INTRODUCTION

Dental phobia may cause recurrent and severe problems for both the patient and the dentist, as it often gives rise to many deleterious effects1). There are reports that people with high dental phobia refuse to keep their dental appointments2,3). The dental injection is one of the causes of their refusal; therefore, in the clinic, the dentist applies a topical anesthetic to minimize pain and fear. Unlike a local anesthetic, the topical anesthetic is used for reducing the pain by numbing a limited area and reversibly blocking the signal flow of the nerve fibers.

The local anesthetic most often used in dentistry is lidocaine hydrochloride [2-diethylamineoacetate-2’, 6’-xylidide]. This anesthetic is available commercially as a 2 or 3% solution with 1:50,000 to 1:100,000 epinephrine4-7). Different forms of lidocaine have been developed for use as topical anesthetics. These forms include ointments, gels and sprays7-9). Despite its convenience, the single application can be washed away quickly by the saliva10).

Currently, there are many studies concerning buccal films that counter the disadvantages of using lidocaine as an oral tablet. Lidocaine in a buccal film form can be easily applied and adheres to the local mucosa. The buccal film has the advantage of not being as uncomfortable as other foreign substances. In this study, we have selected a specially designed buccal film. Ethyl cellulose (EC) was used as the backing layer and hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) were used as the drug-containing layer10-14). Therefore, the aim of this study was to develop a bi-layered strip

consisting of a tooth-adhesive layer and a non-adhesive protective layer to deliver lidocaine. After blending and making the bi-layered film, we examined its sectional image to check its uniformity. Additionally, we evaluated the effect of a fast lidocaine release profile, in vitro and in vivo adhesion times and in vivo drug permeation. The null hypothesis of the study is that there is no difference in the two formulas, PVP/HPMC and HPMC, used for the release of lidocaine.

MATERIALS AND METHODS

MaterialsPolyvinylpyrrolidone (PVP) type Kollidon K30 with a molecular weight of 50,000–55,000 was obtained from BASF (Bask, Ludwigshafen, Germany). Hydroxypropylmethylcellulose (HPMC) type Methocel K4M and ethylcellulose (EC) were obtained from Sigma Chemical (No.E-8003, Sigmaaldrich, MA, USA). Lidocaine was obtained from Supriya Chemicals (Mumbai, India). The bi-layered strip was prepared with a casting-solvent evaporation technique using different concentrations of polymers.

Preparation of the bi-layered filmFor the bi-layered film, the backing layer was made first, and then the adhesive layer was completed. EC was dissolved in 100 g of ethyl alcohol for 5 h, and 1% dibutyl phthalate (DPB) was added as a plasticizer. Ten milliliters of backing solution was poured into a 10 mm petri dish, and the backing solution was evaporated at a controlled rate by covering the petri dish to avoid the blistering effect on dried strips. For the adhesive layer, 5 wt% of PVP and 2 wt% of HPMC were individually

The in vitro and in vivo effects of a fast-dissolving mucoadhesive bi-layered strip as topical anestheticsJiyeon ROH1,2, Mira HAN3, Kyoung-Nam KIM1,2 and Kwang-Mahn KIM1,2

1 BK 21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea2 Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 03722, Korea3 CJ Healthcare, 811 Deokyeong-ro, Majang-myeon, Icheon-si, Gyeonggi-do 17389, KoreaCorresponding author, Kwang-Mahn KIM; E-mail: kmkim@yuhs.ac

To overcome pain on injection, the dentist can apply a topical anesthetic spray. Despite the convenience, it is not easy to apply it locally. So, we developed an oral mucoadhesive bi-layer film containing an anesthetic. We used polyvinylpyrrolidone (PVP)/hydroxypropyl methylcellulose (HPMC) and HPMC-only layer as the drug-containing layer and ethyl cellulose (EC) as the backing layer. The lidocaine released was tested in vitro together with the adhesion time and cytotoxicity of the film. Mucosa permeability was tested in vivo. Statistical analysis was performed, with p at 0.05 taken to be significant. The lidocaine was released significantly faster in the PVP/HPMC than HPMC-only group and 80% of the drug was released within 1 min (p<0.05) and they attached at least 3 h. The test groups showed no toxicity and the drug effectively permeated the mucosa (p<0.05). We suggest this new mucoadhesive anesthetic may reduce dental phobia.

Keywords: Dental phobia, Mucoadhesive, Bi-layered film, Anesthesia, Lidocaine

Color figures can be viewed in the online issue, which is avail-able at J-STAGE.Received Oct 28, 2015: Accepted Mar 1, 2016doi:10.4012/dmj.2015-369 JOI JST.JSTAGE/dmj/2015-369

Dental Materials Journal 2016; 35(4): 601–605

Table 1 Test groups used in this study. The composition of the experimental groups FA and FB was listed.

Code HPMC (g) PVP (g)

FA 10 90

FB 100 0

dissolved in distilled water to create two homogeneous solutions. Then, 10 mL of glycerin was added as a plasticizer (60°C, 24 h) to each of the solutions. The two solutions were mixed in differing amounts under stirring for 12 h (HPMC-PVP). Then, the required amount of lidocaine was slowly added into the HPMC-PVP solution. The HPMC-PVP-LIDO solution was poured onto the dried backing layer and allowed to dry in an oven at 80°C until a flexible bi-layered strip was formed. The strip was cut into 6 mm diameter pieces and stored at (23±2)°C and (50±2)% relative humidity until further analysis was performed.

Bi-layer film analysisThe Fourier Transform Infrared Spectroscopy (FT-IR) spectra of the HPMC-PVP blended thin strips were obtained using a FT-IR spectrophotometer (Nexus-870, Thermo Nicolet, USA). To avoid any moisture effects, the samples were dried overnight in a desiccator. The IR spectra in absorbance mode were obtained in the spectral regions of 1,000–4,000 cm−1. The spectrum of each sample was acquired by accumulation of 32 scans with a resolution of 4 cm−1. To observe the morphology, cross-sections of the bi-layered strips were observed using an image analyzer (HIROX KH-1000, HIROX, Seoul, Korea).

In vitro adhesion time of the bi-layered stripTo measure the in vitro adhesion time and the release of lidocaine, the film was attached to the bottom of a beaker. Under the film, 15 μL of simulated saliva fluid (SBF) was used to hydrate the adhesion surface. After the film attached, the beaker was filled with 800 mL of SBF and kept at 37°C. A 150 rpm stirring rate was applied to simulate the buccal cavity environment, and strip adhesion was monitored for 24 h. The time necessary for complete erosion or detachment of the strip from the beaker surface was recorded.

In vitro lidocaine release from the bi-layered stripThe 100 mL of phosphate buffer solution (PBS, pH 7.0) was stirred at 100 rpm and (37±1)°C. At appropriate time intervals, 1 mL samples were withdrawn and then replaced with the same volume of buffer15). Lidocaine concentrations were calculated spectrophotometrically at a wavelength of 264 nm.

Cytotoxicity test (WST-1) of the filmTo evaluate the cytotoxicity of the film, a WST-1 test was conducted with L-929 mouse fibroblasts. The 5×103/well of fibroblasts were seeded in 96-well microplates for 24 h. To make an extraction solution, we immersed the test groups in cell culture media according to ISO 10993-12. The cell culture media (with no test material) was used as control. After 24 h, the cell culture medium was changed to an extraction solution. After another 24 h of incubation, the supernant solution was replaced to 100 μL of premixed WST-1 solution to each well and then incubated for 4 h. The wells were read using an ELISA reader at 540 nm, and then the % viability was

calculated. For each WST-1 test, the control was defined as 100%.

In vivo testing: adhesion time and lidocaine permeability of the bi-layered stripTo evaluate the mucous adhesion time in vivo, we euthanized two male mongrel dogs, each 18–24 months old and weighing approximately 30 kg. Animal selection, management, preparation, and surgical protocol followed the routine procedure approved by the Animal Care and Use Committee at the Yonsei Medical Center, Seoul, Korea (No.09-125). The bi-layered strips were attached to the canine oral cavity. The surfaces of the strips were moistened with 15 μL SBF, and then the strips (size 6 mm2) were brought immediately into contact with an initial force for 5 s. The entire experiment was performed at room temperature. The adhesion of the strips was monitored for 30 min, and the number of strips that detached from the oral cavity was recorded. To measure the permeation concentration of lidocaine, the mucous membrane was removed and analyzed using a UV/VIS spectrometer.

Statistical analysisThe IBM SPSS 20.0 program (SPSS, Chicago, IL, USA) was used for the statistical analysis. All results were analyzed by one-way ANOVA followed by the Tukey test at a level of significance of 0.05.

RESULTS

The spectrum from FT-IR (A) and the sectioned image of FA (B) are shown in Fig. 1. In Fig. 1A, the FTIR shows homogeneous results for three randomly selected films. The benzene ring and bonded-OH of lidocaine helped produce the signal in the approximately 3,030 cm−1 range. The C=C produced a signal in the 1,450–1,600 cm−1 range. The sectioned surface of the bi-layered film is shown in Fig. 1B. The EC layer was dried in the lower part of the film as the backing layer; the HPMC-PVP layer was dried onto the backing layer as the adhesive layer. Thus, the bi-layered strips had the mechanical and physical combination of the EC layer and the HPMC-PVP layer.

The result of the in vitro test of adhesion time of the bi-layered strip is shown in Fig. 2. The PVP and EC groups, which were made of only one layer, detached immediately from the beaker wall. The FA group showed that 2 out of 3 specimens were detached from the beaker wall after approximately 4 h. In contrast, all FB groups

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Fig. 1 The spectrum of FTIR (A) and a cross-section image of FA (B).

In Fig. 1A, the randomly selected film showed homogeneous and there were same spectrum with lidocaine, HPMC and PVP spectrum. The sectioned surface of the bi-layered film is shown in Fig. 1B. The EC layer was dried in the lower part of the film as the backing layer; the HPMC-PVP layer was dried onto the backing layer as the adhesive layer. There was tightly attached cross-sectional image.

Fig. 2 In vitro adhesion time of bi-layered strip. The mono-layered PVP and EC groups detached

immediately from the beaker wall. The FA group showed that 2 out of 3 specimens were detached from the beaker wall after approximately 4 h. In contrast, all FB groups were only detached after 5 h.

Fig. 3 In vitro lidocaine-release profiles of bi-layered strips in the FA group and the FB group (There were significant differences in all time points).

The lidocaine release profile of the FA was rapid, and 16 mg of lidocaine had been released within the initial 5 min. The FA showed 4 times faster lidocaine-releasing property than in the FB group (p<0.05).

Fig. 4 Cell viability [* means significant difference (p<0.05)].

All experimental groups except the FB group were non-toxic. The highest cell viability was in the EC strip at 94.61%, with a rank order of FA (HPMC-PVP blended strip) and lidocaine. However, there were no significant differences in cytotoxicity (p>0.05).

were only detached after 5 h.The accumulated release of lidocaine is shown in

Fig. 3. The lidocaine release profile of the FA was rapid, and 16 mg of lidocaine had been released within the

initial 5 min. As a result, the FA group with HPMC-PVP showed a fast lidocaine-releasing property that was 4 times faster than in the FB group (p<0.05).

The cytotoxicity results for each film are shown in Fig. 4. All experimental groups except the FB group were non-toxic. The highest cell viability was in the EC strip at 94.61%, with a rank order of FA (HPMC-PVP blended strip) and lidocaine. However, there were no

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Fig. 5 In vivo adhesion time and lidocaine permeability results of the bi-layered strip in the FA group.

[*means significant difference (p<0.05)]. Not all films detached within 30 min. The study showed that 282.21 μg was the highest amount of lidocaine that permeated the alveolar mucosa (p<0.05), compared with 210.16 μg in the buccal mucosa and 196.21 μg in the labial mucosa.

significant differences in cytotoxicity (p>0.05). The FB group showed the lowest cell viability (p<0.05).

Figure 5 shows the amount of lidocaine that permeated through the mucosa. Not all films detached within 30 min. The study showed that 282.21 μg was the highest amount of lidocaine that permeated the alveolar mucosa (p<0.05), compared with 210.16 μg in the buccal mucosa and 196.21 μg in the labial mucosa.

DISCUSSION

Various topical anesthetics including lidocaine have been developed8). The existing topical aesthetics have been used for patients suffering from dental phobia16), but they have had limited effects on reducing patient’s phobia. To reduce this phobia, various methods are being used clinically. Although researchers have introduced a variety of products, their local application has not been long lasting. Therefore, we modified a mucous-adhesive method for the relief of discomfort and fear of injection by designing a fast-release formula17,18).

The bi-layered structure was designed to avoid the strip being washed out by saliva and to deliver drug unidirectionally to the mucosa. In addition, the bi-layered strip is designed to affect only the isolated area corresponding to an injection point and not to affect other unwanted regions. For this design, we used ethyl cellulose, which is hydrophobic, as the backing layer and polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) as the drug-containing layer. PVP is a water-soluble tablet binder, and it simply passes through the body when taken orally. However, PVP lacks the property of adhesion. HPMC is a water-soluble polymer with good film-forming properties. The degree

of substitution, types of functional group substitution, and chain length of this polymer affect its permeability, mechanical properties and water solubility. In a previous study, HPMC showed a slow drug-release behavior19). Therefore, we added various concentrations of PVP in the pilot test and, finally, 90% of PVP was considered the most appropriate for application in this study14). For the mucoadhesion property, we added 10% of HPMC in the FA group.

To make a bi-layered film, we used the solvent evaporation method, which is considered easy and simple. However, depending on the evaporation characteristics, it is not easy to make a uniform film20). In the pilot test, different volumes of adhesion polymer were tested to make a uniform film. The thickness and weight of the polymer samples differed in spite of maintaining a uniform volume. Depending on the volume of the adhesive polymer, the lidocaine content within each specimen also differed. Therefore, we fixed the content in relation to the volume of the adhesive polymer and found the appropriate volume for the proper film lidocaine contents. In terms of uniformity, the formula was confirmed by FTIR. When we randomly made the films, the functional groups of lidocaine and HPMC-PVP were produced at the same peaks, as confirmed by FTIR.

In Fig. 3, it is shown that, in the FA group, the HPMC-PVP-blended strips showed faster drug release than that of the FB group, which had a pure HPMC strip. During the first minute, 16 mg of lidocaine was released, and lidocaine release continued until the film was peeled off. The mucoadhesive film was designed for applying the topical anesthetic before the injection, so we considered 5 min to be sufficient time to allow between applying the film and receiving the injection in a clinical situation. Therefore, the lidocaine-release test was conducted for 5 min. Of course, the two groups had shown clinically acceptable adhesion time in vitro (Fig. 2).

A mucoadhesive film applied to the mucosa tissue might irritate the subject. Therefore, we conducted cytotoxicity tests of each film. There was no toxicity for the backing layer, the FA group and for lidocaine. This lack of toxicity was reported in previous studies21-23). However, the FB group, which had only HPMC, showed a significantly lower cell viability than other groups (p<0.05).

Even though the FA group with low HPMC concentration had the weakest attachment, it adhered for sufficient time to show effectiveness. We chose the blending concentrations of the FA group with the quickest release time, and this combination seemed best for our needs. On the basis of the results, we chose the blending proportion of HPMC:PVP=10:90 when manufacturing mucoadhesive drug layers.

The bioadhesive force of the prepared film specimen was an important property. If film specimens detach from oral mucosa before the clinical application time, they might pose a danger to the patient, who might be anesthetized in unwanted regions. We measured the bioadhesive force indirectly using the in vivo adhesion

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time test. The results showed that the film specimens did not detach within 30 min, which was the expected application time for each film specimen. The permeation of lidocaine was evaluated for the labial, buccal, and alveolar mucosa, which are typical local injection points. The alveolar mucosa was more permeated with lidocaine than the buccal mucosa and labial mucosa. In general, the oral mucosa is classified as a somewhat leaky epithelium with a permeability rank order of alveolar mucosa>buccal>labial mucosa, based on the thickness and degree of keratinization of the tissues.

In conclusion, from the above results, the null hypothesis was rejected, and we confirmed that the prepared strip, for the FA group, showed proper oral mucosa adhesion and fast drug release. Therefore, if the prepared strip is clinically applied, it might show positive effect for the patients with dental phobia. Also, to support its effect, clinical study needed to be assessing in the future.

ACKNOWLEDGMENT

This study was supported by BK 21 Plus project in Yonsei University College of Dentistry.

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