International Journal of Pharmaceutics 511 (2016) 380–389

Development and characterization of crosslinked hyaluronic acidpolymeric films for use in coating processes

Débora Sgorlaa, Andreia Almeidab,c, Claudia Azevedoc, Élcio Jose Bunhaka,Bruno Sarmentoa,b,c,d,*, Osvaldo Albuquerque Cavalcantia,e

aCentro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná, Cascavel, Brazilb i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugalc INEB—Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Porto, PortugaldCESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Instituto Universitário de Ciências da Saúde, Gandra, PortugaleDepartamento de Farmacologia e Terapêutica, Universidade Estadual de Maringá, Maringá, Brazil

A R T I C L E I N F O

Article history:Received 13 June 2016Received in revised form 14 July 2016Accepted 15 July 2016Available online 18 July 2016

Keywords:BiopolymerEthylcelluloseHyaluronic acidPolymeric coatingTrisodium trimetaphosphate

A B S T R A C T

The aim of this work was to develop and characterize new hyaluronic acid-based responsive materials forfilm coating of solid dosage forms. Crosslinking of hyaluronic acid with trisodium trimetaphosphate wasperformed under controlled alkaline aqueous environment. The films were produced through castingprocess by mixing crosslinked or bare biopolymer in aqueous dispersion of ethylcellulose, at differentproportions. Films were further characterized regarding morphology by scanning electron microscopy,robustness by permeation to water vapor transmission, and ability to hydrate in simulated gastric andintestinal physiological fluids. The safety and biocompatibility of films were assessed against Caco-2 andHT29-MTX intestinal cells. The permeation to water vapor transmission was favored by increasinghyaluronic acid content in the final formulation. When in simulated gastric fluid, films exhibited lowerhydration ability compared to more extensive hydration in simulated intestinal fluids. Simultaneously, insimulated intestinal fluids, films partially lost weight, revealing ability for preventing drug release atgastric pH, but tailoring the release at higher intestinal pH. The physiochemical characterization suggeststhermal stability of films and physical interaction between compounds of formulation. Lastly,cytotoxicity tests demonstrated that films and individual components of the formulations, whenincubated for 4 h, were safe for intestinal cells Overall, these evidences suggest that hyaluronic acid-based responsive films, applied as coating material of oral solid dosage forms, can prevent the prematurerelease of drugs in harsh stomach conditions, but control the release it in gastrointestinal tract distalportion, assuring safety to intestinal mucosa.

ã 2016 Elsevier B.V. All rights reserved.

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International Journal of Pharmaceutics

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1. Introduction

The development of systems for modified release of drug, whichprovides delivering of active compounds to specific regions of thebody, is still a major challenge for researchers both from academic

Abbreviations: CHA, cross-linked hyaluronic acid; DSC, differential scanningcalorimetry; EDS, energy dispersive spectrometry; EC, ethylcellulose; XRD, X-raydiffraction; SGF, fluid of gastric simulation; SIF, fluid of intestinal simulation; GIT,gastrointestinal tract; HA, hyaluronic acid; FTIR, infrared spectroscopy; SEM,scanning electron microscopy; SI%, swelling index; TGA, thermogravimetricanalysis; STMP, trisodium trimetaphosphate; WVT, water vapor transmission.* Corresponding author at: Centro de Ciências Médicas e Farmacêuticas,

Universidade Estadual do Oeste do Paraná, Cascavel, Brazil.E-mail addresses: bruno.sarmento@ineb.up.pt, brunocsarmento@hotmail.com

(B. Sarmento).

http://dx.doi.org/10.1016/j.ijpharm.2016.07.0330378-5173/ã 2016 Elsevier B.V. All rights reserved.

community and pharmaceutical industrial. In order to direct thedelivery of drug formulated into tablets, the use of polysaccharidesfor coating processes of solid oral dosage forms can contribute for abetter tailoring of the release of active molecules for the intestinalsites of absorption. Such improvement has been particularlydemonstrated when natural polysaccharides are used in associa-tion with well-established coating materials as ethylcellulose (EC)and its derivatives (Serverino et al., 2011).

Hyaluronic acid (HA) is a linear anionic polysaccharide formedby repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine. HA presents excellent biocompatibility, low toxicityand is fully biodegradable in vivo (El-Aassar et al., 2015; Lee et al.,2015; Oliveira et al., 2016). Moreover, HA is known for being easilymodified with chemical functionalization of functional groups. Italso possesses a natural affinity for specific receptors, such as CD44

D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389 381

(Han et al., 2012; Mero and Campisi, 2014), which makes it apossible agent for target delivery. These receptors are overex-pressed on various epithelial cells, as enterocytes (Misra et al.,2011; Saravanakumar et al., 2013).

Owing to the potential and versatility of this polymer, anincreasing number of biomedical applications have been de-scribed. The HA have been widely used for viscosupplementationof synovial fluid in patients that suffer from arthritis (Lùrati et al.,2015), in intervertebral disc regeneration (Shen et al., 2010), for thetreatment and diagnosis of atherosclerosis (Lee et al., 2015), forcontrolled drug release purposes (Kumar et al., 2015; Kwon et al.,2015; Zhong et al., 2015) and as component of a wide variety ofcosmetics (Pan et al., 2013). The development of HA-basedderivatives with enhanced or modulated properties has also beensuggested (Schanté et al., 2011).

Still, HA is a highly hydrophilic polymer that can absorb a largeamount of water, expanding up to 1000 times its solid volume,forming a loose network (Mero and Campisi, 2014). This featureresults in inability to withstand the conditions founded in theupper gastrointestinal tract (GIT) when used alone in coating filmforming process and may promote a premature release of drugtrapped in the coated tablet. Trying to overcome this limitation, HAcan be chemically modified by crosslinking agents such astrisodium trimetaphosphate (STMP), which is a low toxicity saltthat does not cause adverse effects in humans and reacts withhydroxyl groups of the polysaccharide (Dulong et al., 2004;Prezotti et al., 2012; Souto-Maior et al., 2010).

Thus, the aim of this study was promote the crosslinking of HAwith STMP, with subsequent production of new polymeric materialin free film form. The characteristics of developed films for use inthe coating process of solid oral dosage forms proposed formodified release of drug were also addressed. This crosslinkedalternative intends to decrease hydrophilicity of bare HA, incombination with the mixture of insoluble EC, a classical filmcoating component. Also, it may be possibly to increase theintestinal targeting drug delivery due to the presence of HA(crosslinked or bare) due to its potential ability to bind to CD44receptors (Vafaei et al., 2016).

2. Materials and methods

2.1. Materials

Hyaluronic acid 1.5 MDa (sodium salt, from Streptococcus equisubsp. zooepidemicus) was from Contipro (Czech Republic). Ethyl-cellulose (Surealease1) was a gift from Colorcon (Brazil) andtrisodium trimetaphosphate from Sigma-Aldrich (Brazil). All otherchemicals were of lab grade. Simulated gastric fluid (SGF, pH = 1.2)and simulated intestinal fluid (SIF, pH = 6.8) without addition ofenzymes, were prepared according to United States Pharmaco-poeia (USP).

2.2. Development of crosslinked hyaluronic acid

A solution of 1% HA (200 mL) was prepared in water (pH 12.0),which was stirred for 2 h to allow complete homogenization. Atbasic pH, a complex of di-polymer phosphate ester is formed frompolysaccharide and STMP (Gliko-Kabir et al., 2000). Subsequently,20 mL of 30% STMP solution in water were added at the previousHA solution and stirred for additional 2 h. After this process, theportions corresponding to the concentrations of 5 and 10% of cross-linked polysaccharide were added to the EC dispersion to obtainthe free films (Bunhak et al., 2015).

2.3. Production of films

Conventional solvent evaporation method, also called castingprocess, was used for prepare films in seven different proportions(Rabito et al., 2012; Santos et al., 2013). Different formulationswere obtained varying the concentration of the ethylcellulose and/or HA, maintaining the polymer mass constant at 4% (w/v).Polymeric solutions were prepared by magnetic stirring undervacuum conditions to avoid air incorporation and possible bubblesformation in the films, in a final volume of 10 mL. Then, solutionswere poured onto Teflon1 plates and oven-dry at 60 �C for 12 h(Rosiaux et al., 2013; Yang et al., 2010). Films were removed fromthe plates after drying and placed at desiccator at roomtemperature.

2.4. Macroscopic characteristics and free films thickness assessing

Films were analyzed by their macroscopic features as homoge-neity, presence or absence of air bubbles and cracks. Thesecharacteristics may modify the films integrity and affect furtheranalysis. Thickness of films were determined averaging fiverandom points with a micrometer (Mitutoyo1) (Rabito et al.,2012; Santos et al., 2013). The films were kept at desiccator withsilica gel until the tests accomplishment.

2.5. Water vapor transmission study

Initially, 10 mL of distilled water were added to permeabilitydomes (Braive Instruments1 type), where films samples werefixed. Then, each set (dome + distilled water + film) was weighedand stored in a desiccator containing silica gel at room tempera-ture. The following measurements were made after 24, 48, 72, 96and 120 h. Furthermore, silica was replaced after each weighing.The weight difference of each dome in the respective time intervalswas recorded and Eq. (1) was used to calculate the vaportransmission rate of water transported through the films. TheWVT was standardized for a period of 24 h (Oliveira et al., 2011;Santos et al., 2013).

WVT ¼ g:24t:a

ð1Þ

where g is the weight loss, t is the time (h) during which the weightloss was accompanied and a is the film area (m2). Measurementswere performed in triplicate for each formulation.

2.6. Swelling index determination

The different hydration characteristics of the new polymericmaterial were determined by swelling assessment. Briefly, samplesof films were cut into pieces of about 1 cm2, accommodated in Petridishes and left into an oven at 50 �C for 15 h, to achieve thecomplete loss of moisture. Afterward, films were stored indesiccators containing silica gel for conducting experiments.Samples of the different formulations were initially weighed onanalytical balance to determine the dry mass and immediatelyimmersed in containers containing SGF or SIF for predeterminedperiods of time (1–10, 30, 60, 90, 120 and 150 min) at constanttemperature (37 �C). After, the films were removed from the mediausing tweezers, carefully dried between two sheets of filter paperto remove excess water, and then weighed again. This test wascarried on in triplicate and Eq. (2) was used for SI% quantification(Oliveira et al., 2011; Santos et al., 2013).

SI% ¼ weight of swollen film � weight of dry filmweight of dry film

� 100 ð2Þ

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2.7. X-ray diffraction analysis

Chemical analysis of samples of films were evaluated using anenergy dispersive spectrometer for X-rays (EDS) (SwiftED-3000model) with tungsten filament as the electron source operated at15 keV. X-ray diffraction (XRD) was performed on a Brukerdiffractometer D2 Phaser model with a copper emission source(CuKa = 1.5418 Å) operated at 30 keV, current 10�3 A, workingwindow 7–60� and increase of 0.02�/s.

2.8. Thermal analysis

Differential scanning calorimetry (DSC) was used to evaluatethe thermal behavior of films. Samples of different formulations(about 6 mg) were placed in closed aluminum crucibles andsubjected to a controlled heating program ranging from 25 to280 �C at a heating rate of 10 �C per minute. The tests wereconducted in an inert atmosphere with argon flow of 50 mL perminute, using a DSC Q100 equipment (TA Instruments). For thethermogravimetric analysis (TGA), curves were obtained in thetemperature range of 25–800 �C with the same heating rate (10� Cper minute), using approximately 6 mg of samples in open ceramiccrucibles and inert atmosphere. The argon flow was 100 mL perminute and the equipment employed a TGA 2950 (TA Instruments).

2.9. Scanning electron microscopy

Samples of different formulations were microscopically evalu-ated in dry state and after swelling in SGF and SIF. Subsequently,hydrated films were freeze-dried (�55 �C during 6 h) in Terroni1

lyophilizer LT 1000 model (Bunhak et al., 2015). After this step, thesamples were sputtered with a thin layer of gold/palladium alloyon their surface, placed on tape carbon and analyzed by scanningelectron microscopy with field emission by JEOL JSM-6340-Fequipment, operated at 2 keV.

2.10. Infrared spectroscopy

In order to assess the interaction between components, filmswere analyzed by Fourier transform infrared spectroscopy (FTIR),on a Vertex 70v model (Bruker Corporation, Germany), with thespectral range of 4000–400 cm�1, resolution of 4 cm�1 and 100scans. The infrared absorption spectra were obtained after samplepreparation with KBr.

Fig. 1. Schematic reaction between hyaluron

2.11. Cell culturing

Caco-2 cells (clone C2BBe1) were obtained from American TypeCulture Collection (ATCC, USA), and HT29-MTX cell line was kindlyprovided by Dr. T. Lesuffleur (INSERMU178, Villejuif, France).Dulbecco's Modified Eagle medium (DMEM, Lonza), was supple-mented with 10% (v/v) fetal bovine serum (FBS) (Merck Millipore),1% (v/v) penicillin (100 U/mL, Merck Millipore), streptomycin(100 mg/mL, Merck Millipore) and 1% (v/v) non-essential aminoacids (NEAA, Merck Millipore). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide(DMSO), were obtained from Sigma Aldrich, Triton X-100 1% fromSpi-Chem and culture flasks and 96-well tissue culture plates werepurchased from Corning Inc., USA. The plates were read inmicroplate spectrophotometer (Biotek Synergy 2, USA) and cellswere maintained in an incubator (ESCO CelCulture1 CO2 Incuba-tor, Singapore) at 37 �C temperature and 5% CO2 in a watersaturated atmosphere.

2.12. Cytotoxicity studies

Potential cytotoxicity of EC 00:100 HA, EC 00:100 CHA, EC100:00 HA, EC 95:05 HA, EC 90:10 HA, EC 95:05 CHA and EC 90:10CHA was tested against Caco-2 and HT29-MTX cells, colorectalcancer cells, using MTT reagent. Caco-2 (passage 51–54) and HT29-MTX (passage 41–43) cells grew separately in culture flasks in acomplete medium consisting of DMEM supplemented with 10% (v/v) FBS, 1% (v/v) L-glutamine, 1% (v/v) NEAA, and 1% (v/v) antibiotic-antimitotic mixture (final concentration of 100 U/mL Penicillin and100 U/mL Streptomycin). The culture medium was replaced everyother day. Cells were seeded (200 mL) into wells of 96-well tissueculture test plates with 1 �104 cells/well for HT29-MTX cell lineand 2 � 104 cells/well for Caco-2 and incubated at 37 �C in a 5% CO2

air atmosphere for 24 h; After that, medium was removed and cellswere washed twice with 200 mL of phosphate-buffered saline(PBS). Then was replaced fresh warm medium (200 mL) with twoconcentrations, 0.5% and 1% (m/v) of the films described above. Anegative control (1% Triton X-100) and positive control (mediumwith cell incubation) was included. After 4 h of incubation at 37 �Cin 5% CO2 atmosphere, the medium was discarded and cells arewashed twice with 200 mL of PBS. Finally, was added 200 mL ofMTT solution per well (final concentration of 0.5 mg/mL) and wasincubated for 4 h in dark. After 4 h, was removed and discard theMTT solution, and was added 200 mL of DMSO to dissolve the MTT

ic acid and trisodium trimetaphosphate.

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formazan crystals. The plates were placed in orbital shaker for15 min in the dark, at RT and the absorbance was measured using amicroplate spectrophotometer at 570 nm and 630 nm. The resultswas analysed according to the following equation:

Viability %ð Þ ¼ experimental value � negative controlpositive control � negative control

� 100 ð3Þ

2.13. Statistical analysis

All the experiments were performed in triplicate and arerepresented as mean � standard deviation (SD). Analysis ofvariance (ANOVA) with Bonferroni Multiple/Post Hoc GroupComparisons and Tukey’s multiple-comparison test were per-formed using GraphPad Prism1 software (version 5.03). Thesignificance level was set at probabilities of *p < 0.05, **p < 0.01,and ***p < 0.001.

3. Results and discussion

3.1. Crosslinking of hyaluronic acid

The crosslinking process of HA with STMP was established in astoichiometric ratio of one STMP reactive groups with threehydroxyl pairs of the HA, to obtain a modified material with lowerhydrosolubility. The reaction scheme is shown in Fig. 1.

Fig. 2. Optical images of different formulations. a) Control film of synthetic polymer EClinked polysaccharide EC 00:100 CHA d) EC 95:05 HA e) EC 95:05 CHA f) EC 90:10 HA

3.2. Macroscopic characteristics and films thickness

Macroscopic analysis of morphological characteristics of thefilms is a fundamental evaluation, considering the need tomaintain the integrity of films as coating material. Therefore,films that showed any type of defect must be excluded, as mayaccelerate premature degradation of solid matrices and release ofpayload (Oliveira et al., 2011; Rabito et al., 2012; Santos et al., 2013).As shown in Fig. 2, the different formulations allowed thedevelopment of films with absence of air bubbles and/or crasheson the surface. The homogeneity in the distribution of polymersinvolved was also macroscopically evident, presenting a probablecompatibility in the dispersion of the constituents. However, anincrease in HA or CHA concentration promoted a variation at filmsappearance, such as a decrease in transparency and flexibility tomanual handling. The function of polysaccharide was to providespecificity to the new polymeric material. All these data aresummarized in Table 1.

Regarding the film thickness, changes in different formulationswere also noted, when compared to the control EC and HAfilms (Table 2). Furthermore, the low variability of triplicatesindicates that the method was reproducible, ensuring the filmsformation with homogeneous thicknesses. These are in accordancewith previous reports (Bunhak et al., 2007; Souto-Maior et al.,2010).

100:00 HA b) Control film of polysaccharide EC 00:100 HA c) Control film of cross- and g) EC 90:10 CHA.

Table 1Macroscopic morphological assessing of different formulations.

Formulation Homogeneity Cracks Airbubbles

Transparency Flexibility

EC 100:00 HA 0 0 0 +++ +++EC 00:100 HA 0 0 0 +++ +++EC 00:100CHA

0 0 0 + ++

EC 95:05 HA 0 0 0 ++ ++EC 90:10 HA 0 0 0 + +EC 95:05 CHA 0 0 0 + ++EC 90:10 CHA 0 0 0 + +

0: No changes observed, +: Slight presence, ++: Moderate presence, +++: Strongpresence.

Table 2Mean values of the thicknesses of the films. Each value represents the mean � SD ofthree independent measurements (n = 3).

Formulation Thickness (mm)

EC 100:00 AH 0.0607 � 0.0011EC 95:05 AH 0.0822 � 0.0059EC 90:10 AH 0.0912 � 0.0090EC 95:05 AHR 0.076 � 0.0205EC 90:10 AHR 0.1260 � 0.0109

Fig. 3. Weight loss values of films in WVT study.

384 D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389

3.3. Water vapor transmission study

The evaluation of permeability, applying the WVT technique infree films is considered a simple method, when compared withtraditional diffusion methods. However, it provides importantresults concerning the polymeric material for coating process inmodified drug delivery systems (Souto-Maior et al., 2010).

As depicted in Table 3, vapor transmission rate of water wasaffected by the polymeric composition of films. For EC:HA blendfilms, the WVT increased with increased content of CHA or HA,confirmed by the weight loss of the sets (dome + water + film)—Fig. 3. It was reported that HA films are ineffective as a moisturebarrier due to its hydrophilic nature, as well as konjac gluco-mannan film (Li et al., 2015).

The increase in hydrophilicity of the system was caused by theaddition of the unmodified polysaccharide, which is rich inhydroxyl (��OH) groups that interact with water molecules to formhydrogen bonds. However, when CHA was used for film formationthe permeability was reduced when compared to the sameconcentration of unmodified polysaccharide, being the ratesclosest from control film (EC). This fact was already expectedand can confirm that the natural polymer crosslinking process withSTMP decreased the hydrophilicity of the new material. Theimproved hydrophilicity behavior occurs due to the reduction ofhydroxyl groups available to interact with the water.

Statistical analysis of the data showed that there weresignificant differences (p < 0.05) between different formulationsand control. However, when all of these values was subjected toTukey’s multiple-comparison test only the EC 90:10 HA formula-tion versus EC 100: 00 HA, EC 95:05 HA and CHA and EC 90:10 CHA,

Table 3Weight loss values and WVT rates of different formulations. Each value representsthe mean � SD of three independent measurements (n = 3).

Formulation Weight loss values (g/120 h) WVT (g/m2 24 h) g/h

EC 100:00 HA 1.6767 � 0.0488 335.34 0.0139EC 95:05 HA 2.1517 � 0.1557 430.34 0.0179EC 90:10 HA 2.9004 � 0.0766 580.08 0.0241EC 95:05 CHA 1.6016 � 0.2614 320.32 0.0133EC 90:10 CHA 2.1372 � 0.2118 427.44 0.0178

EC 95:05 HA versus EC 95:05 CHA and EC 95:05 CHA versus EC90:10 CHA presented significant parameters.

Similarly other authors evaluated the pectin addition toKollicoat SR30D and the influence of b-cyclodextrin, an acrylicpolymer, and were observed the same trend (Oliveira et al., 2011;Wei et al., 2009). Furthermore, in another study was concludedthat hydroxyl groups of the pectin had bound to STMP andpromoted a reduction in the hydrophilicity of the films. They wereproduced with an aqueous dispersion of polymethyl methacrylate(Eudragit1 RS 30 D) associated with the phosphate pectin and aprebiotic oligosaccharide. Also, this feature is important to controlthe diffusion of fluids and drugs when trapped in the systems(Souto-Maior et al., 2010).

3.4. Swelling index determination

In this study, the determination of SI% was used to evaluate thehidrophilicity of the films through hydration behavior coming fromthe different polymeric formulations at simulated physiologicalfluids (SGF and SIF). The results of Si% after 150 min of immersion inthe fluids, kept at 37 �C are shown in Fig. 4. It was observed ageneral dependency on the swelling degree, according to filmscomposition and pH of the fluid. In SGF, different formulationsproduced exhibited a lower degree of hydration, which isassociated with the acidic pH that provides greater interlockingof the anionic polymeric network. The carboxyl groups remain inthe protonated form, reducing thereby the water retention by thesystem. Unlike in SIF, whose pH is higher, carboxylate groups(COO��) suffered ionization, which leads to repulsion of the chainsand determines an expansion of polymeric network. These areasthen occupied by water, explain the higher hydration founded. The

Fig. 4. Swelling index of films after 150 min of immersion at SGF and SIF. Valueswere reported as mean � standard deviation (n = 3). (*), (**), and (***) denotes asignificant difference (p < 0.05, p < 0.01 and p < 0.001), respectively.

D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389 385

present results are similar with other works at the same researchline (Souto-Maior et al., 2008, 2010). Furthermore, the samerelation between the SI%, film composition and pH of the mediumwas reported in another research that studied the characteristics offilms produced from Eudragit FS 30 D a pH-dependent polymerand arabinoxylan (Rabito et al., 2012).

This pH-dependent swelling behavior dependent on the choiceof the polysaccharide appears to be quite interesting since it shouldcontribute to avoid premature release of the drug in the upperparts of the GIT, which has low pH. However, there is vulnerable pHranges in the higher distal portion of the GIT (Maior et al., 2008).

In SIF, the films of EC 90:10 HA formulation showed partialdisintegration, which was observed by weight loss betweenswelling intervals. This behavior can be related to polymericexpansion and high water solubility of hyaluronic acid, whichallows its migration to the medium. This partial disintegration wasalso observed in films of EC 90:10 CHA formulation, however to alesser degree. These evidences demonstrate the performance ofSTMP on the polysaccharide during the crosslinking process andconsequent reduction in its hydrophilicity.

3.5. Energy dispersive spectrometer for X-rays

The EDS analyzes provided a qualitatively identified theelements present on the films and to evaluate the homogeneitywith which they were dispersed. The results presented inSupplementary material suggest that there was a good homoge-neity of carbon (relative to the polymer backbone) in EC 100:00 HA,EC 00:100 HA, EC 90:10 HA and EC 90:10 CHA formulations.However, in EC 00:100 CHA, EC 95:05 HA and EC 95:05 CHAformulations it was less homogeneous. Regarding the filmsproduced with CHA there was also phosphorus identificationrelative to STMP, indicating that the crosslinking process promotedthe incorporation of this component into carbon skeleton of thepolymer. Sodium was identified too, and it was associated withSTMP structure or also as waste of base used for pH correction,which was maintained around 12 during the process.

3.6. X-ray diffraction

From the degree of organization of the polymeric material itwas possible to determine its amorphous or crystalline nature, asshown in Fig. 5. According to the XRD spectra films showedamorphous nature, except the EC 90:10 CHA formulation whichpresented a more crystalline nature. It occurs because thisformulation has a bigger quantity of CHA in your compositionand the chemical modification of the polysaccharide with STMPbecame its most organized structure. However, the EC 95:05 CHAand EC 90:10 CHA films did not show the same pattern of behavior.

Fig. 5. XRD diffraction patterns of different formulations.

Moreover, it was evident that the addition of polysaccharidechanged the structure of films.

3.7. Thermal analysis

Two experiments were conducted in order to investigate thethermal stability of both synthetic polymer as well as pure or cross-linked polysaccharide at different formulations. Fig. 6 shows theDSC curves of different films obtained under an inert atmospherein the temperature range of 25–280 �C.

When CHA behavior was compared to unmodified HA, as shownin Fig. 6(a), it was observed that the endothermic peak of CHAdehydration decreased in intensity and shifted to lower temper-atures when compared to HA (120.31 �C and 108.85 �C, respective-ly). This result suggests that the cross-linked polysaccharide withSTMP presents a smaller amount of carboxylic groups available tointeract with water molecules. Because of this reason theincorporation of STMP decreased hydrophilicity of the biopolymer.These evidences are in agreement with similar results obtainedduring evaluation of thermal characteristics of bare pectin andcross-linked (Maior et al., 2008). Moreover, the two DSC

Fig. 6. DSC thermograms of different formulations. a) Control films of natural andcross-linked polysaccharide (EC 00:100 HA and EC 00:100 CHA, respectively) b)Control film of synthetic polymer EC 100:00 HA and different formulations: EC95:05 HA, EC 95:05 CHA, EC 90:10 HA and EC 90:10 CHA.

386 D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389

thermograms showed the presence of an exothermic peak at 234.9and 232.79 �C which are associated with decomposition of HA andCHA, respectively. These results were supported by previousstudies carried out with this polysaccharide (Collins and Birkin-shaw, 2008).

The EC 100:00 AH control film thermogram—(Fig. 6(b)) showedthe presence of an endothermic peak at 95.55 �C, which isassociated with the loss of moisture remaining after the initialdrying procedure. Similar peaks were also observed in differentformulations thermograms at 96.32, 96.32, 82.83 and 84.11 �Cattributable to EC 95:05 HA, EC 95:05 CHA, EC 90: 10 HA and EC90:10 CHA, respectively. Subsequently, the EC 100:00 HA filmcurve demonstrated another endothermic event at 222.98 �C. Itwas quite similar to that was found in another research where itwas reported the same event at around 223 �C, followed by ECdecomposition at around 355 �C (Alves et al., 2009).

The degradation profiles reveal that EC:HA blend films arethermally stable up to 200 �C. So, the new polymeric material canbe used for the coating process since these processes are carriedout at temperatures below 60 �C.

Moreover, regarding the glass transition temperature (Tg) of thecompounds, it was not possible to observe changes in the DSCthermograms probably due to interference attributable to thepresence of moisture.

Fig. 7 shows the TGA curves for the different films under inertatmosphere at 25–800 �C temperature range. According to thisfigure, the pattern of decomposition of the polysaccharide revealsthat the crosslinking promoted an increase of their thermalstability. It was observed by the lower weight loss rates from CHAwhen compared to the HA at the TGA curves of both samples. Onthe other hand, TGA curve of pure EC film showed a decompositionpattern with two weight loss stages, which are associated withinitial and final stages of degradation of the synthetic polymer. Thefirst stage was shown around 319.50 �C and represents apercentage of 78.88% of weight loss. The second thermal eventwas observed at 360.20 �C, which is associated with the finalpolymer degradation, and is responsible for 3.24% of weight loss.These data are in agreement with findings described by otherauthors that report 316 �C as initial decomposition temperature ofEC and the maximum temperature of 363 �C (Lomakin et al., 2011).

The representative curves of thermal decomposition of differ-ent formulations showed an improvement of thermal stability of

Fig. 7. TGA thermograms of different formulations.

EC films after incorporation of the polysaccharide. It wasdemonstrated by the reduced weight loss compared to controlfilm of synthetic polymer. Similar behavior was related afteraddition of HA to 3D composites based on the blends of chitosanand collagen, in other words, it means that the sample was moreresistant to thermal decomposition (Sionkowska et al., 2016).

3.8. Scanning electron microscopy

The results of scanning electron micrographs of films can beobserved in Fig. 8. According to the micrographs of Fig. 8A(a), B(a)and C(a) when dried EC 100:00 AH control film was compared tothe immersion ones at physiological simulation fluids no changeswere observed. They all maintained a smooth and homogeneousappearance in both situations. However, changes in the morpho-logical characteristics were observed when the hyaluronic acid wasadded in the formulations of the films. In SGF (Fig. 8B), aheterogeneous surface was observed, probably related to thepresence of the polysaccharide. It was also possible to see abreakdown of the polymeric network, which is more prominentwhen the samples were immersed in SIF, as shown in Fig. 8C. Thisfigure also demonstrated a more relaxed polymeric network. Thisphenomenon occurs at pH ranges close to basic becausecarboxylate groups suffer ionization and determine an expansionof the polymeric network. Consequently, there is a HA migration tothe medium, as previously discussed in SI% analysis. Furthermore,when the appearance of films produced with HA were compared tofilms produced with CHA it was possible to observe a differentarrangement for formulations produced with the same concentra-tion of natural polymer.

The micrographs of EC 90:10 CHA formulation corroborate theresults found in XRD and DSC tests for this same composition,demonstrating a more ordered structure. Furthermore, due to thearrangement of the structure, it can be observed the presence ofpores after immersion in simulated physiological fluids. However,it is clear that the EC 90:10 CHA formulation appears more swollenin SIF, confirming the findings of the SI% analysis.

3.9. Infrared spectroscopy

Fig. 9 shows the FTIR spectra in the spectral range from 4000 to400 cm�1 for different formulations of films. For EC control film(Fig. 9(d)), the main bands indicate an OH stretching at around3500 cm�1, CH3 complex vibrations at 1375 and 1450 cm�1

corresponding to the small bands related to the vibrations ofCH3 complex, respectively. The COC stretching of cyclic ether at1100 cm�1 is also shown (Fernández-Pérez et al., 2011; Li et al.,2015).

Fig. 9(a) shows absorption bands of pure HA film at 611, 1411,1619 and 3420 cm�1 attributable to C��O��C stretching, C��Ogroup in combination with C¼O, secondary amide group and OHstretching, respectively. These are in accordance with previousreports (Reddy and Karunakaran, 2013). The pure CHA film showsbroad absorption bands at 1049–910 cm�1 revealing the emer-gence of an axial deformation corresponding to phosphate groupsattached to the polysaccharide after cross-linking with STMP asreferred in previous reports that analyzed natural and cross-linkedgum guar (Codagnone et al., 2004). They observed the samedeformation on the same region of number of waves.

Moreover, it was observed that the samples of differentformulations showed similar bands when compared to controlfilms, as shown in Fig. 9(b), (c), (e), (f). These data indicatethe possible existence of only physical mix between theconstituents of different formulations. The absorption spectra ofthese films revealed no displacement or formation of new bandsor peaks. Similar results were described during evaluation of

Fig. 8. Micrographs of different formulations. A) Dried state B) After immersion in SGF and C) After immersion in SIF. a) Control film of synthetic polymer EC 100:00 HA b) EC95:05 HA c) EC 95:05 CHA d) EC 90:10 HA and e) EC 90:10 CHA.

D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389 387

a-glucoligosacharide and Tween1 80 additives in EC filmsformation (Alves et al., 2009).

These evidences are significant because, the films can conservethe properties of two different materials and may use them insynergism both the characteristic of target site-specific of thepolysaccharide as time-dependency of EC, potentially improvingthe controlled drug delivery.

3.10. Cytotoxicity studies

In order to determine the toxicity of the films, the MTT assaywas performed against intestinal cell lines for 4 h, which is themaximum residence time in the gastrointestinal tract, as hasalready been described (Antunes et al., 2013; Araujo et al., 2014).

Fig. 9. FTIR absorption spectra of different formulations. a) Control film of naturalpolysaccharide EC 00:100 HA b) EC 95:05 HA c) EC 90:10 HA d) Control film ofsynthetic polymer EC 100:00 HA e) EC 95:05 CHA f) EC 90:10 CHA and g) Controlfilm of cross-linked polysaccharide EC 00:100 CHA.

388 D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389

As depicted in Fig. 10, the films and individualized materialswere founded safe for intestinal epithelial cells. When comparingthe results obtained with the positive control, which representseach cell line cultured only in complete medium, it is found thatalmost all films have a very similar cell viability (or even higher) of100%, which indicates that these materials are compatible with thebiological organism. On the other hand, there is some statisticalsignificance when comparing the results obtained for a film of 0.5%concentration (w/v) to a concentration of 1.0% (w/v). These resultswould be expected, since the concentration of film duplicates forthe same number of cells. However, with no apparent toxicity, allfilms investigated are compatible with the biological environment,which may be used in controlled drug delivery systems, respectingthe concept of biocompatibility.

This new material is intent to be used on tablets coating,improving the performance of drugs already market, and promotethe delivery of pharmacologically active substances directly totheir target of action. The effect of drug loading into tablets is notdependent on the coating material, but the film coating is able tocontrol the release of the drug, according the degradation kineticsand diffusion rate. Thereby, it is important to improve theefficiency of drug treatments, coupled with the reduction in dose

Fig. 10. Cell viability of EC 00:100 HA, EC 00:100 CHA, EC 100:00 HA, EC 95:05 HA, EC 90cell lines, respectively, at concentrations of 0.5% and 1.0% after incubation for 4 h. Values

significant difference (p < 0:05, p < 0:01and p < 0:001), respectively.

and avoiding side effects. In this case, the targeting of the drugwould be provided by the presence of the polysaccharide and itspotential affinity for the CD44 receptor. From the overall obtainedresults, the EC 90:10 CHA formulation appears to be the mostpromising coating materials to be employed in final dosage forms.

4. Conclusion

Natural polymers have become an important tool, alongsidewith synthetic polymers already established by the pharmaceuti-cal industry, for the development of new polymeric materials foruse in modified drug delivery systems whose the main potentialapplication is the coating of solid oral dosage forms. Moreover, thepresence of the polysaccharide may help to provide targeting drugdelivery to the site of action, due to its affinity by CD44 receptors. Inshort, this study revealed that the crosslinking process of HAimproved its hydrophilic character and provided the developmentof films suitable for planned tests. An increase in polysaccharideconcentration at the films favored the WVT, when compared to thecontrol (EC). When immersed in SGF, different formulationsshowed a lower degree of hydration, however, in SIF, filmsexhibited weight loss. This phenomenon occurred probably due tothe migration of the biopolymer to the medium, as demonstratedby SEM tests, which demonstrated a more expanded polymericnetwork in this pH range. Furthermore, the physicochemicalcharacterization suggested thermal stability of different formula-tions of films and physical interaction between constituents. Thus,these evidences supports that this new polymeric material couldbe used for the coating process, further supported on the absenceof cytotoxicity to intestinal cells. These features can improve theapplication of the new material in the production of promisingcontrolled drug delivery systems for oral administration. Currentongoing research is being performed to evaluate the positive role ofcoating material on dissolution and functionality.

Acknowledgments

Débora Sgorla would like to thank the financial support ofCAPES/FA Program (Chamada Pública 11/2014) and Bruno Sar-mento thanks the financial support of the project of Ciências SemFronteiras Program, Special Visitor Researcher (Chamada deProjetos MEC/MCTI/CAPES/CNPq/FAPs—no 09/2014) both linkedto Pharmaceutical Sciences Postgraduate Research Program (PCF—UNIOESTE/Paraná/Brazil). This work was also financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through theCOMPETE 2020—Operacional Programme for Competitiveness and

:10 HA, EC 95:05 CHA and EC 90:10 CHA films against Caco-2 (A) and HT29-MTX (B)were reported as mean � standard deviation (n ¼ 6). (*), (**), and (***) denotes a

D. Sgorla et al. / International Journal of Pharmaceutics 511 (2016) 380–389 389

Internationalisation (POCI), Portugal 2020, and by Portuguesefunds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework ofthe project “Institute for Research and Innovation in HealthSciences” (POCI-01-0145-FEDER-007274). Also acknowledges Col-orcon company in Brazil by the sample of ethylcellulose—Sure-alease1, Dc. Leandro Freire dos Santos, Dc. Fauze Jacó Anaissi andMe. Philip Quadros Mariani, who contributed to the realization ofcharacterization techniques (EDS and XRD) of this work and PCF—UNIOESTE Supervisor for the support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2016.07.033.

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