Direct Measurement of Interactions between Stimulation

Since some reports have indicated the possibility of the destruction of the polymer layer under a large compression force (>10 nN),22 the compression ...
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Langmuir 2008, 24, 3987-3992

3987

Direct Measurement of Interactions between Stimulation-Responsive Drug Delivery Vehicles and Artificial Mucin Layers by Colloid Probe Atomic Force Microscopy Motoyuki Iijima,*,† Motoyasu Yoshimura,† Tadashi Tsuchiya,† Mayumi Tsukada,† Hideki Ichikawa,‡ Yoshinobu Fukumori,‡ and Hidehiro Kamiya† Institute of Symbiotic Science and Technology, Tokyo UniVersity of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, and Faculty of Pharmaceutical Sciences and Co-operatiVe Research Center of Life Sciences, Kobe Gakuin UniVersity, Minatojima 1-1-3, Chuo-ku, Kobe 650-8586, Japan ReceiVed December 5, 2007. In Final Form: January 20, 2008 A novel thermo- and pH-sensitive nanogel particle, which is a core-shell structured particle with a poly(Nisopropylacrylamide) (p(NIPAAm)) hydrogel core and a poly(ethylene glycol) monomethacrylate grafted poly(methacrylic acid) (p(MMA-g-EG)) shell, is of interest as a vehicle for the controlled release of peptide drugs. The interactions between such nanogel particles and artificial mucin layers during both approach and separation were successfully measured by using colloid probe atomic force microscopy (AFM) under various compression forces, scan velocities, and pH values. While the magnitudes of the compression forces and scan velocities did not affect the interactions during the approach process, the adhesive force during the separation process increased with these parameters. The pH values significantly influenced the interactions between the nanogel particles and a mucin layer. A large steric repulsive force and a long-range adhesive force were measured at neutral pH due to the swollen p(MMA-g-EG) shell. On the other hand, at low pH values, the steric repulsive force disappeared and a short-range adhesive force was detected, which resulted from the collapse of the shell layer. The nanogel particles possessed a pH response that was sufficient to protect the incorporated peptide drug under the harsh acidic conditions in the stomach and to effectively adhere to the mucin layer of the small intestine, where the pH is neutral. The relationships among the nanogel particle-mucin layer interactions, pH conditions, scan velocities, and compression forces were systemically investigated and discussed.

Introduction

* Corresponding author. E-mail address: [email protected] (M. Iijima); Phone & Fax: 81-42-388-7068. † Tokyo University of Agriculture and Technology. ‡ Kobe Gakuin University.

and a poly(methacrylic acid) grafted with a poly(ethylene glycol) monomethacrylate (p(MMA-g-EG)) shell. The p(NIPAAm) hydrogel core is made of a thermosensitive material known for its swelling and collapsing properties below and over 32 °C, respectively.7 The p(MAA-g-EG) shell is made of a pH-sensitive polymer that collapses in acidic media and swells in neutral media.8 In addition, a swollen p(MMA-g-EG) shell possesses a mucoadhesive property9 as well as a proteolytic enzyme inhibitory effect,10 both of which are favorable for enhancing peptide adsorption through the small intestine. By combining these two responsive components into core-shell nanogel particles, the novel designed device for oral peptide deliveries is expected to perform as follows. The peptides can be loaded under mild conditions such as low temperature and neutral pH where both the core and shell components are swollen. In the gastric fluid, which is at the body temperature, both components remain collapsed so that the peptides can be protected from enzymatic degradation. In the small intestine, the swollen p(MMA-g-EG) shell can exhibit both mucoadhesive properties and the peptidetransport enhancing effect in the mucosal cell lining, while the p(NIPAAm) core remains shrunken; this allows for the release of the peptide in a prolonged manner. The nature of the interaction between the drug delivery vehicles and the mucous layer is also expected to be important in terms of the utility as a drug delivery system. Without effective localization of the drug delivery vehicles at the mucous layer,

(1) Edwards, R. J.; Moran, N.; Devocelle, M.; Kiernan, A.; Meade, G.; Signac, W.; Foy, M.; Park, S. D. E.; Dunne, E.; Kenny, D.; Shields, D. C. Nat. Chem. Biol. 2006, 3, 108. (2) Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. Science 1999, 285, 1569. (3) Bromberg, L. E.; Ron, E. S. AdV. Drug. DeliVery ReV. 1998, 31, 197. (4) Pepas, N.; Robinson, J. R. J. Drug Targeting 1995, 3, 183. (5) Davies, N. M.; Farr, S. J.; Hadgraft, J.; Kellaway, I. W. Pharmaceut. Res. 1991, 8, 1039.

(6) Ichikawa, H.; Yamasaki, Y.; Fukumori, Y. In New Trends in Polymers for Oral and Parental Administration from Design to Receptors; Barratt, G., Duchene, D., Fattal, F., Legendre, J. Y., Eds.; Editions DeSante: Paris, 2001; p 257. (7) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (8) Lowman, A. M.; Peppas, N. A. Macromolecules 1997, 30, 2959. (9) Peppas, N. A.; Lowman, A. M. Protein and Peptide Drug Research, Munksgaard 1998, 127, 206. (10) Madsen, F.; Peppas, N. A. Biomaterials 1999, 20, 1701.

Significant advances in biochemistry have led to the discovery of a large number of bioactive molecules such as peptides and proteins.1,2 However, the bioavailability of peptides and proteins as therapeutic agents by oral administration is quite low due to the epithelial barriers of the gastrointestinal tract and gastrointestinal degradation by digestive enzymes. To overcome these problems, numerous researchers have focused on mucoadhesive drug delivery vehicles over the last two decades.3-5 These are designed to exploit the attraction between the mucous layer and the polymer-based drug delivery vehicles to enhance the peptide drug absorption at preferable sites in the gastrointestinal tract. Further, for application to oral administration, the drug delivery system is required to have specific abilities such as protecting the peptides from harsh manufacturing and physiological environments. To achieve such specific functions, Ichikawa et al. previously designed novel thermo- and pH-sensitive nanogel particles with a core-shell structure as functional particulate devices for oral peptide delivery.6 The designed nanogel particles were obtained from a poly(N-isopropylacrylamide) (p(NIPAAm)) hydrogel core

10.1021/la7038043 CCC: $40.75 © 2008 American Chemical Society Published on Web 03/01/2008

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the bioavailability and permeability of the peptide drugs cannot be improved. Various techniques have been used to measure the mucoadhesive properties, such as tensile force measurements,11-13 fluorescence probe measurements,14 flowability measurements of polymer particles in a channel filled with mucous gel,15,16 and rheological methods.17 However, these current methods used to estimate the mucoadhesion properties do not provide direct evidence of the polymer-mucous layer interactions. Cleary et al.18 have measured the mucoadhesive interaction between a mucin layer and a Pluronic-PAA-modified glass microsphere by using a colloid probe atomic force microscopy (AFM) method that can measure the direct interactions. However, they used a model particlesa Pluronic-PAA-modified glass microspheres and only measured the adhesive force that could be detected during the separation process. In terms of pH-sensitive drug delivery vechicles, drastic changes in the steric repulsive force could be expected due to the pH-induced collapsing and swelling properties. Thus, measuring the surface interactions during both the approach and the separation processes by using a colloid probe with drug delivery vehicle particles is necessary to characterize the real surface interactions between the drug delivery vehicles and the mucous layers. In this study, the surface interaction between a p(NIPAAm)/ p(MMA-g-EG) core-shell nanogel particle and an artificial mucin layer was directly measured by using colloid probe AFM techniques. The relationships among the swelling and collapsing properties of p(NIPAAm)/p(MMA-g-EG) nanogel particles, surface interactions during the approach and separation processes, and the experimental conditions such as the compression forces, scan velocities, and pH values were systemically investigated. Experimental Section Materials. N-Isopropylacrylamide supplied by Kohjin Co., Ltd., was recrystallized to remove the impurities. Methacrylic acid (MAA) and sodium dodecyl sulfate (SDS) were purchased from Nacalai Tesque Inc. Methoxy-terminated poly(ethylene glycol) monomethacrylate (PEGMA) with PEG of molecular weight 1000 and tetraethylene glycol dimethacrylate (TEGDMA) were obtained from Polysciences Inc. Mucin (from the stomach), hydrochloric acid (30%), ammonia aqueous solution (28 wt %), ethanol (95%), phosphoric acid (>85%), potassium dihydrogenphosphate (>99.0%), disodium hydrogenphosphate (>99.0%), and sodium chloride (>99.5%) were purchased from Wako Pure Chemical Industry Ltd. 3-Aminopropyltriethoxy silane (APTS), 1-hydroxycyclohexyl phenyl ketone, and mica were purchased from Shin-Etsu Chemical Co., Ltd., Aldrich Chemical Co., Ltd., and Yamaguchi Mica Co., Ltd., respectively. Preparation of p(NIPAAm)/p(MMA-g-EG) Core-Shell Nanogel Particles. The core-shell nanogel particles were synthesized by a semi-continuous two-stage photoinitiated free radical dispersion polymerization technique. The details of the synthesis procedure are reported elsewhere.6 Initially, NIPAAm (1.5 g), TEGDMA (0.06 g), and 1-hydroxycyclohexyl phenyl ketone (an initiator, 0.06 g) were dissolved in 73.38 g of 0.4 mM aqueous SDS solution. This solution was then exposed to ultraviolet light with a wavelength of 365 nm at 70 °C for 30 min with stirring. Subsequently, an aqueous solution (75 g) of MMA and PEGMA (molar ratio ) 1:1) was added dropwise to the resultant colloidal p(NIPAAm) nanoparticles. The employed core-to-shell weight ratio was 55:10. Finally, the as-prepared nanogel particles were dialyzed using cellulose acetate tubing in purified water for 5 days. The concentration (11) Chickering, D. E.; Mathiowitz, E. J. J. Controlled Release 1995, 34, 251. (12) Mortazavi, S. A.; Smart, J. D. Int. J. Pharm. 1995, 116, 223. (13) Ch’ng, H. S.; Park, H. K.; Robinson, J. R. J. Pharm. Sci. 1985, 74, 399. (14) Park, K.; Robinson, J. R. Int. J. Pharm. 1984, 19, 107. (15) Mikos, A. G.; Peppas, N. A. S.T.P. Pharma 1986, 2, 705. (16) Achar, L.; Peppas, N. A. J. Controlled Release 1994, 31, 271. (17) Hassan, E. E.; Gallo, J. M. Pharm. Res. 1990, 7, 491. (18) Cleary, J.; Bromberg, L.; Magner, E. Langmuir 2004, 20, 9755.

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Figure 1. FE-SEM images of (a) nanogel particles collected by freeze drying, (b) nanogel particles collected by spray-freeze drying, and (c) prepared colloid probe. of the nanogel particles was 1.03 wt %. The pH-zeta potential curves and particle size of nanogel particles were measured by a z-analyzer (model 502, Nihon Rufuto Co. Ltd.) and by the dynamic scattering method (HPP5001, Malvern Instruments Ltd.), respectively. Measurement of Surface Interaction between Core-Shell Nanogel Particles and Mucin Layers. A mucin layer was fabricated on a flat mica surface in the following manner: First, APTS (0.06 g) was added dropwise into water (8.4 g), adjusted to pH 4, and then stirred for 30 min. Then, small mica pieces (6.0 g) were added to the APTS aqueous solution, and subsequently, its pH was increased to 12 in order to promote the condensation of APTS on the mica surface. After stirring for 24 h, the mica pieces were collected and dried overnight at 120 °C. Further, mica was rinsed with ethanol twice and dried again at 120 °C for 24 h. Next, mucin powder (0.15 g) was dispersed into ultrapure water (30 g), and the large mucin particles were filtered off by a microporous filter. The APTS-modified mica plates were carefully dipped into the 0.5 wt % mucin powder suspension and slowly stirred for 24 h. Finally, the mica plates were dried under vacuum for 24 h, rinsed with ultrapure water, and then dried under vacuum again. The mica surface with the APTS/mucin layer was examined by AFM imaging. The amount of APTS and mucin was determined by a CHN organic elemental analyzer (JM10 Micro coder, J Science Lab. Co., Ltd.): Mica plates of known weight were injected and combusted at 900 °C, and the carbon content was measured in terms of the CO2 generated by using a differential thermal conductivity detector calibrated with analyticalgrade p-nitroaniline. The nitrogen content was measured in the same manner as the carbon content, except the gas that was detected was N2 (after passing though a reduction chamber). The measured values were corrected by using the backgrounds determined from a blank powder-free air sample. For the preparation of a colloid probe with a nanogel granule, the suspension of core-shell nanogel particles was first diluted to 0.05 wt % by a phosphate buffer aqueous solution (pH ) 3.6). Then, the diluted suspension was sprayed on a copper plate, frozen by liquid nitrogen, and finally dried under vacuum for 24 h. This sprayfreeze drying method was essential to collect the nanogel particles as granules (Figure 1a,b). The freeze-dried granules of the coreshell nanogel particles were adhered to the top of a commercial AFM tip by a micromanipulation system (Figure 1c).19,20 The interactions between the colloid probe carrying nanogel granules and mucin layers in the phosphate buffer aqueous solution were measured by an atomic force microscope (PicoForce, Digital Instrument Co. Ltd.). The scanning speed and the compression force were computer-controlled to between 0.01 and 10 µm/s and 2.18 and 18.7 nN, respectively. The compression force was controlled by monitoring the displacement of the cantilever during the approaching process. When the displacement of the cantilever reached a steady value, the approaching process was arrested and the separation process was initiated.

Results and Discussion Characterization of Core-Shell Nanogel Particles. Figure 2 shows the pH-zeta potential curves of the nanogel particles. (19) Tsukada, M.; Irie, R.; Yonemochi, Y.; Noda, R.; Kamiya, H.; Watanabe, W.; Kauppinen, E. I. Powder Technol. 2004, 142, 262. (20) Iijima, M.; Tsukada, M.; Kamiya, H. J. Colloid Interface Sci. 2007, 307, 418.

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Figure 2. pH-zeta potential curves of prepared nanogel particles. Figure 4. Carbon and nitrogen contents on mica surface without modification (Mica), modified with APTS (AP-Mica), and modified with mucin (M-AP-Mica).

Figure 3. Particle size distributions of core-shell nanogel particles in phosphate buffer solutions measured by the dynamic light scattering (DLS) method.

The isoelectric point (IEP) of the nanogel particles was at nearly pH 4.0 and their surface charge was found to decrease as the pH of the phosphate buffer solution increased. Previous researchers have reported that the IEPs of un-cross-linked PAA and p(MMAg-EG) are 4.86 and 4.56, respectively.21 It is expected that the surface charge on our core-shell nanogel particles is related to the shell layer, particularly due to the dissociation and recombination of carboxyl groups, rather than the core. Figure 3 shows the particle size distribution of the prepared core-shell nanogel particles in a phosphate buffer solution under various pH conditions. Under acidic conditions, the diameter of the particles was nearly 300 nm; however, the diameter gradually increased to 650 nm when the pH was increased to 8.0. As expected, it was estimated that the nanogel particles collapsed under acidic conditions and swelled under neutral conditions. Characterization of Mica Surface Modified by APTS and Mucin. Figure 4 shows the carbon and nitrogen contents on the mica surface before and after surface modification. Before surface modification, no carbon and nitrogen was detected on the mica surface. An increase in the carbon and nitrogen content was observed after each surface modification stepsAPTS treatment and mucin adsorption. Figure 5 shows the AFM images of the mica surface before and after surface modification. While the measurements of the raw mica surface indicated a completely flat surface, the roughness increased on modification with APTS; this is assumed to be due to the condensation of ATPS on the mica surface. This roughness produced by APTS decreased due to the adsorption of mucin. The exact reason for the decrease in roughness on mucin adsorption is unknown, but the phenomenon is consistent with the results of previous reports. Efremova et al. adsorbed mucin on bare mica layers under dilute (0.02 mg/mL)

and dense (0.2 mg/mL) mucin solutions and characterized their surfaces by AFM imaging.22 They reported that a rough surface, which was expected to be a submonolayer or an island of mucin, was measured from mica treated with the dilute mucin solution

(21) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2003, 19, 8675.

(22) Efremova, N. V.; Huang, Y.; Peppas, N. A. Langmuir 2002, 18, 836.

Figure 5. AFM scanning images of mica surface (a) without modification, (b) modified with APTS, and (c) modified with mucin.

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Figure 6. Effect of compression force on the interaction between nanogel particle and mucin layer (a) during approach process and (b) during separation process.

while a flat layer of mucin was obtained when mica was treated with the dense mucin solution. These results suggest that a rough surface due to submonolayer and/or islands of mucin were produced at the very beginning of the adsorption procedure and this surface could be flattened by further adsorption of mucin when the mica is immersed into a dense mucin solution. Since the concentration of the mucin solution in our work is high (5 mg/mL), it is expected that multilayered adsorption of mucin flattened the rough APTS-mica surface. From these elemental analyses and AFM imaging results, we can assert that a flat mucin layer was successfully produced on the mica surface. Interaction between Nanogel Particles and Mucin Layer. Figure 6 shows the effect of the compression force on the surface interaction between the nanogel particles and the mucin layer in a phosphate buffer solution whose pH is adjusted to 6.8, during the approach and separation processes. The compression forces between the nanogel particles and the mucin layer were 2.18, 10.3, and 18.7 nN. As shown in Figure 6a, the compression force did not affect the surface interaction during the approach process. On the other hand, it significantly affected the interaction during the separation process. In Figure 6b, it can be observed that the magnitude of the long-range adhesive force increased with the compression force. Ajuha et al.23 reported that interactions between polymer chains, such as chain linking, strongly depend on the contact stress and contact time. Similarly, in our case, it is expected that the polymer chain linking of the p(MMA-g-EG) (23) Ahuja, A.; Khar, R. K.; Ali, J. Drug DeV. Ind. Pharm. 1997, 23, 489.

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Figure 7. Effect of scan velocity on the interaction between nanogel particle and mucin layer (a) during approach process and (b) during separation process.

shell and mucin is enhanced by increasing the compression force and results in the increase in the adhesive force. Figure 7 shows the effect of the scanning velocity on the surface interaction between nanogel particles and the mucin layer in the phosphate buffer solution whose pH is adjusted to 6.8. The scanning velocity was maintained between 0.01 µm/s and 10 µm/s. Similar to the case of varying the compression forces, the scan velocities did not affect the surface interaction during the approach process (Figure 7a); however, they greatly affected the interactions during the separation process. In Figure 7b, it is observed that the magnitude of the long-range adhesive forces increased by decreasing the scanning velocity. This occurred because decreasing the scanning velocity increased the length of the contact time between the p(MMA-g-EG) shell and the mucin layer, thereby promoting the polymer chain linking. Figure 8a shows the surface interaction between the nanogel particles and mucin layer during the approach process in a phosphate buffer solution at various pH. Since some reports have indicated the possibility of the destruction of the polymer layer under a large compression force (>10 nN),22 the compression force was maintained at 5 nN. Further, in order to easily observe the effect of the pH on the polymer chain linking phenomena, the scan velocity was maintained at 0.02 µm/ssa very low value. The ion concentration was 0.01 mol/L, which is adjusted by using sodium chloride. Figure 8b shows the DLVO curves for each pH value, describing the theoretical electrostatic interactions between the nanogel particles and the mucin layer.

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[(

FEDL ) π0a

)(

2ψpψlκ exp(-κD)

)

1 1 - exp(-κD) 2κ exp(-2κD) (2) (ψp2 + ψl2) 1 - exp(-2κD)

1 + exp(-κD)

1+

(

)]

The theoretical DLVO curves were calculated by the sum of the EDL interaction force and the van der Waals attractive force, which can be expressed as follows:

F ) FEDL -

H Aa 6D2

(3)

Here, HA is the particle-layer Hamaker constant in water. The difference between the measured surface force curves (Figure 8a) and the theoretical DLVO curves (Figure 8b) is related to non-DLVO interactions such as the steric repulsive forces of the polymer chains. From Figure 8a and b, a long-range and large steric repulsive force was detected when the pH values of the phosphate buffer solution were 6.8 and 8.0. It is expected that this large repulsive force was due to the swollen p(MMA-g-EG) shell networks. On the contrary, a short-range and small repulsive force was detected when the phosphate buffer solution was acidic. This small repulsive force is related to the collapsed p(MMAg-EG) shell. Figure 9a shows the surface interaction between the nanogel particles and mucin layer during the separation process in a phosphate buffer solution at various pH. The maximum value of the detected adhesive force for each pH is also shown in

Figure 8. (a) Effect of pH on the interaction between nanogel particle and mucin layer during approach process and (b) theoretical force curves calculated by DLVO theory.

According to Hogg et al.24 and Kemps et al.,25 the electric double layer (EDL) interaction energy between a particle and a layer can be expressed as follows:

[

UEDL ) π0a 2ψpψl ln

(

)

1 + exp(-κD) + (ψp2 + ψl2) ln 1 - exp(-κD)

]

[1 - exp(-2κD)] (1) Here, a is the radius of the particle; 0, the dielectric permittivity of vacuum; , the dielectric constant of water; ψp and ψl, the surface potentials of the particle and the layer, respectively; κ, the inverse Debye screening length; and D, the surface distance between the nanogel particle and the mucin layer. The surface charge of the nanogel particles (ψp) was the value shown in Figure 2 and the surface charge of the mucin layer (ψl) was calculated using the Nernst equation26 with IEP ) 2.6.27 By differentiating eq 1, the particle-layer interaction force due to EDL can be obtained as follows: (24) Hogg, R. I.; Healy, T. W.; Fuerstenau, D. W. Trans. Faraday Soc. 1966, 62, 1638. (25) Kemps, J. A. L.; Bhattacharjee, S. Langmuir 2005, 21, 11710. (26) Healy, T. W.; Fuerstenau, D. W. J. Colloid Sci. 1965, 20, 376. (27) Coltart, D. M.; Royyuru, A. K.; Williams, L. J.; Glunz, P. W.; Sames, D.; Kuduk, S. D.; Schwarz, J. B.; Chen, X-T.; Danishefsky, S. J.; Live, D. H. J. Am. Chem. Soc. 2002, 124, 9833.

Figure 9. (a) Effect of pH on the interaction between nanogel particle and mucin layer during separation process and (b) the magnitude of the maximum adhesive force with various pH values.

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Figure 9b. As seen in Figure 9b, the magnitude of the adhesive force was minimum when the pH was 5.4, and it increased under more neutral or acidic conditions. Interestingly, these pH effects on the magnitude of the adhesive force are completely different from those on Pluronic-PAA, which is a material for non-pHsensitive drug delivery vehicles.18 It has been reported that the adhesive force between Pluronic-PAA and mucin, as measured by the AFM colloid probe method, is highest at approximately pH 5, but decreases under more neutral conditions. Cleary et al. have pointed out that this occurs since the carboxyl groups of PAA and mucin are partially ionized at pH 5, making both the PAA chains and the mucous network extended, and thereby enabling the interpenetration of the polymer chains with the mucin molecules when in contact. They have also reported that the adhesive force is greatly reduced under more neutral conditions because most of the Pluronic-PAA groups are ionized, making both Pluronic-PAA and mucin negatively charged. This reduction in the adhesive force under neutral condition is a problem for drug delivery vehicles because, then, effective localization of drug delivery vehicles in the small intestine, where the pH is neutral, cannot be expected. The use of pH-sensitive p(NIPAAm) core/(p(MMA-g-EG)) shell particles as drug delivery vehicles could be one method to overcome this problem. When the pH values of the phosphate buffer solution were 6.8 and 8.0, since the swollen p(MAA-g-EG) shell network enhanced the crosslinkage with the mucin layer, a long-range adhesive force was detected. The cross-linking phenomena between the swollen p(MAA-g-EG) shell and the mucin layer also increased the magnitude of the adhesive force. On the other hand, when the pH was below 5.4, the p(MMA-g-EG) shell layer collapsed and became rigid; thus, a sharp and short-range adhesive force was detected. Under these conditions, almost no cross-linking phenomena can be expected, and hence, the increase in the adhesive force magnitude must be due to the electrostatic interactions. At pH 5.4, the nanogel particle and mucin layer

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(IEP ) 2.6)27 are both negatively charged so that the adhesive force will be small due to the electrorepulsive force. When the pH is reduced, the surface charge of the nanogel particle and mucin layer will be reduced; this results in a reduction in the electrorepulsive force and an increase in the adhesive force. According to the direct measurement of the interaction force between a nanogel particle and a mucin layer under various pH conditions, the prepared nanogel particles possessed a suitable pH response for application to oral peptide delivery devices. In the case of low-pH regions such as the stomach, the nanogel particles collapsed and possessed a short-range adhesive force, which protects the incorporated peptide drugs from harsh acidic conditions, and no effective adhesion of nanogel particles on the stomach surface will occur. Under neutral conditions such as those in the small intestine, the nanogel particles possessed a long-range adhesive force, which enables them to easily adhere to the mucin layer on the surface of the small intestine; in addition, the p(MAA-g-EG) shell of nanogel particles swelled so that the incorporated peptide drugs can be effectively released.

Conclusion We have successfully measured the direct interactions between a novel mucoadhesive drug delivery vehicle particle and a mucin layer during the approach and separation processes under various conditions. While a neutral pH condition led to the swelling of the nanogel particle and resulted in the appearance of a longrange large steric repulsive force and a long-range large adhesive force, an acidic pH condition led to the collapsing of the nanogel particle and resulted in the disappearance of the steric repulsive forces and appearance of short-range adhesive forces. These measured surface interactions provided significant information for estimating the flowing properties of drug delivery vehicles by oral administration in the small intestine. LA7038043