Film Wound Dressing with Local Anesthetic Based on Insoluble Carboxymethycellulose Matrix

Journal of Applied Biomedicine 15 (2017) 313–320

Contents lists available at ScienceDirect

Journal of Applied Biomedicine

journal homepage: www.elsevier.com/locate/jab

Original research article Film wound dressing with local anesthetic based on insoluble carboxymethycellulose matrix

Lenka Vinklárkováa, Ruta[14_TD$IF] Masteikováa,*, Gabriela Foltýnováa, Jan[15_TD$IF] Muselíka, Sylvie Pavlokováa, Jurga[16_TD$IF] Bernatoniene_ b, David[17_TD$IF] Vetchýa a[18_TD$IF]University of Veterinary and Pharmaceutical Sciences Brno, Faculty[19_TD$IF] of Pharmacy, Department of Pharmaceutics, Brno,[120_TD$IF] Czech Republic b Lithuanian[123_TD$IF] University of Health Sciences, Faculty[124_TD$IF] of Pharmacy, Department of Drug Technology and Social Pharmacy, Kaunas,[125_TD$IF] Lithuania

ARTICLE INFO ABSTRACT

Article history: Received 31 August 2016 The aim of the presented research was to formulate, prepare and evaluate novel film wound dressings Received in revised form 19 May 2017 containing lidocaine hydrochloride. The conversion of partially substituted fibrous sodium carboxy- Accepted 7 August 2017 methylcellulose (CMC) to an acidic form of CMC enabled the formation of an insoluble matrix which Available online 23 August 2017 consequently provided the prepared films with excellent handling properties in their wet state. The drug concentration which was incorporated into an external layer of the film was 5 mg/cm2. The films Keywords: demonstrated satisfactory mass and drug content uniformity as well as an acidic surface pH Carboxymethylcellulose partially advantageous for wound application. An in vitro drug release test proved that the insoluble CMC substituted matrix served as a reliable carrier without slowing down the release of lidocaine hydrochloride more Carboxymethylcellulose acidic form than 90% of the drug was released during the first 15 min, indicating a quick rate of anesthetic action. The Film wound dressing fi fi Topical anesthesia prepared lms could be potential wound dressings for comfortable and ef cient topical anesthesia Lidocaine before/after procedures on the wound. © 2017 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published by Elsevier Sp. z o.o. All rights reserved.

Introduction Separate analgesic strategies may be required for background pain and the pain arising from wound procedures (Orsted, 2010). Wound-related pain can be temporary (acute) or persistent Guidelines for pharmacological wound pain management based on (chronic) (Woo et al., 2008). Acute wound pain can be exacerbated the recommendations by the World Health Organization recom- whenever the wound is handled or manipulated: during dressing mend the use of non-steroidal anti-inflammatory drugs (NSAIDs) removal, wound cleansing or debridement (removing of necrotic or acetaminophen for patients with mild to moderate pain (Briggs tissues). In contrast, persistent (chronic) wound pain is a and Bou, 2002; Woo et al., 2008). NSAIDs provide good pain relief. background symptom that exists at rest and between wound- Moreover, they can positively influence inflammatory processes in related procedures. The level of pain depends on the causes of the the wound, since there is a tendency in chronic wounds for the pain procedural pain (e.g. during dressing removal) and inflammatory response (an important element in the initial operative pain (e.g. wound debridement) are worse than pain at wounding response) to become exaggerated (Boateng and rest (Arroyo-Novoa et al., 2009). Unresolved pain predisposes Catanzano, 2015). The treatment of wound infection, by reducing individuals to stress and associated physiological responses that bacterial load and thereby reducing the inflammatory stimulus to can impair wound healing (Boateng and Catanzano, 2015; Woo, the nervous system, should also result in a reduction in pain 2011), often leading to the need for pharmacological interventions (Boateng and Catanzano, 2015; Sarheed et al., 2016). Unfortunately, for pain prevention. oral use of NSAIDs as well as systemic administration of high antibiotic doses can lead to serious side effects such as gastrointestinal problems, risk of renal failure, prolonged bleeding time due to impaired coagulation, allergic reactions etc. (Meek et al., 2010; Sarheed et al., 2016; Wallace and Vong, 2008). For this * Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, reason, non-pharmacological strategies and topical agents to University of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1946/1, achieve optimal wound-related pain management are an attractive Brno 612 42, Czech Republic. E-mail address: [email protected] (R. Masteiková). solution. Topical agents and correctly selected dressings play a http://dx.doi.org/10.1016/j.jab.2017.08.002 1214-021X/© 2017 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published by Elsevier Sp. z o.o. All rights reserved. 314 L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320 critical role in alleviating wound-related pain (Boateng et al., 2008; removingthe dressing(Sussman,2010).Filmspreparedfromsodium Woo et al., 2008). Recently, an evaluation of the effect of ibuprofen carboxymethylcellulose (NaCMC) possess all these characteristics. in the form of a foam dressing (Biatain Ibu) on persistent and Moreover, carboxymethylcellulose (CMC) is generally regarded as a temporary wound pain underwent clinical trials (Fogh et al., 2012). nontoxic,nonirritant,andbiocompatiblematerialwhichpredestines The ibuprofen foam dressing was shown to consistently relieve it for use in food, cosmetic, pharmaceutical, and biomedical wound pain in exuding wounds of various etiologies (Romanelli applications, including materials for wound care (Rowe et al., et al., 2009), thus, local pain relief by this dressing is possible in the 2015). All of these factors make it a suitable candidate in the most common, painful, exuding, chronic and acute/traumatic preparation of medicated film. A whole range of scientific works wounds and therefore is a safer alternative for systemic pain deals with the preparation and evaluation of medicated CMC films. treatment (Arapoglou et al., 2011). However, these films are mainly intended for buccal/oral drug For the management of procedural or operative pain, local delivery (Gajdziok et al., 2015; Landová et al., 2013; Raju et al., 2011; anesthesia, which includes infiltration or topical application of Ramineni et al., 2013; Saha et al., 2013; Semalty et al., 2010; Vetchý anesthetics, is usually used. Infiltration anesthesia (injection into et al., 2014), and much less for wound therapy (Donnadio et al., 2016; the tissue in and around the wound) per se induces discomfort, Vinklárková et al., 2015). The application properties of film wound may worsen “needle anxiety” in pediatric subjects, and distort the dressings differ quite significantly from those intended for buccal/ wound site (Eidelman et al., 2005). Moreover, Eidelman et al. oral applications. Wound dressings are applied on a much larger (2005) found that the majority of clinical trials demonstrated surface area than buccal preparations. For this reason, good equivalent or superior analgesic efficacy for topical formulations mechanical properties of medicated CMC films are required. compared with conventional lidocaine infiltration. Especially once wetted, they must maintain the cohesiveness that Lidocaine is an essential drug on the World Health Organiza- enables them to be easily manipulated and removed without tion’s essential drug list, and is considered efficacious, safe and residues. One option is to reinforce the film with a supporting cost-effective for any health-care system (Weinberg et al., 2015). material as was done in our previous study (Vinklárková et al., 2015). The efficacy of topical lidocaine alone or in combination with other The other option is to prepare film containing an acidic form of CMC anesthetic agents in the management of acute wound pain has (HCMC), which is insoluble in water, thus more wet resistant. The been confirmed in clinical practice (Cuomo et al., 2015; Desai et al., idea that film containing HCMC would be of increased strength was 2014; Gaufberg et al., 2007; Little et al., 2009; Pasero, 2013). described for the first time by Butler (1962) in his US patent. According to Sussman and Bates-Jensen (2012), lidocaine in the However, the patent contains only the general preparation form of a soak may be recommended as a quick and efficient way to conditions, and lacked precise description of the technology and reduce local wound pain during debridement procedures. Another evaluations methods necessary for the preparation of films with option is EMLA cream (containing a eutectic mixture of lidocaine suitable properties for wound application. Moreover, the technique and prilocaine), which is only intended for contact with intact described inpatent was for non-medicatedfilms only. Films based on adjacent skin. Nevertheless, Blanke and Hallern (2003) found that, insoluble CMC with an active substance have not yet to be prepared on the basis of clinical experience, the topical application of EMLA and evaluated. cream directly to the wound before sharp debridement is efficient, The aim of the presented research was to prepare a novel film economical, safe, and tolerable for the patient. One common wound dressings based on an insoluble carboxymethylcellulose disadvantage of topical analgesics is the amount of time needed matrix with lidocaine hydrochloride as an active compound, and before effect (usually 20–30 and up to 60 min prior to procedures evaluate their physicochemical properties as well as in vitro drug (Sussman and Bates-Jensen, 2012)). For this reason they are more release. suitable for non-emergent procedures. Another deficiency of topical liquid or semisolid anesthetics is the messiness of Material[13_TD$IF] and methods application and removal (Little et al., 2009). Currently, if the anesthetic must be applied directly to the wound topically, there is The partially substituted (degree of substitution 0.34) sodium no other choice than a liquid or semisolid preparation. Thus the carboxymethylcellulose (NaCMC) in the form of non-woven textile development of new, more sophisticated forms is required. Film (Hcel1 NaT) was supplied by Holzbecher, spol. s r.o., Bleaching & wound dressing is a pharmaceutical form without the drawback Dyeing Plant in Zlíc9 (Czech Republic), lidocaine hydrochloride, messy application and removal. Moreover, film application allows macrogol 300, glycerin and hydrochloric acid (all Ph. Eur. grade) more precise dosing of active substances. were purchased from Fagron (Czech Republic). All other chemicals Films, as pharmaceutical dosage forms, are currently used for and reagents used in the study were of analytical grade. oral, buccal or ophthalmic application with a variety of medicated or non-medicated preparations on the market. The film wound Preparation of films dressings on the market are non-medicated ones intended only for protection of the wound against the effects of the surrounding Films based on an insoluble carboxymethylcellulose (CMC) environment or mechanical injury (Sussman, 2010). For this matrix were prepared using an sequential solvent casting method. reason, there have been many studies into how to prepare film A modified technique according to the US patent (Butler,1962) was wound dressings with an active substance (Boateng et al., 2013; used. In order to create the insoluble CMC (HCMC) matrix, the Jridi et al., 2017; Liakos et al., 2013; Pereira et al., 2013; Thu et al., acidification of both polymer dispersion and casted film was 2012; Wang et al., 2012). None of these, however, were dedicated to necessary. Both the acidification of the polymer dispersion without the development of a wound dressing with the rapid release of a any further treatment of the film and the acidification of casted local anesthetic. NaCMC film itself without previously acidifying the dispersion Different polymers may be used to prepare the film. Natural proved insufficient. The acidification of NaCMC film itself did not materials are more friendly on bodily tissues than synthetic ones, achieve required mechanical properties. It is likely that the HCMC and are therefore often studied for wound care applications (Juncu matrix was not completely formed. Similarly, films prepared from a et al., 2016;Maveret al., 2015;Ramli and Wong, 2011;Xu et al., 2007). dispersion acidified to a pH value of 3 or higher without additional An ideal film dressing must be supple and possess homogenous and treatment of casted films were low in quality. Acidification to pH smooth surfaces (Ramli and Wong, 2011). Transparency is another values lower than 3 resulted in precipitation and loss of film- important property allowing for the wound’s assessment without forming properties. The double acidification led to the creation of L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320 315

films with suitable properties. Thus the films were prepared as Surface pH follows: The polymer dispersion was composed of 1% (w/w) NaCMC and 2% (w/w) macrogol 300 or glycerin in purified water. The surface pH of the prepared films was evaluated using a Non-woven sodium carboxymethylcellulose textile (Hcel NaT) was WTW pH 3210 SET 2 pH-meter (WTW, Germany) with a flat glass cut into small pieces and poured over with a solution of macrogol electrode. Alterations of the surface pH in the conditions or glycerin in hot water (80 C). This mixture was heated in a water simulating the wound environment were assessed using an bath to maintain a temperature of 80 C for 3 h, then stirred using a artificial wound model (as described above). 6.25 cm2 Cito-UNGUATOR 2000 dispersing device (SMS Heiztechnik GmbH, (2.5 2.5 cm) samples of the film were cut and put on the surface Deutschland) set on program “Gel”, and heated again for 1 h at the of the wound model. The Petri dish was covered with a lid to same temperature. Then the mixture was left to cool and swell at prevent water evaporation, and surface pH was measured at an ambient temperature for 24 h. The next day the NaCMC determined time intervals in triplicate. dispersion was acidified under continuous stirring using a 5% solution of hydrochloric acid (HCl) until a pH value of 3 was Mechanical properties achieved. Then 25 g of the resulting dispersion was casted on a glass Petri dish 10 cm in diameter and pre-dried in the oven A modified method according to Shidhaye et al. (2008) was (Heratherm, Germany) at 70 C for 1 h followed by 24 h of drying at used to evaluate the mechanical properties of the prepared films in ambient conditions. The dry films were left in the Petri dishes, and dry state and after swelling for 3 h on an artificial wound model. acidification/washing was administered as follows: 20 ml of 5% A CT3 Texture Analyzer (Brookfield, USA) equipped with a 4.5 kg solution of HCl for 20 min ! straining ! rinsing with purified load cell and TexturePro CT software was used to determine the water ! 20 ml of purified water for 20 min ! straining ! 20 ml of tensile strength of the prepared films. Film samples (10 40 mm) purified water for 20 min ! straining ! 20 ml of 5% solution of were held between two clamps of a TA-DGA probe positioned at a macrogol or glycerin for 20 min ! straining. Macrogol or glycerin distance of 2 cm. The lower clamp was held stationary and the was added into the final washing liquid to compensate for these strips of the film were stretched by the upper clamp moving at a plasticizers washed away from the film during the acidification/ rate of 0.5 mm/s until the strip broke. The work done during this washing process. The final layer of film was created from a solution process and the deformation (elongation) of the film at the of lidocaine hydrochloride to obtain the required concentration moment of tearing were both measured. This process was repeated of the drug (5 mg/cm2) 5 g of 6.3% solution for one Petri dish were five times for each film sample. added. Once the final layer was applied, the films were left to dry under ambient conditions. The dried films were peeled from the Uniformity of mass Petri dishes, examined visually for morphological defects (e.g. cracks, shrinking, etc.) which can affect handling, testing and For this evaluation, the adapted test as described in European application as well as aesthetic appearance, and stored in a closed Pharmacopoeia (chapter 2.9.5. Uniformity of mass of single-dose box to await testing. Films without active substance were made in preparations) was applied. 20 units of precisely cut samples from the same manner for comparison of physical properties. random sites of the prepared films (2.5 2.5 cm) were weighed individually, and the average mass SD was determined. The Microscopic properties and thickness individual masses were then compared with the average mass and the percentage of deviation was calculated. The microscopic properties of the prepared films were evaluated using an optical microscope (STM-902 ZOOM, Opting, Uniformity of drug content Czech Republic) and a color digital camera (DFW 700, Sony, Japan). The appearance of the films was observed at a magnifica- For this evaluation, the adapted test as described in European tion factor of 20 and 50. Illustrative digital images were taken at the Pharmacopoeia (chapter 2.9.6. Uniformity of content of single- same time. dose preparations) was applied. 10 units (5 5 cm) were precisely At the measurement of film thickness, a rectangular sample of cut from random sites of the prepared films and dissolved the film was vertically fixed in a holder, the microscope was separately in beakers containing 200 ml of PBS. Standard solutions focused on the edge of the film, and the sample thickness was of 0.15, 0.3, 0.45, 0.6 and 0.75% lidocaine hydrochloride (w/w) were measured at 10 different places along the film. This was repeated prepared using a PBS, and calibration curves using HPLC were 5 times with each film sample. constructed. The HPLC system employed was a YL 9100 chromato- graph (Young Lin Instrument, South Korea), using a variable Swelling property wavelength UV detector set at 210 nm. Analysis was performed on a Polaris C18 column (250 4.6 mm; 5 mm) at temperature 30 C. The swelling properties of the prepared films were measured in A mixture containing 0.2% (w/w) phosphoric acid in water and a physiological buffer solution of pH 7.2 (PBS). For these purposes, acetonitrile (75:25) was used as a mobile phase, at a flow rate of an artificial wound model developed by us was used. This wound 1 ml/min and injection volume of 3 ml. The average concentration model consisted of a Petri dish and absorptive sponge soaked in a of lidocaine hydrochloride in each sample, percentage deviation test liquid. The surface of the sponge was rough resembling the and SD were then calculated. surface of wound. The amount of PBS was 20 ml, which was enough to soak the sponge and create a thin layer of a liquid on the surface In[132_TD$IF] vitro drug release imitating the wound exudate. 6.25 cm2 (2.5 2.5 cm) samples of the film were cut and weighed (Wd). The sample was placed on the A standard paddle dissolution apparatus Sotax AT7 Smart surface of the wound model, the Petri dish was covered with a lid to (Donau Lab, Zurich, Switzerland), as described in the European prevent water evaporation, and swollen films were then weighed Pharmacopoeia, was employed to evaluate drug release. A portion 2 at determined time intervals (Ws). The degree of swelling Sw in the of 25 cm (55 cm) of film was used. A side of the film without the film was calculated as drug was attached using double adhesive tape to an inert glass plate as described by Okamoto et al. (2001), then the vessel was Sw = (W W )/W (1) s d d filled with 500 ml of PBS, and maintained at 32 C (temperature of 316 L. Vinklárková et al. / J. Appl. Biomed.[(Fig._1)TD$IG] 15 (2017) 313–320 skin surface) while stirring at 50 rpm. Two-milliliter samples were collected automatically at predetermined time intervals and replaced with an equal amount of PBS to maintain a constant volume. Lidocaine hydrochloride release was quantified by HPLC as described above and was expressed as a cumulative percent released versus time for the 3-h duration of the study.

Statistical analysis

All experiments were carried out at least in triplicate, and data was expressed as a mean standard deviation (SD). Comparison of the means was performed by analysis of variance (ANOVA) at a significance level of a = 0.05. p[103_TD$IF]-values were used as a decisive criterion for indication of significance. The statistical significance of parameters was confirmed if the p[104_TD$IF]-value was equal to or smaller than a. Statistical evaluation of data was performed by means of QC.Expert software, version 3.2.

Results and discussion Fig. 1. Appearance of wetted ﬁlm after application to small lesion.

Partially substituted microfibrous NaCMC (non-woven textile [(Fig._2)TD$IG] Hcel1 NaT) was used in order to increase the mechanical resilience of the films after wetting. It was assumed that non-substituted fibers would act as fillers. According to Paunonen (2013), if the filler is well dispersed in the polymer matrix, an improvement in the mechanical properties is usually observed. Despite the excellent cohesiveness of the wetted films, thanks to the microfibrous NaCMC and proved in our previous study (Vinklárková et al., 2015), it was still necessary to reinforce them even more to enhance handling properties. For this reason, a technological procedure that enables the creation of an insoluble matrix of the acidic form of CMC (HCMC) was chosen. Macrogol 300 and glycerin were used as plasticizers. The solvent casting method was chosen for film preparation. It is a widely used manufacturing process for making films distinguished by its relative simplicity and the low cost that system setup incurs at a research laboratory scale (Morales and McConville, 2011). In the case of medicated film, an active substance may be incorporated in different ways. Considering the fact that the Fig. 2. Microscopic appearance of the film without drug: (A)[10_TD$IF] surface of film, (B) assumed indication of the film was anesthesia before painful edge of film with easily recognizable fibers (magnification 20, bar 500 mm). intervention on the wound, the onset of action must be rapid. For this reason, lidocaine hydrochloride was incorporated as the of bioadhesive/mucoadhesive properties which are distinctive to external layer on only one side of film in the form of fine films from NaCMC (Vetchý et al., 2014). Nevertheless, this change crystallized, evenly distributed and firmly adhered particles on the of properties would be positive when applied to wounds as a local surface (see microscopic appearance). The concentration of the anesthetic, as it enables very easy and quick removal without drug in the film was expressed as mg/cm2 of film. This expression residues. facilitates dosage since wound dressings are applied to a certain [(Fig._3)TD$IG] surface area. Perumal et al. (2008) even postulate that determination by weight is erroneous because the final dosage form is determined by area instead of weight in the particular case of films. Moreover, expression related to area is independent of the weight of the film which may vary considerably, as films made from hydrophilic polymers tend to fluctuate in terms of moisture content (Paunonen, 2013). A concentration of 5 mg/cm2 was chosen on the basis of doses for topical application of lidocaine proven in clinical practice (Cuomo et al., 2015, Pasero, 2013; Sussman and Bates-Jensen, 2012). Visual examination demonstrated that prepared films were homogenous with a smooth surface. Although the presence of insoluble HCMC did not allow the excellent transparency typical of films from NaCMC, the prepared films were translucent enough to facilitate observation of wound conditions, especially when wetted (Fig. 1). Dry films were firm and resilient, and films with glycerin were more flexible than macrogol ones. After wetting they both became soft and pliable, with good adherence Fig. 3. Microscopic appearance of the film with lidocaine hydrochloride: (A)[1_TD$IF] to the skin. However, conversion of NaCMC to HCMC led to a loss magnification 20, bar 500 mm, (B)[12_TD$IF] magnification 50, bar 100 mm. L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320 317

Table 1 Thickness, mass, mechanical properties and uniformity of drug content of prepared ﬁlms.

Films with macrogol 300 Films with glycerin

without with without with lidocaine drug lidocaine drug Thickness (mm)a 71.73 12.12 101.86 8.14 68.36 4.87 101.8 8.53 Mass of sample 2.52.5 cm (mg)b 44.4 2.9 76.2 5.8 37.9 2.6 72.3 4.0 Mechanical propertiesc – dry ﬁlm tensile strength (N) 5.39 0.26 2.83 0.51 5.84 0.53 3.39 0.23

deformation/elongation (mm) 3.12 0.32 3.26 0.47 2.52 0.21 3.09 0.18

work done (mJ) 9.19 0.91 5.87 1.39 8.01 1.24 5.57 0.23

– swelled ﬁlm after 3 h on the artiﬁcial wound model tensile strength (N) 3.53 0.21 1.66 0.17 3.74 0.64 1.81 0.27

deformation/elongation (mm) 4.22 0.15 4.29 0.13 3.52 0.39 5.35 0.29

work done (mJ) 6.82 0.19 3.27 0.27 6.0 1.56 4.83 0.96

Uniformity of drug content (sample 55 cm)d –– – average content (mg) 126.8 7.02 131.06 8.65 – deviation from the stated content (125 mg/25 cm2) (%) 101.44 104.84 – marginal deviation below average content (%) 95.69 93.16 – marginal deviation above average content (%) 108.6 113.5

Mean SD (an[109_TD$IF]= 50, bn = 20, cn =5, dn = 10).

Observation of microscopic appearance of prepared films as pH value, thus determined swelling values of prepared films confirmed that partially substituted CMC maintained a fibrous could adequately reflect those in a real wound. nature (Fig. 2). Medicated films contained evenly distributed The films exhibited a low degree of swelling (Fig. 4), indicating particles of crystallized lidocaine hydrochloride (Fig. 3). weak holding capacity for the exudate while still maintaining their Film thickness is an important parameter from a technological excellent structural integrity for a reasonable time period. standpoint. Uniform thickness indicates a correct preparation The degree of swelling (Sw) increased only slightly with time. method and a strong likelihood of drug content uniformity as well The higher swelling values were obtained from medicated films as a regular process of drug release. The thickness of prepared films (p[105_TD$IF] < 0.05). Most likely, a lidocaine salt of CMC with a higher was dependent on drug content films with lidocaine hydrochlo- swelling responsiveness was formed during preparation. The ride were thicker (p[105_TD$IF]< 0.05) than lidocaine-free ones (Table 1). The influence of type of plasticizer on swelling properties was not influence of type of plasticizer (macrogol or glycerin) was confirmed. negligible (p[106_TD$IF]> 0.05). Alterations to the surface pH of the films during 3 h in The swelling behavior of the films is an important property for conditions simulating a wound environment are shown in Fig. 5. their practical application. Liquid-uptake of the film prevents dry- The figure demonstrates that surface pH increased with time out and creates conditions for moist wound healing (Pišlová et al., (p[105_TD$IF]< 0.05) probably due to an ion-exchange reaction resulting in 2015). It may be affected by several factors such as pH or the the gradual formation of sodium or potassium salts of the CMC. presence and character of ions. A physiological buffer solution of Nevertheless, all prepared films retained acidic pH values pH 7.2 is similar to wound fluid with regard to ion content as well (pH <4.5). This evaluation is very important because it reflects [(Fig._4)TD$IG]

Fig. 4. Swelling behavior of prepared films. [(Fig._5)TD$IG]318 L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320 soft, flexible, pliable and elastic in order to cope with the stresses exerted by different parts of the body (Boateng et al., 2008). These desirable characteristics can be achieved by investigating their tensile properties to ensure a balance between flexibility and rigidity. Fig. 6 demonstrates comparable mechanical properties of prepared films. Films with glycerin had slightly better mechanical properties than macrogol plasticized ones, but this difference was not statistically significant (p[107_TD$IF]> 0.05), thus the influence of plasticizer was not proved. The greatest statistically significant differences (p[105_TD$IF]< 0.05) were noticeable between films with and without lidocaine or dry and swelled ones. Not surprisingly, lower values of tensile strength, higher elongation and less work necessary to tear the film were all observed in swelled films. The lower tensile strength of medicated films was probably caused by the interaction of anionic HCMC and cationic lidocaine hydrochloride leading to the formation of a compound with worse mechanical properties. The fact that added active compounds, in general, affect mechanical properties of CMC films, is also stated by Paunonen (2013). Nevertheless, both medicated films and lidocaine-free ones manifested excellent handling properties after Fig. 5. Alterations of surface pH in the conditions simulating wound environment. swelling (3 h on wound model), making them extremely beneficial [(Fig._6)TD$IG] in clinical applications. It would be interesting to compare the mechanical properties of prepared films with these described in the patent (Butler, 1962) which gave us this idea. Unfortunately, direct comparison was impossible due to differing units of measurement of tensile strength used in the patent which could not be converted. Nevertheless, a relative comparison of strength loss after wetting seemed to favor our films wet films in the patent were ca. five times weaker than dry ones, whereas the weakening of films prepared by us was less than half of dry value after swelling on the wound model (Table 1). This finding confirms the correctness of the developed technology. According to European Pharmacopoeia, not more than 2 of the individual masses may deviate from the average mass by more than 10% if a pharmaceutical form has an average mass 80 mg or less, and none deviates by more than twice that percentage. All prepared films met these requirements. Films prepared with glycerin were slightly lighter than those with macrogol (p[108_TD$IF]< 0.05), regardless of lidocaine content (Table 1). European Pharmacopoeia does not state the requirements for medicated films regarding drug content. Thus, requirements for transdermal patches or ophthalmic inserts, as the most similar

Fig. 6. Mechanical properties of prepared films: tensile strength and deformation/ [(Fig._7)TD$IG] elongation of film at the moment of tearing (*p[9_TD$IF]< 0.05 film without drug vs. film with lidocaine, [13_TD$IF]p < 0.05 dry film vs. wetted film).

the impact of the wound dressing on the wound environment. It is known that pH plays a significant role in wound healing. In general, lowering pH has shown to result in an improvement of wound healing (inhibition of microbial growth, alteration of activity of matrix metalloproteinases etc.) (Percival et al., 2014). Because the supposed duration of the application of the prepared lidocaine films should be relatively short, immediate influence on wound healing need not be significant. However, inhibition of microbial growth is also important for short application. Likewise, synergy of antimicrobial activity should be hypothesized since both lidocaine hydrochloride (Johnson et al., 2008) and acidic form of CMC (Touati et al., 2013) itself are active against microorganisms, thus prepared films should be especially beneficial when treating infected wounds. The mechanical properties of the prepared films are shown in Table 1. Characterization of mechanical properties is important fi because film dressings are required to be durable, stress resistant, Fig. 7. Release of lidocaine hydrochloride from prepared lms. L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320 319 pharmaceutical forms to film wound dressings, should be applied. Conflict of interests[134_TD$IF] In the case of transdermal patches, the preparation complies with the test of if the average content of 10 dosage units is between 90% The authors declare that there is no conflict of interests and 110% of stated content and if the individual content of each regarding the publication of this paper. dosage unit is between 75% and 125% of the average content. In the case of ophthalmic inserts, the preparation complies with the test Acknowledgement of if each individual content is between 85% and 115% of the average content. The values shown in Table 1 confirm that the This work was supported by finances from the Technology prepared films met the requirements for both transdermal patches Agency of the Czech Republic ALFA program (research project No. and ophthalmic inserts. TA04010065). Not surprisingly, drug release from prepared films was very rapid, because lidocaine hydrochloride is highly water-soluble and References was incorporated as the external layer of the film. More than 90% of the drug released during the first 15 min, and the remaining Arapoglou, V., Katsenis, K., Syrigos, K.N., Dimakakos, E.P., Zakopoulou, N., Tsoutsos, D., Gjedsbel, K., Glynn, C., Schäfer, E., Petersen, B., 2011. Analgesic efficacy of an content of lidocaine was released within an hour (Fig. 7). The data ibuprofen-releasing foam dressing compared with local best practice for painful shows almost identical release profiles both for films containing exuding wounds. J. Wound Care 20, 319–325. macrogol and those containing glycerin. Drug release from the Arroyo-Novoa, C.M., Figueroa-Ramos, M.I., Miaskowski, C., Padilla, G., Stotts, N., fi Puntillo, K.A., 2009. Acute wound pain: gaining a better understanding. Adv. prepared lms was similar to the observations made by Murata Skin Wound Care 22, 373–380. et al. (2010), who investigated fast dissolving films expected to be Blanke, W., Hallern, B.V., 2003. Sharp wound debridement in local anaesthesia using applied in the oral cavity or Vakili et al. (2012), who studied EMLA cream: 6 years' experience in 1084 patients. Eur. J. Emerg. Med. 10, 229– lidocaine hydrochloride release from porous cellulose film 231. Boateng, J., Catanzano, O., 2015. Advanced therapeutic dressings for effective wound matrices. Sustained/extended release may be achieved if lidocaine healing a review. J. Pharm. Sci. 104, 3653–3680. is dispersed in a polymer matrix, as done by Repka et al. (2005) or Boateng, J.S., Matthews, K.H., Stevens, H.N.E., Eccleston, G.M., 2008. Wound healing – Okamoto et al. (2001). However, extended release was not the aim dressings and drug delivery systems: a review. J. Pharm. Sci. 97, 2892 2923. Boateng, J.S., Pawar, H.V., Tetteh, J., 2013. Polyox and carrageenan based composite of our study. We assumed that the drug-containing layer would film dressings containing anti-microbial and anti-inflammatory drugs for dissolve within a short time period and release the drug toward the effective wound healing. Int. J. Pharm. 441, 181–191. wound surface where the local anesthetic action of lidocaine Briggs, M., Torra, I., Bou, J.E., 2002. Pain at wound dressing changes: a guide to management. EWMA Position Document Pain at Wound Dressing Changes. MEP hydrochloride could take effect. The dissolution test proved that Ltd., London, pp. 12–17. the insoluble carboxymethylcellulose matrix served as a reliable Butler, R.W., 1962. Preparation of free acid cellulose ether films. US 3 (064), 313. carrier, without slowing down the release of lidocaine hydrochlo- Cuomo, R., D'Aniello, C., Grimaldi, L., Nisi, G., Botteri, G., Zerini, I., Brandi, C., 2015. EMLA and lidocaine spray: a comparison for surgical debridement in venous leg ride. The result was a dosage form that can be expected to provide a ulcers. Adv. Wound Care 4, 358–361. quick and satisfactory rate of anesthetic action as a prepared film Desai, C., Wood, F.M., Schug, S.A., Parsons, R.W., Fridlender, C., Sunderland, V.B., wound dressing. 2014. Effectiveness of a topical anaesthetic spray as analgesia for dressing changes: a double-blinded randomized pilot trial comparing an emulsion with an aqueous lidocaine formulation. Burns 40, 106–112. Conclusion[13_TD$IF] Donnadio, A., Ambrogi, V., Pietrella, D., Pica, M., Sorrentino, G., Casciola, M., 2016. Carboxymethylcellulose films containing chlorhexidine-zirconium phosphate fi – Film wound dressings based on an insoluble carboxymethyl- nanoparticles: antibio lm activity and cytotoxicity. RSC Adv. 6, 46249 46257. Eidelman, A.E., Weiss, J.M., Enu, I.K., Lau, J., Carr, D.B., 2005. Comparative efficacy cellulose matrix were successfully prepared using a sequential and costs of various topical anesthetics for repair of dermal lacerations: a solvent casting method. Films containing lidocaine hydrochloride systematic review of randomized, controlled trials. J. Clin. Anaesth. 17, 106–116. possess properties making them suitable for use as comfortable Fogh, K., Andersen, M.B., Bischoff-Mikkelsen, M., Bause, R., Zutt, M., Schilling, S., fi fi Schmutz, J.-L., Borbujo, J., Jimenez, J.A., Cartier, H., Jørgensen, B., 2012. Clinically and ef cient topical anesthetics. This is due to their rmness and relevant pain relief with an ibuprofen-releasing foam dressing Results from a resiliency in dry state and excellent handling properties, softness, randomized, controlled, double-blind clinical trial in exuding, painful venous pliability, good transparency, and adherence to the skin after leg ulcers. Wound Repair Regen. 20, 815–821. š Š ž fl fi Gajdziok, J., Hole ová, S., tembírek, J., Pazdziora, E., Landová, H., Dole el, P., Vetchý, swelling. The high exibility of wetted lms provides good D., 2015. 2015. Carmellose mucoadhesive oral films containing vermiculite/ adaptability to the wound or other treated surfaces with disrupted chlorhexidine nanocomposites as innovative biomaterials for treatment of oral skin integrity. The prepared films showed an acidic surface pH infections. BioMed. Res. Int. 15 Article ID 580146. Gaufberg, S.V., Walta, M.J., Workman, T.P., 2007. Expanding the use of topical advantageous for wound application, as well as satisfactory mass anesthesia in wound management: sequential layered application of topical and drug content uniformity. The films exhibited a low degree of lidocaine with epinephrine. Am. J. Emerg. Med. 25, 379–384. swelling, indicating weak holding capacity for the exudate, which Johnson, S.M., John, B.E.S., Dine, A.P., 2008. Local anaesthetics as antimicrobial fi agents: a review. Surg. Infect. (Larchmt) 9, 205–213. is typical of the most polymeric lms. An in vitro drug release study Jridi, M., Sellimi, S., Ben Lassoued, K., Beltaief, S., Souissi, N., Mora, L., Toldra, F., Elfeki, confirmed rapid lidocaine hydrochloride release. The principal A., Nasri, M., Nasri, R., 2017. Wound healing activity of cuttlefish gelatin gels and implication of the developed technology is that it is suitable for the films enriched by henna (Lawsonia[135_TD$IF] inermis) extract. Colloids Surf. A: – preparation of both drug-containing film wound dressings and Physicochem. Eng. Aspects 512, 71 79. Juncu, G., Stoica-Guzun, A., Stroescu, M., Isopencu, G., Jinga, S.I., 2016. Drug release non-medicated ones. Films without an active substance could be kinetics from carboxymethylcellulose-bacterial cellulose composite films. Int. J. used to protect slightly exuding or non-exuding wounds (such as Pharm. 510, 485–492. Landová, H., Danek, Z., Gajdziok, J., Vetchý, D., Štembírek, J., 2013. Mucoadhesive epithelizing wounds) against injury and the growth of micro- films as perspective oral dosage form. Ces. Slov. Farm. 62, 4–11. organisms, thanks to its good mechanical properties and low Liakos, I., Rizzello, L., Bayer, I.S., Pompa, P.P., Cingolani, R., Athanassiou, A., 2013. surface pH values. The described technology for drug incorpo- Controlled antiseptic release by alginate polymer films and beads. Carbohydrate ration, when a highly soluble active substance or combination of Polym. 92, 176–183. fi Little, C., Kelly, O.J., Jenkins, M.G., Murphy, D., McCarron, P., 2009. The use of topical such substances form a layer on only one side of the lm, is anaesthesia during repair of minor lacerations in Departments of Emergency particularly beneficial for preparations with rapid drug release in Medicine: a literature review. Int. Emerg. Nursing 17, 99–107. the case of local anesthesia the combination of several actives Maver, T., Maver, U., Mostegel, F., Griesser, T., Spirk, S., Smrke, D.M., Stana- Kleinschek, K., 2015. Cellulose 22, 749–761. should result in major improvement of effect. These attributes, as Meek, I.L., van de Laar, M.A.F.J., Vonkeman, H.E., 2010. Non-steroidal anti- well as evaluation of long-term stability or in vivo effects, will inflammatory drugs: an overview of cardiovascular risk. Pharmaceuticals 3, however, require further investigations. 2146–2162. 320 L. Vinklárková et al. / J. Appl. Biomed. 15 (2017) 313–320

Morales, J.O., McConville, J.T., 2011. Manufacture and characterization of Sarheed, O., Ahmed, A., Shouqair, D., Boateng, J., 2016. Antimicrobial dressings for mucoadhesive buccal films. Eur. J. Pharm. Biopharm. 77, 187–199. improving wound healing. In: Alexandrescu, V.A. (Ed.), Wound Healing - New Murata, Y., Takashi, I., Kofuji, K., Nishida, N., Kamaguchi, R., 2010. Preparation of fast Insights into Ancient Challenges. InTech, pp. 373–398 (10–5772/61906, https:// dissolving films for oral dosage from natural polysaccharides. Materials 3, www.intechopen.com/books/wound-healing-new-insights-into-ancient- 4291–4299. challenges/antimicrobial-dressings-for-improving-wound-healing). Okamoto, H., Taguchi, H., Iida, K., Danjo, K., 2001. Development of polymer film Semalty, A., Semalty, M., Nautiyal, U., 2010. Formulation and evaluation of dosage forms of lidocaine for buccal administration: I. Penetration rate and mucoadhesive buccal films of enalapril maleate. Ind. J. Pharm. Sci. 72, 571–575. release rate. J. Control. Release 77, 253–260. Shidhaye, S.S., Saindane, N.S., Sutar, S., Kadam, V., 2008. Mucoadhesive bilayered Orsted, M.E., 2010. Wound infection & pain management. Wounds Int. 1, 1–6. patches for administration of sumatriptan succinate. AAPS PharmSciTech 9, Pasero, C., 2013. Lidocaine patch 5% for acute pain management. J. PeriAnaesthesia 909–916. Nursing 28, 169–173. Sussman, C., Bates-Jensen, B., 2012. Management of wound pain. In: Sussman, C., Paunonen, S., 2013. Strength and barrier enhancements of cellophane and cellulose Bates-Jensen, B.M. (Eds.), Wound Care: A Collaborative Practice Manual for derivative films: a review. BioResourses 8, 3098–3121. Health Professionals.. Wolters Kluwer Lippincott Williams & Wilkins Percival, S.L., McCarty, S., Hunt, J.A., Woods, E.J., 2014. The effects of pH on wound Philadelphia, pp. 535–572. healing, biofilms, and antimicrobial efficacy. Wound Repair Regen. 22, 174–186. Sussman, G., 2010. Technology update: understanding film dressings. Wounds Int.1, Pereira, R.F., Mendes, A., Bártolo, P.J., 2013. Novel alginate/aloe vera hydrogel blends 23–25. as wound dressings for the treatment of several types of wounds. Chem. Eng. Thu, H.-E., Zulfakar, M.H., Ng, S.-F., 2012. Alginate based bilayer hydrocolloid films as Trans. 32, 1009–1014. potential slow-release modern wound dressing. Int. J. Pharm. 434, 375–383. Perumal, V., Govender, T., Lutchman, D., Mackraj, I., 2008. Investigating a new Touati, G., Barry, M., Cauet, H., Biendo, M., Strohl, P., 2013. Antibacterial effect of approach to film casting for enhanced drug content uniformity in polymeric modified cellulose: in[139_TD$IF] vitro and clinical study potential applications in the films. Drug Dev. Ind. Pharm. 34, 1036–1047. prevention of surgical site infection. Hojení Ran [Wound Healing] 7, 25–26. Pišlová, M., Kolárová, K., Vosmanská, V., Kvítek, O., Švorcík,9 V., 2015. Preparation of Vakili, H., Genina, N., Ehlers, H., Bobacka, J., Sandler, N., 2012. Using ion-selective polysaccharide films based on chitosan and cellulose. Chem. Listy 109, 942–945. electrodes to study the drug release from porous cellulose matrices. Raju, S., Reddy, S.P., Kumar, A., Deepthi, A., Sreeramulu, R.K., Reddy, M.P.V., 2011. Pharmaceutics 4, 366–376. Flash release oral films of metoclopramide for pediatric use: formulation and in[136_TD$IF] Vetchý, D., Landová, H., Gajdziok, J., Doležel, P., Danek, Z., Štembírek, J., 2014. vitro evaluation. J. Chem. Pharm. Res. 3, 636–646. Determination of dependencies among in[140_TD$IF] vitro and in vivo properties of Ramineni, S.K., Cunningham, L.L., Dziubla, T.D., Puleo, D.A., 2013. Development of prepared mucoadhesive buccal films using multivariate data analysis. Eur. J. imiquimod-loaded mucoadhesive films for oral dysplasia. J. Pharm. Sci. 102, Pharm. Biopharm. 86, 498–506. 593–603. Vinklárková, L., Masteiková, R., Vetchý, D., Doležel, P., Bernatoniene,_ J., 2015. 2015. Ramli, N.A., Wong, T.W., 2011. Sodium carboxymethylcellulose scaffolds and their Formulation of novel layered sodium carboxymethylcellulose film wound physicochemical effects on partial thickness wound healing. Int. J. Pharm. 403, dressings with ibuprofen for alleviating wound pain. BioMed Res. Int. 11 Article 73–82. ID 892671. Repka, M.A., Gutta, K., Prodduturi, S., Munjal M. m Stodghill, S.P., 2005. Wallace, J.L., Vong, L., 2008. NSAID-induced gastrointestinal damage and the design Characterization of cellulosic hot-melt extruded films containing lidocaine. Eur. of GI-sparing NSAIDs. Curr. Opin. Invest. Drugs 9, 1151–1156. J. Pharm. Biopharm. 59, 189–196. Wang, T., Zhu, X.-K., Xue, X.-T., Wu, D.-Y., 2012. Hydrogel sheets of chitosan, honey Romanelli, M., Dini, V., Polignano, R., Bonadeo, P., Maggio, G., 2009. Ibuprofen slow- and gelatin as burn wound dressings. Carbohydr. Polym. 88, 75–83. release foam dressing reduces wound pain in painful exuding wounds: Weinberg, L., Peake, B., Tan, C., Nikfarjam, M., 2015. Pharmacokinetics and preliminary findings from an international real-life study. J. Dermatol. pharmacodynamics of lignocaine. A review. World J. Anaesthesiol. 4, 17–29. Treatment 20, 19–26. Woo, K., Sibbald, G., Fogh, K., Glynn, C., Krasner, D., Leaper, D., Österbrink, J., Price, P., Rowe, R.C., Sheskey, P.J., Quinn, M.E. (Eds.), 2015. Handbook of Pharmaceutical 2008. Assessment and management of persistent (chronic) and total wound Excipients. Pharmaceutical Press, London 888 p. pain. Int. Wound J. 5, 205–215. Saha, S., Tomaro-Duchesneau, C., Daoud, J.T., Tabrizian, M., Prakash, S., 2013. Novel Woo, K.Y., 2011. Exploring the effects of pain and stress on wound healing. Adv. Skin probiotic dissolvable carboxymethyl cellulose films as oral health Wound Care 25, 38–44. biotherapeutics: in[138_TD$IF] vitro preparation and characterization. Expert Opin. Drug Xu, H., Ma, L., Shi, H., Gao, C., Han, C., 2007. Chitosan-hyaluronic acid hybrid film as a Deliv. 10, 1471–1482. novel wound dressing: in[14_TD$IF]97 vitro and in vivo studies. Polym. Adv. Technol. 18, 869– 875.