers randomly observed on the SEM images.The internal morphology of liposome-loaded SF nanofibers was revealed by TEM (H-800，Hitachi，Japan).The attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)was obtained at room temperature using an FTIR spectrophotometer (Avatar380，USA).All spectra were recorded by absorption mode at 2 cm-1interval and in the wavelength range of 500-4 000 cm-1wave numbers.

2 Results and Discussion

2.1 Morphology and characterization of cationic ethosome

As shown in Fig.1，the ethosome particles have a mean diameter of around 78.3 nm，and the polydispersity index is less than 0.165，which can be helpful for the particles to transfect stratum corneum［14］.The TEM image of the ethosome clearly shows the formation of vesicles，which is spherical and multilamellar(Fig.2).

Fig.1 The size distribution of ethosome

2.2 Morphology of ethosome-loaded SF/PEO nanofibers

Fig.2 Negative stain electron micrographs of ethosomes

To optimize the condition of electrospinning，with the concentration of SF and PEO fixed to be 160 g/L and 10 g/L respectively according to our pre-experiment，different amounts of the ethosome (0，1.25%，2.5%，5.0%，and 7.5% by weight based on the content of SF)were added into the blend solution.As shown in Fig.3，the morphology of the ethosomeloaded SF/PEO nanofibers was round with a diameter ranging from 574 to 795 nm.In Fig.3，AD reprents average diameter;SD reprents standard deviation based on the sample.With the increase of the ethosome content，the average diameter of the nanofiber also increased gradually.However，fibers cannot be obtained via electrospinning when the concentration of ethosome increases to 7.5% by weight，due to the high viscosity of the solution.

Fig.3 SEM micrographs and diameter distribution histograms of electrospun ethosome-loaded SF/PEO nanofibers with different contents of ethosome ((a)，(e):0;(b)，(f):1.25%;(c)，(g):2.5%;(d)，(h):5.0% by weight based on the content of SF)

The internal morphology of ethosome-loaded SF/PEO nanofibers was revealed by TEM and the image clearly showed the ethosomes distributing randomly on the SF/PEO nanofibers(Fig.4).

Fig.4 TEM of the ethosome-loaded SF/PEO nanofiber

2.3 Structure analyses of ethosome/SF/PEO nanofibers mats

FTIR was used to study the molecular conformation of the fibers.SF with the structure of random coil or a-helix has characteristic absorption bands at 1 648 cm-1(amide Ⅰ)，1 537 cm-1(amide Ⅱ)，and 1 240 cm-1(amide Ⅲ).While phosphatidyl choline has the characteristic absorption peaks at 2 923，2 852，1 739，1 467，1 237，and 1 066 cm-1;and the characteristic absorption peaks of cholesterol are at 1 467，1 378，1 080，and 530 cm-1.FTIR assay showed the characteristic absorption peaks of phosphatidyl choline (2 923，2 852，and 1 739 cm-1，as indicated with arrows in Fig.5)appeared in the ethosome-loaded SF/PEO nanofibers，while absence in the SF/PEO nanofibers.The FTIR data are consistent with the TEM image，demonstrating the ethosome successfully loaded on the SF/PEO nanofiber through the green electrospinning.

Fig.5 FTIR transmission spectra of (a)electrospun ethosome-loaded SF/PEO nanofibrous mats;(b)ethosome;(c)electrospun SF/PEO nanofibrous mats

3 Conclusions

A novel ethosome-loaded SF/PEO nanofibrous mat was successfully fabricated by a green electrospinning process.The morphology of the ethosome-loaded SF/PEO nanofiber was similar as non-loaded SF/PEO nanofiber.The diameter of the ethosome-loaded SF/PEO nanofiber was about 600-800 nm and increased with the increase of the ethosome content.When the content of ethosome increases to 7.5% by weight，no electrospun fiber can be obtained.Possessing the merits of both ethosome and nanofiber，as-spuned ethosome-loaded SF/PEO nanofibrous mats may have a promising application in transdermal drug delivery.

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