International Journal of Pharmaceutics 452 (2013) 283– 291
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm
ngineering poly(ethylene oxide) buccal films with cyclodextrin: novel role for an old excipient?
gnese Miroa, Ivana d’Angeloa, Antonella Nappia, Pietro La Mannab, Marco Biondia,aura Mayola, Pellegrino Mustob, Roberto Russob, Maria Immacolata La Rotondaa,rancesca Ungaroa, Fabiana Quagliaa,∗
Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, ItalyInstitute of Chemistry and Technology of Polymers, National Research Council of Italy, via Campi Flegrei, 34, Olivetti Buildings, 80078 Pozzuoli, Naples, Italy
a r t i c l e i n f o
rticle history:eceived 11 February 2013eceived in revised form 6 May 2013ccepted 11 May 2013vailable online 22 May 2013
eywords:uccal filmsyclodextrins
a b s t r a c t
Inspired by the multiple roles cyclodextrins can play in polymeric systems, here we engineeredpoly(ethylene oxide) (PEO) films with (2-hydroxypropyl)-�-cyclodextrin (CD) as multipurpose ingre-dient. To shed light on the potential of CD in formulating PEO buccal films for the delivery of poorlywater-soluble drugs, we preliminarily assessed thermal and mechanical properties as well as wettabilityof films prepared at different PEO/CD ratios. PEO/CD platform containing 54% by weight of CD was cho-sen as the optimized composition since it matched acceptable mechanical properties, in terms of tensilestrength and elasticity, with a good wettability. The platform was tested as buccal delivery system for tri-amcinolone acetonide (TrA), a lipophilic synthetic corticosteroid sparely water soluble. Confocal Raman
imaging clearly showed that CD was homogeneously (i.e. molecularly) dispersed in PEO. Nevertheless,homogenous drug distribution in the film without TrA crystallization occurred only in the presence of CD.Finally, CD-containing PEO film placed in simulated buccal fluids provided a useful speed-up of TrA releaserate while showing slower dissolution as compared to PEO film. These results, as well as compliance withquality specifications of pharmaceutical manufacturing products, strongly support the soundness of the
ard fu
strategy and prompt tow
. Introduction
Amid transmucosal delivery routes, buccal delivery remainsarticularly attractive due to high patient compliance and uniquehysiological features (Sudhakar et al., 2006; Patel et al., 2011;earnden et al., 2012; Senel et al., 2012). Research has been devoted
o find strategies to enhance drug permeation through buccalucosa and allow the absorption in the systemic circulation of drug
mounts in the therapeutic range (Hassan et al., 2009). Opposite ishe case of local delivery, where the drug should concentrate in theiseased area through immediate partitioning in the upper layerf epithelium to give a therapeutic response, whereas systemicbsorption should be as less as possible.
Great attention has been recently focused on film dosage formsue to a number of advantages over tablets in term of patient com-liance, possibility to attain either local or systemic effects as well
s great design flexibility (Morales and McConville, 2011; Senelt al., 2012). Mucoadhesive thin films have been marketed in theast few years for transmucosal buccal absorption of class II drugs
(hydrophobic and poorly soluble but highly permeable accordingto Biopharmaceutical Classification Scheme) with a special inter-est into antipain agents (fentanyl buccal soluble film marketedas Onsolys®/Breakyl® based on BEMA® technology, and incomingsuboxone film based on MonoSol Rx’s PharmFilm® technology).Whether extensive literature has been produced on systemic drugabsorption, much less is known on strategies to attain a localeffect while limiting systemic response. This aspect is of key inter-est for local buccal diseases, such as stomatitis/mucositis, surgicalwounds and generic lesions, where pathological areas have differ-ent extension and specific local treatments are not available to thepatient.
Mucoadhesive films are prepared from polymers, which ensureincreased retention time at action site, and plasticizer/s to controlmechanical properties. Indeed, an ideal film should adhere to buccalmucosa for a time long enough to allow drug absorption while with-standing mechanical stresses during residence in mouth. A widearray of mucoadhesive film-forming polymers have been used inthe production of bioadhesive drug delivery systems as recently
reviewed (Andrews et al., 2009; Morales and McConville, 2011).Amid them, high molecular weight poly(ethylene oxide) (PEO) isespecially favorable as bioadhesive and gives films with zero-orderrelease kinetics (Prodduturi et al., 2005; Thumma et al., 2008).
The most widespread technique to produce films is solvent-asting where a drug-polymer solution is prepared, dried, cut andacked to give a thin film (Morales and McConville, 2011). Sincehe early development of drug-loaded film, great concern in robustlm manufacturing has been attributed to content uniformity,hich needs to comply with requirements of Pharmacopeias. This
spect is further complicated by the fact that the incorporation ofoorly water-soluble drugs in mucoadhesive platforms made ofater-soluble polymers is challenging. Different strategies to over-
ome this issue have been proposed so far and thin films wherehe drug is initially solubilized, emulsified or dispersed in a poly-
er/plasticizer solution have been attempted. Amid them, controlf drug solubility along manufacturing process is a valuable option,specially during drying step where drug aggregation or conglom-ration of the ingredients can occur (Yang et al., 2008).
Cyclodextrins are well known complexing agents alreadypproved for human use able, among other things, to enhancerug solubility, to mask undesirable taste and increase chemicalnd physical stability of different bioactive molecules (Kurkov andoftsson, 2012). Incorporation of cyclodextrins in a polymeric drugelivery system has been recently revised as strategy to modulateroperties of drug delivery systems with special regard to the con-rol of release rate (Miro et al., 2011) as well as drug absorptionhrough buccal mucosa (Cappello et al., 2006; Loftsson et al., 2007;
iro et al., 2009).Although some applications have been described in which
yclodextrins or cyclodextrin derivatives have been employed toodify thin polymeric membranes with different purposes, these
tudies have dealt with crosslinked cyclodextrins or covalentlymmobilized cyclodextrins. Very few examples are reported inhe literature where cyclodextrins are simply dispersed in the
embrane to modify overall properties (Fenyvesi et al., 2007;ópez-de-Dicastillo et al., 2010).
Inspired by the multiple roles cyclodextrins can play in poly-eric systems, here we engineered mucoadhesive PEO films with
2-hydroxypropyl)-�-cyclodextrin (CD) as multipurpose ingredi-nt based on its safety in humans and solubility in organic solvent.o shed light on the potential of CD in formulating buccal filmsor the delivery of poorly water-soluble drugs, we preliminarilyssessed thermal and mechanical properties as well as wettabil-ty on PEO films prepared at different PEO/CD ratios. The optimizedlatform composition was tested on triamcinolone acetonide (TrA),
lipophilic synthetic corticosteroid sparely water soluble, whichs used to relieve the discomfort of buccal aphtae. A manufac-uring process taking advantage of peculiar PEO/CD solubility inthanol/water vehicles is proposed. Overall properties of drug-oaded films were then investigated paying particular attentiono drug homogeneity and release rate. Finally, the application ofaman spectroscopy to assess distribution of the drug in the matrix
s here reported for the first time.
. Materials and methods
.1. Materials
Triamcinolone acetonide (TrA) was kindly supplied by Fisio-harma (Italy), (2-hydroxypropyl)-�-cyclodextrin (CD, DS 0.99)as a gift from Roquette Frère (France), whereas NF gradeoly(ethylene oxide) (PEO) (Polyox WSR 205, approximate MW00 kDa; Polyox WSR 301, approximate MW 4000 kDa) was kindlyupplied by Dow Chemical Company (Midland, MI, USA). Polyox
SR 205 and 301 were always employed as blend at 1:1 w/w rationd referred in the following as PEO. Type II mucin from porcinetomach was purchased from Sigma Aldrich (Italy). All the otherhemicals were of analytical reagent grade.
a Melting enthalpies were normalized with respect to the net mass of PEO.b Standard deviations were calculated on at least three repeats.
2.2. Film preparation
Drug-free PEO and PEO/CD films were prepared by solventcasting. PEO films were prepared from 18 mL of a water solutioncontaining PEO (63 mg) added with 6 mL of ethanol. The solutionwas maintained under stirring for 3 h, then placed in a teflon cap-sule (diameter 7.5 cm) and allowed to dry at room temperature upto constant weight. For PEO/CD films, PEO water solution containedalso different amount of CD (0–80% CD, w/w) which gave films withcompositions reported in Table 1.
TrA-loaded PEO and PEO/CD films (PEO/TrA and PEO/CD54/TrA)were prepared analogously at a constant TrA content per surfacearea of 0.07 mg/cm2. To this purpose, 6 mL of an ethanol solutioncontaining TrA (3 mg) were added to 18 mL of water containing PEO(63 mg) or PEO/CD (63 mg of PEO and 75 mg of CD, PEO:CD weightratio 1:1.2, w/w). TrA weight percent in PEO and PEO/CD54 filmswas 4.8% and 2.1% of the total weight, respectively.
Film thickness was assessed by a digital caliper and reported asmean value of three measurements on three different points of asingle dry film.
2.3. Thermal analysis
Thermal analysis was performed to evaluate the effect of CDaddition on the crystallinity of PEO-based films and to assess theinteractions between the polymer and CD in dry films. To this aim,single scans were carried out on unloaded PEO/CD films at differ-ent PEO:CD weight ratios and on TrA-loaded films. Samples wereequilibrated at −20 ◦C and scans were registered up to 100 ◦C witha heating ramp of 10 ◦C/min under an inert nitrogen atmosphere(flow rate: 50.0 mL/min). In the experimental temperature range,the melting of the ethylene oxide units is registered (De Lisi et al.,2007). The heat absorbed during melting was determined by a dif-ferential scanning calorimeter (DSC Q1000, TA Instruments, USA)calibrated with a pure indium standard. The areas of the melt-ing peaks derived from DSC thermograms represent the meltingenthalpies of the platforms and were calculated through the fol-lowing equation:
�Hf = 1m
∫ t2
t1
qdt (1)
where �Hf is the melting heat, m the mass of the sample, t1 and t2the time points defining the opening and closing of the endothermicpeak with respect to the baseline of the thermograms, respectively,q the heat flow as detected by DSC and dt the elementary time inter-
val. Peak maximum corresponds to the melting temperature (Tm)of the platforms. The degree of crystallinity of platforms was cal-culated by normalizing melting enthalpies of the platforms to themelting enthalpy of completely crystalline PEO (188 J/g (Cimmino
l of Ph
ea
Imatitce
wwa
2
eu4fTbfiidtncmwgaf
�
ε
E
htoaid
2
iaaGBaat
A. Miro et al. / International Journa
t al., 1990)) and to the actual PEO mass fraction. Results wereveraged on at least three independent experiments.
DSC tests also enabled an estimate of CD/PEO molar ratio.ndeed, as previously described (Lazzara and Milioto, 2008), the
elting enthalpy per gram of macromolecule was quantified as function of the reciprocal of polymer weight fraction (1/W) inhe film. The reciprocal of the extrapolation of 1/W to zero melt-ng enthalpy represents the weight composition of the polymer inhe complex corresponding to a totally amorphous film. Thus, stoi-hiometry of PEO–CD complex can be estimated by the followingquation:
NCD
NPEO= (100 − WPEO)
MWPEO
WPEOMWCD, (2)
here NCD/NPEO is the molar ratio between CD and PEO, WPEO theeight percentage of the polymer in the film, MWPEO and MWCD
re the molecular weights of PEO and CD, respectively.
.4. Mechanical properties
To investigate the effect of CD addition to PEO on stress andlongation at break of the films, mechanical tests were carried outsing an Instron 4301 tensile testing machine (room temperature,5–50% relative humidity). Experiments were performed on drug-ree PEO, PEO/CD30, PEO/CD54 and PEO/CD70 platforms and onrA-loaded PEO/CD54 films. Stress–strain curves were obtainedy testing 30–40 mm long, 5–7 mm wide and 20–30 �m thicklm strips with no visual defects. Samples were cut and pos-
tioned between two clamps separated by a distance of 30 mm andesigned to avoid strip damaging during experiments. Data fromhe film samples that failed at (and not between) the clamps wereot utilized to evaluate mechanical properties. During mechani-al tests, the lower clamp was held stationary while the upper oneoved at a 5 mm/min strain rate until strip breaking. Grip regionsere reinforced to reduce the possibility of failure. Force and elon-
ation at break were recorded, and the stress (�N) [MPa], elongationt break (εb) and Young modulus (E) [MPa] were calculated by theollowing equations:
N = FL
S0(3)
b = 100�L
L0� (4)
= �L
εL(5)
ere FL is the tensile force at break [N] and S0 the initial cross sec-ional area [m2], while �L and L0 [m] are the length increase and theriginal length of the sample, respectively [m], �L and εL the stressnd strain in the elastic region, respectively. Experiments were runn triplicate and results are expressed as mean value ± standardeviation.
.5. Contact angle measurements
CD effect on platform wettability was investigated by perform-ng contact angle (�) measurements on PEO, PEO/CD30, PEO/CD54nd PEO/CD70 films at room temperature using a drop shapenalysis profile device (DataPhysics OCA-15 plus, DataPhysics,ermany). Ultrapure water drops (0.5 �L; Millipore Corporation,
edford, USA, resistivity: 18 M� cm) were deposited on films using
variable-volume micropipette. The corresponding static contactngle was measured by means of a Young-Laplace drop profile fit-ing. The angles were obtained using the tangent of the drop profile
armaceutics 452 (2013) 283– 291 285
at the triple line and contact angle values were averaged on at leastsix drops per film.
2.6. Solubility of TrA
A weighted amount of TrA (0.5–8 mg) was dissolved in 6 mL ofethanol and added to 18 mL of an aqueous solution of CD (75 mg)or PEO/CD (63 and 75 mg, 1:1.2, w/w respectively). The sampleswere filtered through a 0.45 �m HA filter (Millipore) and analyzedfor TrA content by spectrophotometry (UV–Vis spectrophometerModel 1200, Shimadzu) at � = 238 nm. Linearity of response wasassessed in the range 1–20 �g/mL by analyzing TrA standard solu-tions in ethanol (LOD = 0.4 �g/mL).
2.7. Film homogeneity
TrA amount present in a film section was indicative of filmhomogeneity and evaluated by quantitative analysis and Ramanspectroscopy.
For UV analysis, a round film was cut in eight sections whichwere singularly dissolved in a water:ethanol solution (3:1, v/v, thatis the same ratio employed to prepare films). The amount of TrAdissolved was evaluated in each sample by UV as reported above. Aplot of TrA dissolved as a function of section weight was obtained.
The Raman spectra were collected by a confocal Raman spec-trometer (Horiba-Jobin Yvon Mod. Aramis) operating with a diodelaser excitation source emitting at 532 nm. The 180◦ back-scatteredradiation was collected by an Olympus metallurgical objective(MPlan 50x, NA = 0.75) with confocal and slit apertures both set to200 �m. A grating with 600 grooves/mm was used throughout. Theradiation was focused onto a Peltier-cooled CCD detector (SynapseMod. 354308) operating in the Raman-shift range 2000–200 cm−1.The data gathered by the instrument were converted into ASCIIformat and transferred to the MATLAB computational platform forfurther processing.
The distribution of TrA in the film was investigated by ConfocalRaman imaging making use of the spectroscopic contrast amongthe mixture components. For analytical purposes, provided thatthe Raman scattering cross-section can be considered invariant, theintensity of a Raman line, Iv, can be expressed as:
I = KVCI0
where Kv is a constant for each peak analogous to the molar absorp-tivity in IR spectroscopy, V is the sample volume illuminated by thesource and viewed by the detector, C is the sample concentrationand I0 is the intensity of the exciting radiation. In this formulationthe constant Kv incorporates numerous instrumental factors whichare difficult to estimate; thus absolute Raman intensities are sel-dom measured and quantitative analysis relies on intensity ratiosbetween an analytical and an internal reference peak or betweentwo analytical peaks, depending on the information sought. Thus,the spectroscopic parameter Ii/Ij is proportional to ni/nj, the molarratio between the species i and j, and plotting Ii/Ij as a functionof the position will highlight the relative distribution of the twocomponents in the x–y plane (Hendra, 2002).
The Raman images were elaborated by in-house written pro-grams, making use of the image processing facilities and surfaceinterpolating algorithms of the MATLAB environment.
2.8. Release studies in simulated buccal fluids
Release kinetics of TrA from films were evaluated in differ-ent media and conditions. For sink conditions (TrA solubility0.02 mg/mL), film (1.5 cm2) was placed in a teflon support closedwith a stained net and immersed in 15 mL of simulated saliva
286 A. Miro et al. / International Journal of Ph
0
20
40
60
80
100
50
55
60
65
70
75
0 20 40 60 80
Degree of crystallinity
Melting t emperature
Degre
e o
f cry
sta
llinity
(%
) Meltin
g te
mpera
ture
(°C)
CD (w/w %)
Fr
(otmt
bcadT2ficcfdc
oLdp(adwie
3
3
3
wm�Tc≤sDi
ig. 1. Left y axis: degree of crystallinity of PEO and PEO/CD films (normalized withespect to PEO mass fraction weight percentage). Right y axis: melting temperature.
2.38 g Na2HPO4, 0.19 g KH2PO4, 8 g NaCl per liter adjusted withrthophosphoric acid) at 37 ◦C under stirring. At predeterminedimes, 1 mL of medium was withdrawn and replaced with fresh
edium. The samples were analyzed for TrA content by spec-rophotometry.
TrA release from PEO/TrA or PEO/CD54/TrA films in an artificialuccal mucus and simulated saliva was also evaluated in non-sinkonditions. Briefly, 10 mL of artificial buccal mucus were prepareddding 50 mg of porcine mucin to 10 mL of simulated saliva. Theispersion was stirred until a homogenous mixture was obtained.hen, 0.5 mL of artificial buccal mucus were added to each well of a4-well plate and a circular section of a PEO/TrA or PEO/CD54/TrAlm (1 cm2) was placed on top. At different time points, mucus wasompletely withdrawn from wells, diluted with 2 mL of ethanol andentrifuged for 20 min at 9000 rcf. The supernatant was collectedor each sample and analyzed in order to evaluate TrA amountissolved. TrA dissolution in simulated saliva was carried out asontrol.
TrA was analyzed by HPLC in order to avoid mucin interferencen quantitative analysis. A Shimadzu apparatus equipped with aC-10ADvp pump, a SIL-10ADvp autoinjector, a SPD-10Avp UV–Visetector and a C-R6 integrator was employed. The analysis waserformed on a Luna 5 �m, C18 column (250 mm × 4.6 mm, Å)Phenomenex, USA). The mobile phase was a 40:60 (v/v) mixture ofcetonitrile and water pumped at a flow rate of 1 mL/min. The UVetector was set at 234 nm. A calibration curve for TrA in ethanolas constructed in the concentration range 0.3–30 �g/mL. Exper-
ments were run in triplicate for each time point and results arexpressed as percentage of TrA dissolved ± SD over time.
. Results
.1. PEO/CD films
.1.1. Thermal propertiesThermograms of PEO/CD films prepared at different PEO:CD
eight ratio (supplementary material S1) displayed an endother-ic melting peak allowing the calculation of Tm, peak areas andHm as explained in Section 2.3. Results reported in Fig. 1 and
able 1 showed that melting peak areas, and hence the degree ofrystallinity, were roughly constant for platform with a CD content
60% w/w. On the other hand, PEO/CD75 and PEO/CD80 platforms
howed a drastic decrease of the degree of crystallinity. Moreover,SC results showed a weakly decreasing trend of platform Tm with
ncreasing CD weight fraction.
armaceutics 452 (2013) 283– 291
3.1.2. Contact angle measurementsResults of contact angle measurements performed on PEO-
based films are reported in Table 1. In the case of pure PEO films,the water drop caused a partial swelling of the polymers, and thusa lower reliability of the test. Thus, an accurate recoding of the pre-swelling contact angle required a high number of measurements.To the best of our knowledge, few contact angle measurementson PEO networks are reported in the literature, probably due tothis phenomenon (Hu et al., 2008; Sagle et al., 2009). An increaseof CD content in the film led to a significant reduction of platformswelling. Contact angle results presented a relatively high standarddeviation indicating an intrinsic low precision of measurement interms of test repeatability. Nevertheless, the methods allowed toindividuate a trend of contact angle values as a function of CD con-tent. In particular, a drop of about 30◦ of contact angle values wasobserved when CD content was above 30%, w/w. This indicates athreshold CD content between 30 and 54%, w/w, at which surfaceproperties of the film significantly change.
3.2. Mechanical properties
Stress–strain curves of drug-free PEO-based films (PEO,PEO/CD30, PEO/CD54) are presented in Fig. 2, while in Fig. 3 �MAX,εb and E values obtained from these curves are reported. In thepresence of CD, stress–strain curves displayed a linear region up to1% elongation, while afterwards a plastic behavior, showing a yieldpoint and necking, was envisaged. Actually, the effect of CD additionin PEO films (0–54%) on mechanical properties was an increase ofboth �MAX and E, and a decrease of εb. In the case of PEO/CD70 plat-form, no flexible film formation was allowed. Indeed, the absenceof a plastic region and a very low elongation at break were noticed,hence resembling the behavior of a brittle material unsuitable forclinical use (data not shown). For PEO, PEO/CD30 and PEO/CD54platforms, maximum stress was in the 4.6–8.5 MPa range. Actu-ally, in literature, �MAX values span two orders of magnitude (from0.3 to 20 MPa) (Peh and Wong, 1999). Thus, �MAX presented byPEO, PEO/CD30 and PEO/CD54 platforms are potentially suitablefor clinical applications.
3.3. TrA-loaded films
3.3.1. Formulation conditionsPEO/CD54 platform was selected as the optimal composition to
be tested for further studies since it matched acceptable mechan-ical properties in terms of tensile strength and elasticity with agood wettability. A preliminary evaluation on films loaded with apoorly water soluble lipophilic dye suggested that dye was welldistributed in PEO/CD54 films while forming macroscopic aggre-gates in PEO film (supplementary material, Fig. S2). On the basisof these results PEO/CD54 was loaded with TrA and referred to asPEO/CD54/TrA in the following.
TrA solubility in the water/ethanol casting solutions used toprepare films was preliminarily assessed (supplementary material,Fig. S3). TrA was highly soluble in ethanol/water (1:3, v/v) whereasits solubility in water was as low as 0.3 mg/mL. Addition of CD in theethanol/water mixture produced an increase of TrA solubility andamounts of TrA up to 5 mg could be dissolved. The addition of PEOin the solution at the same concentration as that employed in thecasting solution was effective in increasing further TrA solubilityup to 10 mg (Fig. 4).
3.3.2. Thermal and mechanical properties
To assess the influence of TrA within platforms, DSC and
mechanical tests were repeated on TrA-loaded films, and resultsare reported in Table 2. We found out that TrA presence into thefilms caused a slight reduction of both Tm and �Hm, thus suggesting
A. Miro et al. / International Journal of Pharmaceutics 452 (2013) 283– 291 287
Fig. 2. Stress–strain curves of PEO-based films. Inset: zoom of the same curves at low deformation.
Fig. 3. Left y axis: tensile strength (�MAX) and Young modulus (E) of
Fig. 4. TrA solubility in ethanol/water (1:3, v/v) containing CD ( ) or PEO and CD( ). CD and PEO concentration in water was 3.5 and 4.2 mg/mL, respectively. TrAwas dissolved in ethanol. Three measurements for each point were carried out. SDwas always lower than 4%.
PEO-based films. Right y axis: percent elongation at break (εb).
that the drug behaves as an impurity. Similarly, TrA addition intoPEO/CD54 films caused negligible changes of tensile strength, andYoung modulus.
3.4. Film homogeneity
Drug distribution in the film was evaluated by quantitative anal-ysis of TrA in film sections (Fig. 5) and by Raman spectroscopy(Fig. 6).
As it can be seen, for PEO film no relationship was found betweensection weight and TrA amount actually measured (Fig. 5a). Con-
trariwise, a linear relationship was found for PEO/CD/TrA films, thussuggesting that CD provides for a homogeneous distribution of thedrug at macroscopic level (Fig. 5b).
Table 2Results of thermoanalytical and mechanical experiments carried out on PEO-basedfilms with/without TrA.
a Standard deviations were calculated on at least three repeats.b TrA was 4.8% of the total weight.c TrA was 2.1% of the total weight.
288 A. Miro et al. / International Journal of Pharmaceutics 452 (2013) 283– 291
secti
taeiPtsapftsabsite
PotbTrfiPtp
3
daavu(afIfaif1
Fig. 5. Relationship between the amount of TrA and the weight of film
In the system under investigation the absence of inelastic scat-ering in the wavenumber range 1550–1800 cm−1 for both PEOnd CD allows the detection of interference-free signals from TrAven when it is present in very low amounts. The fact that TrA wasnsoluble in PEO is clearly evidenced by the Raman images of aEO/TrA film at 0.07 mg/cm2 of TrA (Fig. 6a) where the TrA crys-alline domains are readily identified. A closer inspection of thepectra reported in the insets, reveals that the TrA signal is presentlso in the PEO regions, albeit its intensity is barely detectable. Inarticular, the peak shape of the 1670 cm−1 signal is distinctly dif-erent from that observed in the TrA domains and resembles closelyhe profile observed in a diluted TrA/chloroform solution and in theolid TrA/CD complex (Miro et al., 2012). This is an indication that
small amount of TrA is actually solubilized in PEO, this fractioneing readily detected by Raman spectroscopy. The very low inten-ity of the 1670 peak in the PEO matrix as compared to that detectedn the case of the PEO/CD54/TrA complex (vide infra) demonstrateshat the amount of TrA which can be directly solubilized in PEO isxtremely small.
In Fig. 6b and c the Raman image of a ternary compositionEO/CD54/TrA at 0.07 mg/cm2 of TrA, which corresponds to anverall TrA content in the system of 1.0 wt%, is reported. In par-icular, Fig. 6b has been obtained by considering the intensity ratioetween the CD peak at 1320 cm−1 and the PEO peak at 362 cm−1.he uniform color (I1320/I362 = 0.4 ± 0.05) indicates a constant molaratio of the two components in the investigated area and con-rms a homogeneous, molecular level dispersion of CD into theEO matrix. An equally uniform pattern is observed by consideringhe intensity ratio between the TrA peak at 1670 cm−1 and the PEOeak at 362 cm−1 (Fig. 6c).
.4.1. TrA release in simulated buccal fluidsTrA release kinetics from films in simulated saliva (sink con-
itions) are reported in Fig. 7. As it can be seen in Fig. 7a, theddition of CD increased the release rate of the drug and allowed
complete delivery of TrA within 10 min. To better mimic inivo conditions, TrA release from films was followed also in sim-lated saliva and artificial buccal mucus in non-sink conditionsFig. 7b). TrA release was strongly influenced by both film typend release medium composition. Release in simulated saliva wasaster as compared to artificial mucus for both formulations tested.n both media, an increase of TrA released percentage was observedor CD-containing film. In the presence of mucin, TrA percent-
ge released from PEO/TrA film was 7% after 5 min and did notncrease over time. On the other hand, TrA percentage releasedrom PEO/CD54/TrA film progressively increased reaching 47% after0 min.
on (8 sections for each film). PEO/TrA film (a); PEO/CD54/TrA film (b).
An estimate of dissolution rate of PEO and PEO/CD54 films wasderived from the visual evaluation of the time needed for Nile-redloaded films to dissolve in water or in mucin (multimedia data S4).A slower dissolution was observed for PEO/CD54 films as comparedto PEO films in both media tested.
4. Discussion
Although film dosage forms are emerging as a treatment modal-ity in different pathologies, it is still very challenging for the finalproduct to comply with quality requirements. A great concern infilm manufacturing relies on drug content uniformity of the prod-uct obtained. Nonetheless, this aspect has been poorly addressedin the literature so far, with special regard to the evaluation ofdrug distribution in the polymer platform. It is not clear what thispoor drug distribution in the film depends upon. Some authorsinvoke the monolayerd structure of the film, others suggest thatingredient self-aggregation along the drying process is a key fac-tor (Yang et al., 2008). Homogeneity, in term of drug distributionin the platform, is crucial for lipophilic drugs due to their chemicalnature. In fact, incorporation of a poorly water soluble moleculein a hydrophilic film-forming agent is hard to attain due to lackof mutual solubility. Despite the possibility to find suitable con-ditions for drug/polymer co-solubilization in the casting solution,upon drying drug can form crystalline clusters in the matrix, withobvious effects on drug dissolution rate. From a biopharmaceuticalpoint of view, poor dissolution of a drug loaded in a polymer thinfilm can turn as a critical parameter for realizing extensive and fastdrug adsorption at mucosal site and avoid washing-out of undis-solved drug particles. Thus, smart strategies are strongly needed toobtain homogenous film able to dissolve fast the drug at mucosallevel while withstanding adequate permanence at administrationsite.
Starting from the general assumption that prevention of drugcrystallization along the manufacturing process could serve to solveboth processing and biopharmaceutical issues, we tried to explore ifCD, combined with PEO in specific conditions, could be successfullyemployed to produce a film for mucosal delivery of the poorly watersoluble drug TrA.
We preliminarily assessed the thermal/mechanical propertiesand wettability of unloaded PEO/CD films to investigate CD as atool to engineer macroscopic properties of PEO platforms. Hydroal-coholic solutions containing different PEO/CD weight ratios werelimpid in a wide range of water/ethanol ratios, did not incorporate
air bubbles due to their low viscosity, and could be easily castedto give thin film. Thermograms of dried films showed that, withincreasing CD content, platform Tm progressively decreased, whichwas associated to CD intercalation between the PEO polymeric
A. Miro et al. / International Journal of Ph
Fig. 6. Raman image of PEO-film (a) and PEO/CD54/TrA (b) films at 0.07 mg/cm2
of TrA. The insets represent spectra collected at the indicated positions. The Ramanimages were obtained by considering: (a) the intensity of the TRA peak at 1670 cm−1;(b) the intensity ratio I1320/I362; (c) the intensity ratio I1668/I362.
armaceutics 452 (2013) 283– 291 289
chains and hence to an increase of free volume in the platform(plasticizing effect). The corresponding peak areas, and hence thedegrees of crystallinity, were roughly constant while CD contentwas ≤60%, w/w. These results indicate that the envisaged increaseof platform free volume did not interfere with the formation ofpolymeric crystalline clusters. More specifically, below 60%, w/wCD content, PEO macromolecules can organize into crystallineclusters, while CDs tend to self-associate within the amorphousregions of PEO. When CDs content was quantitatively predominant(>60%, w/w), extensive amorphous regions are formed and theformation of PEO crystals is strongly hampered. The proposedmechanism of PEO/CD interaction at different ratios is reportedin Fig. 8. In particular, supramolecular PEO/CD structures tend toform within the amorphous regions of PEO with a stoichiometrywhich can be calculated by extrapolating �Hm to zero (Lazzaraand Milioto, 2008). In the studied platforms, a EO/CD molar ratioaround 7.4 was found out.
It must be also underlined that CD addition in PEO films stronglyaffects the macroscopic mechanical properties, in terms of anincreased modulus and a decreased elongation at break. This canbe interpreted in terms of CD–PEO interactions through hydrogenbonds. In particular, hydroxyl groups of CDs are able to form sec-ondary bonds with ether groups of PEO, thus establishing a sortof physical network, which reduces molecular mobility and there-fore the resistance to deformation. Here we demonstrate that CDsthemselves act as modulators of film mechanical properties, andcan be therefore considered as multipurpose excipients. Indeed,the formation of supramolecular structures between PEO and CDscaused a drop of elongation at break and an increase in elastic mod-ulus, thus causing the formation of an increasingly brittle materialwith increasing CD content. Thus, mechanical properties could beoptimized by properly choosing the CD content in the film whichmatches high �MAX and εb. In particular, CD addition leaded to ahigher rigidity of the linear elongation region, which was asso-ciated to the behavior of glassy amorphous CD domains. Aboveapproximately 1% elongation, the platforms qualitatively displayedthe elastic behavior of pure PEO, due to the presence of extensivecrystalline polymeric regions.
Moreover, it is necessary to stress that, along with mechanicalproperties, film adhesion to mucosa is initiated by hydration whichensures a successful application and permanence of the dosageform. We found out that contact angles were lower at CD con-tent higher than 54%, thus showing that CD addition enhances theoverall hydrophilicity of films improving wettability.
On the basis of these results, PEO platform prepared at 54% CD(1:1.2 PEO/CD weight ratio) was considered to meet the require-ments of elasticity and wettability, and loaded with TrA as a modelof sparingly water soluble drug. Concerning the preparation of thecasting solution, we found previously that the presence of ethanoland CD in specific ratios was efficient in increasing water solubilityof TrA (Miro et al., 2012). Here we found that PEO exerted a coop-erative effect with ethanol and CD, producing a two-folds increaseof TrA solubility thus suggesting its significant contribution to drugsolubilization process.
Confocal Raman imaging clearly showed that CD was homo-geneously (i.e. molecularly) dispersed in PEO films. In a previouscontribution, it was demonstrated that a complex is formedbetween TrA and CD, both in solution and in the solid state, andits molecular details spectroscopically investigated (Miro et al.,2012). The complex formation brings about a modification of theRaman spectrum of the guest molecule in the 1550–1750 cm−1
region which makes it distinctly different from the spectrum of
crystalline TrA and affords the unambiguous identification of thecomplex in mapping experiments. Thus, uniform patterns wereobtained in Raman images collected on a TrA/CD sample having a1/7 molar ratio (the same employed to prepare the film developed
290 A. Miro et al. / International Journal of Pharmaceutics 452 (2013) 283– 291
F PEO/r l) (b).
iPCt
Fpsuis
ig. 7. TrA release from films in simulated buccal fluids. Release kinetics of TrA fromeleased over time in artificial buccal mucus (simulated saliva is reported as contro
n the present paper) (Miro et al., 2012). The Raman spectra ofEO/CD54/TrA ternary system revealed the formation of the above
D/TrA complex which was stable in the PEO matrix and promotedhe solubilization of TrA in the polymeric platform up to a content
ig. 8. Proposed mechanism of PEO–CD interaction. CD-free PEO shows both amor-hous and crystalline regions (a). With increasing CD content, supramoleculartructures are formed in amorphous regions of the polymer (b) and (c) until sat-ration occurs around 60%, w/w CD content (d). Above this CD content, extensive
nvasion of the polymeric matrix takes place, causing loss of the original crystallinetructures of PEO (e).
TrA film ( ) and PEO/CD54/TrA film (�) in simulated saliva (a). Percentage of TrAFilm area was 1.5 cm2.
of around 1% by weight, avoiding the precipitation of the drug inthe form of crystallites, as it occurs in the absence of CD.
Morphology analysis, constant PEO/TrA ratio in film sec-tions of different weight together with microscopic analysis ofPEO/CD54/TrA mutual solubility in the solid state demonstrated byRaman strongly support the soundness of the strategy proposed,indicating also that PEO/CD54/TrA film complies with specifica-tions of Pharmacopeias in term of uniformity of dosage units. On anapplicative standpoint, this could allow the patient to cut the filmat a size suitable to cover a desired area.
As a final consideration, the process of in vivo drug transportin a tissue covered with a mucosa first involves drug dissolutionfrom the film in the mucus. If we consider a buccal applicationof the film developed here, the knowledge of TrA release profilein simulated buccal fluids is crucial to provide a solid rationalefor a therapeutically effective delivery system. Notably, CD addi-tion in PEO films accomplished a useful speed-up of TrA releaserate in simulated saliva in all the conditions tested and in agree-ment with previous findings (Ammar et al., 2008; Jug et al., 2009).The decrease of drug crystallinity due to CD addition suggested byRaman analysis likely contributed to its fast and complete dissolu-tion. This drug solubilizing effect was accompanied by a retardationof platform dissolution in aqueous media which can be ascribedto PEO/CD interactions in the solid state. As a consequence, TrAwas released in approximately the same time frame of platformsolubilization which is ideal for a drug delivery system. The pro-moting effect of CD on TrA dissolution was further evidenced inartificial mucus. Indeed, data highlighted how release rate maybe strongly reduced in mucin-reach environment that resemblescloser in vivo conditions and how the presence of CD can be ofhelp.
5. Conclusions
In this work, we demonstrated the usefulness of hydroxypropyl-�-cyclodextrin as a multi-purpose agent in the design of PEO-basedbuccal films. Indeed, due to their peculiar composition, PEO/CDfilms developed allow to load the sparingly soluble drug TrA ham-pering drug crystallization in the drying step of manufacturing, theachievement of optimal mechanical properties/wettability and afast drug dissolution in simulated mucus. This result is of greatconvenience from a biopharmaceutical standpoint to promoteextensive drug absorption at administration site and for patients
who can subdivide the film before use and adapt its shape to theextension of a lesion. In perspective, CD-containing films can beconsidered as a therapeutic option in the local treatment of diseasesinvolving an accessible mucosa, especially buccal cavity.
l of Ph
A
f
A
f2
R
A
A
C
C
D
F
H
H
H
H
A. Miro et al. / International Journa
cknowledgement
This paper is dedicated to the memory of our colleague andriend Brunella Cappello.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.013.05.030.
eferences
mmar, H.O., Salama, H.A., El Nahhas, S.A., Elmotasem, H., 2008. Design and evalu-ation of chitosan films for transdermal delivery of glimepiride. Curr. Drug Deliv.5, 290–298.
ndrews, G.P., Laverty, T.P., Jones, D.S., 2009. Mucoadhesive polymeric platformsfor controlled drug delivery. Eur. J. Pharm. Biopharm. 71, 505–518.
appello, B., De Rosa, G., Giannini, L., La Rotonda, M.I., Mensitieri, G., Miro, A., Quaglia,F., Russo, R., 2006. Cyclodextrin-containing poly(ethyleneoxide) tablets for thedelivery of poorly soluble drugs: potential as buccal delivery system. Int. J.Pharm. 319, 63–70.
immino, S., Di Pace, E., Martuscelli, E., Silvestre, C., 1990. Evaluation of theequilibrium melting temperature and structure analysis of poly(ethyleneoxide)/poly(methyl methacrylate) blends. Die Makromol. Chem. 191,2447–2454.
e Lisi, R., Lazzara, G., Milioto, S., Muratore, N., 2007. Laponite clay in homopoly-mer and tri-block copolymer matrices – thermal and structural investigations.J. Therm. Anal. Calorim. 87, 61–67.
enyvesi, É., Balogh, K., Siró, I., Orgoványi, J., Sényi, J., Otta, K., Szente, L., 2007. Per-meability and release properties of cyclodextrin-containing poly(vinyl chloride)and polyethylene films. J. Inclusion Phenom. Macrocyclic Chem. 57, 371–374.
assan, N., Ahad, A., Ali, M., Ali, J., 2009. Chemical permeation enhancers for trans-buccal drug delivery. Expert Opin. Drug Deliv. 7, 97–112.
earnden, V., Sankar, V., Hull, K., Juras, D.V., Greenberg, M., Kerr, A.R., Lockhart, P.B.,Patton, L.L., Porter, S., Thornhill, M.H., 2012. New developments and opportu-nities in oral mucosal drug delivery for local and systemic disease. Adv. DrugDelivery Rev. 64, 16–28.
u, Z.K., Chen, L., Betts, D.E., Pandya, A., Hillmyer, M.A., DeSimone, J.M., 2008. Opti-cally transparent, amphiphilic networks based on blends of perfluoropolyethersand poly(ethylene glycol). J. Am. Chem. Soc. 130, 14244–14252.
armaceutics 452 (2013) 283– 291 291
Jug, M., Becirevic-Lacan, M., Bengez, S., 2009. Novel cyclodextrin-based film formu-lation intended for buccal delivery of atenolol. Drug Dev. Ind. Pharm., 1–12.
Lazzara, G., Milioto, S., 2008. Copolymer-cyclodextrin inclusion complexes inwater and in the solid state. A physico-chemical study. J. Phys. Chem. B 112,11887–11895.
Loftsson, T., Vogensen, S.B., Brewster, M.E., Konradsdottir, F., 2007. Effects ofcyclodextrins on drug delivery through biological membranes. J. Pharm. Sci. 96,2532–2546.
López-de-Dicastillo, C., Gallur, M., Catalá, R., Gavara, R., Hernandez-Munoz, P., 2010.Immobilization of a-cyclodextrin in ethylene-vinyl alcohol copolymer for activefood packaging applications. J. Membr. Sci. 353, 184–191.
Miro, A., Rondinone, A., Nappi, A., Ungaro, F., Quaglia, F., La Rotonda, M.I., 2009.Modulation of release rate and barrier transport of diclofenac incorporated inhydrophilic matrices: role of cyclodextrins and implications in oral drug deliv-ery. Eur. J. Pharm. Biopharm. 72, 76–82.
Miro, A., Ungaro, F., Balzano, F., Masi, S., Musto, P., Lamanna, P., Uccello-Barretta,G., Quaglia, F., 2012. Triamcinolone solubilization by (2-hydroxypropyl)-�-cyclodextrin: a spectroscopic and computational approach. Carbohydr. Polym.90, 1288–1298.
Miro, A., Ungaro, F., Quaglia, F., 2011. In: Bilensoy, E. (Ed.), Cyclodextrins as SmartExcipients in Polymeric Drug Delivery Systems. John Wiley & Sons, Inc., Hobo-ken, New Jersey (USA), pp. 65–89.
Morales, J.O., McConville, J.T., 2011. Manufacture and characterization of mucoad-hesive buccal films. Eur. J. Pharm. Biopharm. 77, 187–199.
Patel, V.F., Liu, F., Brown, M.B., 2011. Advances in oral transmucosal drug delivery.J. Control. Release 153, 106–116.
Peh, K.K., Wong, C.F., 1999. Polymeric films as vehicle for buccal delivery: swelling,mechanical, and bioadhesive properties. J. Pharm. Pharm. Sci. 2, 53–61.
Prodduturi, S., Manek, R.V., Kolling, W.M., Stodghill, S.P., Repka, M.A., 2005. Solid-state stability and characterization of hot-melt extruded poly(ethylene oxide)films. J. Pharm. Sci. 94, 2232–2245.
Senel, S., Rathbone, M.J., Cansiz, M., Pather, I., 2012. Recent developments in buccaland sublingual delivery systems. Expert Opin. Drug Deliv. 9, 615–628.
Sudhakar, Y., Kuotsu, K., Bandyopadhyay, A.K., 2006. Buccal bioadhesive drug deliv-ery – a promising option for orally less efficient drugs. J. Control. Release 114,15–40.
Thumma, S., ElSohly, M.A., Zhang, S.Q., Gul, W., Repka, M.A., 2008. Influence ofplasticizers on the stability and release of a prodrug of �9-tetrahydrocannabinol
incorporated in poly (ethylene oxide) matrices. Eur. J. Pharm. Biopharm. 70,605–614.
Yang, R., Fuisz, R., Myers, G., Fuisz, J. Thin film with non-self-aggregating uniformheterogeneity and drug delivery systems made therefrom. US Patent 7425292(16-9-2008).
