Jie Gong,Fei Tong,Bin Wang,Di Ma,Chunyong Zhang,Jinlong Jiang,Lixiong Zhang*

1 College of Chemical and Environmental Engineering,Jiangsu University of Technology,Changzhou 213001,China

2 Jiangsu Provincial Key Laboratory of Palygorskite Science and Applied Technology,Faculty of Chemical Engineering,Huaiyin Institute of Technology,Huai’an 223003,China

3 State Key Laboratory of Materials-Oriented Chemical Engineering,College of Chemistry and Chemical Engineering,Nanjing Tech University,Nanjing 210009,China

Keywords:Zeolite A Hybrid film Water absorption Antimicrobial activity

ABSTRACT Zeolite A/CS xerogel hybrid films were prepared by in-situ crystallization method with uniform structure and good strength.The hybrid films prepared from the precursor films dried at 50 °C showed zeolite A was well crystallized.The hybrid films show high water absorption,good elastic strength and Young modulus.The antimicrobial ability of the hybrid films was investigated after Ag+-ion exchanged.The Ag+-exchanged hybrid films with 35%(mass)content of zeolite A showed the highest antimicrobial activity,which could reduce the concentration of the microbial to zero after 7 h.

1.Introduction

Traditional medical bandaging materials are composed of filmforming polymer and antibacterial content,they must have properties of non-toxic,flexibility and biocompatibility with the skin [1].Meanwhile,the medical bandaging materials should not only inhibit the accumulation of the secretion on the wound,but also prevent the incursion of bacteria and dust [2,3].Chitosan (CS) has many advantages in medical applications because of its biocompatibility,biodegradability,and non-toxicity,which enables CS as a suitable biological material for wound repair [4–6].

Zeolites are widely used in the fields of adsorption,separation and catalysis.At the same time,zeolites also have many applications in the fields of antibacterial and antiviral drug delivery due to their good biological activity,stability and compatibility [7–9].Therefore,zeolite/CS composite materials can be ideal medical bandage materials.Nowadays,many researchers pay their attention to the preparation of zeolite/CS composite materials for bone tissue engineering,drug release and medical bandage[10–13].The zeolite/CS composite materials have unique morphology,structure and property.Most zeolite/CS composite materials are prepared by mixing zeolite and CS gel,and then the ion exchange method is used to obtain the products.But the procedures are tedious and complicated.So an easy and effective way to obtain the zeolite/CS hybrid materials is needed.In our previous work,zeolite/CS composite microspheres were successfully prepared,which showed good property of gas adsorption,separation and heavy metals adsorption [14–16].

In the present work,zeolite A/CS hybrid films were prepared by the in-situ crystallization method,the content of zeolite could be controlled easily by changing the composition of the synthesis solution.The zeolite A/CS hybrid films exhibited good water absorption ability,mechanism property,and antibacterial property,which may have potential application as medical bandaging materials.

2.Experimental

2.1.Preparation of zeolite A/CS xerogel hybrid films

Zeolite A/CS xerogel hybrid films were prepared as follows:First,2.07 g of silica sol (40% (mass),Zhejiang Yuda Chemical Industry Co.,Ltd.)and 1.2 g of CS(CS,90%deacetylated,Sinopharm Chemical Reagent Co.,Ltd.) were dispersed into 15 g of deionized water,followed by adding 1.6 ml of acetic acid (CH3COOH) aqueous solution (36% (mass),Shanghai Lingfeng Chemical Reagent Co.) dropwise under vigorous stirring for 6 h to obtain a homogeneous silica/CS mixture.Poured 10 g of the silica/CS mixture into Petri dish (diameter of 10 cm) homogeneously and submerged at room temperature for 30 min to remove air bubbles inside,followed by drying at 50 °C in the oven overnight.Thus the silica/CS xerogel film,which attached onto the bottom of the Petri dish,was obtained.Then an aqueous solution composed of 26.60 g of deionized water,2.59 g of sodium hydroxide and 0.80 g of sodium aluminate was added into the above Petri dish slowly and submerged the film for 24 h.The precursor film was completely exfoliated from the bottom of the Petri dish.Finally,the system was sealed and hydrothermally treated at 80 °C for 5 h.Zeolite A/CS xerogel hybrid film was obtained after washing and drying at room temperature for 24 h.As theoretical calculated,the content of zeolite A in the hybrid film was 55% (mass) (this film was denoted as S-55).To obtain xerogel hybrid films with different contents of zeolite A,the amounts of silica sol and NaAlO2were simultaneously reduced with the same ratio (mass) of 25%,50% and 75%while the amount of NaOH was reduced by 10%,25% and 50%,respectively.The obtained hybrid films with zeolite contents(mass) of 45%,35% and 20%,they were denoted as S-45,S-35 and S-20,respectively.Pure CS xerogel film (denoted as S-0) was also prepared following the above procedure without adding silica sol,soaking in the NaAlO2solution and hydrothermal treatment.

2.2.Water uptake

S-n samples(n=0,20,35,45,55)with the size of 2 cm× 2 cm were weighted (W1) and immersed at 37 °C into phosphate buffer solution of pH 7.4 for a certain time.According to Italian Pharmacopoeia XII,phosphate buffer solution was composed of 2.38 g sodium phosphate dibasic,0.19 g potassium phosphate monobasic,8.0 g sodium hydrochloride and 1000 ml deionized water.At predetermined time,the films were removed,carefully bolted with a filter paper and weighed (W2).The water absorption capacity was calculated by the following equation:

2.3.Antimicrobial testing

The silver ion-exchange was performed by inserting the zeolite A/CS film in a 0.01 mol∙L-1solution of AgNO3in a polypropylene bottle at room temperature for 2 h with gentle agitation.Then the film was taken out of the solution,washed with deionized water for several times,and soaked in deionized water for 1 h before it was used for antimicrobial testing.

The antimicrobial testing of the film was performed as follows.Individual colonies of E.coli grown on Luria-Bertani (LB) plates were transferred with sterile toothpicks to sterile test tubes containing LB broth and incubated at 37 °C overnight with shaking.The Ag+-exchanged zeolite A/CS hybrid film was exposed to ultraviolet light for 20 min to ensure sterility prior to biocidal testing.Then it was placed into the conical flask and 0.5 ml of the E.coli suspension with optical density(OD)of 0.104 was added and incubated at 37°C at a constant relative humidity of 60%for 0,2,5,7,9 and 12 h,respectively.The buffer solution was diluted with distilled water and test the optical density of E.coli suspension.By contrast,the antimicrobial test of zeolite A/CS hybrid films without Ag+-exchange was also examined.For further examination,the Ag+-exchanged zeolite A/CS xerogel hybrid film with 35% zeolite,was placed into the conical flask.0.5 ml of the E.coli suspension containing greater than 1 × 106colony-forming units (CFU) was added and incubated at 37 °C at a constant relative humidity of 60 % for 2,5,and 7 h.After the E.coli suspensions were spread on LB agar plates and incubated at 37°C for 24 h for visual enumeration.The CFUs were counted visually and back-calculated to determine the number of CFUs surviving on the samples after each incubation time [10].

2.4.Characterization

X-Ray diffraction(XRD)patterns were collected on a Bruker D8 Advance powder diffractometer using a Ni-filtered Cu Kα radiation source at 40 kV and 100 mA.Digital camera(Canon 1000D,China)and scanning electron microscope (SEM,Hitachi S-4800 and Quanta 200)were used to investigate the particle size,morphology and microstructure of the films.Thermogravimetric (TG) analysis was carried out on the calcined samples in air up to 800 °C using a Netzsch STA 409 instrument with a heating rate of 10 °C∙min-1.Stress–strain mechanical tests were carried out using a Frictionabrasion testing machine(MPX-2000,China).The Young’s modulus(the slope of the stress–strain curve in the elastic deformation region),tensile strength (the tensile stress at the breaking point of the specimen),and elongation at break(the percentage increase in length that occurs before the sample breaks)were measured on rectangle shaped film stripes,obtained using a cutting machine,length and width of which were 100 mm and 5 mm,respectively.Growth curves of E.coli were obtained by measuring the optical density using ultraviolet spectrophotometer with a wavelength of 600 nm.

3.Results and Discussion

3.1.Characterization of zeolite A/CS xerogel hybrid film

Silica/CS xerogel film was prepared by drying silica/CS mixture at 50 °C in an oven overnight.After water evaporation,silica/CS mixture was transformed into the silica/CS xerogel film attached onto the bottom of the Petri dish.It is convenient to get the precursor film from the Petri dish completely.As shown in Fig.1a,the precursor film is transparent and has the same size and the shape with the bottom of the Petri dish.After crystallization,the transparent precursor film turned into a white and sub-transparent xerogel hybrid film(Fig.1b).The surface of the xerogel hybrid film is not smooth and flat as that of the precursor film because CS may contract affected by temperature and basicity [13].In Fig.1c,the silica/CS xerogel films with different silica amounts show the broad diffraction peak at 2θ of 20°,indicating the precursors are amorphous.Fig.1d and e shows the XRD patterns of the xerogel hybrid films with four different zeolite contents.Before calcination,the XRD patterns of the hybrid composites exhibited diffraction peaks corresponding to zeolite A,but with a slight bell baseline,probably due to the present of CS (Fig.1d).With the increase of zeolite contents,the diffraction peak intensity increases obviously and the baseline of XRD patterns became flatter.After calcination in air at 550°C for 300 min,no other diffraction peaks were found in XRD patterns (Fig.1e),indicating formation of pure zeolite A.These results indicate that xerogel hybrid films are composed of zeolite A with high crystallinity.The theoretical mass of zeolite in the hybrid films is 55%,45%,35%and 20%,respectively.In order to measure the exact zeolite contents,TG analysis was conducted on these samples,including the pure CS xerogel film (S-0) in air.The TG curve of S-0(Fig.1f)shows that pure CS film can be nearly decomposed,leaving a residue content of about 1.9%.The TG curves of the other hybrid films (Fig.1f) are similar,indicating a three-stage weight loss.From the weight loss,the exact zeolite contents of S-n (n=55,45,35,20) can be calculated as 43.6%,37.9%,28.2% and 6.7%,respectively.

Fig.1.Optical pictures of the SiO2/CS precursor film dried at 50°C(a)and the corresponding zeolite A/CS after crystallization(b).XRD patterns of the silica/CS xerogel films with different silica amounts(c).XRD patterns of zeolite A/CS films with different zeolite contents before(d)and after calcination(e).TG curves of the pure CS film and zeolite A/CS films with different zeolite contents (f).

The morphologies and surface structures of the S-n (n-55,45,35,20) are observed by SEM.The top-view of S-55 (Fig.2a) shows a uniform structure without cracking.Closer observation of the surface (insert in Fig.2a) reveals that the average size of zeolite A cubic crystal is about 1–2 μm.No CS can be observed as the surface is covered with dense intergrown zeolite A crystal layer.Fig.2b shows the cross-section of the film with a thickness of about 50 μm.The high resolution image of the cross-section shows that the film is filled with zeolite A while these particles are not well intergrown (Fig.2c).The fracture surface is composed of zeolite A and interconnected by fibrous materials.These fibrous materials can be identified as CS as indicated in our previous report[14].It is clearly that zeolite A inside the film is not as dense as that on the surface,and some zeolite particles are even not well crystallized.The difference might be contributed to that the external surface was fully immersed into the synthesis solution and leading to well crystallize.However,synthesis solution was difficult to penetrate into the inside and leading to form spare zeolite A.Fig.2d,e,f are the images of zeolite A/CS film with 45%,35%,20%zeolite contents,respectively.Zeolite A with a size of 1–2 μm can be observed from all the images.

3.2.Water absorption capacity

Water absorption capacity is an important property for a wound dressing material as it absorbs wound exudates,avoiding maceration and enhancing wound healing [17].It also effects the diffusions of nutrients,cells and biological active molecules [18].The water absorption capacities of the hybrid films of S-n are examined by immersing 2 cm × 2 cm hybrid films in 10 g phosphate buffer solution for 15–1440 min.The results in Fig.3 indicate that water absorption capacities of S-0 and S-20 reach a maximum value of about 310% and 170%,while the water absorption capacities of S-35,S-45 and S-55 are nearly 180%,145% and 135%.Obviously,the hydration percentage of the xerogel hybrid films is decreasing as increasing the amount of zeolite A,which means adding zeolite A is not benefit to improve water absorption capacity.It is possibly attributed to the morphologies and structures of xerogel hybrid films.As analysis in Fig.2,the external surface appears more compactly as the amount of zeolite A increases,retarding water absorption into the inside.In addition,S-35,S-45 and S-55 spent only 120 minutes to reach the maximum value of water absorption capacity compared to 240 minutes of S-0 and S-20.The shorter absorption time may be explained by the unique porosity and hydrophilicity of zeolite A [19],in which diffusion rate of water is very fast.

Fig.2.SEM images of(a)top-view,(b)cross section and(c)high-resolution of the cross-section of zeolite A/CS film with 55%zeolite content,and SEM images of the top-view of zeolite A/CS film with (d) 45%,(e) 35%,and (f) 20% zeolite content.

Fig.3.Water absorption capacities of zeolite A/CS films with different zeolite contents.

Water absorption ability can be effected by factors,like hydrophilicity,morphology,porosity and synthesis conditions.Compared with some commercially available PU membrane dressings and other polymer films,which exhibit low water absorption ability in the range of 31%–87%(mass)[20,21],and some composite films,which even reaches a high water absorption ability of more than 1000% (mass) [22],the water absorption ability of the prepared hybrid films is placed in a medium level.Actually an ideal wound dressing normally present water absorption in the range of 100%–900% (mass) [20].As a result,the xerogel hybrid films prepared in the present work are all appropriate for the wound dressings.

3.3.Mechanical ability

Fig.4.Tensile strength (a),Young’s modulus (b) and elongation (c) of zeolite A/CS films with different zeolite contents.

Based on some reports,the wound dressing should be flexible,stable and strong enough to cover the wound surface during healing period[23,24].The mechanical properties of zeolite A/CS xerogel hybrid films with different zeolite contents were investigated at room conditions (T=20 °C,RH=50%).As shown in Fig.4,the presence of zeolite A has an effect on the tensile strength,the Young’s modulus and the elongation derived from the analysis of the stress–strain curves.The plot shows that the tensile strength and the Young’s modulus are enhanced first and then reduced as the contents of zeolite increases.The intermolecular hydrogen bonding between chitosan and zeolite A makes the loaded stress easily transferred to zeolite A,which will increase the mechanical properties.But excessive zeolite A is not beneficial to improve the tensile strength and the Young’s modulus obviously.It may be attributed to a partial re-aggregation of zeolite A caused by the phase separation between chitosan and silica particles while preparing silica/CS xerogel films[22].S-35 and S-45 have the maximum value of the tensile strength and the Young’s modulus,respectively,and the values are comparable to those of SBA-15/CS films [21,22,25].In addition,the elongation strength of the xerogel hybrid films decreases with increasing the contents of zeolite A (Fig.4c).This phenomenon can be explained that the hydrothermal process at 80 °C for 5 h will reduce the stability of chitosan and have a low elongation strength.Though the elongation strength is bad compared to the pure chitosan film,it is still superior to that of some reported wound dressing films[25],which can meet the demand for medical bandaging materials.

3.4.Antimicrobial ability

Fig.5.Growth curves of E.coli in presence of zeolite A/CS films with different zeolite contents without the treatment of Ag+-exchanged with silver(a)and with the treatment of Ag+-exchanged with silver (b).

To investigate the antimicrobial ability of the zeolite A/CS and Ag+-exchanged zeolite A/CS hybrid film,the antimicrobial experiment was carried out by measuring optical density of E.coli solution (with initial OD value of 0.104) under a certain time.Fig.5a exhibits the growth curves of E.coli treated with zeolite A film containing different amount of zeolites.The curve of pure CS film without zeolite shows an obviously lead in the value of OD.All the values of OD increase as prolonging the time until the experiment was carried on for 12 h (the values are ranging from 4.0 to 5.5).Meanwhile,when the Ag+-exchanged zeolite A/CS hybrid film were used,all the values of OD,which are ranging from 1.0 to 1.2,are the highest at the time of 5 h (Fig.5b).As dead E.coli would release lysozyme to dissolve itself,all the curves decline with the extension of time,which means the Ag+enhances the antimicrobial ability significantly.It is also can be seen from Fig.5 that all the curves of films containing zeolites are lower than the curve of pure CS film,further indicating zeolite has a good antimicrobial activity [26].The E.coli Solution treated for 12 h with Ag+-exchanged zeolite A/CS xerogel hybrid film with 35% zeolite displayed the lowest OD value.These results indicate that introducing appropriate amount of zeolite into the CS film is beneficial for its antimicrobial ability.

For further examination,the E.coli suspension (an initial concentration of 1 × 106CFU),which was treated with the Ag+-exchanged zeolite A/CS xerogel hybrid film with 35% zeolite for 2,5,and 7 h,were spread on LB agar plates and incubated at 37 °C for 24 h for visual enumeration.The antimicrobial testing results shown in Fig.6a indicate that the concentration of E.coli is reduced to 43.7 × 103CFU∙ml-1after 2 h of incubation time,and further reduced to 2.7×103CFU∙ml-1after 5 h(Fig.6b).After 7 h of incubation,the concentration of E.coli is nearly reduced to zero,suggesting all the bacteria is killed(Fig.6c).These results further suggest that the Ag+-exchanged zeolite A/CS xerogel hybrid film exhibits high antimicrobial activity.Zeolite/organic composite films own the good antimicrobial property due to the enhanced adsorption ability by electrostatic or van der Waals force [27].Zeolite A are aluminosilicate crystals with unique pore size and hydrophilic property,which provide suitable location for bacterial,resulting in absorbing more microbial.Under the circumstance,the Ag+on the film could kill the bacterial efficiently.Compared with zeolite/organic composite films reported in literatures [28–30],in which it takes 6–24 h to kill all the bacterial at a similar test condition,the films prepared in the present work show a higher antimicrobial activity.

4.Conclusions

In the present work,the in-situ crystallization method was used to prepare zeolite A/chitosan xerogel hybrid films.The hybrid films have uniform structure and good strength with crystallization time of 5 h at 80 °C.Zeolite A particles covered on them had a rather small size of 0.5–1 μm.The water absorption and mechanical property were tested,respectively.The results revealed that the S-35 reached its highest water absorption ability of 180% and the best elastic strength,and S-45 showed the largest Young modulus.The antimicrobial ability of the xerogel hybrid films was investigated after Ag+-ion exchanged.The 35% Ag+-exchanged xerogel hybrid films showed the highest antimicrobial activity,which could reduce the concentration of the E.coli to zero after 7 h.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig.6.The images of the E.coli colonies incubated at 37°C for 24 h.Before incubation,the E.coli suspension was treated with the Ag+-exchanged zeolite A/CS xerogel hybrid film for different times:(a) 2 h,(b) 5 h,(c) 7 h.

Acknowledgements

We are grateful for financial support from the National &Local Joint Engineering Research Center for Deep Utilization Technology of Rock-salt Resource(SF201804),Opening Topic of Key Laboratory of Attapulgite Resources Utilization in Jiangsu Province(HPK201804) and Jiangsu Province Industry University Research Project (BY2019133).