Antibiofouling Zwitterionic Gradational Membranes with Moisture

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Anti-biofouling Zwitterionic Gradational Membranes with Moisture Retention Capability and Sustained Anti-microbial Property for Chronic Wound Infection and Skin Regeneration Yunbo Feng, Qian Wang, Min He, Xiang Zhang, Xiaoling Liu, and Changsheng Zhao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00629 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Biomacromolecules

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Anti-biofouling Zwitterionic Gradational Membranes with Moisture

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Retention Capability and Sustained Anti-microbial Property for

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Chronic Wound Infection and Skin Regeneration

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Yunbo, Fenga†, Qian Wanga†, Min Hea, Xiang Zhanga, Xiaoling, Liua* and

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Changsheng, Zhaoa**

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a

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Materials Engineering, Sichuan University, Chengdu 610065, People’s Republic of

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China

College of Polymer Science and Engineering, State Key Labo ratory of Polymer

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* Corresponding author. Tel: +86-28-85400453, Fax: +86-28-85405402, E-mail:

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[email protected] or [email protected] (C.S. Zhao), [email protected]

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(X.L. Liu)

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ACS Paragon Plus Environment

Biomacromolecules 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

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ABSTRACT

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Non-adherent wound dressings with moisture management and long-lasting

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antibacterial properties have great significance for wound healing clinically. Herein, a

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novel multicomponent zwitterionic gradational membrane is fabricated by co-

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electrospinning method to realize low biofouling and favorable moisture control as well

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as long-acting antibacterial properties during chronic wound healing process. The

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obtained membrane possesses excellent anti-biofouling performance that effectively

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resists protein, bacteria and cell adhesion according to in vitro anti-fouling evaluation.

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Furthermore, gradational co-electrospinning method grants the composite membrane

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with moisture retention capability which could effectively absorb wound exudate and

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maintain moisture healing environment. Additionally, in vivo and in vitro antibacterial

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investigations reflect that the composite membrane has excellent long-acting

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antibacterial property. Moreover, in vivo wound healing assessment confirms that the

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prepared membrane significantly reduces the complete wound healing time than

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commercial wound dressing. These results highlight such zwitterionic gradational

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membrane as an advanced wound dressing to meet the various requirements for chronic

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wound infection and skin tissue regeneration in clinical applications.

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INTRODUCTION

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Covering the opened wound with a dressing could not only simply insulate injured

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tissue from external environment but also maintain a moderate healing state, which

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promote epithelialization and tissue regeneration.1-3 However, biofouling is one of the

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clinical issues for traditional medical gauze and cotton, which brings about severe

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wound adhesion and may cause secondary damage during dressing removal.4 Moreover,

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inadequate moisture management and insufficient of antibacterial abilities also severely

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hamper the normal healing process especially during chronic wound recovery.5 Thus,

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these requirements motivate us to search for multi-functional wound dressings with the

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following healing functions for chronic wound: (i) effectively prevent surface

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biofouling to achieve painless removal; (ii) fully absorb wound exudate and maintain

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moisture healing environment (iii) avoid bacterial infections and provide stable and

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long-lasting antibacterial functions.6

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Hydrophilic

polymers

such

as

poly-(ethylene

glycol),

polyelectrolyte,

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polysaccharide and zwitterion are used for hydrophilic modification to improve the

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biocompatibility of the matrix and endow wound dressings with antifouling

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properties.7-11 Among them, zwitterionic polymer is especially popular due to its super-

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hydrophilic and ultralow biofouling performance.12 Recently, some studies have

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devoted to fabricating zwitterionic hydrogels and exploring the potential applications

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in non-adherent wound dressings.13-17 Whereas, compared with hydrogels, zwitterionic

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nanofiber membrane possess higher water absorption rate and gas permeability due to

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its high porosity structures and high surface-area-to-volume ratio.4, 18-19 However, few



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reports involve preparing zwitterionic nanofiber membranes for wound dressing

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applications considering the challenges of severely swelling and water stability of the

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nanofiber membrane. Although UV-crosslinking method has recently been reported,

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the problems of complicated material preparation and poor crosslinking efficiency

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severely limit the practical applications.4,

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method to prepare water stabled zwitterionic nanofiber membrane and optimize their

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anti-biofouling property is of great significance in clinical surgery.

20-21

Thus, search a simple and feasible

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Removing superfluous wound exudate and maintaining relatively moist healing

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environment are the pervasive requirements during wound healing.5 However,

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electrospun wound dressing prepared from traditional hydrophobic substrates generally

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exhibits a lower absorption capacity of the wound fluid, resulting in excessive hydration

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of the wound.2, 22-24 Although hydrophilic modification or directly using hydrophilic

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substrate could improve exudate adsorption of the wound dressing, but it

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simultaneously accelerates the evaporation of water from the wound surface to the

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outside, which brings about fast wound drying and hindering tissue repairing.1

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Thus, in order to optimize the wound healing environment as far as possible, there is an

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urgent requirement to search a preparation method that endows the fabricated wound

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dressing with both high wound fluid absorption capacity and moisturizing property

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synchronously.

4 9 25

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Chronic wound infection is another serious problem during healing process.

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Continuous bacterial infection wound hamper damaged tissues to fast healing seriously,

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worse still, lead to scarring and pain even disability or death.24 In order to effectively


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diminish infection and facilitate the wound healing as soon as possible, much effort

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have been made to develop drug-loading membranes by blending26-27 or coating

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method28. Nevertheless, burst release during initial period usually make it difficult to

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meet the demand of long-term antibacterial requirement.29 Although special

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electrospinning methods such as coaxial30 and emulsion electrospinning31 could

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overcome the defect of the burst release, instability as well as poor biocompatibility

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caused by emulsifiers also limit the wide range of medical applications for such

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membrane.32-33 Halloysite clay nanotubes as a kind of biocompatible natural nano-

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materials could encapsulate molecules including drugs, proteins, and DNA.34-35

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Particularly, the drug-loaded halloysite composite would successfully suppress the

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initial drug burst release and exhibit excellent long-term release ability.24,

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Therefore, it is a feasible and effective way to endow wound dressings with long-lasting

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and controllable drug release profile by embedding drug-loaded halloysite nanotubes

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into the nanofibers.

32-33, 36-37

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In this study, we report an innovative gradational co-electrospinning method to

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fabricate drug-loaded zwitterionic electrospun composite membrane for the purpose of

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realizing excellent anti-biofouling performance, moisture retention capability and long-

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acting antibacterial abilities during wound healing process. Polycaprolactone served as

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hydrophobic component within the membrane could effectively reduce moisture

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evaporation from the wound surface and prevent the invasion of external

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microorganisms simultaneously. Hydrophilic amino-modified zwitterionic poly

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(sulfobetaine methacrylate) P(SBMA-r-AMA) nanofibers endow the composite



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membrane with excellent anti-biofouling property and exudate absorption capability as

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well as outstanding water stability after crosslinking with genipin. Additionally, drug-

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loaded halloysite as sustained release nanocarrier is doped into above mentioned

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P(SBMA-r-AMA) nanofibers and gradationally dispersed within the composite

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membrane to realize long-lasting antibacterial functions. These gradient structures not

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only maximize the hydrophilicity requirements in different directions but also further

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prolong the path of drug diffusion to satisfy the anti-microbial requirements for chronic

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wound infection and skin regeneration.

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MATERIALS AND METHODS Synthesis and Characterization of Zwitterionic P(SBMA-r-AMA) Copolymer.

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Sulfobetaine methacrylate (SBMA) monomer was synthesized according to our

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previous report and the final yield was 90%.38 Zwitterionic P(SBMA-r-AMA) was

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synthesized by free radical copolymerization with SBMA and 2-aminoethyl

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methacrylate (AMA) under nitrogen protection.39 The detail procedures were clearly

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presented in Supporting Information.

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Surface Modification and Drug Loading Process of Halloysite Nanotubes.

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Surface silane coupling of halloysite nanotubes (HNTs) could maintain stable and

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homogeneously dispersion in electrospinning solution without agglomeration during

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the electrospinning process.36 The details were presented in Supporting Information.

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For drug loading process of HNTs, multiple vacuum drug-loading were carried out to

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maximize loading content of tetracycline hydrochloride (TCH) inside the lumens of

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HNTs and the mass ratio between HNTs and TCH was 1:5 to achieve the best drug

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loading effect.

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Gradational Co-electrospinning of Nanotube Doped Membrane Asymmetric

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gradational nanofiber membranes were fabricated via co-electrospinning process. Poly

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(ε-caprolactone) (PCL) solution with a concentration of 8wt% was prepared by

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dissolving 2.4g PCL in 27.6g trifluoroethanol (TFE) at room temperature and used as

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second electrospun component in each samples. Zwitterionic polymer solution with a

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concentration of 8wt% was obtained by dissolving 2.4g P(SBMA-r-AMA) in 27.6g

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trifluoroethanol under same condition. Modified halloysite with percentage of 0, 1, 5,



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10, 20wt% (according to amount of zwitterionic polymer) were added into above

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mentioned zwitterionic polymer solutions respectively and kept stirring for 2 days to

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form uniform dispersed electrospinning solutions. Additionally, genipin was added into

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the P(SBMA-r-AMA) solution 12h before electrospinning to form a flowable polymer

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mixture. The composition of zwitterionic P(SBMA-r-AMA)/HNTs electrospinning

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solutions were listed in Table 1.

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Table 1 Zwitterionic P(SBMA-r-AMA) electrospinning solutions Sample

P(SBMA-r-AMA)

Solvent(TFE)

HNT

Genipin

Electrospinning

(g)

(g)

s

(mg)

type

(mg) HNTs-0

2.4

27.6

0

240

Gradational

HNTs-1

2.4

27.6

24

120

Gradational

HNTs-5

2.4

27.6

120

48

Gradational

HNTs-10

2.4

27.6

240

24

Gradational

HNTs-20

2.4

27.6

480

12

Gradational

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The scheme of gradational co-electrospinning was exhibited in Scheme 1C.

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Firstly, four syringes were loaded with 5ml of the above mentioned PCL solution

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respectively and electrospinning at speed of 0.3mm/min for initial 1h synchronously to

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form PCL layer. Then, one of the initial PCL solution was replaced with 5ml P(SBMA-

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AMA)/HNTs mixture (The other three syringes still loaded with PCL solutions) and

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co-electrospinning for another 1h under same injection speed. Subsequently, the


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residual PCL electrospinning solutions were gradually replaced with P(SBMA-

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AMA)/HNTs mixture and the co-electrospinning was carried out under same

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electrospinning time interval and injection speed respectively, which gradually formed

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the gradational intermediate layer. Finally, all the initial four PCL solutions were

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replaced with P(SBMA-AMA)/HNTs mixture and electrospinning for last 1h to

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establish the outer layer of P(SBMA-AMA)/HNTs. Optimized high voltage (18-20KV)

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was applied between the needle and the roller type collector and the distance between

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the needle and collector was 15cm.

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In Vitro Biocompatibility evaluation of HNTs and HNTs doped membranes

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Cytotoxicity of the modified HNTs and HNTs doped membranes were characterized

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by CCK8 assay with L929 cells.40 The detailed descriptions were presented in

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Supporting Information.

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Anti-biofouling Measurements of the Membranes. Static protein adsorption of

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the electrospun membranes was measured by Protein Assay Reagent Kit (PIERCE) and

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ELIASA (Thermo-Fisher, Multiskan GO) at 562nm.41 Surface protein fluorescence

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analysis of the membranes was carried out by fluorescein isothiocyanate labeled BSA

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(FITC-BSA, 5mg/L).12 Bacterial adhension on the membranes was evaluated according

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to our earlier study by BacLight viability Kit.42 The samples (1 cm × 1 cm) were

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incubated in 2ml of 106 cfu ml-1 bacterial suspension respectively at 37℃ for 12h and

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imaged with fluorescence microscopy after treating with fluorochrome. The attachment

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and proliferation of L929 cells on the different halloysite doped membranes were

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assessed by CCK-8 assay and measured with a microplate reader (Model 550, Bio-Rad)



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at 450 nm.

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In Vitro Anti-bacterial and Zone of Inhibition. In order to investigate the long-

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term bacteriostatic ability and bactericidal efficiency, different drug-loaded membranes

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were evaluated by inhibition zone and the optical degree of co-cultured solution.43

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Moreover, colony counting method was also used to intuitively characterize the

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bactericidal effect of the composite membranes according to a previous report.44 The

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details refer to Supporting Information, and Escherichia coli (E.coli, gram negative) as

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well as Staphylococcus aureus (S.aureus, gram positive) bacteria were served as the

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model bacteria during in vitro anti-bacterial characterization.

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Drug Release Measurement of composite membranes. Drug-release

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measurement of different samples were carried out to get the drug release profile.

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Gradational membranes with different drug-loading modes were cut into 1 cm × 1 cm,

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accurate weighted, and incubated in 2ml of deionized water at 37℃ with continuous

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shaking. At predetermined time intervals, 2ml of soaking solution was taken out and

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the samples were then soaked with another 2ml of fresh deionized water under same

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conditions. The drug-release amounts at different time intervals were detected with UV-

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1750 spectrophotometer (Shimadzu Co., Ltd., Japan) at 400nm and the release amount

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was calculated by standard curve as mentioned above.

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In Vivo Anti-bacterial Assessment. Infection model was set up according to

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previous study.22 Briefly, Male SD rats (8weeks, 300g) were anesthetized via injection

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of 0.3ml pentobarbital (3%) intraperitoneally and a round wound with 7mm in diameter

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were made. All the SD rats were randomly divided into four groups with which different


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dressings were covered after inoculating S.aureus suspension (10μL, 107 CFU in

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culture medium) respectively. After 3 days observation, the wound were rinsed with

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10μL normal saline repeatedly and each leacheate was cultured on agar plates to

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investigate the antibacterial ability.

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In Vivo wound healing Examination. In Vivo wound healing was invested via

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SD rats. After anesthetization, 7mm round skin injury were made from the back of the

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rats. All the rat samples were divided into four groups. Briefly, group A was control

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sample with nothing dressed after injured, group B received 3M Tegaderm film, group

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C was dressed with M-TCH-G nanofiber membranes, group D was treated with M-

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HNTs/TCH-TCH-G nanofiber mats. After 3,7,10 and 14 days’ supervision, the wound

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area was imaged by digital camera and the wound size was measured based on eq1

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Wound size (%) = [W(3,7,10,14) / W0]×100%

(1)

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Where W(3,7,10,14) represent wound area (mm2) after 3,7,10 and 14 days respectively and

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W0 represent the initial wound area.

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Animal welfare: All procedures involving the use of animals in this study were

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prospectively reviewed and approved by the Institutional Animal Care and Use

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Committee. This study was conducted in accordance with the National Institutes of

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Health Guide for the care and use of laboratory animals (NIH Publications No. 8023,

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revised 1978). This experiment conformed to the legal requirement in China and was

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approved by the ethical committee (No. K2016027) of West China Hospital, Sichuan

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University. During the experimental period, the animals had free access to water and

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food.



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RESULTS AND DISCUSSION

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Fabrication and characterization of Gradational Composite Membranes.

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P(SBMA-r-AMA) was prepared by random copolymerization in aqueous solution

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under nitrogen protection (Scheme 1A). The successful synthesis of P(SBMA-r-AMA)

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was confirmed by 1H-NMR and 13C-NMR (Figure S1).39 The introduction of amino

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group provided the crosslinking point which could further react with genipin to cope

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with stability requirement of membranes under in vivo complex environment. The

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surfaces of halloysite nanotubes were modified with aminopropyl triethoxysilane

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(APTES) to enhance the distribution ability in electrospinning solutions (Scheme 1B).

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The successful surface amino modification was verified according to FT-IR, XPS, and

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TGA analyze (Figure S2). After surface treatment, the dispersion uniformity of

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halloysite in electrospinning solution was improved, and no markedly agglomeration

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was observed during the electrospinning process at the same time.

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The composite nanofiber membranes were fabricated by co-electrospinning

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method (Scheme 1C). The received membrane possessed two surfaces with different

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hydrophilicity and a continuous gradational intermediate transition layer, in which two

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different nanofibers interdigitated with each other to improve the uniformity and

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stability of the composite membranes. The received gradient composite membranes

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with different HNTs content of 0, 1, 5, 10, 20 wt% were labeled as M-HNTs-0, M-

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HNTs-1, M-HNTs-5, M-HNTs-10, M-HNTs-20, respectively.



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Scheme 1. (A) General procedure for the synthesis of random polymer P(SBMA-r-AMA); (B) Schematic

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illustration of the surface modification process of halloysite nanotubes; (C) Schematic overview of the

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gradational co-electrospinning procedure.

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The prepared membranes exhibited uniform diameter distributions and smooth

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nanofiber morphology without beads formed (Figure 1A). The mean diameters of the

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different halloysite doped P(SBMA-r-AMA) nanofibers were range from 0.93 to

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1.15μm, compared with 0.27μm of PCL nanofibers. Halloysite nanotubes didn’t

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obviously change the morphology of the nanofibers even reach at content of 20% wt.

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Meanwhile, according to FT-IR spectral (Figure 1B), characteristic adsorption peaks

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of P(SBMA-r-AMA) at 1038 cm-1 and 1540cm-1 corresponding to -SO3- and amideⅡ

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were appeared in all halloysite doped membranes, which indicates that the

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incorporation of nanotubes had no obvious influence on the chemical composition of

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P(SBMA-r-AMA). TGA measurements of different membranes (Figure 1C) presented

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two thermal decomposed temperatures at 310 ℃ and 380 ℃ respectively, which


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confirmed the successful incorporating of both composition in the composite

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membranes. Moreover, halloysite doped membranes showed higher onset

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decomposition temperature compared with control sample (M-HNTs-0), indicating that

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halloysite could improve thermal stability of the composite membranes at certain extent.

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What's more, residual weight of the membranes were increased with the incorporation

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rate of the nanotubes, which also illustrated the successful incorporation of halloysite

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into the composite membranes.

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Figure1. Physical and chemical characterization of different halloysite doped membranes: (A) SEM and

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diameter distribution of the membranes; (B) FT-IR measurement for different halloysite doped

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membranes; (C) thermo gravimetric profiles of the membranes; (D) tensile strength of the composite

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nanofiber membranes at direction of vertical and parallel to the collector.

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Mechanical strength of the nanofiber membranes was depended on mechanical



1

property of individual fiber and fiber arrangement.45 The mechanical strength of wet

2

state at different directions (parallel or vertical to the collect roller) was studied (Figure

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1D). On the parallel direction, the tensile strength of the membranes significantly

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increased with the addition of halloysite. According to previous report, inorganic

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halloysite could parallel to the fiber direction during nanofiber formation, which

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effectively reinforced the paralleled mechanical strength for the membranes.36 In

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contrast, on the vertical direction, the tensile strength gradually decreased with the

8

addition of halloysite, particularly, the tensile strength significantly dropped when the

9

amount of halloysite reached at 20% wt. At such direction, insufficient of organic-

10

inorganic interfacial binding force would become a major point, which explained the

11

reason why mechanical strength slowed down dramatically with the halloysite

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embedded on the vertical direction. Furthermore, tension-Deformation curve of the

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composite membranes reflected this trend as well. (Figure S3).

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Pre-crosslinking Process and Water Stability of the Composite Membranes.

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Super hydrophilicity is not only the advantage of zwitterionic material but also the

16

biggest limitation for its application. Nanofiber membranes applied in the field of

17

wound dressings must possess good wet stability to maintain the morphology of

18

nanofiber and pore structure during long-term healing process. Genipin as natural

19

biomaterial crosslinking regent (reacted with amino) reveals time-dependent and

20

temperature-dependent crosslinking property.46 Therefore, crosslinking degree, which

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would further affect the spinnability of the electrospinning solutions, could be

22

controlled by crosslinking reaction time. The mechanism of pre-crosslinking


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electrospinning method was shown in Figure 2. Halloysite modified with amino could

2

act as crosslinking points between P(SBMA-r-AMA) molecules. After 12h pre-

3

crosslinking with genipin, crosslinking structures were established between P(SBMA-

4

AMA) molecules and halloysite. Moreover, it was worth noting that the electrospinning

5

solutions were still flowable after 12h pre-crosslinking due to low intermolecular

6

crosslinking density of P(SBMA-AMA) (Figure 2B). Under such crosslinking degree,

7

electrospinning process was not affected by crosslinking structures. However, after

8

sufficient reaction for 1 week, the molecules chains were completely restricted by

9

genipin and such gel network cannot flow nor electrospinning (Figure 2C). Therefore,

10

fabricating the pre-crosslinked electrospinning solution into nanofibers membranes

11

during electrospinning range and further thorough crosslinking were the main ideals in

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this method.

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Figure 2. Pre-crosslinking electrospinning process: (A) P(SBMA-r-AMA) electrospinning solution with

15

20% HNTs and 0.5% genipin 12h before electrospinning; (B). electrospinning solution after 12h

16

crosslinked; (C) electrospinning solution after sufficient crosslinked for 7 days.



1

The cyclic water swelling test and SEM were carried out to verify the stability of

2

the received membranes. As Figure 3A presented, surface of P(SBMA-r-AMA)

3

exhibited dark blue after thoroughly crosslinking with genipin. There was no obvious

4

shrinkage or decomposition when completely soaked in water for 7 days. The soaking-

5

drying recycle was repeated for four times (12h for each cycle in water), and no distinct

6

quality change was detected after each cycle (Figure 3B). Moreover, the composite

7

membranes possessed 340% of water adsorption, which illustrating the excellent

8

wound exudate adsorption ability of such composite membranes. Additionally,

9

compared with dry state membranes (Figure3C), nanofiber had a certain degree

10

collapse due to strong solvation after completely exposed to water environment for 24h.

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However, there was no significant change in porosity after water treatment, which met

12

the requirement for gas exchange in the wound healing process. These results verified

13

the water stability of such zwitterionic composite membranes.

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Figure 3. Water stability of zwitterionic composite membranes: (A) membranes with 20% HNTs and

3

immersion in water for 7 days; (B) result of cyclic water swelling test; SEM of P(SBMA-r-AMA) surface

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before (C) and after (D) soaking in water for 24h.

5

Gradient wetting property and moisture retention profile of the membranes.

6

Fast moisture evaporation was one disadvantage for traditional wound dressings, which

7

made wound surface desiccate and worsen the healing conditions. Moist healing

8

environment was conducive to the regeneration of the granulation tissue and epithelial

9

regrowth.4 In this study, gradational distribution of the hydrophobic PCL nanofiber was

10

acted as “water-stopper” within the composite membranes, which could not only

11

prevent external pollutant such as water contamination and microorganism but also

12

block the moisture from injured tissue and maintained a favorable moist wound healing

13

conditions. Additionally, gradational composition of super-hydrophilicity P(SBMA-r-

14

AMA) nanofiber could fully absorb the wound exudate, importantly, drain the wound

15

effusion to the intermediate layer to prevent excessive wound hydration (Figure 4A).



1

The apparent hydrophilicity difference between the two sides of the membrane was

2

showed in Figure 4C. Moreover, compared with the side of PCL nanofiber, P(SBMA-r-

3

AMA) side exhibited lower initial WCA (below 60 degrees) and declined rapidly within

4

6s, which also verified the excellent hydrophilic and water adsorption ability of the

5

composite membranes (Figure 4B). Moreover, bilateral wetting property of the

6

obtained composite membranes were also evaluated, and normal saline solution with

7

1mg/ml FITC-BSA was used to simulate wound tissue exudate (WTE) during the

8

wetting experiment. As showed in Figure 4D, when the P(SBMA-r-AMA) side was in

9

contacted with the WTE, the droplet spread rapidly on the zwitterionic side within the

10

first three seconds and gradually infiltrated the surface. However, it was obvious that

11

only little proportion of surface infiltration on the other side (PCL side) was detected

12

even after 60 minutes of infiltration. Meanwhile, the difference in fluorescence intensity

13

on both surfaces also visually reflected this phenomenon, which indicated that most of

14

the exudate was absorbed by the P(SBMA-r-AMA) side and then blocked by the

15

hydrophobic PCL nanofiber layer. On the other hand, when WTE was infiltrated from

16

the PCL side firstly (Figure 4E), the droplet couldn’t spread on the surface and,

17

importantly, the WTE was unable to infiltrate to the other side (zwitterionic surface).

18

Bilateral wetting evaluation indicated that the composite membranes with such

19

asymmetry structure possessed triple moisture management functions of exudate

20

absorption and moisturization capability as well as anti-infiltration property, which

21

could provide a relatively isolated and moist healing environment for wounded tissue.


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1 2

Figure 4. (A) Scheme of gradient wetting property; (B) WCA variation of different HNTs doped

3

membranes at different time interval; (C) WCA images of HNTs embeded membranes at initial time;

4

(D) bilateral wetting evaluation of P(SBMA-r-AMA) side and (E) PCL side.

5

Anti-Biofouling Property of the Membranes. Before evaluating the anti-fouling

6

properties and other biological performance of the composite membranes, it was critical

7

to ensure that the fabricated membranes possesses good biocompatibility especially cell

8

compatibility.24 Therefore, cytotoxicity of the halloysite nanotubes and halloysite

9

doped composite membranes were evaluated via extraction method with L929 as model

10

cell (Figure S5 and S6). The results indicate that the halloysite and halloysite-loaded

11

nanofiber membranes exhibited good cell compatibility with no cytotoxicity.

12

Protein adsorption on wound dressings surface could lead to many bio-reaction

13

and cause activation of coagulation pathways, which often arouse wound adhesion in



Page 22 of 54

1

clinical practice.41 Bovine serum albumin (BSA), a kind of protein that apt to adsorb

2

on various surface, was used as fouling reagent in this study.47-48 In order to evaluate

3

the protein resistance ability of the wound-contacted side of P(SBMA-r-AMA)

4

nanofiber membranes, static protein adsorption and surface fluorescently evaluation of

5

FITC-BSA were investigated. For the control sample of PCL nanofiber membrane,

6

static BSA adsorption amount increased approximately 10 times (about 9.38 μg/cm2)

7

compared with all the P(SBMA-r-AMA) samples (1.44, 0.90, 0.96, 0.95, 0.99 μg/cm2

8

for M-HNTs-0 to M-HNTs-20 respectively) according to the result of static BSA

9

adsorption (Figure 5A). Each O.D. value of desorption solution at 562 nm was also

10

echoed for such trend as Figure 5B demonstrated. Protein contamination on material

11

surfaces was mainly caused by hydrophobic interactions between substrate and the

12

protein molecule.49 The hydrophobic property of PCL nanofiber could easily lead to

13

adhesion of large amount of protein onto the membrane. As evidently shown in Figure

14

5C, majority of FITC-BSA absorbed on the surface of PCL nanofibers and obvious

15

fluorescence fiber morphology were detected. In contrast, the fluorescence intensity

16

obviously decreased for each sample of HNTs doped P(SBMA-r-AMA) nanofiber

17

membranes. As previous discussed, zwitterionic PSBMA have strong hydration due to

18

the interaction between charged groups in their pendant and water molecules.4,

19

Therefore, protein adsorption result confirmed the supper hydrophilic property of the

20

zwitterionic composite nanofiber membranes and the excellent ability to resist initial

21

protein adhesion.


20

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1 2

Figure 5. (A) Static BSA adsorption amounts; (B) O.D. at 562nm of each SDS solutions; (C) surface

3

fluorescently images of different samples after exposure to FITC-BSA.

4

Microorganisms adhesion onto the surface of the material is a precondition for its

5

proliferation, which finally caused biofilm formation and infection in practical medical

6

application including wound dressings.50 Thus, it is particularly important to reduce or

7

prevent the initial bacterial adhesion onto the surface of wound dressings. In this study,

8

E.coli and S.aureus were selected as model bacteria to investigated the bacterial

9

resistance ability of the composite membranes (Figure S4).The result indicates that the

10

surface of different halloysite doped P(SBMA-r-AMA) nanofiber membranes exhibited

11

lower bacterial adhesion ratio than that of PCL nanofiber surface after 12 hours co-

12

culture for both E.coli and S.aureus. What’s more, electrospun membranes possess



1

rough and porous surface structures which were easier for bacteria or other microbial

2

to adhere or trap within the nanofibers.4 Even so, the prepared zwitterionic composite

3

membranes still revealed outstanding anti-bacterial adhesion property for both E.coli

4

and S.aureus. Wound dressing with good anti-bacterial adsorption property has lower

5

replacement frequency, which further reduces the frequency of wound exposure as well

6

as the risk of outside bacterial invasion.

7

Cell adhesion is another challenge for wound dressings considering the

8

disturbance for new growth skin tissues during dressing removal process. In this study,

9

cell adhesion was characterized using CCK-8 cell counting assay with L929 fibroblast.

10

Compared with TCP and PCL control membrane, different halloysite doped gradational

11

composite membranes presented lower O.D. value (0.4 to 0.6) and basically remained

12

unchanged after 3 days incubation (Figure 6A). Furthermore, even after 7 days’

13

incubation, the cell adhesion still remained a low rate for the zwitterionic composite

14

membranes (Figure 6B). Moreover, fluorescence microscopy images of L929 cell

15

adhered on the membranes of PCL and P(SBMA-r-AMA) after 7 days incubation were

16

presented in Figure 6C. The zwitterionic composite membranes exhibited extremely

17

low surface cell adhesion even after 7 days incubation. However, for PCL membrane,

18

distinct cell adhesion and proliferation were detected. Therefore, combine with the

19

biocompatibility evaluation, the result of cell adhesion assessment fully confirmed the

20

anti-cell adhesion property of the zwitterionic P(SBMA-r-AMA) nanofiber membranes.

21

Therefore, excellent anti-fouling performance for protein, bacteria as well as L929 cell

22

of such super-hydrophilic zwitterionic composite nanofiber membranes would provide


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1

potential application value for non-adherent wound dressing applications.

2 3

Figure 6. Cell adhesion evaluation of gradational composite membranes: (A) O.D. value at 450nm after

4

culturing for 1, 3, 5, and 7 days respectively; (B) O.D. value after 7days incubation; (C) Fluorescence

5

microscopy images of L929 cell which adhered on PCL and P(SBMA-r-AMA) membranes after 7 days

6

incubation.

7

Long-lasting Antibacterial ability. Wound bacterial infection would cause

8

chronic inflammation which could further engender severe scars or skin tissue defect

9

for patients. Therefore, dressing with strong bactericidal effect and long-term inhibition

10

of bacterial growth ability has far-reaching application value in clinical. Considering

11

the metabolic type of clostridium tetani which commonly proliferated around wound,

12

facultative anaerobic type of E.coli was chosen as model bacterium to investigate in

13

vitro bacterial inhibition capacity of the drug-loaded membranes. Gradational



1

composite nanofiber membranes with different drug loading modes were fabricated by

2

respectively incorporating: (1) HNTs, (2) TCH, (3) drug loaded halloysite (HNTs/TCH),

3

(4) TCH and HNTs/TCH into P(SBMA-r-AMA) solutions. PCL served as hydrophobic

4

second gradient component within all the samples and the whole drug loading content

5

in all membranes was set as 20 wt% related to mass of P(SBMA-r-AMA). Additionally,

6

in order to study the effect of the gradational structure on drug release profile, we

7

prepared gradational composite membranes and general planar composite membranes

8

for each sample in the meantime. The obtained samples were labeled as M-HNTs/TCH,

9

M-HNTs/TCH-G; M-TCH, M-TCH-G; M-HNTs/TCH-TCH, M-HNTs/TCH-TCH-G

10

respectively (“G” means composite membrane was fabricated by gradational co-

11

electrospinning method).

12

The unidirectional drug-control mechanism of gradient structure was presented in

13

Figure 7A. Gradational nanofiber structure could increase the path and resistance

14

power of drug diffusion in gradient direction compared with planar structure. As shown

15

in Figure 7B and C, bacterial inhibition zones were observed in all drug-loaded

16

membranes except control sample after first 24 hours incubation. Moreover, the M-

17

TCH sample exhibited largest initial inhibition zone than other membranes because of

18

initial burst drug diffusion. However, with the TCH fast releasing from the nanofiber,

19

inhibition zone of M-TCH gradually declined within 5 days. After 7 day’s incubation,

20

bacteria proliferated into the original inhibition zone, which reflected the antibacterial

21

inefficacy of the M-TCH. Nevertheless, for M-HNTs/TCH composite membranes, the

22

initial drug released rate was effectively slowed down but the inhibition zone could


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1

retain for at least 5 days due to the controllable release effect of the halloysite

2

nanocarrier. However, after 7 days’ incubation, same antibacterial inefficacy was still

3

detected for M-HNTs/TCH membranes. Compared with M-TCH and M-HNTs/TCH,

4

the inhibition zone of gradational drug-loaded membranes (M-HNTs/TCH-G) was

5

clearly visible and the diameter of effective bacteriostatic zone almost kept unchanged

6

after 7 days continuously incubation. These results indicate that gradational drug-

7

loading structure of the composite membrane was more conducive to sustained drug

8

release, which would fully meet the long-acting antibacterial requirement during skin

9

regeneration period.



1 2

Figure 7. In vitro antibacterial evaluation of different drug-loaded composite membranes: (A)

3

Unidirectional drug-control release mechanism of gradient structure membranes; (B) Inhibition zone

4

images after 1, 3, 7 days’ incubation; (C) Diameter changes of the inhibition zone for different

5

membranes; (D) Initial antibacterial ability of the composite membranes.

6

Additionally, initial stage of epidermal injury is a high-risk period of wound


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1

bacterial infection.24, 36 It is of great significance for wound dressing to increase the

2

primal drug release amount appropriately while ensuring the long-term antibacterial

3

effect. Therefore, the initial anti-bacterial property was measured by bacterial co-

4

culture methods, then S.aureus and E.coil were both evaluated considering the high risk

5

of infection during incipient period. As shown in Figure 7D, 100% of bacterial

6

clearance was achieved within the first 12h for M-HNTs/TCH-TCH-G and M-TCH-G,

7

compared with M-HNTs/TCH-G (78.3% for E.coil and 87.1% for S.aureus after 12h

8

incubation). Additionally, the results of colony count and bacterial O.D. value after 12h

9

incubation also confirmed the high initial antibacterial ability of M-HNTs/TCH-TCH-

10

G membranes (Figure S7). In summary, combined with the results of long-lasting

11

bacteriostatic evaluation and initial antibacterial assessment, gradational composite

12

membranes incorporated with both TCH and HNTs/TCH could exhibit both long-acting

13

and high initial antibacterial ability, which would more adequately cope with the

14

antibacterial requirement during chronic wound recovery process.

15

Release profiles of halloysite nanotubes and drug-loaded composite

16

membranes. According to previous study, cationic tetracycline hydrochloride (TCH)

17

was not only encapsulated into the lumens of nanotubes but also adsorbed on external

18

surface of HNTs by electrostatic interactions simultaneously.32 A filamentous structure

19

which perpendicular to the axial direction of halloysite was clearly detected after

20

loading with TCH and the boundary of the HNTs/TCH became blurred compared with

21

pristine HNTs (Figure 8A). Drug release investigation of HNTs/TCH and different

22

drug-loaded membranes were showed in Figure 8B. Almost 90% of loaded TCH was



1

burst released within initial 8h in water environment. For the M-TCH-G samples, about

2

95% of loaded TCH was released from membranes within 4 days followed by a low-

3

rate releasing for subsequent 2days. Although the hydrophilic nanofiber membrane

4

exhibited a certain degree of sustained drug release property compared to HNTs/TCH,

5

the drug release rate still dissatisfied the long-term antibacterial requirement in the

6

wound healing process. However, M-HNTs/TCH-G presented a sustained drug release

7

process for at least 20 days and the initial burst release rate was significantly shut down

8

compared with HNTs/TCH and M-TCH-G. According to previous study, such long-

9

lasting drug release process attributed to a two stages drug movement mechanism.36

10

Furthermore, nanofiber matrix partially clogs the two ends of the inner HNTs, which

11

also increased the resistance to drug molecular migration. According to Figure 8B, the

12

M-HNTs/TCH-TCH-G sample presented same total drug release time (about 20 days)

13

as M-HNTs/TCH-G. However, the initial drug release rate was dramatically accelerated

14

due to part of directly loaded TCH within the composite membrane. Therefore, this

15

drug-loading model, which can maintains a relatively high initial release as well as

16

long-lasting release profile, is the most reasonable drug-loading method for chronic

17

wound regeneration.


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Biomacromolecules

1 2

Figure 8. Cumulative TCH release profiles: (A) TEM of HNTs before and after drug loading; (B) TCH

3

released ratio of HNTs/TCH and different drug-loaded gradient membranes and released mass for

4

gradational membranes with different drug loading methods.

5

In Vivo antibacterial evaluation. In order to evaluate the antibacterial activity of

6

the drug-loaded nanofiber membrane in vivo, S.aureus-infected wound model was

7

established (Figure 9A). After 3 days observation, the wound covered with various

8

dressings (PBS, 3M Tegaderm film, M-TCH-G, M-HNTs/TCH-G) were imaged

9

(Figure 9B), and the wound exudate was cultured respectively after diluting with saline.

10

After S.aureus infection, PBS and 3M Tegaderm film covered wound samples

11

presented obvious tissue redness, and yellow inflammatory exudate was detected on the

12

wound surface, indicating that 3M Tegaderm film (a commercial wound dressing)

13

exhibits inefficient antibacterial ability. Meanwhile, the wound treated with PBS was

14

obviously dry due to long-term wound exposure which seriously interfered the normal

15

healing process. However, for M-TCH-G and M-HNTs/TCH-G covered samples, no

16

obvious wound infection was observed and wound surface still maintained moderate

17

moist. Moreover, according to bacterial culture evaluation (Figure 9C), wound exudate



1

extracted from M-TCH-G and M-HNTs/TCH-G samples presented significant

2

difference in O.D. values (0.057 and 0.073 for M-TCH-G and M-HNTs/TCH-G)

3

compared with PBS and 3M Tegaderm film treated samples (0.492 for PBS and 0.276

4

for 3M Tegaderm film) after 12h bacterial incubation. Additionally, the results of

5

bacterial plate counts (Figure 9D) also confirm that the wound treated with PBS and

6

3M Tegaderm film present significant bacterial proliferation and low bactericidal

7

performance (12.23×107 and 10.35×107 CFU respectively), while samples covered

8

with M-THC-G and M-HNTs/TCH-G exhibit strong antibacterial ability (0.06 and 0.18

9

×107 CFU) which attributed to TCH release from the membranes.


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1 2

Figure 9. In vivo antibacterial evaluation: (A) S.aureua infected model; (B) images of S.aureus-infected

3

rat wound dressed with PBS, 3M Tegaderm film, M-TCH and M-HNTs/TCH after 3 days infections; (C)

4

O.D. value and colony count derived from different samples; (D) bacterial plate counts of different

5

samples.

6

In Vivo Wound Healing investigation. To further investigate wound healing

7

ability of such composite membranes in complexed in vivo environments, we

8

established an epidermal tissue healing model to simulate the wound healing process.

9

M-TCH-G and M-HNTs/TCH-G nanofiber membranes were evaluated respectively

10

(3M Tegaderm film as well as blank sample were employed as control). After 3, 7, 10,



1

and 14 days observations, each wound sample was imaged and calculated (Figure 10A).

2

The initial wound defect exhibited a certain degree of convergence for M-HNTs/TCH-

3

G sample on day 5. However, obvious wound size reduction was appeared on the day

4

9 for the untreated sample and on day 7 for 3M Tegaderm film. Meanwhile, untreated

5

wound surface presented a desiccative wound appearance due to fast moisture

6

evaporation. In contrast, M-TCH-G and M-HNTs/TCH-G samples exhibited a moist

7

but not overhydrate healing environment. Therefore, significant difference in wound

8

size between M-HNTs/TCH-G and control sample was detected after 7 days

9

observation due to distinction of healing environment and antibacterial activity. After

10

10 days treatment, newborn epidermal tissue had basically covered the original wound

11

for M-HNTs/TCH-G sample compared with other dressings. Wound samples with

12

which M-HNTs/TCH-G membranes covered had completely healed on day 14 and the

13

original skin defects had been totally covered with smooth palingenetic epidermal tissue.

14

However, wound dressed with 3M Tegaderm film and control samples still presented

15

an obvious unhealed skin defect after 14 days healing evaluation. Furthermore, the

16

wound size variation of different samples (Figure 10B) also reflects the best wound

17

healing ability for such composite membranes. Meanwhile, complete wound healing

18

time (Figure 10C) also confirm that M-HNT/TCH-G sample possesses the shortest

19

wound healing time compared with other dressings. These in vivo wound healing

20

results indicate that the prepared M-HNTs/TCH-G membrane could maintain a good

21

healing environment for the wound and effectively promote the wound recovery.


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1 2

Figure 10. In vivo wound healing investigation: (A) Images of rat skin wound dressed with different

3

materials after 0, 3, 7, 10, and 14 days observation; (B) Wound size for different samples at different

4

time intervals; (C) Complete wound healing time (CWHT) of different samples after injured.

5



1

CONCLUSIONS

2

Novel multi-functional zwitterionic gradational membranes were successfully

3

fabricated using a gradational co-electrospinning method for conquering wound

4

infection and promoting wound healing. By capitalizing on zwitterionic P(SBMA-r-

5

AMA), the obtained zwitterionic composite membranes exhibited the desired

6

hydrophilicity as well as excellent anti-biofouling property for protein, bacteria and

7

L929 cell. Moreover, the pre-crosslinking and gradational co-electrospinning method

8

endowed the composite membranes with wet stability and moisture retention ability

9

which could effectively retain the wound moisture and adsorb wound exudate.

10

Additionally, gradational composite nanofiber structures could prolong unidirectional

11

drug release time, which presented effective and long-acting antibacterial activity

12

against S.aureus and E.coil both in vivo and in vitro. Furthermore, in vivo wound

13

healing evaluation also indicated that the prepared zwitterionic gradational membranes

14

could significantly promote wound healing than commercial 3M Tegaderm film. Such

15

multi-functional wound dressing owned significant clinical application potential in the

16

field of chronic wound healing. More importantly, this approach of fabricating the

17

gradational composite electrospun membranes could be extended for different

18

electrospinning application fields.

19


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Biomacromolecules

1

ASSOCIATED CONTENT

2

Supporting Information

3

Materials. The detailed description of experiment and characterization methods.

4

1H-NMR

and 13C-NMR of SBMA monomer and P(SBMA-r-AMA). Characterization

5

of halloysite nanotube before and after modification. Tension-Deformation graph of

6

HNTs doped membranes. Fluorescence microscopy images of S.aureus and E.coil

7

which attatched on the different side of composite membranes. In vitro cell cytotoxicity

8

of HNTs and HNTs doped membranes. Bacterial colony count and bacterial O.D. value

9

after initial 12h incubation.

10

AUTHOR INFORMATION

11

Corresponding Author

12

**E-mail: [email protected], [email protected] (C.S. Zhao).

13

*E-mail: [email protected] (X.L. Liu).

14

ORCID

15

Changsheng Zhao: 0000-0002-4619-3499

16 17

Author Contributions

18

† These

19

Notes

20

The authors declare no competing financial interst.

authors contributed equally to this work.



1

ACKNOWLEDGMENTS

2

We acknowledge that this work was financially sponsored by the National Natural

3

Science Foundation of China (No. 51433007, 51673125, 51773127, 51803131,

4

51803134 and 51873115), the State Key Research Development Programme of China

5

(Grant Nos. 2016YFC1103000 and 2018YFC1106400), and Science and Technology

6

Program of Sichuan Province (2017SZ0011 and 2019YJ0132). We also thank Ms. H.

7

Wang, of the Analytical and Testing Center at Sichuan University, for the SEM

8

micrographs and our laboratory members for their generous help.

9

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10

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J. 2016, 287, 640-648.

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(10) Venault, A.; Liou, C.-S.; Yeh, L.-C.; Jhong, J.-F.; Huang, J.; Chang, Y., Turning Expanded

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Poly(tetrafluoroethylene) Membranes into Potential Skin Wound Dressings by Grafting a Bioinert

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TOC figure



Scheme 1. (A) General procedure for the synthesis of random polymer P(SBMA-r-AMA); (B) Schematic illustration of the surface modification process of halloysite nanotubes; (C) Schematic overview of the gradational co-electrospinning procedure.


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Figure1. Physical and chemical characterization of different halloysite doped membranes: (A) SEM and diameter distribution of the membranes; (B) FT-IR measurement for different halloysite doped membranes; (C) thermo gravimetric profiles of the membranes; (D) tensile strength of the composite nanofiber membranes at direction of vertical and parallel to the collector.



Figure 2. Pre-crosslinking electrospinning process: (A) P(SBMA-r-AMA) electrospinning solution with 20% HNTs and 0.5% genipin 12h before electrospinning; (B). electrospinning solution after 12h crosslinked; (C) electrospinning solution after sufficient crosslinked for 7 days.


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Figure 3. Water stability of zwitterionic composite membranes: (A) membranes with 20% HNTs and immersion in water for 7 days; (B) result of cyclic water swelling test; SEM of P(SBMA-r-AMA) surface before (C) and after (D) soaking in water for 24h.



Figure 4. (A) Scheme of gradient wetting property; (B) WCA variation of different HNTs doped membranes at different time interval; (C) WCA images of HNTs embeded membranes at initial time; (D) bilateral wetting evaluation of P(SBMA-r-AMA) side and (E) PCL side.


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Figure 5. (A) Static BSA adsorption amounts; (B) O.D. at 562nm of each SDS solutions; (C) surface fluorescently images of different samples after exposure to FITC-BSA.



Figure 6. Cell adhesion evaluation of gradational composite membranes: (A) O.D. value at 450nm after culturing for 1, 3, 5, and 7 days respectively; (B) O.D. value after 7days incubation; (C) Fluorescence microscopy images of L929 cell which adhered on PCL and P(SBMA-r-AMA) membranes after 7 days incubation.


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Figure 7. In vitro antibacterial evaluation of different drug-loaded composite membranes: (A) Unidirectional drug-control release mechanism of gradient structure membranes; (B) Inhibition zone images after 1, 3, 7 days’ incubation; (C) Diameter changes of the inhibition zone for different membranes; (D) Initial antibacterial ability of the composite membranes.



Figure 8. Cumulative TCH release profiles: (A) TEM of HNTs before and after drug loading; (B) TCH released ratio of HNTs/TCH and different drug-loaded gradient membranes and released mass for gradational membranes with different drug loading methods.


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Figure 9. In vivo antibacterial evaluation: (A) S.aureua infected model; (B) images of S.aureus-infected rat wound dressed with PBS, 3M Tegaderm film, M-TCH and M-HNTs/TCH after 3 days infections; (C) O.D. value and colony count derived from different samples; (D) bacterial plate counts of different samples.



Figure 10. In vivo wound healing investigation: (A) Images of rat skin wound dressed with different materials after 0, 3, 7, 10, and 14 days observation; (B) Wound size for different samples at different time intervals; (C) Complete wound healing time (CWHT) of different samples after injured.


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Antibiofouling Zwitterionic Gradational Membranes with Moisture

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