Chitosan nanocomposite films based on halloysite nanotubes modification for potential biomedical applications

Journal Pre-proofs Chitosan nanocomposite films based on halloysite nanotubes modification for potential biomedical applications Mei Xie, Kaibing Huang, Fan Yang, Ruina Wang, Lei Han, Han Yu, Ziru Ye, Fenxia Wu PII: DOI: Reference:

S0141-8130(19)37024-2 https://doi.org/10.1016/j.ijbiomac.2019.10.154 BIOMAC 13655

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International Journal of Biological Macromolecules

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31 August 2019 16 October 2019 17 October 2019

Please cite this article as: M. Xie, K. Huang, F. Yang, R. Wang, L. Han, H. Yu, Z. Ye, F. Wu, Chitosan nanocomposite films based on halloysite nanotubes modification for potential biomedical applications, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.154

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Chitosan nanocomposite films based on halloysite nanotubes modification for potential biomedical applications

Mei Xie1, Kaibing Huang1*, Fan Yang2, Ruina Wang3, Lei Han3, Han Yu1, Ziru Ye1, Fenxia Wu4,5 1

College of Materials Science and Engineering, Hunan University, Changsha, 410082, PR China 2

Technology Center, China Tobacco Henan Industrial Co.,Ltd, Zhengzhou, 450000, PR China

3

Technology Center, China Tobacco Guizhou Industrial Co.,Ltd, Guiyang, 550009, PR China;

4

Changsha Loyal Chemical Technology Company Limited, Changsha, 410082, PR China 5

Hunan Engineering Research Center of Eco-friendly Water Based Adhensive Materials, Changsha, 410081, PR China.

E-mail address Mei Xie: [email protected] Kaibing Huang: [email protected] (Corresponding author) Fan Yang: [email protected]

Ruina Wang: [email protected] Lei Han: [email protected] Han Yu: [email protected] Ziru Ye: [email protected] Fenxia Wu: [email protected] Telephone number: +86-0731-89720945

Abstract Chitosan is attracting increasing attention for biomedical applications because of its biocompatibility. In the present study, raw halloysite nanotubes (RHNTs) were functionalised with (3-aminopropyl) triethoxysilane (APTS) and then a sequence of novel chitosan biofilms were prepared by adding amino-modified halloysite nanotubes (HNTs-NH2) as a reinforcing material and ethylene glycol diglycidyl ether (EGDE) as a cross-linking agent. The reaction between the APTS and the RHNTs was demonstrated through characterisation of the HNTs-NH2. Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD) results confirmed the interaction of HNTs-NH2 with chitosan and EGDE. Scanning electron microscopy (SEM) showed a transformation of the surface morphology of the chitosan films. Measurement of the mechanical and thermal properties showed that the nanocomposite films exhibited substantial improvements in tensile strength, elongation at break and thermal stability

compared with those of the pure chitosan films. However, the swelling rate of the nanocomposite films decreased upon incorporation of the HNTs-NH2 and EGDE. In addition, the water vapour transmission rate (WVTR) of the nanocomposite films was also improved. Given the aforementioned results, chitosan nanocomposites are promising biomedical materials. Keyword：Halloysite nanotubes; Chitosan; Nanocomposite film; Solvent intercalation; Physical properties

Fig. 1. Graphical abstraction for the formation of nanocomposite films

1. Introduction To meet growing clinical needs, chitin, chitosan, protein, gelatine, starch and other natural polymers are attracting increasing attention on the field of biomedicine because of their excellent properties, high sustainability and low cost [1]. They have found various applications ranging from tissue engineering to drug delivery, gene delivery, cosmetics, medicine, agriculture, pharmaceuticals, wound healing and bone tissue engineering, among others [2]. The good biocompatibility of natural polymers,

which originates from their structural similarity to extracellular matrix components, is one of the main reasons for their prevalence because it promotes cell adhesion and proliferation [3]. Chitosan is a natural polymer polysaccharide obtained by partial deacetylation of chitin in a concentrated solution of a base such as sodium hydroxide (NaOH) [4]. Because of their high tensile strength, good toughness, simple preparation, easy film formation,

good blood compatibility, cell compatibility, degradability and

antibacterial capability, chitosan films are widely used in packaging, agricultural products, water treatment, tissue engineering, biomedicine, pharmaceuticals, etc. [5,6]. In general, ideal biomaterials should not only possess appropriate physical and mechanical properties to prevent secondary infection but also provide a good bionic environment to promote cell adhesion, proliferation and differentiation [7,8]. However, because of their poor mechanical strength, limited water sensitivity and lack of stability in acidic media, chitosan films easily flocculate in aqueous solutions, which limits their application in clinical practice. Therefore, the chemical and mechanical stability of chitosan films needs to be enhanced through chemical cross-linking on hard surfaces or immobilisation in various substrates [9]. The addition of nanofillers to polymer blends enables the development of new polymers with unique mechanical and chemical properties that greatly improve physical defects while maintaining the original biological properties of the matrix [2,10]. Ethylene glycol diglycidyl ether (EGDE) is a diepoxy cross-linking agent that can react with hydroxyl, amino, carboxylic acid and sulfhydryl groups [11]. On the one

hand, EGDE is high reactivity because of the high energy in its three-membered rings [12,13]. On the other hand, EGDE is more soluble in water than other dioxygen compounds and it is cheaper than most other crosslinkers. So, EGDE may soon become a popular polymer crosslinker [14]. In previous studies, EGDE has been used to crosslink cytokeratin, chitosan or similar materials containing a living group, thereby improving their physical properties [15,16]. Furthermore, EGDE exhibits acceptable cytotoxicity and its biological safety ensures that it can be used in biomedical applications [17]. The ideal wound coating material should have characteristics that include good porosity, good ventilation/oxygen permeability and protection of the wound surface from infection [18,19]. Often, the addition of reinforcing fillers can not only meet this requirement but also increase the mechanical strength and thermal stability of polymers [20]. Notably, halloysite nanotubes (HNTs) are natural clay silicate minerals with nanostructures similar to those of carbon nanotubes (CNTs); they possess inner gibbsite octahedral sheet groups (Al–OH) and external siloxane groups (Si–O–Si), which form a positively charged cavity and a negatively charged shell over a range of pH values [21,22]. HNTs, as environmentally friendly 1D natural nanofillers, exhibit good biocompatibility, eco-sustainability and non-toxicity [23]. In addition, the high surface area, large aspect ratio and unique hollow nanotubular structure of HNTs endow them with excellent mechanical and adsorption properties and high thermal stability [24,25]. They are often used as effective fillers to enhance polymers. Huang et al. [26] reported the preparation of sodium alginate (SA)/HNTs composite

hydrogels for bone tissue engineering. They found that the addition of HNTs not only increased the compressive mechanical strength of the resultant SA/HNTs composite hydrogels but also improved cell adhesion and proliferation compared with the use of sodium alginate alone. Liu et al. [27] combined solution-mixing and freeze-drying techniques to develop novel chitosan/HNTs nanocomposite (NC) scaffolds. Measurements of the NC scaffolds’ mechanical and thermal properties revealed a substantial improvement in compressive strength, compressive modulus and thermal stability compared with those of the pure chitosan scaffold. Therefore, the preparation of nanocomposites with HNTs as inorganic fillers has shown great prospects in many applications [28]. Nevertheless, the properties of HNTs are affected by the size effect, surface electron effect and hydrogen bond formation of their surface hydroxyl groups, which promote the agglomeration of HNTs, thereby making dispersion in the polymer matrix difficult [29]. This difficulty has greatly affected the application of HNTs; thus, HNTs are often modified before use. In the present study, the solution intercalation method is proposed for the preparation of chitosan/halloysite NCs. Ammoniated HNTs-NH2 was prepared with (3-aminopropyl) triethoxysilane (APTS) as a modifier. Chitosan was selected as the polymer matrix with different ratios of the HNTs-NH2 filler, and EGDE was added to the formulations as a cross-linking agent. The structure of the composites was characterised by various methods, including Fourier transform infra-red (FTIR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). In addition, the effects of the EGDE and HNTs-NH2 concentrations on the swelling ratio,

water vapour transmission rate (WVTR), thermal stability and mechanical properties of the composites were investigated. This paper contributes to the development of sustainable nanocomposites with promising properties for industrial and biomedical applications.

2. Materials and methods 2.1. Materials HNTs were obtained from Guangzhou Runwo Material Technology Co., Ltd. (Guangzhou, China). APTS was purchased from Sahn Chemical Technology (Shanghai) Co., Ltd. (Shanghai, China). Deionised water was homemade. Sodium hydroxide, acetic acid and alcohol were acquired from Tianjin Hengxing Chemical Reagent Co., Ltd. (Tianjin, China). Chitosan was obtained from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). EGDE was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). 2.2. Preparation of Amino Modified Halloysite nanotubes Surface treatment of raw HNTs (RHNTs) with APTS was carried out using the following procedures. Ethanol and deionised water in a ratio of 5:1 were shaken to form a homogeneous solution [30]. 8 g of APTS was added to the solution and hydrolysed for 1 h with stirring at room temperature. Then, 2 g of RHNTs was added to the solution and the resulting mixture was ultrasonicated for 30 min to disperse the solid; the mixture was then reacted at 80°C for 24 h. After the reaction was completed, the APTS-grafted HNTs were separated by centrifugation with deionised water and

ethanol and dried. Ethanol was used as solvent for Soxhlet extraction for 36 h and the product was dried to obtain aminated modified HNTs-NH2. 2.3. Preparation of Chitosan/HNTs-NH2 Nanocomposite Films A 2% aqueous solution of acetic acid was prepared and stirred at room temperature; chitosan was added to this solution (2%, v/v) to obtain a chitosan solution (2%, w/w). In a separate container, an appropriate amount of HNTs-NH2 (0, 5, 10, 15 or 20 wt% relative to chitosan) was dispersed in 2 ml of deionised water to form a HNTs-NH2 clay suspension. The clay suspension was stirred with the chitosan solution under constant stirring until complete mixing. EGDE was added at various concentrations (0, 10, 20, 30 and 40 wt% relative to chitosan) and stirring was continued for 6 h. Next, 15 g of each solution was poured into a glass Petri dish and dried in an oven at 40°C. After membrane formation, each dish was filled with NaOH (0.1 M) and allowed to stand for 30 min to neutralise any residual acetic acid. Thereafter, the membranes were rinsed with distilled water and immersed in distilled water for 30 min to remove NaOH. Finally, the membranes were peeled from the dishes. Correspondingly, Scheme 1 demonstrates the preparation process and Table 1 shows the solution compositions of the membranes.

Scheme 1. Schematic of the preparation processes of (a) functionalised HNTs and (b) chitosan/HNTs-NH2 nanocomposite films Table 1 Compositions of the prepared chitosan nanocomposites

a

HNTs-NH2 (wt%)a EGDE (wt%)

Samples

Cs (%, w/w)

H0E0

2

0

0

H0E10

2

0

10

H5E10

2

5

10

H10E0

2

10

0

H0E20

2

0

20

H5E20

2

5

20

H10E20

2

10

20

H10E30

2

10

30

H15E30

2

15

30

H15E40

2

15

40

H20E40

2

20

40

x wt% indicates the weight percentage of the reagent relative to the total weight of chitosan in the solution.

2.4. Characterisation Techniques 2.4.1. Fourier transform infrared (FTIR) spectroscopy The chemical structures of the HNTs and NC films were characterised using a Bruker Tensor 27 FTIR spectrometer (Bruker, Karlsruhe, Germany). The samples were scanned from 4000 to 400 cm−1. 2.4.2. X-ray photoelectron spectroscopy (XPS) The surface composition and elemental change of different HNTs before and after modification were also characterised by the Thermo Scientific XPS (Escalab 250Xi, AXIS Supra, Thermo Fisher, England). 2.4.3. Thermogravimetric analysis (TGA) The thermal stability of the HNTs and NC films was investigated by thermogravimetric analysis (STA449F5, NETZSCH, Bavaria, Germany) at a heating rate of 10°C min−1 from room temperature to 800°C under an argon atmosphere. 2.4.4. Dynamic light scattering (DLS) The DLS analysis of HNTs was carried out on a Zetasizer Nano ZSE instrument (Malvern Instruments, England). 2.4.5. Transmission electron microscopy (TEM) The structure and morphology of HNTs were characterised by TEM (Tecnai G20, FEI) at an accelerating voltage of 200 kV.

2.4.6. X-ray diffraction (XRD) XRD analysis of the nanocomposite films was further visualised on an X-ray diffractometer (30 kV, 30 mA, λ = 0.154 nm) from 5° to 70°. 2.4.7. Scanning electron microscopy (SEM) The surfaces of nanocomposite films were observed by SEM (Hitachi S480, Tokyo, Japan). The samples were coated with a fine gold layer and examined using an accelerating voltage of 5 kV. 2.4.8. Water vapour transmission rate (WVTR) WVTR of the NC membranes was measured by the gravity method at 25°C. First, calcium chloride particles were placed in an oven at 140°C for 24 h, and then the dried calcium chloride particles were packed in a weighing bottle with dimensions 60 mm × 30 mm. The test film was fixed to the mouth of the bottle and sealed with paraffin. Finally, the weighing bottle was placed in a sealed container containing a saturated NaCl solution; the container conditions were maintained at 25°C and 75% relative humidity, and the container was kept in an incubator for 24 h. The rate of change in the mass of water in each test cup was measured. The WVTR was calculated by the following formula: WVTR(g·m−2 day−1)=G×24/A·T

(1)

where G represents the amount of absorbed water, T represents the test time (days), and A represents the area of the water vapour transmission zone, that is, the area of the

opening of the weighing bottle. 2.4.9. Swelling ratio study Each group of sample films was cut into a shape of 1 cm × 1 cm and weighed accurately with analytical balance after vacuum drying. The dried sample was immersed in distilled water (pH 6.2) at 37°C for 24 h. The samples were removed the swelling medium every few times and wiped with a paper towel to record the weight of the film. All swelling tests were performed in triplicate. The following formula was used to calculate the swelling ratio of the sample: Swelling ratio (%) =

final film weight−initial film weight initial film weight

× 100%

(2)

2.4.10. Mechanical testing of the films Tensile tests were carried out at 25°C and 75% relative humidity using a universal testing machine (WDW-5E, Jinan Shijin Group, Jinan, China). The chitosan nanocomposite films were cut into dumbbell-shaped pieces with a total length of 150 mm and a width of 20 mm in the elongation region. The elongation rate was 5 mm/min. The reported value for each sample represents the average at least three measurement results.

3. Results and Discussion 3.1. Characterisation of Amino-Modified Halloysite The FT-IR spectra of RHNTs (a) and HNTs-NH2 (b) are compared in Fig. 2. In the spectrum of RHNTs, the absorption peaks at 3697 and 3621 cm−1 originate from the

stretching vibrations of the inner Al–OH and outer Si–OH groups of HNTs [31]. The weak absorption peaks at approximately 1639 cm−1 may be due to the deformation of –OH groups of water adsorbed onto the surface of the HNTs [32]. The bands at approximately 1110 and 1028 cm−1 are related to the symmetric and asymmetric stretching vibrations of Si–O–Si bonds, and the peak at 912 cm−1 is attributed to the bending vibration of the Al–OH bonds on the internal surface of HNTs [33,34]. In comparison with the spectra of the RHNTs, the spectra of the APTS-modified samples, HNTs-NH2 (b), show some new FTIR peaks, including the tensile N–H vibration band near 3448 cm−1 and the deformation C–H vibration at 2888 cm−1. In addition, the broad peaks in the range from 1700 to 1200 cm−1 are related to the stretching or bending vibrations of C–H, N–H and C–N [35]. Furthermore, the decrease in intensity of the absorption peak at 3620 cm−1 indicates that the hydroxyl group is consumed. All of these observations indicate the presence of the APTS in the modified HNTs.

Fig. 2. FT-IR spectra of RHNTs and HNTs-NH2

As shown in Fig. 3, the XPS spectrum of RHNTs is mainly composed of many peaks (O1s, Si2s, Si2p and Al2p), consistent with chemical composition of HNTs mentioned in the literature [21,36]. A new N1s peak (401 eV) appeared in the XPS spectrum of the HNT-NH2 compared with the spectrum of the RHNTs, which was caused by the N element of APTS. As supporting evidence, the C1s (286 eV) peak intensity of HNTs-NH2 is stronger than that of RHNTs, which is due to the carbon from APTS [33]. The aforementioned FTIR and XPS results confirm that APTS was successfully grafted onto the HNTs’ surface.

Fig. 3. XPS spectra of RHNTs and HNTs-NH2 The thermal stability of the RHNTs and HNTs-NH2 is shown in Fig. 4. In all curves, the initial mass loss step, involving the evaporation of the adsorbed water, occurred in the temperature range 20–100°C. For the weight loss curve of RHNTs, there are

mainly two steps. The first stage, which mainly occurred between 25 and 400°C, was attributed to the evaporation of adsorbed water on the surface of RHNTs and to the gradual loss of residual interlayer water [21]; the mass loss was approximately 3.8 wt%. The second stage was in the range 400–600°C, mainly because of the dehydroxylation of the structural aluminium alcohol group (Al–OH) [33]; the mass loss was approximately 16.9 wt%. Finally, the total weight loss reached 18.5 wt% when the sample was heated to 800°C. Although the curve of the modified HNTs-NH2 was similar to that of the RHNTs, the HNTs-NH2 exhibited a greater mass loss in the temperature range 400–800°C. Zhu [37] reported a similar conclusion. The mass loss of HNTs-NH2 in the second degradation stage reached 21.7 wt%, and the mass loss at 800°C reached 24.6 wt%, which was related to the additional thermal loss of APTS organics grafted onto HNTs.

Fig. 4. TGA curves of RHNTs and HNTs-NH2

The DLS instrument used the CONTIN algorithm method; this algorithm performs an inverse Laplace transform on the autocorrelation function to fit the relevant data and uses the Stokes–Einstein equation to calculate the hydration diameter. Fig. 5 shows the size distribution of RHNTs and HNTs-NH2. The particle size distributions of RHNTs and HNTs-NH2 are relatively narrow, and the particle size of halloysite nanotubes changes before and after modification. The average diameter of the HNTs-NH2 is 452.37 nm, which is 108 nm larger than that of RHNTs. RHNTs and HNTs-NH2 less than 300 nm accounted for 40.07% and 28.74%, respectively.

Fig. 5. DLS result of RHNTs and HNTs-NH2 TEM images of the RHNTs and HNTs-NH2 under different magnifications are shown in Fig. 6. The images reveal both some similarities and some differences between the materials. The images show that the halloysite particles have a cylindrical shape and contain a transparent central area that runs longitudinally along the cylinder, revealing that the nanotubular particles are hollow and open-ended, consistent with previous research [33,38]. For the RHNTs, the tubes have smooth and uniform outer

surfaces. However, after grafting by APTS, the outer surface of HNTs-NH2 became rough and was covered by some irregular substances, which might increase the diameter of the HNTs. Moreover, the dispersion of the HNTs-NH2 in aqueous/organic solution was ultimately improved compared with that of the RHNTs, which exhibited large-scale agglomeration. This difference in dispersion behaviours may be due to the fact that the long chain of the modifier APTS imparts hydrophobic character to the surface of the HNTs-NH2, and part of the APTS is grafted onto the positively charged inner cavity of the HNTs, altering the overall HNT charge towards more negative values [39,40]. Thus, the modified HNTs exhibit better colloidal stability. This observation could further to provide direct evidence for the reaction between APTS and RHNTs.

Fig. 6. TEM images of the RHNTs ( A1, A2) and HNTs-NH2 (B1, B2) sample. Scale bar in (A1, B1) images represents the length of 0.2 μm and in (A2, B2) images is 50

nm. 3.2. Characterisation of Chitosan/HNTs-NH2 Nanocomposite Films Fig. 7 shows the FTIR spectrum of the chitosan/HNTs nanocomposite films. As depicted in Fig. 7(c), the composite film of H10E0 was prepared by adding filler HNTs-NH2 into chitosan. The broad band centred at 3302 cm−1 in the 3100–3500 cm−1 region is a tensile vibration absorption band in which O–H and N–H groups overlap, and the respective characteristics peaks found at 2941 and 2854 cm−1 are owing to C–H stretching of CH2 and CH3 groups. The band at 1644 cm−1 is attributed to the amide band (amide I and amide II), and the characteristic peaks at approximately 1555 and 1410 cm−1 are the bending vibrations of C–H and N–H, respectively. In addition, the bands at 998 and 902 cm−1 are due to the C–O bond tension vibration of C-3 of chitosan (secondary-OH) and C–O bond tension vibration of C-6 of chitosan (primary-OH), respectively [41,42]. The presence of characteristic peaks of Al–OH at 3694 and 3620 cm−1 indicates the presence of HNTs-NH2, i.e. HNTs-NH2 was successfully added to the polymer network. When EGDE was added, the FTIR characteristic peak of the composite membrane was observed in the spectrum of H10E20 (d). The characteristic peak of Al–OH disappeared at 3694 and 3620 cm−1, which may be attributed to hydrogen bonding between Al–OH and chitosan or EGDE [19]. At the same time, the broadband absorption peak near 3303 cm−1 was strengthened. Moreover, we can also see that the absorption intensity of the characteristic peaks at 1533 and 1408 cm−1 is remarkably

enhanced. This enhancement is attributed to the epoxy groups in EGDE reacting with the amino groups of chitosan to form more C–H and C–N bonds. The peak of the amide III at 1247 cm−1 and the characteristic peak of C–O at 1067–1020 cm−1 were broader and more prominent, indicating that the added crosslinker EGDE interacted with the filler and chitosan [43].

Fig. 7. FT-IR spectra of H10E0 and H10E20 Fig. 8 displays the TGA and DTG curves of the nanocomposites. According to the results, the weight loss process of pure chitosan H0E0 under nitrogen progresses in four stages. The first stage, involving the loss of water and some volatile substances, occurred in the temperature range 30–150°C. The second stage occurred in the temperature range 150–215°C, which was assigned to the deamination and dehydration reaction of chitosan. The third stage took place in the range of 215–

382°C, involving the breakdown of the main chain of chitosan and the breakdown of C–O–C glycoside bonds [44,45]. The fourth stage occurred at 382 to 800°C and mainly involved the oxidative degradation of carbon residues formed in the third stage. With the addition of EGDE and HNTs-NH2, the thermal stability of the composites changed. DTG curves show that the maximum rate change point in the weight loss curve decreased to a great extent and that the final mass loss was less than that of pure chitosan. The results further confirmed that the cross-linking reaction between EGDE and chitosan occurred. The addition of HNTs-NH2 had a similar effect on the degradation stage of the membrane. The well dispersed HNTs-NH2 restricts the long-range-chain mobility of chitosan and can act as a barrier to hinder the permeation of volatile degradation products out of the material [46]. The tubular structure plays a ‘physical shielding effect’, resulting in an effective delay in mass transfer [47,48]. This evidence indicates that chitosan is miscible with HNTs-NH2 and that interactions occur between main groups of chitosan, HNTs-NH2, and EGDE in nanocomposites [49]. Therefore, the structure of the chitosan film was strengthened and the thermal stability was improved after EGDE cross-linking and HNTs-NH2 filler strengthening.

Fig. 8. (A) TGA and (B) DTG thermograms of chitosan nanocomposite films To study the dispersion state of HNTs-NH2 in chitosan films and the microstructure of the composites, the XRD diffraction patterns of HNTs-NH2 and the composites with different clay and cross-linking agents were recorded (Fig. 9). The diffraction peaks of HNTs-NH2 at 2θ = 12°, 20° and 25° are respectively attributed to the (0 0 1), (0 2, 11) and (0 2 0) crystal planes of HNTs-NH2, and this information is consistent with previous research results [50-52]. In the XRD pattern of the pure chitosan film H0E0, inconspicuous broad diffraction peaks appear only in the vicinity of 2θ = 8.6°, 11.6°, 18.6°, 23.5°, indicating that the chitosan film exhibits certain but underdeveloped crystallinity due to the distribution of numerous hydroxyl and amino groups as well as some N-acetylamino groups in the macromolecular chain of chitosan. The composition is relatively complicated and random, which leads to its inability to crystallise. However, intermolecular hydrogen bonds are formed in the molecule because of its high degree of deacetylation, which tends to make the molecular chain structure regular, thereby forming a partially crystalline region [53].

Upon addition of HNTs-NH2, no obvious HNTs-NH2 diffraction peaks were observed in the XRD pattern of H10E0, indicating uniform mixing of chitosan and HNTs-NH2. The only changes are that the diffraction peaks at 2θ = 12° and 18.6° became sharper, and that new shoulders appeared at around 2θ = 20.5° and 24.4°. Nevertheless, compared with the diffraction peaks of HNTs-NH2, the strength of the shoulder peak is decreased markedly and the peak width becomes larger. We concluded that most of the layered structure was broken to form a random structure and that a small part of the wall sandwich structure was retained [54]. When the crosslinker EGDE was also added, the diffraction peaks of the H10E20 film weakened or even disappeared. This result indicates that the added EGDE reacted with the HNTs-NH2, which destroyed the relationship between the HNTs-NH2 and chitosan and made the dispersion of HNTs-NH2 in chitosan film more disordered, resulting in loss of crystallinity.

Fig. 9. XRD patterns of (a) HNTs-NH2, (b) H0E0, (c) H0E20, (d) H10E0, (e)H10E20

The microstructure of the films was analysed by SEM to identify film homogeneity, voids, and surface smoothness [55]. Fig. 10A presents the smooth morphology and compact structure of the surface of the pure H0E0 film. Fig. 10B shows a top-view image of the cross-linked H0E20 biofilm, which reveals that the outer surface of the H0E20 biofilm was more homogenous, regular and smooth than that of the H0E0 biofilm. Similarly, in Fig. 10C, the surface morphology of the H10E0 film changed little because chitosan covered the HNTs-NH2. Only a small amount of HNTs-NH2 aggregates were exposed to the surface of the composite film and transformed into structures with fewer micropores. In the film shown in Fig. 10D, samples H10E20 and HNTs-NH2 have tubular structures and were uniformly dispersed in the matrix; the interface between the HNTs-NH2 and chitosan matrix was thus blurred. This blurring is attributable to the interfacial interactions between the HNTs-NH2 and EGDE and chitosan, as previously described [56]. Moreover, the skin-layer of the H10E20 membrane exhibited more pores because of the interaction of the added nanoparticles HNTs-NH2 with chitosan and EGDE in the mixing process. This greater interaction promotes the exchange between the solvent phase and the nonsolvent phase and demonstrates the role of the porogen, thus affecting the phase-inversion process [57]. Interestingly, the presence of these pores increases the specific surface area of the composite, which will contribute to cell grafting and value addition.

Fig. 10. SEM images of (A) H0E0, (B) H0E20, (C) H10E0 and (D) H10E20 The moisture permeability of materials is usually affected by their hydrophilicity and is also related to their surface structural features such as pores and cracks [58]. In medical applications, wound dressing materials require good air permeability and a high rate of water loss on the wound surface. To provide proper moisture levels for normal wound healing and prevent excessive dehydration, ideal wound dressing materials typically require WVTRs in the range 2000–2500 g·m−2 day−1 [59]. The WVTR values of all the composite films at 25°C and 75% relative humidity are shown in Table 2. The pure chitosan film has a WVTR of 1950 ± 60 g·m−2 day−1, which shows a fairly good WVTR. With the addition of EGDE, the WVTR value of the composite films is reduced because of the cross-linking between chitosan and the increase in the degree of cross-linking, which reduces the porosity of water vapour

permeation. This structure makes the path of water molecules more tortuous and reduces the diffusion rate [60]. When HNTs-NH2 was continuously added to the composite films, the WVTR value of the composite films is improved, possibly because of the hollow tubular structure of the HNTs-NH2, resulting in a porous structure on the surface of the composite films. However, when the amount of HNTs-NH2 is excessive, the barrier effect and agglomeration behaviour of nanotubes begin to play roles that lower the WVTR value of the composite films. Therefore, the addition of the inorganic filler HNTs-NH2 and the crosslinker EGDE affect the WVTR values of the nanocomposite films. Table 2 The WVTRs of the chitosan composites Sample code

Thickness

Water absorption

WVTR

(mm)

(g)

(g·m-2 day-1)

H0E0

0.048 ± 0.006

0.916 ± 0.031

1950 ± 60

H0E10

0.042 ± 0.007

0.897 ± 0.042

1910 ± 80

H5E10

0.044 ± 0.007

0.903 ± 0.025

1920 ± 50

H10E0

0.041 ± 0.002

0.954 ± 0.023

2030 ± 50

H0E20

0.046 ± 0.009

0.881 ± 0.017

1870 ± 40

H5E20

0.047 ± 0.003

0.934 ± 0.043

1980 ± 90

H10E20

0.048 ± 0.006

1.148 ± 0.054

2440 ± 110

H10E30

0.043 ± 0.009

1.126 ± 0.027

2390 ± 60

H15E30

0.045 ± 0.008

1.135 ± 0.047

2410 ± 100

H15E40

0.045 ± 0.009

1.027 ± 0.033

2180 ± 70

H20E40

0.049 ± 0.009

1.009 ± 0.029

2140 ± 60

To study the effect of HNTs-NH2 and EGDE on the swelling properties of chitosan films, we conducted swelling experiments on all of the nanocomposite samples; the

results are shown in Fig. 11. The pure chitosan membranes were completely dissolved after 24 h of immersion in deionised water with a pH of 6.2; only a few microflocculations were observed. When 10% of EGDE was added, the composite films exhibited swelling behaviour, indicating that cross-linking occurred between the EGDE and the chitosan. The swelling ratio decreased with increasing concentration of the EGDE. Because the crosslink density of the composite films is gradually increasing, the increase in the crosslink density causes the space among the water molecular networks to become small [49]. At the same time, the addition of the inorganic filler HNTs-NH2 also substantially reduces the swelling property of the membranes. Because of the limited water absorption of nano-clay, the water absorption of the polymer networks is believed to play a major role in the water absorption of the nanocomposite films. However, it has a function with EGDE and chitosan in the composite films in that it forms two types of network structures—a soft and tight polymer network and a hard but loose inorganic network—leading to increased crosslink density of the polymer. When the content of HNTs-NH2 increases, its presence has a certain shielding effect on the swelling of the membranes. At this time, the water absorption of the polymer network plays a major role and the swelling ratio is gradually lowered.

Fig. 11. The swelling ratio of chitosan nanocomposite films The mechanical properties of the chitosan/HNTs-NH2 nanocomposite films were evaluated; the results are depicted in Table 3. The tensile strength of the pure chitosan film was 11.42 ± 0.26 MPa, and its elongation at break was 5.30 ± 0.36%. After 10% EGDE was added, the tensile strength and elongation at break increased to 15.87 ± 0.54 MPa and 6.32 ± 0.27%, respectively. These results demonstrate that EGDE mainly acts like a chain extender, increasing the molecular weight of chitosan, thereby enhancing and toughening the composite films. The table clearly shows that the tensile strength of chitosan/HNTs-NH2 nanocomposite films increases with a certain range of halloysite nanotubes, consistent with the mechanical properties of the micro/nano-clay particle chitosan films reported by Casariego et al. [61]. They also demonstrated that the tensile strength of the later films increased due to possible strain-induced alignment of the clay particle layers in the polymer matrix and

hydrogen bonding interactions between the polymer and the clay mineral. Therefore, the same phenomena might have occurred in the case of as prepared chitosan/HNTs-NH2 nanocomposite films. Similar results have been presented by other authors [62,63]. When the amount of EGDE is increased to 20%, the slight but sustained increase in strength can be attributed to the formation of intermolecular crosslinks between adjacent chitosan chains by the reaction of difunctional epoxide molecules [64]. The maximum tensile strength value of all the samples was 23.52 ± 0.51 MPa for H10E20. At higher concentrations of EGDE and HNTs-NH2, the tensile strengths of the nanocomposites decreased. This observation shows that more than 20% of the extra EGDE acts as a plasticiser. Similarly, the increased HNTs-NH2 results in a high interfacial area and energy; thus, the HNTs-NH2 clay layers may interact with each other to form accumulated clay throughout the chitosan matrix, thereby reducing the aspect ratio, effective modulus and the reinforcing effect of the clays [65]. We concluded that the improvement in the mechanical properties of the nanocomposites is attributable to numerous factors, including appropriate interaction and bonding between the polymer matrix and the nanoparticles (mostly hydrogen bonding) and the formation of the resulting network and suitable and effective stress transfer efficiency of HNTs-NH2 particles [19].

Table 3 Tensile properties of the chitosan composites Samples

Tensile strength (MPa)

Elongation at break (%)

H0E0

11.42 ± 0.26

5.30 ± 0.36

H0E10

15.87 ± 0.54

6..32 ± 0.27

H5E10

16.32 ± 0.15

5.95 ± 0.42

H10E0

17.53 ± 0.40

5.88 ± 0.39

H0E20

18.38 ± 0.28

8.79 ± 0.43

H5E20

19.63 ± 0.37

8.18 ± 0.57

H10E20

23.52 ± 0.51

7.92 ± 0.66

H10E30

21.35 ± 0.46

7.22 ± 0.45

H15E30

17.11 ± 0.62

6.60 ± 0.30

H15E40

16.21 ± 0.17

5.54 ± 0.34

H20E40

14.97 ± 0.32

5.47 ± 0.83

4. Conclusions A series of novel chitosan nanocomposite membranes were successfully prepared by blending with different dosages of HNTs-NH2 using the conventional solution casting method. FTIR, TGA, XPS, DLS and TEM analyses confirmed the graft reaction between RHNTs and APTS. HNTs-NH2 exhibited better homogeneous dispersion in a chitosan membrane matrix, and the improvements in the microstructure of the films could be observed by SEM and XRD. FTIR analysis also confirmed the interaction between chitosan, EGDE and HNTs-NH2. The formation of a three-dimensional network enhanced the stability of the composites. The high aspect

ratio and thermal insulation properties of the HNTs-NH2 improved the properties of the composites. The addition of EGDE and HNTs-NH2 to chitosan improved the physical performance of the material, including its water resistance, thermal stability, WVTR and mechanical properties. Together, the results in this study indicate that the chitosan/HNTs-NH2 bio-nanocomposites are potential candidates for biomedical applications.

Acknowledgements The authors would like to gratefully acknowledge Hunan University for providing necessary research facilities to carry out this work.

Conflicts of interest There is no conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.

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