AAPS PharmSciTech ( # 2016) DOI: 10.1208/s12249-016-0676-y

Research Article In Vitro and Ex Vivo Evaluation of Novel Curcumin-Loaded Excipient for Buccal Delivery Flavia Laffleur,1,3 Franziska Schmelzle,1 Ariane Ganner,1 and Stefan Vanicek2

Received 15 August 2016; accepted 18 November 2016

This study aimed to develop a mucoadhesive polymeric excipient comprising Abstract. curcumin for buccal delivery. Curcumin encompasses broad range of benefits such as antioxidant, anti-inflammatory, and chemotherapeutic activity. Hyaluronic acid (HA) as polymeric excipient was modified by immobilization of thiol bearing ligands. L-Cysteine (SH) ethyl ester was covalently attached via amide bond formation between cysteine and the carboxylic moiety of hyaluronic acid. Succeeded synthesis was proved by H-NMR and IR spectra. The obtained thiolated polymer hyaluronic acid ethyl ester (HA-SH) was evaluated in terms of stability, safety, mucoadhesiveness, drug release, and permeation-enhancing properties. HA-SH showed 2.75-fold higher swelling capacity over time in comparison to unmodified polymer. Furthermore, mucoadhesion increased 3.4-fold in case of HA-SH and drug release was increased 1.6-fold versus HA control, respectively. Curcumin-loaded HASH exhibits a 4.4-fold higher permeation compared with respective HA. Taking these outcomes in consideration, novel curcumin-loaded excipient, namely thiolated hyaluronic acid ethyl ester appears as promising tool for pharyngeal diseases. KEY WORDS: buccal; curcumin; hyaluronic acid; thiomer; wound healing.

INTRODUCTION Dysfunction of the oral and pharyngeal cavity is very disabling and affects severely the quality of life. Disorders are described as aphthae (1), gingivitis, herpes varicella, or candidosa (2). Due to its efficacy and its safety for human use, curcumin has gained conspicuous interest as a potential therapeutic agent for the prevention and/or treatment of mucosal disorders (3). Curcumin is remarkably non-toxic, antioxidant, anti-inflammatory, antiseptic, and chemotherapeutically active (4) and has limited bioavailability due to its poor absorption from the gastrointestinal tract (5). The physicochemical properties, the short half-life of 1.39 h, and its low molecular weight render it suitable for buccal employment (6). Furthermore, curcumin traditionally is used as protectant in oral health exhibiting wound healing agent (7). Curcumin was chosen as active compound highlighting a new use of a very welldescribed material, hyaluronic acid for buccal delivery. The intraoral or buccal mucosa represents a promising platform for the application of dosage form in order to treat oral and pharyngeal disorders. Buccal delivery is associated 1

Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria. 2 Institute for Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria. 3 To whom correspondence should be addressed. (e-mail: Flavia.Laffleur@uibk.ac.at)

with benefits such as circumventing the first pass effect, avoiding acidic degradation and ease of administration (8). However, the mucosa’s properties, surface area, as well as loss of the dosage form due to the salivary turnover limit the buccal route. One approach is the use of multifunctional excipients with thiomers as one representative (9). Thiomers are thiolated polymers with immobilized thiol groups on their polymeric backbone (10). Several features in particular stability, mucoadhesion, and permeation enhancement arise based on those thiol groups (11). Hyaluronic acid (HA) is an anionic and versatile natural occurring, water soluble, polysaccharide comprising disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, forming a linear polymer (12). HA is applied in wound healing, tissue engineering, and drug delivery (13). On these grounds, the overall goal of this study was to combine the candidate curcumin with tailor made mucoadhesive and lubricative hyaluronic acid conjugate as potential agent for the treatment of oral and pharyngeal diseases. MATERIALS AND METHODS Materials Hyaluronic acid sodium salt from Streptococcus equi (1.5 to 1.8 *106 Da), L-cysteine ethyl ester hydrochloride, Nhydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) 1530-9932/16/0000-0001/0 # 2016 American Association of Pharmaceutical Scientists

Laffleur et al. carbodiimide hydrochloride (EDAC), disodiumhydrogenphosphate (Na2HPO4), potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4), 5,5′-dithiobis-(2-nitrobenzoic acid), resazurin salt, sulforhodamine, and curcumin were purchased from SigmaAldrich. All other reagents used were of analytical grade.

the 6.3.1.0134 (Perkin Elmer, Waltham, USA). The spectra were measured at 22°C. The scans were recorded in a range from scans at 4000 to 600 cm−1 and a resolution of 4 cm−1. Moreover, 1H-NMR spectra were taken on a Bruker DRX 300 MHz spectrometer. Data acquisition and processing were recorded with Bruker Topspin 2.1. All NMR experiments were recorded at 298 K (25°C).

Synthesis of Hyaluronic Acid Cysteine Ethyl Ester Immobilization of thiol bearing ligand to the polymeric backbone was achieved by a method described previously by our research group (14). Briefly, 0.4% (w/v) solution of hyaluronic acid (HA) was adjusted to pH 5.5 with 1 M HCl. By adding EDAC (50 mM) and NHS (50 mM), respectively, L-cysteine (1:1 molar ratio) was covalently attached to the polymeric backbone. Dialysis (cellulose hydrate tubing, cutoff 12.000 Da, Carl Roth, Karlsruhe, Germany) started after 6 h of incubation. Conjugates were three times dialyzed against demineralized water containing 1% NaCl followed by twice against demineralized water. HA served as control. The purified thiolated polymer was lyophilized (Christ, Gamma 116 LSC, Osterode am Harz, Germany) and kept stored at 4°C until further use. Characterization of Hyaluronic Acid Cysteine Ethyl Ester Ellman’s assay quantifies the amount of free thiol groups on the polymeric backbone via spectrophotometric determination where an aliquot of 100 μL was transferred to a 96well microplate and measuring the absorbance at 450 nm (Tecan infinite M200). Moreover, disulfide bond assay was conducted after the addition of NaBH4 and Ellman’s reagent as previously reported by Habeeb (15). Furthermore, attenuated-total-reflectance Fourier transform infrared spectroscopy (ATR-FTIR)) spectra of unmodified HA and thiolated HACYS were obtained from a Perkin Elmer Spectrum 100 ATR-FTIR spectrometer (Perkin Elmer, Waltham, USA) in combination with a Spectrum software version

Cell Viability Investigations The synthesized polymer conjugates thiolated hyaluronic acid cysteine ethyl ester (HA-SH) and unmodified HA, respectively, were explored in terms of cell viability. Caco-2 cells were cultured at 95% humidity, 37°C, and in atmosphere of 5% CO2. Resazurin assay was conducted within the passages 20–30. Preparation of Minimum Essential Medium (MEM) with Earle’s salts, 2.2 g/L NaHCO3 and stable glutamine supplemented with 100 mg/mL streptomycin, 100 mg/mL penicillin, and 10% fetal bovine serum was disposed. The cells were treated with the testing conjugates for 12 h, followed by incubation with 250 μL resazurin solution (44 μM/well) was laced and lasted for 3 h. Cell viability assay was performed with resazurin salt. After adding resazurin to viable cells, a reduction of resazurin to resorufin takes place, whereby resorufin’s fluorescence could be detected via fluorescence measurements at 540-nm excitation and 590-nm emission wavelength (16). Shortly, 0.5% (w/v) HA-SH and HA solutions were prepared and cells were treated, respectively. Minimum essential medium and Triton-X® (1% (v/v)) served as negative and positive control, respectively. The incubation period was 12 h; afterwards, resazurin solution was added in order to determine the safety potential of the polymeric excipients. Cell viability rates were measured at excitation wavelength of 540 nm and emission wavelength of 590 nm followed by a calculation according the equation:

Cell viability ½% ¼ ðAverage absorbance value of each sampleÞ=ðAverage absorbance value of low controlÞ  100

Evaluation of Water Uptake Capacity

Ex Vivo Evaluation of Mucoadhesiveness

Flat-faced discs (30 mg, 5.0 mm diameter) were prepared of lyophilized HA-SH as well as HA, respectively, by compressing with a single punch eccentric press, 12-kN compaction pressure (Paul Weber, Remshalden-Grunbach, Germany). Following a gravimetric method, swelling behavior was investigated. HA-SH and HA discs were mounted on a needle hanging in a vessel containing simulated saliva fluid (SSF) pH 6.75 at 37 ± 0.5°C. At predetermined time points, swollen test discs were taken out and reweighed until constant weight was reached. The amount of water uptake was calculated according to the following equation:

In order to perform the mucoadhesiveness, buccal porcine mucosa was separated and cut into small pieces. The tissue was fixed on a steel cylinder of the dissolution apparatus (diameter 4.4 cm; height 5.1 cm; apparatus fourcylinder, USP XXIII). The polymer conjugates were attached to the buccal mucosa and submersed in vessels containing simulated saliva fluid. USP type apparatus (Erweka DT 700, Heusenstamm, Germany) was set at 37 ± 0.5°C whereas the cylinder rotate and the detachment of the polymers from the mucosa were visually determined (Laffleur et al., 2015a).

Water uptake ½mg ¼ Wu−W0=W0 Mucoadhesive Strength Assay where Wu expresses the weight of uptaken water at time t and W0 expresses the initial weight of the disc.

Quantitative mucoadhesive studies were performed using excised porcine buccal mucosa (17). In brief, testing

Novel Curcumin-Loaded Excipient for Buccal Delivery polymers were fixed to a stainless steel flat disc (10 mm in diameter), hanging from a laboratory stand with a nylon thread (15 cm). Buccal mucosa was glued on a platform placed in a beaker filled with simulated saliva. Then, polymer and mucosa were brought in contact. By manually switching the knob provided with the mobile platform, the mucosa was detached down from the test disc at a rate of 0.1 mm/s after 20-min incubation time. Data was collected by the computer software (SartoCollect V 1.0; Sartorius AG, Germany) connected with the balance with integrated interface. Data was transferred to EXCEL 2007 (Microsoft, USA) and the force versus displacement curves were analyzed to determine the maximum force of detachment (MDF) and the total work of adhesion as the area under the curve in accordance with the trapezoidal rule. Permeation Through Buccal Mucosa Curcumin, HA-SH, and HA 0.5% (w/v) solution were prepared, respectively, in order to evaluate the potential permeation-enhancing effect of thiolated HA-SH through freshly excised buccal mucosa. 2 × 2 cm2 tissues were mounted on Ussing-type chambers with a surface area of 0.64 cm2 (18). After an incubation of 15 min with simulated saliva fluid, 0.5% (w/v) HA and HA-SH, respectively, with curcumin in a final concentration of 0.5% were applied to the donor compartments. At prescheduled time points, samples of 100 μL were drawn over 6 h. The samples were immediately measured at 428 nm and concentration was calculated according to the standard calibration curve. Permeated curcumin was detected spectrophotometrically and apparent permeability coefficients (Papp) were calculated regarding following equation: Papp ¼ Q=ðA  c  t Þ where Q exhibits total amount permeated throughout 6 h (μg), A represents the diffusion area (0.64 cm2), c shows the initial concentration of curcumin in the donor compartment (μg/cm3), and t exhibits the time of permeation study(s). Transport enhancement ratios were determined by Papp values as following: R ¼ PappðsamplesÞ=PappðcontrolsÞ

Appraisal of Sulforhodamine Release Sulforhodamine release of HA-SH and HA was investigated with dissolution study. For this purpose, 0.001% curcumin was incorporated while preparing a drug solution and adding to the polymer mixtures. The polymer-drug mixtures were incubated for 3 h and lyophilized. Lyophilized polymer-drug mixtures were compressed into discs and release behavior was investigated. Vessels containing 50 mL of simulated saliva fluid were prepared, discs were placed into the vessels, and samples of 100 μL were taken at predetermined time points over 3 h. The disc sulforhodamine release was determined by spectrophotometrically detection (19). Statistical Data Analysis For statistical analysis, analysis of variance was used. A probability of less than 0.05 (P < 0.05) was considered statistically significant. All results are presented as mean ± SD. RESULTS AND DISCUSSION Synthesis and Characterization of Thiolated Hyaluronic Acid Conjugate Figure 1 shows the formation of amide bond between the thiol bearing ligand and the hyaluronic acid. Moreover, free thiol groups of thiolated hyaluronic acid-cysteine ethyl ester (HA-SH) were determined by Ellman’s assay. Ellman’s results exhibit 145.52 ± 4.91 μmol per gram of hyaluronic acid resulting in 5.14% degree of substitution by thiol ligand. Additionally, disulfide bond content was found to be 57.14 ± 31.54 μmol per gram of hyaluronic acid. HA-SH was white and of fibrous structure. Furthermore, as seen in Fig. 2a, H-NMR spectra of the product exhibits the ethyl group of the successful attachment of the ligand L-cysteine ethyl ester. Moreover, IR Spectra shown in Fig. 2b prove the succeeded synthesis. Safety Consideration Caco-2 cells were incubated with HA and HA-SH for 12 h in order to evaluate their in vitro cytotoxic effect. Results are shown in Fig. 3. Indicating a cell viabiltiy over 90% for both, HA and HA-SH, after 12 h, render these polymeric

Fig. 1. Schematic pathway of the synthesis. Hyaluronic acid was modified by covalent attachment of L-cysteine ethyl ester via amide bond formation mediated by carbodiimide

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Fig. 2. a H-NMR spectra of educt and product. b IR Spectra of HA (blue line) and HA-SH (red line)

excipients not harmful to the cells. Polymers were tested in a final concentration of (0.5%; m/v), due to the fact that concentration higher than 0.5% (w/v) led to high viscosity of the sample. In addition, the chosen concentration admits comparing cell viability of the well-established modified polymers. The herein found results of Resazurin assay are in good agreement with previously published work by Laffleur et al. convincing once more in safety of the synthesized thiolated polymer (18).

Fig. 3. Comparative cell viability rates of hyaluronic acid-cysteine ethyl ester (HA-SH) and unmodified hyaluronic acid (HA) after incubation period of 12 h on Caco-2 cells. All indicated values represent an average of at least three experiments (±S.D.)

Swelling Behavior Swelling and capillarity processes have a great impact on the mucoadhesion (20). Within the study, water-absorbing capacity was gauged by a gravimetric method as depicted in Fig. 4. Thiolated hyaluronic acid conjugate expressed a 2.75fold higher swelling behavior compared to corresponding unmodified hyaluronic acid over the time. Given that, HA-SH exhibit higher swelling ratio in comparison to unmodified

Fig. 4. Overview of the water uptake capacity of hyaluronic acidcysteine ethyl ester (HA-SH) (black dots) and unmodified hyaluronic acid (HA) (white dots) during 10 min. All indicated values represent an average of at least three experiments (±S.D.) (significant difference HA-SH and unmodified HA (*p < 0.05))

Novel Curcumin-Loaded Excipient for Buccal Delivery HA; reasons could be found in the covalent attachment of cysteine ethyl ester. Mueller showed a water uptake behavior of less than 350 mg with chitosan derivatives whereas HACYS in this study (21) showed an uptake of 600 mg. The electrostatically driven reaction between chitosan and mucin can be interpreted as the process of complex coacervation. However, the molecular mass, conformation, and overall flexibility of chitosan (the latter determined by the charge density, i.e., the degree of acetylation) also play a significant role (22). Moreover, it is reported that the reaction is entropydriven and that pH is a significant factor for mechanical strength of mucoadhesion and that hydration and swelling are contributing factors too (23). The swelling behavior is a complex phenomenon. In case of the unmodified polymer, polymeric chains are flexible and only hydrogen bonds are built. Due to a hydrate shell, the water uptake profile is limited. The water uptake capacity is restricted by the high solubility resulting in the disintegration of the polymer matrix. In case of the modified hyaluronic acid, the polymeric chains are flexible and due to the newly immobilized sulfhydryl group, now, disulfide bonds were built inter and intramolecular. Acting as a three-dimensional frame builder, modified HA keeps the incorporated water within the matrix. Operating like a gelling agent, modified HA represents a stiff gel solid with a rigid scaffold leading to a higher water uptake capacity. HA molecules assume an expanded random coil structure in solution. Finally, the degree and rate of polymer swelling influence the case of solid dosage forms since mucoadhesion reaches a maximum at an optimal water content and the over-hydration reduces adhesion (24). Mucoadhesive Study Mucoadhesive polymers need to exhibit characteristics facilitating interactions with mucins. Suitable chain flexibility at the pH and ionic strength of the mucus are required for these polymers whereas increase of chain flexibility is expected to favor interpenetration of mucus layer leading to enhanced mucoadhesion to form entanglements with mucins (25). Mucoadhesion is comprehensively described in the literature (26). Five theories delineate the phenomenon of mucoadhesion. Adsorption, diffusion, electronic, wetting, as well as fracture theory provide a deep insight in mucoadhesive mechanism (27). The wetting theory explains adhesiveness by designing adhesive anchors. Mucoadhesive systems require an intimate and prolonged contact with the mucosal surface in order to provide a longer absorption period (28). The resulting mucoadhesiveness of thiolated hyaluronic acid cysteine ethyl ester and unmodified hyaluronic acid, respectively, is displayed in Fig. 5. Findings showed the detachment of unmodified HA after 7 h whereas thiolated hyaluronic acid cysteine ethyl ester revealed a remarkable adhesion over 24 h. Reasons for the prolonged adhesion time in case of HA-SH is based on disulfide bond formation between free immobilized thiol groups on the polymeric backbone and cysteine-rich substructure of mucus glycoproteins. The performed tensile strength studies were in good agreement with previously published tensile strength studies on

Fig. 5. Assessment of mucoadhesive strength of thiolated hyaluronic acid-cysteine ethyl ester (HA-SH) and unmodified hyaluronic acid (HA) on the rotating cylinder. Indicated values are means (±SD) of at least three experiments (significant difference between HA-SH and unmodified HA (*p < 0.05))

buccal mucosa (18). Mucoadhesive strength assay further underpinned that HACYS was more pronounced mucoadhesive in comparison unmodified HA. Mueller et al. compared thiolated chitosans and their mucoadhesive and water uptaking properties. The mucoadhesive strength study showed more pronounced adhesive strength in case of HACYS with 600 μJ compared to chitosans with values below 480 μJ reported by Mueller et al. (21). Compared to other linear mucoadhesive polymers, HA exhibits a higher detachment work due to the HA/mucus interpenetration resulting in the strengthened mucus layer (29).

Permeation Through Buccal Mucosa Permeation studies through porcine buccal mucosal surface were investigated with Ussing-type chambers. Curcumin 0.5% (w/v) was embedded in thiolated hyaluronic acid-cysteine ethyl ester (0.5%) and hyaluronic acid (0.5%), respectively. Curcumin without carrier was regarded as control. Findings of the permeation study were shown in Fig. 6. The chosen pH was 6. Stability of curcumin is crucial to maintain its physiological activities. In acidic and neutral solutions (pH 2.5–7.0), curcumin emits a bright yellow hue while it turns red at pH above 7. The decomposition of curcumin is pH-dependent and it degrades more rapidly at neutral-basic conditions. In acidic conditions, degradation of curcumin is slow, with <20% of curcumin decomposed after 1 h. Curcumin undergoes rapid hydrolytic degradation at physiological pH or greater which becomes a significant disadvantage in its therapeutic use as reported by Wing-Hin Lee et al. (30). The permeation was 4.4-fold enhanced in case of thiolated hyaluronic acid-cysteine ethyl ester in comparison to respective unmodified hyaluronic acid. Deducing from the results obtaining by the buccal permeation, it could be assumed that thiolated polymer has an exalted bearing on the permeation of curcumin. The rectified impact of permeation enhancement is attributed to inhibition of the protein tyrosine phosphatase (PTP). The underlying mechanism being involved in the inhibition of protein tyrosine phosphatase (PTP) is based on a glutathione reaction. Available reduced glutathione (GSH), exhibiting a thiol group, interacts with PTP and influences

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Fig. 6. Investigation of the permeation through buccal mucosa via Ussing-type chambers (0.64 cm2 surface area). Curcumin was incorporated and examined in the presence of the test compounds 0.5% (w/v) unmodified hyaluronic acid (black dot) and 0.5% (w/v) thiolated hyaluronic acid-cysteine ethyl ester (HA-SH) (black triangle), respectively. Indicated values are means (±SD) of at least three experiments (significant difference between HA-SH and unmodified HA (*p < 0.05))

the permeation enhancement. Moreover, glutathione is known to be available in the reduced (GSH) and oxidized (GSSG) form at the apical side of mucosa. Thiomers could further reduce GSSG and increase the amount of GSH (31). PTP inhibition is carried into execution by disulfide bond formation between free thiol groups on the polymeric backbone and those of PTP (32). Moreover, it was also reported that HA is able to enhance the penetration of drugs, such as acyclovir, in an extent comparable to chitosan hydrochloride (33). Release of Sulforhodamine Matrix systems and their mechanisms of drug release involve solvent penetration, hydration and swelling of the polymer, diffusion of the dissolved drug in the matrix, and erosion of the gel layer as reported by Salomon et al.

(34). A major impediment of intraoral delivery poses the non-uniform distribution of drugs within saliva. Furthermore, the wash off and loss of the dosage form leads to uncontrolled drug release. By dint of a sustained drug release, a prolonged therapeutic level of drugs will be maintained which diminished the application frequency leading to a better patient’s compliance. During 6 h, HACSY compassing curcumin exhibit a 1.6-fold controlled release in comparison with unmodified HA as shown in Fig. 7. The stability of polymeric matrix occurs due to the formation of inter and intra chain disulfide b o n d s . C o n s e q u e n t l y, a s u s t a i n e d dr u g r e l e a s e of sulforhodamine was indemnified over 6 h. Other previous reported anionic polymers such as Carbopol® have many advantages in the design of sustained-release delivery systems, such as good gel-forming ability and mucoadhesive properties resulting due to its ionic nature

Fig. 7. Comparative study of release profile of sulforhodamine. Thiolated hyaluronic acidcysteine ethyl ester (HA-SH) (black triangle) and unmodified hyaluronic acid (HA) (black dots) were investigated over 6 h. The indicated release time represents an average of at least three experiments (±SD) (significant difference between HA-SH and unmodified HA (*p < 0.05))

Novel Curcumin-Loaded Excipient for Buccal Delivery and high sensitivity to the pH of the medium. Carbopol® carboxyl groups do not dissociate at pH 1.2, resulting in a tightly enclosed matrix. However, almost all Carbopol® carboxyl groups dissociate at pH 6.8, resulting in the formation of a swollen gel. Therefore, Carbopol® can be expected to exhibit both pH-sensitive and mucoadhesive features (35). These results correlate with previously published studies concerning SRH release out of polymeric matrices reported by Laffleur et al. (36). CONCLUSION Within this study, a new use of a very well-described material was highlighted. Hyaluronic acid was successfully thiolated with cysteine ethyl ester. Due to this thiolation, distinctive features such as permeation enhancement and mucoadhesion augmentation enfolded compared to unmodified polymer. Mucoadhesive studies on the buccal mucosa revealed a 3.4-fold higher adhesion compared to corresponding hyaluronic acid. By virtue of controlled release, these dosage forms could be applied where prolonged drug retention is required. However, more studies are needed to prove that curcumin has the potential of an effective agent in the treatment of various mouth disorders. ACKNOWLEDGMENTS The authors thank Josef Mayr in Mayr, Natters, Austria for the porcine mucosa. COMPLIANCE WITH ETHICAL STANDARDS Declaration of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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