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Adhesion of Polyether-Modified Poly(acrylic acid) to Mucin John Cleary,† Lev Bromberg,‡ and Edmond Magner*,† Materials and Surface Science Institute and Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland, and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received April 21, 2004. In Final Form: August 17, 2004 Pluronic-PAA, a thermogelling copolymer composed of side chains of poly(acrylic acid) (PAA) grafted onto a backbone of Pluronic copolymer, is of interest as a vehicle for the controlled release of compounds. An important feature of such a vehicle is its bioadhesive/mucoadhesive properties, which in the case of Pluronic-PAA are significant due to the presence of the PAA side chains. An atomic force microscopy (AFM) method has been developed and utilized to investigate the interactions between a Pluronic-PAA-modified microsphere and mucous substrates. The bioadhesive force was successfully measured, and trends were observed under conditions of varying pH and ionic strength. Pluronic-PAA exhibits significant mucoadhesion over a range of pH values, with mucoadhesion being optimal at pH 4-5 (adhesive force ∼80 mN/cm2) and dropping sharply at higher pH, to a value of ∼20 mN/cm2 at pH 8. The mucoadhesive force decreased with increasing ionic strength, from a value of ∼80 mN/cm2 in 0.025 M NaCl to ∼25 mN/cm2 in 1.0 M NaCl. These results have been interpreted in terms of the effect of changing pH and ionic strength on electrostatic interactions and swelling of the polymer and mucin layers. Tensiometric force measurements indicated that hydrophobic interactions, as well as hydrogen bonding and electrostatic interactions, were significant in the mucoadhesion of Pluronic-PAA copolymers. Experiments with a range of Pluronic-PAA copolymers with varying PPO contents in the Pluronic segments showed that increasing the overall PPO content increased the hydrophobicity of the polymer solutions. This was reflected in the increases in the advancing contact angles with the mucin layer, indicating that hydrophobic interactions play a role in the adhesion of Pluronic-PAA to mucin.
Introduction Triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) with the structure PEOPPO-PEO are known generically as poloxamers and under the tradenames Pluronic or Synperonic. Pluronicg-poly(acrylic acid) (Pluronic-PAA) is a thermogelling polymer formed by free-radical polymerization of acrylic acid (AA) with chain transfer to the Pluronic resulting in grafting of poly(acrylic acid) (PAA) side chains onto the Pluronic backbone.1 At low temperatures, 1-3 wt % aqueous solutions of Pluronic-PAA are free-flowing. At temperatures above 20 °C, a rapid 10-103-fold increase in viscosity occurs over a range of several degrees, resulting in the formation of a viscoelastic hydrogel.2 The onset of gelation coincides with the formation of uniformly spaced micellar aggregates which are formed by entropy-driven aggregation of the poly(propylene oxide) (PPO) segments and act as physical cross-linkers for gelation.3 The aggregates consist of a spherical core of dehydrated PPO segments, surrounded by an “inner corona” of more hydrated PEO segments and an “outer corona” of ionized PAA segments.4 The hydrophobic cores allow hydrophobic materials to be solubilized in aqueous solutions of PluronicPAA. Due to this combination of properties, Pluronic-PAA is a material of considerable promise as a vehicle for the * Corresponding author. Phone: (353) 61202629. Fax: (353) 61213529. E-mail:
[email protected]. † University of Limerick. ‡ Massachusetts Institute of Technology. (1) romberg, L. J. Phys. Chem. B 1998, 102, 10736-10744. (2) Bromberg, L. Macromolecules 1998, 31, 6148-6156. (3) Ho, A. K.; Bromberg, L. E.; O’Connor, A. J.; Perera, J. M.; Stevens, G. W.; Hatton, T. A. Langmuir 2001, 17, 3538-3544. (4) Huibers, P. D. T.; Bromberg, L. E.; Robinson, B. H.; Hatton, T. Macromolecules 1999, 32, 4889-4894.
controlled release of pharmaceutically active compounds. Investigations on the solubilization and release of various drugs and model compounds by Pluronic-PAA solutions and hydrogels have shown that these materials have the ability to significantly retard the release of various solutes ranging from low-MW hydrophobic materials to proteins.3,5-7 The mucoadhesive nature of Pluronic-PAA is also important in terms of its utility as a drug delivery system. Mucoadhesive polymers have a number of advantages as controlled release drug delivery systems. Localization at a specific region in the body helps improve drug bioavailability at the site of interest. Mucoadhesion also increases the intimacy of contact between a drug-containing polymer and a mucous surface, which can enhance the permeability of the drug. This is especially important for protein and peptide drugs, as well as for charged species. Prolonged residence times are also attained, leading to less frequent dosing and improved patient compliance. Mucoadhesive polymers can also protect the active compound from enzymatic degradation. Mucoadhesion of Pluronic-PAA is due mainly to the presence of chains of PAA, which have well-characterized mucoadhesive properties.8-13 It (5) Cleary, J.; Bromberg, L. E.; Magner, E. Langmuir 2003, 19, 91629172. (6) Bromberg, L; Temchenko, M. Langmuir 1999, 15, 8627-8632. (7) Bromberg, L.; Magner, E. Langmuir 1999, 15, 6792-6798. (8) Tamburic, S.; Craig, D. Q. M. Thermochim. Acta 1997, 294, 99106. (9) Degim, Z.; Kellaway, I. W. Eur. J. Pharm. Sci. 1996, 4, S145. (10) Mortazavi, S. A. Int. J. Pharm. 1995, 124, 173-182. (11) Mahrag Tur, K.; Ch’ng, H.-S. Int. J. Pharm. 1998, 160, 61-74. (12) Riley, R. G.; Smart, J. D.; Tsibouklis, J.; Young, S. A.; Hampson, F.; Davis, A.; Kelly, G.; Dettmar, P. W.; Wilber, W. R. Int. J. Pharm. 2002, 236, 87-96. (13) Kockisch, S.; Rees, G. D.; Young, S. A.; Tsibouklis, J.; Smart, J. D. J. Controlled Release 2001, 77, 1-6.
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has been suggested14 that for mucoadhesion to take place the following stages must occur: (a) development of an intimate contact between the bioadhesive polymer and the mucous tissue, (b) penetration of the polymer into the mucous surface or interpenetration of the polymer and mucin chains, and (c) formation of secondary chemical bonds, such as electrostatic and hydrophobic interactions, hydrogen bonding, and van der Waals interactions. A variety of techniques have been used to measure bioand mucoadhesion in vitro, including the Wilhelmy plate method15 and tensile force measurements.16-21 Other methods examined the movement of polymer particles in a flow of fluid along a strip of biological tissue22 or a channel filled with mucus gel23,24 or utilized fluorescence probe measurements25 or the staining of mucin with colloidal gold.26 Hassan and Gallo27 developed a rheological method for evaluating mucoadhesion of polymers, on the basis that the viscosity of polymer/mucin mixtures is the net result of the resistance to flow exerted by individual chain segments, physical chain entanglements, and noncovalent intermolecular interactions, which are the same as the interactions involved in the process of mucoadhesion. However, there is no standard test method established for bioadhesion, and consequently the data obtained are often subjective and difficult to compare due not only to the different parameters used as measures of bioadhesive force, but also to the fact that the results obtained depend on the experimental conditions, such as applied force and duration of contact in tensile testing, or polymer concentration and ionic strength in the rheological method. Atomic force microscopy (AFM) has been widely used in investigations of bio- and mucoadhesion. In many cases, AFM imaging has been used to examine the amount of material adhering to a surface.28-32 The colloid probe technique developed by Ducker and co-workers33,34 has been commonly used to measure adhesive forces. This (14) Duchene, D.; Touchard, F.; Peppas, N. A. Drug Dev. Ind. Pharm. 1988, 14, 283-318. (15) Smart, J. D.; Kellaway, I. W.; Worthington, H. E. C. J. Pharm. Pharmacol. 1984, 36, 295-299. (16) Gurny, R.; Meyer, J. M.; Peppas, N. A. Biomaterials 1984, 5, 336-340. (17) Blanco-Fuente, H.; Anguiano-Igea, S.; Otero-Espinar, F. J.; Blanco-Me´ndez, J. Int. J. Pharm. 1996, 142, 169-174. (18) Chickering, D. E.; Mathiowitz, E. J. Controlled Release 1995, 34, 251-262. (19) Santos, C. A.; Jacob, J. S.; Hertzog, B. A.; Freedman, B. D.; Press, D. L.; Harnpicharnchai, P.; Mathiowitz, E. J. Controlled Release 1999, 61, 113-122. (20) Mortazavi, S. A.; Smart, J. D. Int. J. Pharm. 1995, 116, 223230. (21) Ch’ng, H. S.; Park, H. K.; Robinson, J. R. J. Pharm. Sci. 1985, 74, 399-405. (22) Teng, C. L. C.; Ho, N. F. H. J. Controlled Release 1987, 6, 133149. (23) Mikos, A. G.; Peppas, N. A. S.T.P. Pharma 1986, 2, 705-716. (24) Achar, L.; Peppas, N. A. J. Controlled Release 1994, 31, 271276. (25) Park, K.; Robinson, J. R. Int. J. Pharm. 1984, 19, 107-127. (26) Park, K. Int. J. Pharm. 1989, 53, 209-217. (27) Hassan, E. E.; Gallo, J. M. Pharm. Res. 1990, 7, 491-495. (28) Patel, D.; Smith, J. R.; Smith, A. W.; Grist, N.; Barnett, P.; Smart, J. D. Int. J. Pharm. 2000, 200, 271-277. (29) Kakoulides, E. P.; Smart, J. D.; Tsibouklis, J. J. Controlled Release 1998, 54, 95-109. (30) Denis, F. A.; Hanarp, P.; Sutherland, D. S.; Gold, J.; Mustin, C.; Rouxhet, P. G.; Dufreˆne, Y. F. Langmuir 2002, 18, 819-828. (31) Cunliffe, D.; de las Heras Alarco´n, C.; Peters, V.; Smith, J. R.; Alexander, C. Langmuir 2003, 19, 2888-2899. (32) Deacon, M. P.; McGurk, S.; Roberts, C. J.; Williams, P. M.; Tendler, S. J. B.; Davies, M. C.; Davis, S. S.; Harding, S. E. Biochem. J. 2000, 348, 557-563. (33) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239-241. (34) Ducker, W. A.; Senden, T. J. Langmuir 1992, 8, 1831-1836.
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Figure 1. A typical force/distance profile as measured between a Pluronic-PAA-modified glass microsphere and a mucous surface in phosphate buffer solution as the tip approaches the surface (- - -) and retracts from the surface (-). Point A corresponds to the reference zero, where cantilever deflection is independent of tip-surface separation. B is the point where the tip contacts the surface. Between B and C, the force increases as the tip presses against the surface. From C to D, the tip is pulling away from the surface until at point D the contact is broken and the tip springs back to its original deflection at E.
Figure 2. (A) A 20 µm glass microsphere attached to an AFM cantilever. (B) Schematic of the method used to measure bioadhesive forces between Pluronic-PAA and a mucin-functionalized surface.
technique involves attaching a colloidal-sized particle (most commonly a sphere) of a material of interest to an AFM cantilever. Force-distance mode AFM is then used to measure the forces between this “colloid probe” and the surface of interest. Figure 1 shows a typical force-distance profile and illustrates some of the major features of such profiles. In this study, a variation of the colloid probe method has been utilized to measure the bioadhesive force between Pluronic-PAA copolymer and mucin-coated surfaces as a function of pH and ionic strength. A 20 µm glass microsphere covalently modified with a layer of Pluronic-PAA copolymer was attached to the AFM cantilever to form the colloid probe (Figure 2). A mucous surface was prepared by attaching a layer of mucin to an epoxy adhesive layer deposited on a glass plate. Adhesive forces between the probe and the mucous surface were then measured using force-distance mode AFM. Interactions between the Pluronic-PAA copolymers and mucin enriched
Adhesion of Polyether-Modified PAA to Mucin
by sialic acid were studied using dynamic contact angle technique and rheology. The effect of hydrophobic interactions on the mucin-copolymer interactions was examined by using Pluronic-PAA copolymers with varying PPO contents.
Langmuir, Vol. 20, No. 22, 2004 9757 Scheme 1. Reaction Scheme for the Attachment of Pluronic-PAA to Glass Surfacesa
Experimental Section Materials. Pluronics were obtained from BASF Corp. and were used without further treatment. Poly(ethylene oxide)-bpoly(propylene oxide)-b-(poly(ethylene oxide))-g-poly(acrylic acid) was synthesized by dispersion/emulsion polymerization of acrylic acid along with simultaneous grafting of poly(acrylic acid) onto Pluronic backbone as described in detail elsewhere.1-3,7,35-38 Weight-average molar masses of the Pluronic-PAA were in excess of 106. The polymer consisted of about 45% Pluronic F127 and 55% poly(acrylic acid) as measured by FTIR and NMR spectroscopy.44 To prepare solutions of Pluronic-PAA, the polymer samples were dispersed in distilled water and gently stirred at 4 °C for 48 h. The pH was adjusted with 5 M NaOH as needed. The solutions were filtered through filters with pore diameters of 0.8 µm and stored at 4 °C. The polymer concentration in the solutions was controlled within (0.005%. The following chemicals and materials were used as received: mucin from bovine submaxillary glands (Sigma), N-hydroxysuccinimide (NHS) (97%, Aldrich), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (99%, Fluka), N-(2-hydroxyethyl)piperazineN′-(2-ethanesulfonic acid) sodium salt (HEPES) (99.5%, Sigma), (3-aminopropyl)trimethoxysilane (97%, Aldrich), methanol (99.8%, Aldrich), hydrochloric acid (37%, Aldrich), Araldite slow-setting epoxy adhesive (Farnell), borosilicate glass microscopes slides (Sigma), 20 µm borosilicate glass microspheres (19.9 ( 1.4 µm, Structure Probe Inc.), toluene (99.8%, anhydrous, Aldrich). Purified water (18.2 MΩ) was obtained from an Elgastat Maxima water purification system. For the tensiometric experiments, mucin from porcine stomach (Sigma) was fractionated as described previously.45 The fraction containing 20 wt % of total proteins and 13 wt % of sialic acids (N-acetylhexosamines) was used. The amino acid content of the mucin was lysine 1.1, histidine 0.4, arginine 3.1, aspartic 1.7, threonine 13.0, serine 19.6, glutamic 5.7, proline 6.4, glycine 20.1, alanine 14.2, cystine 0.8, valine 8.0, methionine 0.2, isoleucine 2.9, leucine 1.1, tyrosine 0.8, phenylalanine 0.7, tryptophan 0.1 mol %. Methods. To develop a method of covalently attaching Pluronic-PAA to glass substrates, glass microscope slides were modified using the following procedure. The glass slide was first cleaned using the method recommended by Cras et al.,41 that is, immerse for 30 min in 1:1 methanol:HCl, rinse in H2O, dry under N2, immerse for 30 min in H2SO4, rinse in H2O, dry under N2. The glass surface was then silanized by refluxing for 12 h with 3-aminopropyltrimethoxysilane (10 wt % solution in toluene) producing an amine-functionalized glass surface (Scheme 1A). This in turn was coupled with the -COOH groups of the PluronicPAA using the EDC/NHS coupling system (Scheme 1B). The reaction was carried out at 70 °C for 4 h (HEPES buffer, pH 7.5), after which the slides were rinsed extensively in DIW to remove any noncovalently bound material. AFM imaging was used to characterize the surface of the slides. An identical procedure (35) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197-221. (36) Bromberg, L. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: New York, 2001; Vol. 4: Solid Thin Films and Layers, Chapter 7, pp 369-404. (37) Bromberg, L. Ind. Eng. Chem. Res. 2001, 40, 2437-2444. (38) Bromberg, L. Langmuir 1998, 14, 5806-5812. (39) Bromberg, L. Ind. Eng. Chem. Res. 1998, 37, 4267-4274. (40) Bromberg, L. E.; Orkisz, M. J.; Ron, E. S. Polym. Prepr. 1997, 38, 626. (41) Cras, J. J.; Rowe-Taitt, C. A.; Nivens, D. A.; Ligler, F. S. Biosens. Bioelectron. 1999, 14, 683-688. (42) Hilal, N.; Bowen, W. R. Desalination 2002, 150, 289-295. (43) Hutter, J. J.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 18681873. (44) Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum: New York, 1985; Vol. 1, pp 249-292. (45) Semenov, A. N.; Rubinstein, M. Macromolecules 2002, 35, 48214837.
a In step A, the hydroxyl groups on the glass surface react with 3-aminopropyltrimethoxysilane to give an NH2-functionalized surface. In step B, the NH2 groups are coupled with the COOH groups of Pluronic-PAA using the carbodiimide coupling agent EDC, with NHS as activator.
was used to modify the glass microspheres. Modified microspheres were attached to AFM cantilevers with an epoxy adhesive, using a procedure similar to that described by Hilal and Bowen.42 The mucous substrate was prepared by allowing a thin layer of epoxy adhesive to spread over an area of approximately 1 cm in diameter. As the adhesive was initially relatively free flowing it formed a smooth surface, which was allowed to dry for 15-30 min at which point it was slightly tacky. Mucin (as received in the form of fibrous portions) was then placed on the surface of the adhesive and was left in position for several hours until the glue was completely set. The slide was then rinsed thoroughly, first under a flow of DIW and then by immersion in gently stirred DIW for 2 h, to remove any mucin that was not firmly attached to the epoxy substrate. The surface of the mucin layer was examined by AFM imaging, and the thickness of the mucin layer was estimated by taking an image covering the edge of the mucin layer and carrying out a line analysis to determine the “step height”. The AFM instrument used was a Topometrix Explorer AFM. Contact-mode AFM images were obtained in air or buffer solution as appropriate using Topometrix 1520-00 cantilevers with square pyramidal silicon nitride tips (height 4 µm, tip radius 30 mM)58 and that such a change in mucin conformation must strongly reduce the potential for mucoadhesion. The fact that it was still possible to measure significant adhesion forces at high salt concentrations such as 1.0 M suggests either that the mode of attachment constrained the ability of the mucin layer to change its conformation or that hydrophobic interactions between the mucin and the PPO segments of Pluronic-PAA make a significant contribution to the adhesive force. Assessment of the Colloid Probe Technique for Measuring Mucoadhesion. The colloid probe technique described here has both advantages and limitations as a method for assessing mucoadhesion of copolymers. It involves functionalization of both the colloid probe (glass microsphere) and the planar surface (glass slide) and is therefore more complicated than some other methods. Its reproducibility is limited by factors including variations in the thickness and topography of the polymer and mucin layers, as well as the radius of the microsphere. On the other hand, it provides a direct measurement of mucoadhesive forces on the microscale, and it is possible to clearly distinguish trends in the mucoadhesive force as a function of pH and ionic strength. The dependence of the measured force on sampling speed could be seen as a limitation in terms of obtaining an absolute value of mucoadhesive force; however, it also provides a means of assessing the rate with which viable mucoadhesive joints are formed in a given system. Tensiometry. Typical Wilhelmy force loops obtained when the mucin-coated plates were immersed into and removed from 1 wt % Pluronic (F127)-PAA solutions are presented in Figure 8. A large force hysteresis was observed, resulting from the wetting of the mucin layers by the polymer solution. Advancing angles were measured to be 88° ( 5° and 100° ( 4° at 37 and 20 °C, respectively, while receding angles were -1° at both temperatures, indicating that the mucin layers were hydrated. The force required to lift the plate from solution was much higher at 37 °C than at 20 °C. This observation can be explained by the fact that the solution of the F127-PAA is a freeflowing liquid at 20 °C and a viscoelastic gel at 37 °C.3 The formation of physically cross-linked gels leads to large polymer layers that are interconnected and thus need to be lifted together when the interfacial gel layers adhere to mucin. Judging by the lower advancing contact angle observed at 37 °C, the formed gel appears to be less (58) Bastardo, L.; Claesson, P.; Brown, W. Langmuir 2002, 18, 38483853.
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Figure 8. Wilhelmy plate plots of glass slides coated with mucin in contact with 1 w/v% aqueous solution of Pluronic (F127)-PAA at pH 7.0.
hydrophobic than the solution of the same polymer. This observation can be explained by the fact that the gel is formed due to the formation of the micelle-like aggregates, where the hydrophobic PPO segments are buried in the inner cores of the aggregates, exposing hydrophilic PAA and PEO segments.4 The effect of hydrophobic interactions on the mucincopolymer interactions was examined by varying the PPO content of the copolymer, the only hydrophobic segment in Pluronic-PAA. The bulk content and length of PPO have been shown to be essential factors for critical micellization temperatures, gelation threshold, cohesion, and other properties of the Pluronic-PAA solutions and hydrogels.3,54 The overall Pluronic content in each copolymer was set at 45-49 wt %, while the content of PPO segment varied more than 4-fold. Significant increases in contact angles were observed as the PPO content increased (Figure 9). All of the copolymers were capable of wetting the mucin layers, with the receding angles not exceeding 6° (data not shown). The Pluronic L121-PAA coating effectively repelled water (advancing angle of 117° as compared to 109° for a hydrophobic polymer such as poly(tetrafluoroethylene)). The overall PPO content increased the hydrophobicity of the polymer solutions, as reflected in the increases in the advancing contact angles with the mucin layer. These results indicate that hydrophobic interactions play a role in the adhesion of Pluronic-PAA to mucin. Conclusions The colloid probe technique has been shown to provide a direct measurement of mucoadhesive forces on the microscale. The advantages and limitations of the technique as a means for assessing mucoadhesion of polymeric materials have been discussed. In the case of Pluronic-PAA adhesion to mucous surfaces, the polymer exhibits significant adhesive forces over a range of conditions. Hydrogen bonding, electrostatic forces, interpenetration of the Pluronic-PAA and mucin layers, and hydrophobic interactions all play a role in the adhesion process, with the relative importance of each depending on the prevailing conditions. While hydrophobic interactions between PPO segments and mucin, as well
Figure 9. Effect of bulk PPO content of Pluronic-PAA copolymers on advancing (1,2) and receding (3) contact angles. Systems studied were glass plates coated on both sides with Pluronic-PAA immersed into and removed from water (pH 7.0, 20 °C) (1, 3) or glass plates coated with mucin and immersed into and removed from 1 wt % Pluronic-PAA aqueous solution (pH 7.0, 37 °C) (2). Filled squares and circles show the advancing and receding contact angles, respectively, for the mucin-coated glass plates immersed into and removed from water.
as interpenetration of the polyether segments with the mucin layer, have been shown to contribute to the overall mucoadhesive force, the PAA segments are predominantly responsible for the mucoadhesion Pluronic-PAA copolymers. The adhesive forces are at a maximum in the pH range 4-5, intermediate at lower pH, and drop sharply at higher pH. The observed trends have been explained in terms of the ionization and extension of the copolymer and mucin layers, both of which are affected by varying pH. At a constant pH of 5.0, the adhesive forces decreased with increasing ionic strength. We ascribe this to the screening of intramolecular repulsions between the negative charges on the Pluronic-PAA carboxyl groups at increased ionic strength. This results in a more compact conformation of the PAA segments, the main contributors to mucoadhesion of Pluronic-PAA, and hence to lower mucoadhesive forces. Tensiometric measurements using a Wilhelmy balance apparatus showed that the force required to lift the plate from the solution was much higher when the PluronicPAA was in the viscoelastic gel phase (37 °C) rather than in the solution phase (20 °C), due to the formation of large, interconnected polymer layers at the higher temperature. The effect of overall PPO content on the mucin-copolymer interactions was examined by using different Pluronics in the Pluronic-PAA copolymers. Significant increases in contact angles were observed as the PPO content increased, indicating that hydrophobic interactions participate in the adhesion of Pluronic-PAA to mucin. Acknowledgment. This research was funded by the Higher Education Authority through the Program for Research in Third Level Institutions (1999-2000). LA048993S
