A. David Woolfson

	Pharmaceutical dosage forms contain both pharmacologically-active compounds and excipients, which have been added to aid the formulation and manufacture of the dosage form. The properties of the final dosage form are highly dependent on the excipients chosen, their concentration and interaction with both the active compound and each other. Excipients cannot be regarded as inert or inactive ingredients and a detailed understanding of their physiochemical properties, safety and handling, and regulatory status is essential for pharmaceutical scientists.

	There has been increasing interest in the use of bioadhesive polymers as excipients in the design of drug delivery systems. One of the advantages of using these materials is that they can maintain contact with mucosal surfaces for longer periods than nonbioadhesive polymers. Because polymers possessing bioadhesive properties can retain drugs in close proximity to membranes rich in underlying vasculature, they may offer a solution to the poor bioavailability of some drugs and a method to avoid enzymatic degradation of others.

	Film formulations based on poly(methylvinylether/maleic anhydride [PMVE/MA]) are known to possess moisture-activated bioadhesive properties.

 Bioadhesive films cast from aqueous blends of PMVE/MA have diverse uses, such as a means of establishing an electrically conducting interface for bioelectrodes and as an adhesive drug delivery matrix.

 In structural terms, the five-membered anhydride ring of PMVE/MA contributes two carbon atoms to the polymer backbone and, therefore, confers rigidity on the system. On hydrolysing this anhydride moiety to the corresponding free acid form (PMVE/MAC [Figure 1]), a reduction in glass transition temperature (T

) of only 10 °C from the relatively high T

 of the dry powder (151 °C) is observed because of the increased flexibility of the free acid structure.

 Films cast from aqueous blends of the free acid are, consequently, very brittle and of little use in formulating films for drug delivery purposes. Therefore, these systems require the inclusion of a second excipient to act as a plasticizer in the film casting blend.

	Plasticizers used for PMVE/MAC film formulations are, typically, water-miscible, polyhydric alcohols, such as glycerol. These plasticizers are suspected of cross-linking PMVE/MAC on heating or on prolonged standing at room temperature.

 This study investigates the influence of two different plasticizers on the properties of films cast from aqueous blends of PMVE/MA and potential mechanisms by which the polymer may cross-link on storage. The aim is to produce a stable film that maintains its adhesion and conformability properties on storage, rendering it suitable for use as a topical drug delivery system.

 Gantrez AN-139, a copolymer of MVE and MA was provided by ISP Co. Ltd (UK). Glycerol, tripropyleneglycol methyl ether (TPM [Dowanol]) and methylene blue were obtained from Sigma Aldrich (UK). All other chemicals used were of analytical reagent quality.

 Aqueous polymer blends were prepared as previously described using the required weight of PMVE/MA, which was added to ice-cooled water (reagent grade 1) and stirred vigorously.

 The mixture was then heated and maintained between 95 and 100 °C until a clear solution was formed. Upon cooling, the required amount of plasticizer was added, the pH adjusted to 4.5 using 10 M NaOH and the casting blend adjusted to final weight with water. Bioadhesive films were prepared by slowly pouring the aqueous blend (30 g) into a mould consisting of a release liner (FL2000 PET 75 μ 1S; Rexam Release BV, The Netherlands), siliconized side-up, secured to a Perspex base plate using a stainless steel clamp. Once assembled, the internal dimensions available for casting were 100 mm by 100 mm (Figure 2). The mould was placed on a levelled surface to allow the blend to spread evenly across the area of the mould. The cast blend was dried under a constant airflow at 25 °C.

	Films cast from blends containing PMVE/MA alone, PMVE/MA with glycerol and PMVE/MA with TPM were stored at 25 °C in a sealed vessel with a relative humidity of 65% maintained by the presence of a saturated solution of sodium nitrite. Films were removed from storage at 7-day intervals and their bioadhesive, tensile and swelling characteristics investigated.

	The bioadhesive properties of all films were evaluated quantitatively using a TA-XT2 texture analyser (Stable Microsystems, UK) in tensile mode. Full thickness, shaved, neonate porcine skin was attached with cyanoacrylate adhesive to a lower platform. Film segments (1 cm

) were attached to the probe of the texture analyser using double-sided adhesive tape. Adhesion was initiated by adding a defined amount of water (10 μL) over an exposed skin sample (1 cm

) and immediately lowering the probe with attached film.

	Upon contact, a force of 5 N for 30 s was applied before the probe was moved upwards at a speed of 0.1 mm s

. Adhesion was recorded as the force required to detach the sample from the surface of the excised skin. The distance to separation of a test film from the skin substrate, that is, the normal displacement from the skin surface that the probe had travelled at the instant the film and substrate lost contact with each other, was also recorded to provide some measure of the cohesion within the film sample. Results were reported as the mean (± standard deviation [SD]) of five replicates.

	The tensile strength and percentage elongation at break of films were determined using the texture analyser. Film strips of 5 mm width were grasped using an upper and lower flat-faced metal grip laminated with a smooth rubber grip. The distance between the grips was set at 20 mm and this distance, therefore, represented the length of film under stress. A cross-head speed of 6 mm s

 was used for all measurements. The resultant force–time profiles were analysed using propriety software (Dimension 3.7E; Stable Microsystems, UK). Only results from films that were observed to break in the middle region of the test strip during testing were used. The percentage elongation at break, E

, of tested films was determined using Equation 1.

	Where E is the extension to break of the film and L

 is its original length. The break strength, B, of tested films was determined using Equation 2.

	Where F is the break force of the film and AR is its cross-sectional area. Results were reported as the mean (±SD) of five replicates.

	The swelling behaviour of films was investigated by immersing dry, preweighed film segments (1 cm

) in distilled water (30 mL) at 25 ºC. The segments were allowed to swell to equilibrium and then reweighed following careful blotting with absorbent paper to remove surface water. The equilibrium weight swelling index (SI) of the films was defined as the ratio of the weight of the swollen film (W

	Results were reported as the mean (±SD) of five replicates. Where appropriate, results were analysed using a one-way analysis of variance (ANOVA). Post-hoc comparisons were made using Fisher's protected least significant difference (PLSD) test where p <0.05 was taken to represent a statistically significant difference.

	Films cast from aqueous blends of PMVE/MA with no plasticizer were brittle and unsuitable for use as the basis of a conformable, bioadhesive delivery system. Addition of TPM caused a significant reduction in T

 of cast films, as determined using differential scanning calorimetry (DSC [DSC 2920 with refrigerated cooling system; TA Instruments, UK, data not shown]). T

 significantly decreased as TPM contents increased.

Tensile strengths of films cast from aqueous blends containing 10% w/w PMVE/MA decreased significantly (p <0.0001) with the addition of TPM to the casting blend, while the percentage elongations at break showed significant (p <0.0001) increases (Table 1). Glycerol addition to the casting blend produced similar, significant (p <0.0001), reductions in tensile strengths and similar, significant (p <0.0001) increases in percentage elongations at break.

	Neither glycerol (p=0.5964) nor TPM addition (p=0.0516) had a significant effect on the bioadhesion of freshly prepared films to shaved neonate porcine skin (Table 2). Initially, freshly prepared films plasticized with glycerol showed significant (p <0.0001) increases in distance to separation relative to unplasticized films, whereas similar changes were not observed in films plasticized with TPM.

	Films cast from blends containing 5% w/w glycerol lost their bioadhesive strength over time (Figure 3). There were significant decreases in adhesion after only 7 days' storage (p <0.0001). By contrast, the bioadhesive properties of films cast from blends containing 5% w/w TPM remained unaltered over a similar time frame. Films cast from blends containing 10% w/w glycerol also showed significant reductions in adhesion after 6 weeks' storage (p <0.0001), an effect not seen for films cast from blends containing 10% w/w TPM or unplasticized films.

	All films containing glycerol showed pronounced decreases in distance to separation over time, with significant decreases being noted in each case after 7 days' storage. For example, for films cast from blends containing 10% w/w glycerol, the mean distance to separation had already decreased significantly (p <0.0001) after only 7 days. Further significant decreases in distance to separation occurred with increasing storage times. Unplasticized films did not display this effect, while only slight decreases were noted for films containing TPM (data not shown).

	Storage was found to have a profound effect on the tensile properties of films containing glycerol in particular. Films cast from blends containing 5% w/w glycerol showed increases (p <0.0001) in break strength over time, significant changes being observed after only 7 days' storage. Films cast from blends containing 10% w/w glycerol also showed significant increases in break strength over time. After 42 days' storage these films had a mean break strength of 0.424×106 N m

. The original mean break strength of these films was 0.04×106 N m

. Films cast from blends containing 5% w/w TPM showed significant increases in break strength after 6 weeks' storage (p <0.0001). Importantly, films cast from blends containing 10% w/w TPM showed unaltered tensile strengths after 6 weeks' storage (p=0.9368).

An examination of the elongation at break for films cast from blends containing 10% w/w glycerol showed significant decreases on storage (Figure 4). For example, after 6 weeks' storage, the mean percentage elongation at break of films cast from blends containing 10% w/w glycerol had decreased significantly (p <0.0001). A similar pattern was observed for films cast from blends containing 5% w/w glycerol. By contrast, films cast from blends containing 10% w/w TPM showed no significant decreases in percentage elongation at break after 6 weeks' storage (p=0.4455).

	Films cast from blends containing 5% w/w TPM did, however, show significantly decreased percentage elongations at break after 6 weeks' storage, which had mean values of zero (p <0.0001). The original mean percentage elongation at break of these films was 592.2%.

Films cast from aqueous blends of PMVE/MA, with and without plasticizer, dissolved in water initially. Films cast from blends containing 5% w/w glycerol became insoluble in water after 7 days' storage, showing significant increases in equilibrium weight swelling indices (p <0.0001). The swelling indices (Figure 5) then decreased progressively, and films cast from blends containing 10% w/w glycerol displayed a similar pattern. However, the initial rise in the swelling index was much more pronounced in the latter case. No such effect was observed for films plasticized with TPM.

	Plasticized bioadhesive films based on hydrolysed PMVE/MAC, are potentially useful drug delivery platforms for a range of topical applications, including local delivery to moist mucosal epithelial tissue. Such films have excellent initial adhesion and conformability properties.

 However, this study demonstrates the loss of adhesion and concomitant changes in mechanical properties that occurs during storage. Consequently, a detailed investigation has been made of the fundamental behaviour of the plasticized PMVE/MAC film system to allow drug delivery films with stable adhesion and mechanical properties to be formulated.

	Polyhydric alcohols, such as propylene glycol, glycerol, and poly(ethylene glycol) 400, have previously been shown to act as plasticizers of PMVE/MAC films.

 Glycerol is most typically used as a plasticizer for PMVE/MAC.

	However, films cast from blends plasticized with glycerol were observed to lose their adhesion progressively on storage (Figure 3). Distance to separation during tensile mode adhesion testing also decreased. The flexibility of glycerol-plasticized films was reduced concomitantly over time, with films becoming insoluble and showing a marked reduction on water swelling indices after only 7 days' storage (Figure 5). These observations are all consistent with a progressive increase in film cross-linking density during storage.

	TPM was shown to be an effective plasticizer for PMVE/MA-based films intended for drug administration purposes, progressively lowering the T

 of the bioadhesive polymer in direct proportion to the plasticizer concentration in the casting blend. Films cast from PMVE/MA blends containing 5% w/w of TPM maintained their bioadhesive properties (Figure 3) and remained water-soluble after 6 weeks' storage. Elongation at break was also maintained in stored films plasticized with TPM (Figure 4). Solid-state nuclear magnetic resonance (NMR) studies revealed the formation of ester bonds between the two excipients, PMVE/MAC and glycerol (Varian UNITY Inova spectrometer with a 7.05 T Oxford Instruments magnet and a 7 mm standard MAS probe; Doty Scientific Instruments, Columbia, SC, USA, data not shown).

	In contrast to previously reported plasticizers of PMVE/MAC

 TPM is monohydric, possessing only a terminal hydroxyl group. It was chosen as an alternative plasticizer in this study because it could not cross-link PMVE/MAC chains by means of an interaction with the carboxylic acid groups on the bioadhesive polymer, for example, by forming ester linkages. Films plasticized with 5% w/w TPM displayed a significant loss of flexibility on storage. Notwithstanding this, films remained sufficiently pliant for topical application. Whether this reduction in flexibility was a result of enhanced hydrogen bonding, a progressive increase in physical entanglements or to limited ester formation leading to increased interchain cohesion was not determined. However, films cast with higher levels of TPM (10% w/w) displayed mechanical properties similar to those found immediately after drying, the increased excipient content presumably aiding retention of water within the film structure.

	The time-dependent decrease in adhesion and distance to separation seen with glycerol-plasticized films are indicative of a polymer system that is becoming more restricted in movement by the formation of a tighter network of cross-links.

 Hence, film cohesion increases and distance to separation falls. In addition, the loss of functional groups, such as -COOH, which are capable of hydrogen bonding to a biological substrate, may contribute to the observed reduction in bioadhesion.

 In contrast, films containing TPM showed decreased distances to separation, but maintained their bioadhesive strength, suggesting an increased interchain cohesive attraction with preservation of chemical residues capable of hydrogen bonding.

	The swelling indices (Figure 5) of 5% w/w glycerol-plasticized films decreased progressively, suggesting a system that is becoming increasingly organized and of tighter disposition and can, therefore, accommodate less water within its network.

 A similar pattern was found in films cast from blends containing 10% w/w glycerol, but the initial rise in swelling index was much more pronounced. This may arise from the high glycerol content leading to greater interchain separation and formation of a looser network. All unplasticized films and those containing 5% and 10% w/w TPM remained water-soluble throughout the study period.

	The swelling characteristics observed in films cast from blends containing glycerol, however, together with their gradual insolubilization in water, clearly suggest that there is a slow chemical alteration, typically occurring over about 7 days, in the glycerol-plasticized PMVE/MAC film system. It is possible that glycerol is participating in a cross-linking reaction as a result of ester formation with the free carboxylic acid moieties formed on hydrolysis of PMVE/MA. Because glycerol is polyhydric, attachment could occur at more than one point on the bioadhesive polymer chain, thereby contributing to formation of a three-dimensional PMVE/MAC network.

	Hydrogen bonding to the hydroxyl groups on the free acid form of PMVE/MAC is unlikely to be the predominant cross-linking mechanism, however, because TPM has four equivalent oxygen atoms capable of hydrogen bonding in a similar way to those found in glycerol. The formation of a physically cross-linked system because of entanglements between neighbouring chains is, again, unlikely, as this would be more feasible in unplasticized systems because of the reduced interchain separation.

 This may also explain the increasing rigidity of the TPM plasticized polymer films, which are now becoming internally, rather than externally plasticized, because internal plasticization is known to be less efficient than external plasticization.

	As a result of the observed negative impact of excipient–excipient interactions between glycerol and PMVE/MAC on film properties, we have subsequently used TPM as the plasticizing excipient in all bioadhesive films prepared. These films have been employed as topical drug delivery systems for 5-aminolevulinic acid used in photodynamic therapy (PDT),

 which is a technique whereby a combination of a photosensitizing drug and visible light causes destruction of selected cells via singlet oxygen production.

	We have also made use of the observed excipient–excipient interaction to produce PMVE/MAC films cross-linked with poly(ethylene glycol [PEG] 6000). We postulated that, as cross-linking is known to retard drug release,

 an extensively cross-linked system should be able to immobilize a drug compound within the film matrix. Immobilization of a photosensitizer within a film surface coating should inhibit microbial growth on that surface under ambient lighting by generation of toxic singlet oxygen.

We prepared films as previously described from aqueous blends of PMVE/MA (20% w/w) and PEG 6000 (10% w/w). The final film contained 10 mg cm

 of the antimicrobial photosensitizer methylene blue, which had been added in appropriate amounts to the aqueous blend. Films were stored for 72 h in a desiccation chamber at 60 °C to force the cross-linking reaction. When drug release experiments (modified Franz cell; Crown Glass Co. Inc., Sommerville, NJ, USA) were performed immediately upon film preparation, approximately 90% of the methylene blue loading was released after 3 h. However, following cross-linking, the amount of drug released over a similar period was virtually undetectable (Figure 6). Discs of the crosslinked films incubated in 96-well plates under ambient lighting with 10

 MRSA 180 completely resisted biofilm growth.

	In summary, we have identified a detrimental excipient–excipient interaction between copolymer and plasticizer in pharmaceutical systems intended for topical application. As a result, we have employed an alternative plasticizer in films used subsequently in the clinic. However, discovery of the nature of the interaction has been exploited to produce photoactive materials containing immobilized photosensitizer with potential as anti-infective surface coatings for use in hospitals.

	1. R.C. Rowe, P.J. Sheskey and P.J. Weller, Eds., 

Handbook of Pharmaceutical Excipients, 4th Edition

 (Pharmaceutical Press, London, UK, 2003).

	4. A.D. Woolfson, D.F. McCafferty and G.P. Moss, 

	5. K.H. Chung, C.S. Wu and E.G. Malawer, 

	6. Gantrez AN-139 production bulletin,ISP Corp, Wayne, NJ, USA (1995).

	8. G.W. Radebaugh, in J. Swarbrick and J.C. Boylan (Eds), 

Encyclopedia of Pharmaceutical Technology

 (Marcel Dekker, New York, NY, USA, 1992) pp 1–28.

Pharmaceutics: The Science of Dosage Form Design

 (Churchill Livingstone, Edinburgh, UK, 2003).

 is a lecturer in pharmaceutical chemistry

 is the chair in pharmaceutics, all at the School of Pharmacy, Queen's University Belfast, UK.

Articles

Bioadhesive films cast from aqueous blends of PMVE/MA have diverse uses, such as a means of establishing an electrically conducting interface for bioelectrodes and as an adhesive drug delivery matrix.