Carboxymethylcellulose films containing chlorhexidine–zirconium phosphate nanoparticles: antibiofilm activity and cytotoxicity

Abstract

In this paper sodium carboxymethylcellulose (SCMC) films containing chlorhexidine (CLX) loaded into layered zirconium phosphate (ZrP) nanoparticles were prepared with the aim of obtaining wound dressings with antibiofilm activity but reduced cytotoxicity. CLX, which is a broad spectrum antimicrobial agent, unfortunately with high cytotoxicity, was intercalated between the layers of ZrP in order to prolong and localize its release and thus to improve its safety. The intercalated product, ZrP(CLX), was characterized by X-ray powder diffraction (XRPD), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) which revealed a good drug loading (ca. 50% w/w). CLX release from the intercalated product was evaluated as well. Then SCMC composite films containing CLX were prepared and characterized. All films (both films containing neat CLX and intercalated CLX) showed good antimicrobial and antibiofilm activity. As concerns cytotoxicity, it was evaluated using human keratinocytes and fibroblasts. When CLX was intercalated in ZrP, its cytotoxicity was significantly reduced, confirming the potential use of ZrP in drug delivery advanced medical applications.

---

Introduction

Bacterial biofilms are aggregates of microbial cells that colonize and grow on both living and inanimate surfaces.¹ Bacteria in biofilms have physiological and metabolic properties different from those of planktonic cells and have less susceptibility to antibiotics.^2,3 Biofilms can be associated with various microbial infections in the body such as infections of chronic and acute dermal wounds. These kinds of infection are the main cause of delayed healing in surgical wounds, traumatic and burn wounds, and chronic skin ulcers.^3,4 Thus the prevention and control of infections are of primary importance for good wound management and treatment with antimicrobial agents is often required. The systemic administration of antibiotics presents many disadvantages. In fact antibiotic doses required to achieve sufficient systemic efficacy often result in toxic reactions; moreover the poor blood circulation at the extremities in diabetic foot ulcers results in the risk of ineffective systemic antibiotic therapy. Thus the delivery of antibiotics to local wound sites may be a preferred option to systemic administration. The use of dressings to deliver antimicrobials to wound sites can provide a continuous release of the antiseptic agent at the wound surface with long-lasting antimicrobial action preventing biofilm formation in combination with maintenance of a physiologically moist environment for healing. Chlorhexidine (CLX) is a broad-spectrum antibacterial agent, which is active against yeasts such as Candida albicans, and viruses as well. It is a cationic chlorophenyl bisbiguanide (C₂₂H₃₀Cl₂N₁₀) which binds to negatively charged surfaces such as epithelial cells and oral cavity mucosa. Thus, CLX is widely used in clinical dentistry to prevent dental plaque formation and gingival disease, in skin cleaning after an injury and before surgery. Unfortunately CLX has been shown to be cytotoxic in vitro to mammalian cell types present in the wound-bed.^5–7 Moreover, it has been proven that CLX concentrations of 0.001% decreased the viability of primary human dermal fibroblasts in vitro after a 24 h incubation, while a 0.0025% solution resulted in total cell death. The use of CLX formulations therefore has been limited solely to skin cleaning with short-term wound treatments. To improve safety of CLX and to allow its application in long-term solutions, a system able to give a local and prolonged release of CLX at non-toxic but active concentrations would be required.^8–10 The use of inorganic layered nanomaterials in nanomedicine has been growing over the last decade.^11–13 Some layered materials are excellent candidates for use to this scope since their lamellar structures provide a suitable interlayer spacing for drug molecules which can be incorporated, by a process such as ion-exchange. The control of the particle size and intercalation chemistry make them attractive as nanocarriers for delivery of antimicrobial agents. Among them, zirconium phosphate (Zr(HPO₄)₂) is known as host capable of incorporating different types and sizes of guest molecules, which can interact strongly and directly with the layers, taking advantage of its chemical and thermal stability under biological conditions, not cytotoxic, good ion exchange capacity (6.64 meq g⁻¹). Moreover, the peculiar nano-platelet shape should show better adhesion and binding properties in comparison with spherical or cylindrical particles. Recently inorganic layered materials have been proposed for oral and topical administration, for gene therapy and biomedical applications.^14–16 But till now low attention has been focused on layered zirconium phosphate and only a few papers on its use as drug carrier have been reported.^17–23 Zirconium phosphate has a layered structure and each α-layer is made of planes of Zr atoms bonded, on both plane sides, to monohydrogen phosphate groups. Each phosphate group is bonded to three Zr atoms of the plane, while each zirconium is octahedrally coordinated by six oxygens of six monohydrogen phosphate groups. The water molecules can sit in the interlayer region forming a hydrogen bonding network with the phosphate groups.^24,25 Intercalation of small cations via direct ion exchange into α-ZrP is possible due to the exchange of the protons of the phosphate groups. Nevertheless, intercalation of large molecules into α-ZrP via direct ion exchange is extremely slow.²⁴ On the other hand, the use of gels of nanosized ZrP intercalation compounds in aliphatic alcohols prepared by a one pot synthetic procedure²⁶ gives the possibility to intercalate large cations easier, thus making the ZrP nanoplatelets potential nanocarriers for drug delivery.¹⁷ Among the polymers used to obtain hydrocolloid dressings, sodium carboxymethylcellulose (SCMC) is one of the most used.^1,2 It is ether cellulose characterized by carboxymethylcellulose substitution. It is widely used in oral and topical pharmaceutical formulations and in wound treatment.^27–30 Wound hydrocolloid films are used to both moist and dry wounds and have a particular behavior. In fact at the beginning of the application they are impermeable to water vapor but are able to absorb the wound exudates and upon exudate absorption change their physical state with the formation of a gel covering the wound. Thus they become more permeable to water and air. Another advantage is their painless removal with patient compliance improvement. Moreover, SCMC hydrocolloid dressings resulted able to immobilize Pseudomonas aeruginosa and Staphylococcus aureus within the swollen fibres with consequent reduction of harmful bacteria in wounds.³⁰ Hydrocolloids can be used also for the local delivery of active agents such as antimicrobials for infection prevention and treatment. This paper describes the preparation, characterization and antimicrobial activity of carboxymethylcellulose composites films loaded with CLX intercalated ZrP (hereafter ZrP(CLX), intended for microbial wound infection control). Cytotoxicity of prepared films has been evaluated as well.

Experimental section

Materials

Medium molecular weight SCMC (2% solution viscosity of 400–900 mPas) was purchased from A.C.E.F S.p.A. (Fiorenzuola D'Arda, Italy), glycerol 85% was from Sigma-Aldrich Chemical (Milan, Italy). CLX diacetate (C₂₂H₃₀Cl₂N₁₀·2C₂H₄O₂) was kindly supplied by Degussa AG (Hanau-Wolfgang, Germany).

Zirconyl propionate (ZrO_1.26(C₂H₅COO)_1.49, FW = 220 Da) was supplied by Magnesium Elektron Ltd., England. Concentrated orthophosphoric acid (85%, 14.8 M) was supplied by Fluka. Propanol was purchased from Carlo Erba. All other reagents and solvents were purchased from Sigma-Aldrich Chemical (Milan, Italy) and used without further purification.

Synthesis of CLX loaded zirconium phosphate

A gel of alpha-ZrP nanoparticles in ethanol, which contained ca. 8 wt% ZrP, was synthesized following the procedure previously reported.²³ Briefly, 3.3 mmol of zirconyl propionate was dissolved in 10 mL of anhydrous ethanol. Concentrated phosphoric acid was added, at room temperature under stirring, to the above solution so that the H₃PO₄/Zr molar ratio (R) was 6, respectively, and [H₃PO₄] < 2 M. Clear solutions were obtained just after mixing which turned into gels in a few minutes. The gels thus obtained were washed three times with ethanol in order to remove any excess of reagents (phosphoric acid) or by product (propionic acid).

The intercalation process was performed as follows. 1 mol of ZrP gel was added to an ethanol solution of CLX 0.1 M at different molar ratios CLX : ZrP, hereafter R (loading levels). The typical loading levels used were R = 1, 2, 4. The mixture was stirred for three days at room temperature. The suspension was washed three times with abundant ethanol and dried in an oven at 60 °C for one day; then the solid sample was washed three times with water and dried at 60 °C for characterization.

Film preparation

A 1.5% (w/w) SCMC dispersion containing 0.1% v/v glycerol was prepared with stirring at room temperature for ca. 6 hours. Then, after air bubble removal with vacuum, 20 g of the dispersion were cast in a Petri dish (12 cm diameter) and dried at 45 °C for 24 hours. Films containing ZrP(CLX)-x (where x is 1.0, 2.5, 5.0 wt% of ZrP(CLX)) or CLX-x (where x = 0.5, 1.25, 2.5 wt% of CLX) were prepared following the same procedure. For comparison a film containing ZrP 5 wt% was prepared as well. In particular the resulting SCMC dispersion was mixed with a proper weighed amount of the ZrP(CLX) gel or CLX powder to obtain the different loadings. The mixture was stirred overnight and then cast onto a Petri dish. After solvent evaporation (45 °C for 24 h), the composite membrane was peeled off. All composite membranes (hereafter SCMC/ZrP(CLX)-x, where x is the filler loading) were 50 μm thick.

Characterization

The samples were characterized using several analytical methods.

XRPD analysis

X-ray powder diffraction (XRPD) patterns were collected by using a Philips X'Pert PRO diffractometer and a PW3050 goniometer equipped with an X'Celerator detector using a CuKα radiation source with a 2θ step size of 0.017 and a step scan of 60 s. The LFF ceramic tube was operated at 40 kV and 40 mA. To minimize preferred orientations, the powder samples were carefully side-loaded onto a glass sample holder, and rectangle-shaped film stripes were loaded onto an aluminum sample holder.

Thermal gravimetric analysis

Thermal gravimetric analysis (TGA) was performed by using a NETZSCH STA 449 Jupiter thermal analyzer connected to a NETZSCH TASC 414/3A controller at a heating rate of 10 °C min⁻¹ with under a flow of air of approximately 30 mL min⁻¹ up to 1200 °C.

Electron microscopy analysis

Transmission electron microscopy (TEM) analysis was performed by a Philips 208 transmission electron microscope, operating at an accelerating voltage of 100 kV. Powders were rapidly diluted in ethanol and sonicated for a few minutes, then supported on copper grids (200 mesh) precoated with Formvar carbon films, and quickly dried.

Scanning electron microscopy (SEM) images were collected by a Zeiss LEO 1525 FE SEM. The composite films were fractured in liquid N₂. All the samples were coated with a thin layer of chromium before SEM analysis on the fracture surface.

FT-IR spectroscopy

FT-IR spectra were recorded in air, at room temperature, in KBr solid dispersion (Jasco model FT/IR-410, 420 Herschel series – Jasco Corporation Tokyo, Japan) by using the EasiDiff™ Diffuse Reflectance Accessory. Samples were prepared by gently grounding the powders with KBr.

Moisture uptake (MU) and water vapor transmission rate (WVTR)

Film samples (2 cm × 2 cm) were conditioned in a climatic chamber at 37 °C and 75% RH for 48 h and weighed (W₁). Then samples were conditioned in a desiccator under P₂O₅ until constant weight and reweighed (W₂). MU was determined by using the following formula:

% MU = [(W₁ − W₂)/W₂] × 100

WVTR across the films was determined according to the ASTM E96-90 method, Procedure D, with a Payne permeability cup.³¹ Films were mounted on the mouth (3 cm diameter) of a Payne cup containing 20 g of deionized water and this system was weighed, thermostated at 37 °C at 40% relative humidity and finally reweighed after 24 hours. The WVTR was calculated by using the following formula:

WVTR = [(W_i − W_f)/A] × 10⁶ g per m² per day

where A is the area of the cup mouth (706.5 mm²), W_i and W_f are the weights of the cup before and after being placed in the oven, respectively.

In vitro CLX release study from ZrP(CLX)

In vitro CLX release from ZrP(CLX) and a physical mixture of ZrP and CLX was performed by immersing a weighted amount of the samples in 500 mL of a saline solution constituted by NaCl (8.29 g/1000 mL) and CaCl₂ (0.27 g/1000 mL) in sink conditions, under stirring (100 rpm) at 37 °C. CLX release was monitored for 15 days and compared to CLX powder. Four milliliters of dissolution fluid was removed from the vessel at predetermined intervals and replaced by the same volume of fresh dissolution medium. The samples were filtered and CLX content was determined by UV spectrophotometry using an Agilent 8453 UV-Vis spectrophotometer at a maximum absorbance of λ_max = 255 nm. All experiments were done in triplicate and the error was expressed as standard deviation (SD).

In vitro CLX release from CLX loaded films

Film SCMC/ZrP(CLX)-1% and film SCMC/CLX-0.5% were subjected to drug release measurements in flow through Franz diffusion cells (PermeGear, Inc., Bethlehem, Pennsylvania; diameter 20 mm) constituted by a water-jacketed receptor chamber (15 mL) and a donator chamber. The two chambers were separated by a cellulose filter membrane (Filter paper Whatman 41, 20–25 μm; Whatman GmbH, Dassel, Germany). The saline solution with the composition described above was used as a receptor phase, maintained at 37 °C and stirred at 600 rpm. Each film (ca. 35 mg) was loaded on the upper donor chamber and was successively sealed with (Parafilm® Carlo Erba Reagenti S.p.A., Arese, Milano, Italy). At regular time intervals, samples of the receiving phase were withdrawn and CLX content was determined spectrophotometrically at λ_max 255.0 nm by using a UV-Vis spectrophotometer (model 8453; Agilent). The experiments were carried out in sink conditions, data are reported as an average of three measurements, and the error is expressed as ±SD.

Microrganisms

Quantitative analyses were performed in triplicate for three bacterial species: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Streptococcus pyogenes (ATCC20565) and Pseudomonas aeruginosa (ATCC 15692). Microbial inocula were prepared by subculturing bacteria into Muller Hinton Broth (MHB).

Antimicrobial susceptibility test

Antimicrobial susceptibility test was performed using the Kirby–Bauer disk diffusion method. Bacterial sensitivity was tested for the films, gentamicin was used as positive control. Films were cut in 5 mm diameter disks and sterilized under UV rays for 60 minutes. Muller Hinton agar plates were used for testing different bacteria. After adjusting the bacterial suspension to the concentration of 0.5 McFarland, 10 μL inoculum was spread on the agar plate. Film were placed on the top of the culture plates and incubated for 24 h at 37 °C. The guidelines of clinical and laboratory standard institute (CLSI) was followed to determine the disk zone diameters.³²

SCMC films effect on biofilm formation

The in vitro static biofilm assay was performed using a 96-well microtiter plate as previously described with some modification.³³ To grow biofilms, overnight cultures of S. aureus, S. epidermidis, S. pyogenes and P. aeruginosa were diluted 1 : 100 into 15 mL of growth medium (TSB supplemented with 2% sucrose) in presence or in absence of different films. Cultures were incubated at 37 °C for 24 h in static conditions. After incubation, SCMC films were removed and the microbial biofilm developed in each well was washed twice with 200 μL of sterile PBS and then dried for 45 minutes. In each well, 100 μL of 0.4% crystal violet were added for 15–30 minutes. After this procedure, the wells were washed twice with PBS and immediately discolored with 200 μL of ethanol. After 45 minutes 100 μL of discolored solution was transferred to a well of a new plate and the crystal violet measured at 570 nm in a microplate reader (Tecan). The amount of biofilm formed was measured comparing the absorbance values of the film treated wells versus untreated control wells. Biofilm formation bioassays were performed in triplicates in at least two independent experiments.

Cell viability assay

The cytotoxicity was tested by the determination of the cell ATP level by ViaLight® Plus Kit (Lonza). The method is based upon the bioluminescent measurement of ATP that is present in all metabolically active cells. The bioluminescent method utilizes an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer. All films were tested on human dermis fibroblast (HuDe) and human skin keratinocytes (NCTC2544) cells, which were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 10 000 units penicillin and 10 μg streptomycin per mL overnight to confluence. Monolayer cells were treated for 24 h at 37 °C with films sterilized by UV light exposure for 60 minutes. After incubation plates were left at room temperature to cool for 10 minutes and then the cell lysis reagent was added to each well to extract ATP form the cells. Next, after 10 minutes the AMR Plus (ATP Monitoring Reagent Plus) was added and after 2 more minutes the luminescence was read using a microplate luminometer (TECAN). Results are expressed as % relative light units (RLU) considering untreated cells as 100.

Statistical analysis

Statistical significance was determined using t test. Data are expressed as the mean ± SD of three different experiments. A value of P < 0.05 was considered significant.

Results and discussion

ZrP(CLX) synthesis and characterization

Intercalation of CLX was performed in a one-pot reaction by equilibrating ethanol intercalated ZrP nanoparticles with CLX solutions in ethanol. The solid recovered (ZrP(CLX)) was characterized by TEM, TGA, XRPD and FTIR.

Fig. 1 shows the TEM images of pristine ZrP and ZrP(CLX) samples. It can be observed that the ZrP(CLX) particles are similar to those of the ZrP starting materials, with size of some tens of nanometers, and differ from them for a certain degree of agglomeration.

Fig. 1 TEM images of pristine ZrP (on the left) and ZrP(CLX) (on the right).

The weight loss curves of ZrP(CLX) samples are reported in Fig. 2, together with that of pristine ZrP, obtained after drying the corresponding ZrP gel in ethanol and storing the solid over 53% RH. The overall weight loss of ZrP(CLX) samples was much larger than that of pristine ZrP material for all R values.

Fig. 2 TGA of pristine ZrP and intercalation compounds at different molar ratios R = CLX/ZrP.

Specifically, ZrP showed two main weight losses: the loss of hydration water occurred in the range 20–200 °C, while the step between 200 and 800 °C was attributed to the loss of water due to the condensation of the HPO₄ groups. This reaction leads to the formation of cubic zirconium pyrophosphate (PDF# 085-0896) as confirmed by the XRDP pattern of the solid recovered after the TGA analysis (Fig. S1†). The thermogravimetric curves of the ZrP(CLX) samples showed again two steps in the ranges 20–200 °C and 200–800 °C. As for unmodified ZrP, the first step is attributed to the loss of water, while the second step, starting above 200 °C and arising from two overlapped weight losses, is associated with the thermal decomposition of CLX and the condensation of the phosphate groups to produce the pyrophosphate. From the weight loss corresponding to the second step of the TG curve, it was possible to calculate the CLX moles (x) per Zr atom after subtracting the water coming from the phosphate condensation: the maximum amount of CLX was around 0.5 mol per ZrP formula unit, which was about 50% of the weight of the intercalation compound.

The XRPD patterns for the ZrP gel and the ZrP(CLX) gels prepared at different CLX/ZrP molar ratios (R) are reported in Fig. 3a. In both cases, the patterns essentially showed only one strong reflection at low 2θ values, associated with the layer separation (d), and some weak higher order effects. The first reflection in the pattern of the ZrP gel (2θ = 6.2°) is associated with the layer separation (d = 14.3 Å) of the starting ethanol–intercalated ZrP and shifts to 2θ = 4.4° (d = 20.1 Å) when the gel is equilibrated with CLX. The increase of the interlayer distance from 14.3 Å to 20.1 Å proves the successful intercalation of CLX. This is confirmed by the different evolution of the XRPD patterns when the ZrP and ZrP(CLX) gels are dried (Fig. 3b). In the first case, the first reflection shifts to higher theta values and the interlayer distance decreases from d = 14.3 Å to d = 7.6 Å as a consequence of ethanol de-intercalation. In the second case the position of the first reflection does not change: this is consistent with the presence of the bulky CLX molecules in the ZrP interlayer region.

Fig. 3 XRPD patterns of the ZrP gel and of the ZrP(CLX) gel compounds (a) and XRPD patterns of the ZrP and of the ZrP(CLX) compounds dried at 60 °C (b) at different CLX/ZrP molar ratios (R).

The peak broadening observed after drying ZrP(CLX) gel can be attributed to the loss of a small amount of co-intercalated ethanol which can cause some structural disorder in the interlayer region, without altering the interlayer distance of ZrP(CLX).

Taking into account that the planar dimension the phenyl ring in CLX is 5 Å (ref. 34) and the thickness of the layer of the ZrP is around 9 Å (ref. 24 and 35) it can be suggested that the CLX was intercalated parallel to the layer to form a double film with the phenyl ring perpendicular to the layer. According to this simple structural model, the interlayer distance of the intercalation compound was expected to be around 19 Å, which is very close to the d value for the first peak in the XRPD patterns (20.1 Å).

To get information about the CLX–phosphate interaction, the FTIR spectrum of ZrP(CLX) is compared with the spectrum of ZrP (Fig. 4). According to ref. 36 and 37 the strong band centered around 1050 cm⁻¹ appearing in the ZrP spectrum is associated with the stretching vibrations of the monohydrogen phosphate groups of ZrP. This band is shifted to lower frequencies by 50–80 cm⁻¹ when deprotonated phosphate group are formed as a consequence of ion exchange or intercalation reactions.³⁸ The presence of a similar shift in the spectrum of ZrP(CLX) suggests that protons are transferred from the monohydrogen phosphate to the amino groups of intercalated CLX.

Fig. 4 FT-IR spectra of ZrP (a) and ZrP(CLX) (b).

In vitro CLX release from ZrP(CLX)

The in vitro release profile of CLX from ZrP(CLX) is reported in Fig. 5. CLX release from ZrP/(CLX) was compared to those from neat crystalline CLX and from the ZrP and CLX physical mixture (ZrP–CLX), obtained by softly mixing CLX and ZrP with a spatula. While the dissolution of CLX and the release of CLX from ZrP–CLX was very fast and it took about 10 minutes for a complete drug release, CLX release from ZrP(CLX) was very slow and reached about 11% after 8 hours and about 40% after 11 days. This showed that, as a consequence of CLX intercalation between the ZrP lamellae, CLX release was modified and prolonged. This result could be explained with the mechanism of CLX release which was due to ion exchange between CLX and the ions (Na⁺ and Ca²⁺) present in the release medium which consisted of a solution of sodium and calcium hydrochloride. It is noteworthy that in vitro release experiments, carried out in a phosphate buffer containing Na⁺ and K⁺, no significant CLX release was observed. This could be explained taking into account that the affinity of Ca²⁺ for ZrP is higher than that of monovalent cations Na⁺ and K⁺.³⁹

Fig. 5 The in vitro release profile of CLX from ZrP(CLX).

Sodium carboxymethylcellulose composite film characterization

ZrP(CLX) samples were used to prepare composite membranes based on SCMC. The SEM analysis on the fracture section for the films of neat SCMC and for the composite films is shown in Fig. S2.† The neat SCMC film revealed a homogeneous and smooth surface. However, the homogeneity and smoothness of the film surface decreased with the addition of inorganic particles forming agglomerates of micrometric size. For filler loadings up to 5 wt%, these agglomerates have size in the range 1–3 μm and are quite well dispersed over the cross-section of the polymer.

The XRPD patterns of the SCMC/ZrP(CLX)-x composite membranes are shown in Fig. 6. For comparison the patterns of the films of neat polymer and that containing only 2.5% CLX are reported. Since the CLX was nearly X-ray amorphous, the patterns of the composites showed the SCMC characteristic broad reflection centered at 2θ = 20°,⁴⁰ that was unchanged also in the presence of the filler. It is important to point out that the patterns of the composite films containing ZrP(CLX)-x showed the reflection due to 0 0 2 crystallographic planes (2θ ≈ 4.4°) of the filler and that the intensity increased with increasing filler loading.

Fig. 6 XRPD patterns for a neat SCMC film (a), SCMC/CLX-2.5% film (b) and for SCMC/ZrP(CLX)-x composite film with 1 wt% (c), 2.5 wt% (d), 5 wt% (e) filler loadings.

Moisture uptake (MU) and water vapor transmission rate

Once hydrocolloid dressings come in contact with moisture or exudates, they change their physical state and turn into semisolid gels and become progressively more permeable to water and air. Thus with the aim of evaluating the effects of the filler on the SCMC film behavior, the moisture uptake was evaluated and results are reported in Table S1.†

The water percentage adsorption after 48 h was 30.0 wt% for the SCMC film and the presence of the filler did not change the capacity of adsorbing water.

The water vapor transmission rate was determined only for films SCMC and SCMC/ZrP(CLX)-5% and results were 1316 and 1217 g per m² per day respectively, showing only a slight decrease of water vapor transmission rate.

In vitro CLX release from films

CLX in vitro release from film SCMC/ZrP(CLX)-1% was evaluated and compared with that from film SCMC/CLX-0.5% (Fig. 7). CLX release from film SCMC/CLX-0.5% was slow as film swelling and conversion into gel was necessary to permit CLX diffusion. Besides CLX release could be further decreased by the interaction between the positively charged CLX and the negatively charged carboxylic group of SCMC.⁴¹

Fig. 7 In vitro cumulative release of CLX from SCMC/CLX-0.5% film (a) and from SCMC/ZrP(CLX)-1% composite film (b).

As expected, CLX release resulted even slower when it was intercalated between zirconium phosphate lamellae (film SCMC/ZrP(CLX)-1%), because in this case, in addition to film swelling, a further step was necessary before drug release, that is ion exchange between positive charged CLX and the cations of the release medium.

Antimicrobial activity

The antibacterial properties of films were determined using the disk-diffusion Kirby–Bauer test according to directions of Clinical Laboratory Standards Institute. SCMC–CLX films showed a good antibacterial activity against Gram positive bacteria Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes and the Gram negative Pseudomonas aeruginosa (Table 1). The zone of inhibition (ZOI) increased as a function of the CLX concentration. When CLX was intercalated in ZrP the antimicrobial activity was significantly less than in film containing SCMC and CLX for all bacteria except Streptococcus pyogenes that showed a greater variability than the other microorganisms. This effect could be explained by the slow release of CLX intercalated into ZrP. Films made of neat SCMC and containing ZrP (5 wt%) without CLX did not have any anti-microbial activity.

Table 1 Antimicrobial activity of chitosan films versus Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes. Results are expressed as zone of inhibition (ZOI) measured in mm. Data are the mean ± SD of three different experiments. *P < 0.05 (antimicrobial activity of SCMC/ZrP(CLX) film versus antimicrobial activity of SCMC/CLX)

ZOI diameter (mm)	Pseudomonas aeruginosa	Staphylococcus epidermidis	Staphylococcus aureus	Streptococcus pyogenes
SCMC 1.5%	—	—	—	—
SCMC/CLX 0.5%	9.8 ± 0.8	15.8 ± 0.5	15 ± 0.8	22.5 ± 4.0
SCMC/CLX 1.25%	10 ± 0.5	17 ± 0.8	16.1 ± 0.6	23.7 ± 5.5
SCMC/CLX 2.5%	10 ± 0.5	18.6 ± 0.5	17.8 ± 1.0	23 ± 6.1
SCMC/ZrP 5%	—	—	—	—
SCMC/ZrP(CLX)-1%	9.2 ± 0.6*	13 ± 0.8*	12.3 ± 0.5*	18.8 ± 5.4
SCMC/ZrP(CLX)-2.5%	8.7 ± 0.3*	13.4 ± 1.3*	12 ± 0.8*	19 ± 3.6
SCMC/ZrP(CLX)-5%	9.0 ± 0.0*	14.4 ± 1.1*	13.3 ± 1.0*	19.5 ± 4.9
Gentamicin	20.3	30 ± 0.0	21 ± 1.8	27.3 ± 5.8

---

Antibiofilm activity

To analyze in depth the antimicrobial properties of SCMC films, we examined the ability of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Pseudomonas aeruginosa to form biofilm in absence or in presence of SCMC, SCMC/ZrP, SCMC/CLX and SCMC/ZrP(CLX) films. Since the films with different concentrations of CLX had shown a similar antimicrobial activity, only the film with the lowest CLX concentration was tested in the biofilm inhibition assay. Biofilms were grown in static condition in the presence of films and biofilm formation was measured by determining the mass of biofilm using crystal violet staining. All films showed a good antibiofilm activity against the Gram positive bacteria Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes and the Gram negative Pseudomonas aeruginosa (Fig. 8). No difference was found between the film containing SCMC/CLX and SCMC/ZrP(CLX). This phenomenon could be due to the fact that at the concentrations reached during the antibiofilm tests both with SCMC/CLX and SCMC/ZrP(CLX) films, bacterial cells are damaged and the effect on biofilm formation reaches the plateau. In fact, even if CLX is released in a different manner from SCMC/CLC and SCMC/ZrP(CLX) films, CLX concentrations from both films are above 1.5 μg mL⁻¹ which is the concentration of CLX able to induce a reduction of 99.9% in the viable bacterial counts.⁸ As a matter of fact, on the basis of the behavior described in the in vitro CLX release tests, a concentration of ca. 8.25 μg mL⁻¹ from CLX film (about 55% of CLX release) and ca. 2.04 μg mL⁻¹ from ZrP/CLX film (about 10% of CLX release) can be reached after 24 h and both concentrations are above the reported value (1.5 μg mL⁻¹). These results suggested that these films in addition to their ability to inhibit microbial growth were also able to reduce the formation of biofilms which are considered the base of the non-healing and chronic wounds. To our knowledge, this is the first report describing the SCMC/ZrP(CLX) film activity against microbial biofilm formation.

Fig. 8 Films effect on biofilm formation. Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes. *P < 0.05, **P < 0.01 (SCMC/CLX 0.5% treated microorganisms versus SCMC 1.5% treated cells; SCMC/ZrP(CLX) 1% treated microorganisms versus SCMC/ZrP 5% treated cells).

Cell viability

The biocompatibility of ZrP nanoplatelets and their use in drug delivery has been reported.¹⁵ For the potential use of films as advanced wound dressing, the cytotoxicity of SCMC film loaded with ZrP and CLX in HDF human dermal fibroblast cells (HuDe) and human keratinocyte (NCTC2544) cell lines was tested. Cell monolayers 37 °C cell viability was determined by ATP level measurement. The results reported in Fig. 9 showed that the film of SCMC and that containing ZrP did not result as cytotoxic, whereas SCMC/CLX resulted in being significantly toxic for HuDe fibroblast (Fig. 9A) and keratinocyte (Fig. 9B) cells lines. When CLX was intercalated in SCMC/ZrP the cytotoxicity resulted significantly reduced maybe because the prolonged CLX release maintained low CLX concentration. These results confirm the potential use of ZrP in drug delivery advanced medical application.

Fig. 9 Cytotoxicity of film on Human Dermal Fibroblasts (HuDe) (A) and human keratinocytes (NCTC2544) (B). Cell viability was detected after 24 h of contact of film with cell monolayers. *, P < 0.05 cell viability of film treated cells versus untreated cells, #, P < 0.05 SCMC/ZrP(CLX) treated cells versus SCMC/CLX treated cells.

Conclusions

In this paper the preparation of hybrid composite films with antimicrobial and antibiofilm activity, but with reduced cytotoxicity is described. They were prepared by the use of the hydrocolloid SCMC and CLX loaded ZrP. This inorganic matrix, whose use as drug carrier has been proposed recently and only in a few papers, resulted able (i) to intercalate large molecules in a convenient one pot reaction without using preintercalators, by direct ion exchange, (ii) to release CLX in a prolonged manner and (iii) to formulate composite films as potential wound dressings with antimicrobial and antibiofilm activity and with reduced cytotoxicity. In conclusion the prepared films can represent a promising tool to prevent bacterial colonization and biofilm formation on wound surface.

References

1. J. S. Boateng, K. H. Matthews, H. N. E. Stevens and G. M. Eccleston, J. Pharm. Sci., 2008, 97, 2892.
2. L. I. F. Moura, A. M. A. Dias, E. Carvalho and H. C. de Sousa, Acta Biomater., 2013, 9, 7093.
3. R. A. Cooper, T. Bjarnsholt and M. J. Alhede, J. Wound Care, 2014, 23, 570.
4. C. G. Echague, P. S. Hair and K. M. Cunnion, Adv. Skin Wound Care, 2010, 23, 406.
5. G. Faria, C. R. B. Cardoso, R. E. Larson, J. S. Silva and M. A. Rossi, Toxicol. Appl. Pharmacol., 2009, 234, 256.
6. E. Hidalgo and C. Dominguez, Toxicol. In Vitro, 2001, 15, 271.
7. S. Rossi, M. Marciello, G. Sandri, F. Ferrari, M. C. Bonferoni, A. Papetti and C. Caramella, Pharm. Dev. Technol., 2007, 12, 415.
8. A. Agarwal, T. B. Nelson, P. R. Kierski, M. J. Schurr, C. J. Murphy, C. J. Czuprynski, J. F. McAnulty and N. L. Abbott, Biomaterials, 2012, 33, 6783.
9. B. Jiang, G. Zhang and E. M. Brey, Acta Biomater., 2013, 9, 4976.
10. U. Costantino, V. Ambrogi, M. Nocchetti and L. Perioli, Microporous Mesoporous Mater., 2008, 107, 149.
11. S.-J. Choi, G. E. Choi, J.-M. Oh, Y.-J. Oh, M.-C. Park and J.-H. Choy, J. Mater. Chem., 2010, 20, 9463.
12. X. Wang, Y. Du and J. Luo, Nanotechnology, 2008, 19, 1.
13. M. Nocchetti, A. Donnadio, V. Ambrogi, P. Andreani, M. Bastianini, D. Pietrella and L. Latterini, J. Mater. Chem. B, 2013, 1, 2383.
14. V. Ambrogi, M. Nocchetti and L. Latterini, Langmuir, 2014, 30, 14612.
15. C. Aguzzi, P. Cerezo, C. Viseras and C. Caramella, Appl. Clay Sci., 2007, 36, 22.
16. A. Díaz, V. Saxena, J. González, A. David, B. Casañas, C. Carpenter, J. D. Batteas, J. L. Colón, A. Clearfield and M. D. Hussain, Chem. Commun., 2012, 48, 1754.
17. V. Saxena, A. Diaz, M. D. Hussain, A. Clearfield and J. D. Batteas, Nanoscale, 2013, 5, 2328.
18. A. Díaz, A. David, S. E. Wark, P. Zhang, R. Pérez, M. L. González, A. Clearfield, A. Báez and J. L. Colón, Biomacromolecules, 2010, 11, 2465.
19. J. Ai, Y. Chen, H. Jing, X. Gao, J. Liu, K. Ma, J. Suo, S. Yu and S. Song, Sci. Adv. Mater., 2014, 6, 2603.
20. G. Fardella, G. Grandolini, V. Ambrogi and I. Chiappini, Acta Technol. Legis Med., 1997, 8, 153.
21. G. Fardella, G. Grandolini, V. Ambrogi and I. Chiappini, Acta Technol. Legis Med., 1997, 8, 125.
22. A. Díaz, M. L. González, R. J. Pérez, A. David, A. Mukherjee, A. Báez, A. Clearfield and J. L. Colón, Nanoscale, 2013, 5, 11456.
23. A. Clearfield and U. Costantino, in Comprehensive Supramolecular Chemistry, ed. G. Alberti, and T. Bein, Pergamon Elsevier Science Ltd., New York, 1996, vol. 7, ch. 4.
24. A. Clearfield and G. D. Smith, Inorg. Chem., 1969, 8, 431.
25. M. Pica, A. Donnadio, D. Capitani, R. Vivani, E. Troni and M. Casciola, Inorg. Chem., 2011, 50, 11623.
26. N. A. Ramli and T. W. Wong, Int. J. Pharm., 2011, 403, 73.
27. S.-F. Ng and N. Jumaat, Eur. J. Pharm. Sci., 2014, 51, 173.
28. T. W. Wong and N. A. Ramli, Carbohydr. Polym., 2014, 112, 367.
29. M. Walker, J. Hobot, G. Newman and P. Bowler, Biomaterials, 2003, 24, 883.
30. H. Namazi, R. Rakhshaei, H. Hamishehkar and H. S. Kafil, Int. J. Biol. Macromol., 2016, 85, 327.
31. L. Ruiz-Cardona, Y. D. Sanzgiri, L. M. Benedetti, V. J. Stella and E. M. Topp, Biomaterials, 1996, 17, 1639.
32. Clinical and Laboratory Standards Institute (CLSI), Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, Wayne, PA, 8th edn, 2009, M07–A8.
33. T. Iwase, Y. Uehara, H. Shinji, A. Tajima, H. Seo, K. Takada, T. Agata and Y. Mizunoe, Nature, 2010, 465, 346.
34. N. Meng, N.-L. Zhou, S.-Q. Zhang and J. Shen, Int. J. Pharm., 2009, 382, 45.
35. M. Casciola, D. Capitani, A. Donnadio, G. Munari and M. Pica, Inorg. Chem., 2010, 49, 3329.
36. S. E. Horsley, D. V. Nowell and D. T. Stewart, Spectrochim. Acta, Part A, 1974, 30, 535.
37. M. Casciola, A. Donnadio, F. Montanari, P. Piaggio and V. Valentini, J. Solid State Chem., 2007, 180, 1198.
38. M. Casciola, U. Costantino and S. D'Amico, Solid State Ionics, 1986, 22, 127.
39. G. Alberti, M. G. Bernasconi, M. Casciola and U. Costantino, J. Chromatogr., 1978, 160, 109.
40. Q. Zhao, J. Qian, Q. An, Z. Gui, H. Jin and M. Yin, J. Membr. Sci., 2009, 329, 175.
41. V. Ambrogi, L. Perioli, F. Marmottini, M. Moretti, E. Lollini and C. Rossi, Journal of Pharmaceutical Innovation, 2009, 4, 156.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04151e

---