Development and characterization of k-carrageenan platforms as periodontal intra-pocket films

Purpose: To prepare emulsion-based Intrapocket polymeric films for the treatment of periodontitis. Method: Films were fabricated by dehydration of an emulsion containing k-carrageenan (KC) in aqueous phase and Compritol 888 ATO (Compritol) or Dimodan UJ (DU) or different ratios of both. The resulting films were characterized by mechanical texture analyser to determine Young’s modulus and tensile strength. Glass transition temperature (Tg) of the films was evaluated by dynamic mechanical and thermal analyser while surface morphology was evaluated using scanning electron microscope. In-vitro drug release was conducted in pre-warmed phosphate buffer. Bacterial adherence was assessed after 24 h. Results: Young’s modulus was highest for KC films to which no lipid was added (5.33 ± 0.38 GPa) and decreased following lipid incorporation. Tg was highest in KC films (106.25 ± 4.53 ° C) but decreased upon addition of lipids. The surface of KC was smooth but roughness increased with increasing Compritol load. Drug release from KC films was complete (99.80 ± 8.43 %) after 2 h; however, upon adding lipid, the release was extended 8 h and was affected by lipid type and ratio. Microbiologic assay demonstrated noticeable reduction in viable count compared to control and was affected by lipid type and ratio. The film formulated from a combination of DU® and Compritol® in a ratio of 80:20 was strong, flexible and reduced microbial adherence. Moreover, it showed a smooth surface and extended release for over 8 h. Conclusion: Intra-pocket films were prepared by drying emulsion-based films. Resulted films were strong, flexible, prolonged drug release over 8 h and could lower bacterial growth. The prepared film may offer efficient treatment in periodontitis patients.


INTRODUCTION
Periodontitis is a chronic disease [1] characterized by the formation of periodontal pockets deeper than 4 mm [2]. Periodontal disease is associated with degeneration of gum and may extend to alveolar bones or gingiva [3]. Prescribing antimicrobial agents is used in the treatment of periodontitis. However, it is deemed necessary to employ high oral doses to achieve effective drug levels in the gingival crevicular fluid [1]. The limitation observed with high doses microbicide necessitated the need to develop localized delivery systems [4]. The development of intra-pocket delivery systems offer great potential because periodontal pockets represent a reservoir for the microbial organisms [5]. Kcarrageenan (KC) was employed in pharmaceutical industry as an orodispersible film carrier and a common gelling agent [6]. KC forms fast dissolving films and thus may not be suitable for long periods of administration. As a result, an emulsion-based KC film would delay the fast dissolving nature of KC and subsequently retard drug release. Lipid excipients can serve two pronged approaches, firstly modify the drug release and secondly improve the brittleness observed in KC films. Kulkarni proposed that the addition of various types of lipids may affect the properties of the films and control the rate of drug release from its matrices [7]. As a result, it was decided to incorporate two lipids; Compritol® 888 ATO (Compritol ® ) and Dimodan ® UJ (DU ® ) into KC films.
The films were loaded with metronidazole (MNZ) as an antimicrobial agent that has been used for the treatment of periodontitis in the forms of gels and films [5]. Clinical manifestation demonstrated slower rate of relapse of clinical parameters associated with administration of MNZ loaded films [8]. Subsequently, the films composed from KC, MNZ and lipids were subjected to further characterization. Films were prepared with or without the addition of varying amount of Compritol ® and DU ® followed by mechanical testing, drug release testing, and microbial assays to evaluate their use as potential intrapocket films.

Preparation of dried film hydrogel
Films were produced using the modified procedure of Kulkarni et al [9]. Aqueous layer was prepared by stirring 10 mL KC (2 % w / w) with 5mg MNZ at 60°C. The lipid layer was prepared by stabilizing 500 mg molten lipid in 10 mL of Pluronic ® F127 (0. 5 w/v %). To prepare drug-loaded lipid particles, 500 mg molten lipid was added to a 20 mL glass vial containing a stabilizer solution (prepared separately by dissolving 0.5 % Pluronic ® F127 in 100 mL water) to reach a mass of 1 g. The mixture was ultrasonicated (Omni Sonic Ruptor 400, Omni International, Inc, USA) for 10 min with a continuous pulse at 35% power. MNZ was loaded by stirring 5 mg for 15 min to ensure the drug was dissolved into the "milky" emulsion.
The composition of each of the prepared is illustrated in Table 1. KC solution (4 %) was prepared by stirring 0.4 g KC powder in 9.6 mL water at 60 °C for 20 min. A molten solution was combined with an equal volume of drug loaded in lipid particle dispersion and gently stirred for 20 min while heating until homogenization was visually achieved. The resultant gel contained 2 % KC. KC gel (without lipid particles) was prepared by stirring 5 mg MNZ with 2% KC solution at 60°C. Afterward, the gels were poured in Petri dishes (7 cm diameter) and left to dry overnight. The now dried films (intra-pocket films) were weighed three successive days to ensure complete dryness to ensure no water residual was present. The dried films were removed from the Petri dish and kept in plastic bags for further investigation.

Determination of glass transition temperature
Measurements of the glass transition temperature (T g ) of the intra-pocket (dried) films was performed using the Q800 DMTA (TA, USA), the mode selected was tensile and the selected oscillatory frequency was 1Hz to T g . A heating rate of 3° C min -1 was employed over a temperature range of -20 °C to 140 °C. Dried films (n=3) were cut into rectangular shapes (30 mm length of the films, 5 mm width of the films and 0.11 mm and measurement of the thickness was recorded via a digitalized calliper). The T g was detected from the peaks of the tan δ lines [10] .

Evaluation of mechanical features
The film's tensile properties were analysed using a Stable Micro Systems TA-XT2 texture analyser (Goldaming, Surrey, UK). Dried films (3 cm length × 0.5 cm width x 0.11 cm thickness, n = 5) were clamped between the static (lower) and moveable (upper) grips, to make sure the stressed films are kept under constant length of 20 mm. Inducing fracture on the films was done by raising the upper clamp at a fixed rate of 0.5 mms -1 . The mechanical features were retracted from the resulting plot of the stress-strain. The measurements of the various mechanical features were calculated as described by McAvoy et al [11], including the Young's modulus, the elongation (%) recoded at the break point and the Ultimate Tensile Strength (UTS) [11].

In vitro drug release
In-vitro drug release was determined by immersing dried films (average dimensions 30 mm length x 5 mm width x 0.11 cm thickness, n = 3) in 10 mL of pre-warmed phosphate buffer (pH 7.4) at 37°C in glass vials. At certain determined time intervals; media (10 mL) were taken and a fresh 10 mL warmed buffer was added.
The resulted amounts were measured using a previously plotted curve of r 2 value of > 0.99. Plots of measured concentration of released drug against time were plotted as previously recommended [12] using the mechanism of releasing was depicted by implementing the Korsmeyer-Peppas equation [13] as in Eq 1.
where Mt/M ∞ is the fractional solute release, k is a kinetic constant, t is the release time, and n is the release exponent relates the pattern involved in drug release, where Fickian diffusion is suggested when reaching a value of around 0.5, while non Fickian or anomalous diffusion are denoted by values within the range of 0.5 -1.0 [13]. The release study was continued to 24 h to ensure more than 80 % of the drug release in all formulations.

Microbiological assay
The ability of the prepared formulations to minimize microbial growth on their surfaces was studied using two standard microorganisms: Candida albicans (ATCC 10231) and Streptococcus oralis (ATCC 6249). The dried films were exposed to high concentration of microbial growth (circa 1 X1 0 9 CFU / mL, confirmed by viable count) and were left for 24 h, then the adhered bacteria were dislodged and viable count was computed.
Microbial adhesion experiment was done according to Jones et al with modifications. Dried films (n = 6, mean disc thickness of 0.11 mm and mean diameter of 2.8 mm) were swabbed with microorganism suspensions and immediately placed on an Agar Petri dish and left for 24 h. Afterwards, the loose microorganisms were washed out using Quarter Strength Ringer Solution (QSRS) after 3 x washing of 20 mL for 30 s. Dislodgement of adherent microbes was done by sonicating the films separately in sealed test tubes filled with 10 mL QSRS. The films were removed and subsequent calculation for the viable microbes was conducted after serially diluting of the liquid phase, and the measures were quantified as CFU/mm 2 . Each film was compared to a control containing similar formulation components but devoid from MNZ [12].

Assessment of film morphology
Surface topography of the dried films was examined by SEM using a FEI Quanta FEG 450 (ThermoFisher Scientific, USA). Samples were mounted on slides and gold-coated, and images were taken using a 10 keV acceleration voltage of the secondary electron (SE) mode.

Statistical analysis
Simple results were expressed as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) was implemented for advanced statistical analysis, and the Tukey Kramer test was performed for all post hoc tests. A statistical value of p < 0.05 was denoted significant for all types of statistics.

Mechanical properties
The mechanical properties (Young's modulus, UTS and % elongation) of the medical films are presented in Table 3 In vitro drug release Figure 1 illustrates the in-vitro release profile of MNZ from the prepared dried films. Clearly, the polymer type and lipid ratio dictated the release profile. The KC films F1 (with no lipids incorporated) exhibited complete drug release (99.8 ± 8.43 %) in the first hour of the experiment. The incorporation of lipids extended the drug release; the initial stage of the release experiment exhibited a burst in the lipid containg films, where more than 25 % of the drug was released after one hour in all formulations. Furthermore, the lipid type had significantly affected the rate of the drug release, where the incrorporation of Compritol ® to the KC films showed a slower release rate compared to F1 KC film and films containing DU ® .
For example, 6.40 ± 0.63 % MNZ was released from F6 (100 % of Compritol ® ) at 10 minutes, whereas F2 (100 % of DU ® ) illustrated a release of 9.87 ± 0.81 % at the same time point (p < 0.05). After 8 h, the observed release in F6 was 57.16 ± 3.7 % and for F2 the MNZ release was   Table 4 demonstrates that the release exponent (n) was found to be within the range of 0.47 to 0.81 suggesting that the release mechanism was non-Fickian dominated by erosion and diffusion. The release study was continued to 24 hours to ensure more than 80 % of the drug was released in all formulations. Considering C. albicans, neat KC films (F1) disintegrated to gels and were not eligible for viable count procedure therefore; it is not presented in Figure 2. Clearly all dried films had lower growth compared to their control. Considering the prepared films, the highest candida growth was observed with increasing the load of Compritol ® in F6 (loaded with 100 % Compritol ® ) and F5 (loaded with 80 % Compritol ® ) having a growth of 14070 and 9323 CFU / mm 2 , respectively. On the other hand, increasing DU ® ratio demonstrated lower growth.

Microbiologic properties
As observed in the F1 films mixed with C. albicans, KC films F1 and F2 had disintegrated to gel and were not eligible for viable count. The addition of Compritol ® improved the integrity of the films. However, raising the amount of added Compritol ® resulted in an increase in the number of viable S. oralis (Figure 3). For example, the loading of 100 % Compritol ® (F6) demonstrated growth of 7055 CFU/mm 2 . On the other hand, the lowest bacterial growth was observed with the highest employed DU ® loading (F3, 80 % DU ® , P < 0.05 compared to all formulations).

Film morphology
The micrograph illustrates the surface of KC neat film F1 (Figure 4 -A) where it appears smooth. Figures 4-B and 4-C illustrate the SEM micrographs of F2 (100% DU ® ) and F3 (80% DU ® ); both surfaces appear smooth. However, the employment of higher loadings of Compritol ® led to a rough surface. For example, Figure 4-D Micrographs were taken at 5000 X magnification and 5.00 kv which represents F4 (50% Compritol®) shows a unique morphological charac-teristics of the film as the surface appeared rough with sharp objects. Furthermore, films loaded with a higher load of Compritol ® F5 (80 % Compritol ® ) and F6 (100 % Compritol ® ) in Figure 4-E and 4-F, respectively illustrate a rough surface with sphere-like particles that may be developed throughout the cooling process.

DISCUSSION
Following successful preparation of the intrapocket films, different characterization techniques were carried out to evaluate their efficiency. C. albicans viable count of tested films was significantly lower than their corresponding control (P < 0.05 in all cases). KC neat films (F1) turned into gel and therefore it was difficult to dislodge the organisms. Furthermore, increasing the load of Compritol ® increased viable count as observed in F6. The ability of MNZ to lower the viable count may have an impact on reducing the complications of chronic periodontitis as explained by Larsen [8]. S. oralis adherence was significantly lower than control (p = 0.000, supplementary data). KC neat film disintegrated to gel after 24. Raising the amount of added Compritol ® resulted in marked increase in the adherence of bacteria.
Wake et al was able to grow dental biofilms and found the viable count after 24 h incubation ranging from 1 x1 0 9 to 10 10 CFU/mL [14]. KC lipid-loaded films exhibited an exceptional reduction in viable count, which was 10 6 times lower compared to the reported values at the same time point.
Rough surfaces would encourage the attachment of microbes to adhere better [15]. The SEM micrographs illustrated a smooth surface for KC neat and DU ® -containing KC films ( Figure 4). Conversely, Compritol ® exhibited a rough surface. The variation in melting point between DU ® and Compritol ® may attribute to the roughness of the surface, as Compritol ® has a higher melting point which means it solidifies slower than DU ® and this may suggest that compritol lipid particles have a higher probability to arrange themselves into spheres. SEM micrographs suggested employing Compritol ® alone may not be proper due to rough surfaces. In addition, the high number of microbes that grew on the surface of F6 suggested that the addition of Compritol ® in low ratio compared to DU ® in KC films is preferred. The incorporation of lipids significantly affected T g of KC films. T g of F1 was 106 ° C consistent with the value reported in the literature (108.9 ° C) [16]. The incorporation of lipids significantly reduced T g of KC (P<0.05) from 106.25 ± 4.53 °C to a T g range (62 to 86 ° C). The reduction in T g was influenced by the type of lipid and melting temperature [17]. This reduction is attributed to the lower melting point of the lipid that will allow the lipid molecules in their melted form to penetrate between the polymer chains and create more void leading to a lowering in T g as supported by Omelczuk et al [18].
The mechanical properties of polymers are strongly dependent on their T g [18]. Films with lower T g are more flexible and demonstrate higher percentage of elongation. [19]. High T g is observed with high Young's modulus of elasticity [19]. KC neat films demonstrated the highest Young's modulus value. The further decrease in Young's modulus was consistent to the addition of DU ® and Compritol ® which acted like plasticisers. The inclusion of the aforementioned lipids led to an increase in the void volume, thus rendering the polymer chain more flexible (increased % elongation) and weaker (decreased tensile strength). These observations are compatible with findings reported by Oladzadabbasabadi and collegues [20].
The employment of Compritol ® to retard drug release was extensively investigated in the literature [21]. Lipid-containing formulations released more than 25% of drug during the first hour. The release at the 8 h time point has reached 80%. The intra-pocket films contain approximately 250 µg of MNZ. Considering that 25 % of drug content was released (corresponding to 62.5 µg) in the first hour and the remaining drug continued to elute over the next 8 h indicates that MIC was reached during the entire period of administration. The drug release experiments illustrated significant differences between lipid-containing KC films. F6 was able to retard drug release more than F2 and demonstrated the lowest release constant K as observed in Table 4. In addition, the high T g of the KC films loaded with Compritol ® would retard the release of the drug as the gelation and erosion of the KC chains will be delayed.

CONCLUSION
This work reports the successful preparation of Intra-pocket films for the treatment of periodontitis. The films have been fabricated by drying an emulsion-based KC gel. The films showed variation in their characteristics: ability to reduce microbial growth, surface morphology, mechanical properties and drug release. The use of DU ® : Compritol ® 80: 20 in the emulsion based dried films met the demand of this project where the resulting films are strong, flexible, prolonged the drug release over 8 h and reduced the growth of micro-organisms. This indicates potential for further investigation as a commercial product.