Bioadhesive properties and biodistribution of cyclodextrin–poly(anhydride) nanoparticles

European Journal of Pharmaceutical Sciences 37 (2009) 231–240

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Bioadhesive properties and biodistribution of cyclodextrin–poly(anhydride) nanoparticles Maite Agüeros a , Paloma Areses b , Miguel Angel Campanero a , Hesham Salman a , Gemma Quincoces b , b ˜ Ivan Penuelas , Juan Manuel Irache a,∗ a b

Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Apartado 177, 31008 Pamplona, Spain Radiopharmacy Unit, Department of Nuclear Medicine, Clínica Universitaria de Navarra, University of Navarra, 31008 Pamplona, Spain

a r t i c l e

i n f o

Article history: Received 17 December 2008 Received in revised form 10 February 2009 Accepted 15 February 2009 Available online 28 February 2009 Keywords: Nanoparticles Cyclodextrin Bioadhesion Gantrez® AN Poly(anhydride) Oral 99m Tc-radiostudies

a b s t r a c t This work describes the preparation, characterization and evaluation of the nanoparticles formed by the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN) and cyclodextrins, including ␤-cyclodextrin (CD) hydroxypropyl-␤-cyclodextrin (HPCD) and 6-monodeoxy-6-monoamino␤-cyclodextrin (NHCD). The cyclodextrin–poly(anhydride) nanoparticles were prepared by a solvent displacement method and characterized by measuring the size, zeta potential, morphology and composition. For bioadhesion studies, nanoparticles were fluorescently labelled with rhodamine B isothiocianate (RBITC). For in vivo imaging biodistribution studies, 99m Tc-labelled nanoparticles were used. Nanoparticles displayed a size of about 150 nm and a cyclodextrin content which was found optimal under the following experimental conditions: cyclodextrin/poly(anhydride) ratio of 0.25 by weight, 30 min of incubation time between the cyclodextrin and the polymer. Moreover, the oligosaccharide content was higher with CD than with NHCD and HPCD. Overall, cyclodextrin–poly(anhydride) nanoparticles displayed homogeneous bioadhesive interactions within the gut. The intensity of these interactions was higher than for control nanoparticles. The high bioadhesive capacity was observed for HPCD-NP and NHCD-NP which can be related with their rough morphology and, thus, a higher specific surface than for smooth nanoparticles (CD-NP). Finally, from in vivo studies, no evidence of translocation of distribution to other organs was observed when these nanoparticles were orally administered. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The oral route is attractive for drug administration because it is associated with patient convenience and compliance and lower costs. However, a number of drugs remain poorly available when administered by this route. Among other reasons, this important fact can be related to, at least, one of the following effects that can be suffered by many drugs: (i) a low mucosa permeability or a permeability restricted to a specific region within the gut; (ii) a poor solubility in the lumen’s aqueous mediums of the gastrointestinal tract; or (iii) a lack of stability within the gut (Longer et al., 1985; Sakuma et al., 1999; Des Rieux et al., 2006). In order to solve or minimise these drawbacks, a significant number of studies have demonstrated the potential of polymer

∗ Corresponding author. Tel.: +34 948425600; fax: +34 948425649. E-mail address: [email protected] (J.M. Irache). 0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2009.02.010

nanoparticles as delivery systems for the oral administration of poorly absorbed drugs, including insulin (Damge et al., 1988; Babu et al., 2008), cyclosporin-A (El-Shabouri, 2002; Italia et al., 2007), 5-fluorouridine (Arbos et al., 2004) and gemcitabine (Reddy and Couvreur, 2008). All of these findings appear to be supported by the physicochemical and biopharmaceutical properties of these carriers. Among others, their ability to develop bioadhesive interactions within the gut would be one of the key factors influencing their ability to promote the oral absorption of the loaded drug. Thus, using gliadin nanoparticles containing a model molecule, it was demonstrated that it exists a direct relationship between bioadhesion and drug absorption (Arangoa et al., 2000, 2001). In fact, the development of adhesive interactions (between nanoparticles and mucosa) would induce the immobilisation of these drugs delivery devices in intimate contact with the absorptive membrane. This fact would facilitate the establishment of a concentration gradient of the loaded drug from the “reservoir” (nanoparticle) to the circulation and, as a consequence, an increase of it absorption and bioavailability (Ponchel and Irache, 1998; Woodley, 2001).

232

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

In the last years, poly(anhydride) nanoparticles have revealed an exceptional ability to establish bioadhesive interactions within the gut. In addition, the surface of these carriers can be easily modified by simple incubation between the polymer (or the plain nanoparticles) and molecules showing hydroxyl or amino groups. These “decorated” nanoparticles display new abilities which can be of interest for drug delivery purposes. Thus, the coating of poly(anhydride) nanoparticles with bovine serum albumin yielded carriers able to mainly interact with the stomach mucosa (Arbos et al., 2003). When the anticancer agent 5-fluorouridine, substrate of the CYP3A4, was encapsulated in these nanoparticles, the resulting bioavailability was found to be close to 80% (Arbos et al., 2004). In fact, the tropism of these carriers for the stomach was a good strategy to avoid the effect of the CYP3A4 which is mainly localised in the small intestine (Gu et al., 1998; Shen et al., 1997; Mckinnon et al., 1995). On the other hand, the coating of poly(anhydride) nanoparticles with flagellin of Salmonella enteritidis enabled us to design a system (Salman et al., 2005) with a distribution within the gut similar to that described by the whole bacterial cell (Robertson et al., 2003). Similarly, the specific interactions within the gut have been described for poly(anhydride) nanoparticles coated with mannosamine (Salman et al., 2006), thiamine (Salman et al., 2007) or vitamin B12 (Salman et al., 2008). Cyclodextrins are cyclic oligomers of glucose presenting a cagelike supramolecular structure, with lipophilic inner cavities and hydrophilic outer surfaces. They can form water-soluble inclusion complexes with small molecules and portions of large compounds. The inclusion of a guest drug can improve its apparent solubility, physical and chemical stabilities, dissolution rate and bioavailability (Duchêne and Vauthion, 1987; Daoud-Mahammed et al., 2007). Chemically, cyclodextrins consist of at least six d-(+)glucopyranose units attached by ␣-(1, 4)glucosidic bonds. The three natural cyclodextrins, ␣-, ␤-, and ␥-CDs (with 6, 7, or 8 glucose units, respectively), differ in their ring size and solubility (Loftsson and Brewster, 1996). Apart from these naturally occurring cyclodextrins, many derivatives have been synthesised in order to enhance their aqueous solubility, physical and microbiological stability, and/or to reduce the parenteral toxicity of natural cyclodextrins (Szejtli, 1991, 1994; Matsuda and Arima, 1999). ␤-cyclodextrin and its derivatives are the most accessible, the lowest-priced and generally the most useful for pharmaceutical applications because of its ready availability and cavity size suitable for the widest range of drugs (Dass and Jessup, 2000). Furthermore, in the last years, cyclodextrins have also been proposed to increase the drug loading of nanoparticles, liposomes and microparticles (Kihara et al., 2003; Uekama et al., 1998; Duchene et al., 1999a,b). The general objective of this study was to design the combination between poly(anhydride) nanoparticles and cyclodextrins in order to increase the incorporation of lipophilic drugs in these carriers. More particularly, in this work we report the preparation and physico-chemical characterization of these carriers as well as the evaluation of their bioadhesive properties and behaviour within the gut of laboratory animals.

2. Materials and methods 2.1. Chemicals Poly(methyl vinyl ether-co-maleic anhydride), or poly(anhydride) (Gantrez® AN 119; MW 200,000) was kindly gifted by ISP (Barcelona, Spain). Rhodamine B isothiocyanate (RBITC) was supplied by Sigma (Madrid, Spain). ␤-cyclodextrin (CD) was provided by Sigma–Aldrich (Steinem, Germany), 2-hydroxypropyl-

␤-cyclodextrin (HPCD, substitution degree 0.8) by RBI (Massachusetts, USA) and 6-monodeoxy-6-monoamino-␤-cyclodextrin (NHCD, substitution degree 1) was purchased from Cyclolab, (Hungary). 99 Mo generator (Drytec, GE Healthcare Bio-science, England) was eluted following the manufacturer instructions. SnCl2 ·2H2 O and HCl were from Panreac (Barcelona, Spain), 0.9% NaCl from Braun (Barcelona, Spain). Acetone and ethanol were obtained from VWR Prolabo (Fantenay sous Bois, France). All other chemicals used were of analytical grade and obtained from Merck (Darmstadt, Germany). 2.2. Animals Male wistar rats, average weight of 250 g (Harlam, Barcelona, Spain) were housed under normal conditions with free access to food and water. The in vivo studies were performed after approval by the responsible Ethical Committee of the University of Navarra in strict accordance with the European legislation in animal experiments (86/609/EU). 2.3. Preparation of cyclodextrin–poly(anhydride) nanoparticles Cyclodextrin–poly(anhydride) nanoparticles were prepared by a solvent displacement method previously described (Arbos et al., 2002b) with some minor modifications. Briefly, 25 mg of the selected cyclodextrin (CD, HPCD or NHCD) were dispersed in 2 ml of acetone. Then, 100 mg of poly(methyl vinyl ether-co-maleic anhydride) [poly(anhydride); Gantrez® AN 119] were dissolved in 3 ml of acetone and added to the cyclodextrin suspension. The mixture was incubated under magnetic stirring for different times at room temperature. Then, the nanoparticles were obtained by the addition of an ethanol/water mixture (1:1, v/v) and the organic solvents were eliminated by evaporation under reduced pressure (Büchi R-144, Switzerland). All the nanoparticulate batches were purified by centrifugation at 17,000 rpm for 20 min and the supernatants collected to quantify the unbound cyclodextrin. The pellet was then dispersed in 3 ml aqueous solution containing 5% sucrose as cryoprotector. The purification procedure was repeated twice and, finally, the formulations were freeze-dried in a Genesis 12EL apparatus (Virtis, USA). Control nanoparticles were prepared as described above without using cyclodextrins. For in vivo bioadhesion studies, nanoparticles were fluorescently labelled by incubation with 1.25 mg RBITC for 5 min at room temperature prior their purification and lyophilization. 2.4. Characterization of CD-Gantrez nanoparticles formulations 2.4.1. Size, zeta potential, surface morphology and elemental analysis The particle size and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments, UK). For this purpose, samples were diluted with deionized water and measured at 25 ◦ C with a scattering angle of 90◦ . The morphology of the nanoparticles was examined and photographed using a scanning electron microscope (Zeiss DSM 940A SEM; Oberkochen, Germany) with a digital imaging capture system (DISS, Point Electronic GmBh; Halle, Germany). For this purpose, freeze-dried nanoparticles were resuspended in ultrapure water and centrifuged at 27,000 × g for 20 min at 4 ◦ C. Then, supernatants were rejected and the pellets were mounted on a glass plate adhered with a double-sided adhesive tape onto metal stubs and dried under hot flow air. Finally, the particles were coated with a thin layer of 12 nm of gold using an Emitech K550 sputtering device (Emitech, UK).

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

Elemental microanalyses were carried out on vacuum-dried samples using an Elemental Analyzer (LECO, CHN-900 Elemental Analyzer; Wicklow, Ireland). 2.4.2. Cyclodextrin quantification The amount of either CD or HPCD associated to the nanoparticles was determined in the supernatants collected from the purification step of nanoparticles, using a HPLC method (Agilent model 1100 series LC, Waldbronn, Germany) with evaporative light scattering detection (ELSD) (Alltech, IL, USA) previously described (Agueros et al., 2005). For the quantification of the amino derivative of ␤-cyclodextrin, a modification of the method described above was used. Thus, separation was carried out at 40 ◦ C on a reversed-phase NH2-Zorbax (Agilent 4.6 mm × 150 mm, 5 ␮m) obtained from Agilent Technologies (Waldbronn, Germany). ELSD conditions were optimized in order to achieve maximum sensitivity: the drift tube temperature was set at 71 ◦ C, the nitrogen flow was maintained at 1.9 ml/min and the gain was set to 1. The mobile phase composition was a mixture of methanol:water (80/20, v/v) at a flow-rate of 1 ml/min. Each sample was assayed in triplicate and results were expressed as the amount of cyclodextrin per mg nanoparticle. These chromatographic conditions were also used to calculate the poly(anhydride) content in the nanoparticles and the yield of the process was estimated as follows: Yield = (Qinitial − QPVM/MA − QCD ) × 100

(1)

where Qinitial is the initial amount of poly(anhydride) and cyclodextrin added. QPVM/MA and QCD are the amount of the copolymer and cyclodextrin quantified in the supernatants, respectively. 2.4.3. RBITC quantification The amount of the RBITC loaded into the nanoparticles was determined by colorimetry at wavelength 540 nm (Labsystems iEMS Reader MF, Finland). The RBITC loading was estimated as the difference between its initial concentration added and the concentration found after total hydrolysis of 2 mg of nanoparticles in 0.1 N NaOH (24 h, 37 ◦ C). For quantification, standard curves of RBITC in 0.1 N NaOH were used (concentration range of 5–30 ␮g/ml; r > 0.999). 2.5. In vitro release of RBITC from nanoparticles To ensure that the detected fluorescence within the gut mucosa and histological cryo-sections prepared after oral administration of fluorescent particles, was due to RBITC-loaded particles, the release of the fluorescent marker was determined at different times after incubation of 10 mg particles in 1 ml of simulated gastric medium (pH 1.2) at 37 ◦ C for 1 h followed up by 24 h of incubation in 1 ml of simulated intestinal medium (pH 7.4). These media were prepared as described in USP XXIX. At each time interval, 1 ml of the nanoparticle suspensions (10 mg RBITC-NP) were introduced in Vivaspin® tubes with a membrane 10,000 MWCO PES (Vivascience, Sartorius, Hannover, Germany), and centrifuged at 5000 rpm for 15 min. The amount of RBITC released was measured in the supernatants by colorimetry at wavelength 540 nm (Labsystems iEMS Reader MF, Finland). Release profiles were expressed in terms of cumulative release in percentage and plotted versus time. 2.6. Labelling of nanoparticles with 99m Tc Poly(anhydride) nanoparticles were labelled with 99m technetium by reduction with stannous chloride. Briefly, 1 mCi of freshly

233

eluted 99m Tc-pertechnetate was reduced with 0.03 mg/ml stannous chloride and the pH was adjusted to 4 with 0.1 N HCl. Then, 10 mg of freeze-dried nanoparticles (either control nanoparticles or HPCD-nanoparticles) in 1 ml of water and 99m Tc were added to prereduced tin, the mixture vortexed for 30 s and incubated at room temperature for 10 min. The overall procedure was carried out in helium-purged vials using helium-purged solutions to minimise oxygen content and avoid oxidation of pre-reduced tin. The radiochemical purity was examined by a double-solvent instant thin layer chromatography (ITLC) system using silica gel coated fiber sheets (Polygram® sil N-RH, Macherey-Nagel, Düren, Germany) with methyl ethyl ketone (first solvent) and 18% sodium acetate (second solvent) as mobile phases. The labelling yield was always over 90%. 2.7. Gastrointestinal transit studies The gastrointestinal transit studies were carried out using the protocols described previously (Arangoa et al., 2000, 2001; Arbos et al., 2002a). Twelve hours before the experiment, the animals were placed in metabolic cages and drink provided ad libitum. Rats were fed with 1 ml aqueous suspensions of the different formulations, containing 10 mg nanoparticles (around 45 mg particles/kg body weight). The animals were sacrificed by cervical dislocation at 0.5, 1, 3 and 8 h post administration. The abdominal cavity was opened and the gastrointestinal tract removed. Then, the gut was divided into six anatomical regions: stomach, small intestine (I1, I2, I3, I4), and caecum. Each mucosa segment was rinsed with PBS in order to eliminate the non-adhered fraction. Then, each rinsed mucosa segment was digested in 1 ml 3 M NaOH for 24 h. RBITC was extracted with 2 ml methanol, vortexed for 1 min and centrifuged at 5000 rpm for 10 min. Aliquots (1 ml) of the obtained supernatants were diluted with water (3 ml) and assayed for RBITC content by spectrofluorimetry (GENios,TECAN, Austria) at ex 540 nm and em 580 nm, to estimate the fraction of adhered nanoparticles to the mucosa. Standard curves were prepared by addition of RBITC-solutions in 3 M NaOH (0.5–10 ␮g/ml) to control tissue segments following the same steps of extraction (r > 0.996). 2.8. Kinetic curves and parameters of bioadhesion For each nanoparticulate formulation, the total adhered fraction in the whole gastrointestinal tract was plotted versus time. From these curves, the parameters of bioadhesion (Qmax , AUCadh , Tmax , MRTadh and Kel ) were estimated from 0–8 h post-administration as described previously (Arbos et al., 2002a) and calculated using WinNonlin 5.2 software (Pharsight Corporation, Mountain View, USA). Qmax (Damge et al.), or the maximum amount of nanoparticles adhered to the gut surface, is related to the capacity of the material to develop adhesive interactions. AUCadh (mg h) or the area under the curve of the adhered nanoparticles versus time, represents the intensity of the bioadhesion phenomenon. MRTadh (h) or the mean residence time of the adhered fraction of the nanoparticles to the mucosa, is related with the relative duration of the adhesive interactions. Finally, Kel (h−1 ) is the terminal elimination rate of the adhered fraction of nanoparticles in the gastrointestinal mucosa. 2.9. Tissue proceeding The presence of RBITC-loaded poly(anhydride) nanoparticles in the gastrointestinal mucosa was visualised in a microscope (Olympus CH-40, Japan) with fluorescence source (Olympus URFLT50, Japan). Mucosa portions of about 2-cm treated with the tissue-proceeding medium O.C.T.TM (Sakura, Netherlands), were

234

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

immersed in melting isopentane (Fluka, Buch, Switzerland) and frozen in liquid nitrogen. Each sample was cut into 5-␮m sections on a cryostat (2800 Frigocut E, Reichert-Jung, Germany), attached to glass slides and stored at −20 ◦ C before fluorescence microscopy visualization. 2.10. In vivo biodistribution studies with radiolabelled NP Prior to the experiment, animals were placed in metabolic cages and drink provided ad libitum. Radiolabelled nanoparticles (1 mCi, 10 mg) were dispersed in 1 ml water, filtered through a 0.45 ␮m Millipore filter and given by oral gavage to the animals. Animals were anesthetized with 2% isofluorane and placed in prone position on the gammacamera. The Gammagraphic studies were performed in a E.cam Dual-Head Variable-Angle System gammacamera (Siemens Medical Systems, USA) with an intrinsic spatial resolution ≤3.8 mm in the centre of the field of view. For image acquisition the gammacamera was programmed to reach 500.000 events with a static program. The images acquired after the administration of the radiolabelled nanoparticles every 30 min for 8 h.

Fig. 1. Influence of the different cyclodextrins used and the incubation time on the oligosaccharide loading (␮g cyclodextrin/mg nanoparticles). Data express the mean ± SD (n = 8). Experiments were carried out at a cyclodextrin/poly(anhydride) ratio of 0.25 (w/w) for () CD, (䊉) HPCD or () NHCD.

2.11. Statistical analysis

a short time of incubation between the polysaccharide and the polymer, the degree of association (␮g cyclodextrin/mg nanoparticles) increased by increasing the time of incubation between both compounds. Thus, an incubation time of 30 min between both compounds produced nanoparticles with a cyclodextrin content of about 1.2- (for CD), 1.4- (for NHCD) and 3- (for HPCD) times higher than without incubation. However, the prolongation of the incubation time negatively influenced the cyclodextrin loading in nanoparticles. On the basis of these results, nanoparticles were prepared at a cyclodextrin/poly(anhydride) ratio of 0.25 by weight and an incubation time of 30 min. In addition, under these experimental conditions, the overall yield of this preparative process of nanoparticles was calculated by HPLC to be close to 90%.

The bioadhesion data and the physico-chemical characteristics were compared using the nonparametric Mann–Whitney U-test and Student t-test respectively. p Values of <0.05 were considered significant. All calculations were performed using SPSS® statistical software program (SPSS® 10, Microsoft, USA). 3. Results 3.1. Optimization of the preparative process of nanoparticles As a preliminary study, the optimization of the preparative process of cyclodextrin–poly(anhydride) nanoparticles was performed. For this purpose, three main parameters were selected: (i) nature of the oligosaccharide (CD, HPCD and NHCD); (ii) the poly(anhydride)/cyclodextrin ratio; and (iii) the time of incubation between the cyclodextrin and the polymer before the formation of nanoparticles by desolvation. Concerning the cyclodextrin/poly(anhydride) ratio, a ratio of 0.25 by weight allowed us to obtain stable and homogeneous batches of nanoparticles with a high yield of production (close to 90%). On the contrary, a ratio higher than 0.30 by weight, induced the formation of large amounts of aggregates. A ratio lower than 0.20 by weight, significantly decreased the amount of cyclodextrin incorporated in the nanoparticles (data not shown). Fig. 1 shows the influence of the time of incubation between the poly(anhydride) and the oligosaccharide prior the formation of nanoparticles by desolvation on the incorporation of the cyclodextrin in the resulting nanoparticles. In spite of the fact that cyclodextrin–poly(anhydride) nanoparticles can be produced after

3.2. Characterization of cyclodextrin–poly(anhydride) nanoparticles Table 1 summarizes the main physico-chemical properties of these nanoparticle formulations. Overall, cyclodextrin nanoparticles displayed a slightly smaller mean size than for control nanoparticles (180 versus 140 nm). Nevertheless, in all cases, the batches were highly homogeneous with a very low polydispersity (data not shown). Similarly, all the nanoparticle formulations displayed a similar negative surface charge. The morphological analysis by scanning electron microscopy (Fig. 2) showed that poly(anhydride) nanoparticles consisted of a homogeneous population of spherical particles with a size similar to that obtained by photon correlation spectroscopy. Nevertheless, some differences

Table 1 Physico-chemical characteristics of the different cyclodextrin–poly(anhydride) nanoparticles (mean ± SD, n = 12). Experimental conditions: poly(anhydride): 100 mg; cyclodextrin: 25 mg; incubation time: 30 min. NP: control nanoparticles prepared without cyclodextrins. CDNP: ␤-cyclodextrin–poly(anhydride) nanoparticles. HPCD-NP: 2-hydroxypropl-␤-cyclodextrin–poly(anhydride) nanoparticles. NHCD-NP: 6-monodeoxy-6-monoamino-␤-cyclodextrin–poly(anhydride) nanoparticles. Size (nm) NP CD-NP HPCD-NP NHCD-NP a b

179 144 140 151

± ± ± ±

2 6 7 7

Zeta potential (mV)

Yield (%)

−48.1 −51.1 −52.1 −49.3

91.3 94.4 91.1 86.2

± ± ± ±

0.8 8.8 3.7 2.4

Amount of cyclodextrin associated to the nanoparticles (␮g/mg nanoparticles). Rhodamine B isothiocyanate content in ␮g/mg nanoparticles.

± ± ± ±

3.1 5.3 4.1 3.9

Cyclodextrin contenta (␮g/mg) – 88.4 ± 9.9 68.4 ± 4.3 71.2 ± 8.4

RBITC contentb (␮g/mg) 10.9 13.3 12.4 11.8

± ± ± ±

0.3 2.1 1.3 0.7

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

235

Fig. 2. Scanning electron microscopy (SEM) microphotographs from lyophilized nanoparticles. (A) NP. (A.1) Magnification of a section of photograph (A). (B) CD-NP. (B.1) Magnification of a section of photograph (B). (C) HPCD-NP. (C.1) Magnification of a section of photograph (C). (D) NHCD-NP. (D.1) Magnification of a section of photograph (D).

were observed during the analysis of the surface of these carriers. Thus, the surface of NP and CD-NP were found to be smooth whereas HPCD-NP and NHCD-NP displayed a rough surface. On the other hand, the amount of cyclodextrin associated to nanoparticles was found to be dependent on the type of the oligosaccharide used. Thus, under the experimental conditions described here, ␤-cyclodextrin showed an apparent higher ability to associate to poly(anhydride) than HPCD or NHCD. The presence of cyclodextrin in the resulting nanoparticles was also confirmed by elemental analysis (Table 2). The cyclodextrin–poly(anhydride) nanoparticles displayed a higher oxygen content than control nanoparticles (51% versus 42%) associated with a lower carbon content (42% versus 52% for NP). Finally, concerning the incorporation Table 2 Elementary analysis of nanoparticles. NP: control nanoparticles; CDNP: ␤cyclodextrin–poly(anhydride) nanoparticles; HPCD-NP: 2-hydroxypropl-␤cyclodextrin–poly(anhydride) nanoparticles; NHCD-NP: 6-monodeoxy-6monoamino-␤-cyclodextrin–poly(anhydride) nanoparticles.

NP CD-NP HPCD-NP NHCD-NP

C (%)

H (%)

O (%)

52.52 42.37 41.27 43.12

5.09 5.94 5.92 5.78

42.46 51.61 52.84 51.17

of RBITC to nanoparticles, the presence of cyclodextrin did not significantly (p < 0.05) influence the fluorescent marker loading into the resulting nanoparticles (Table 1). 3.3. In vitro release of RBITC from nanoparticles To ensure that the fluorescence intensity determined in the gastrointestinal mucosa was due to the RBITC-associated nanoparticles, in vitro release of RBITC was firstly examined. Fig. 3 shows the release profile of RBITC from nanoparticle formulations after their incubation in both simulated gastric and intestinal fluids. The percentage of RBITC released after 1 h incubation in simulated gastric fluid (SGF) followed by 23 h in simulated intestinal fluid (SIF) was quite similar for all the nanoparticles formulations tested. In all cases, the total amount of RBITC released from nanoparticles in 24 h was found to be lower than 10%. Interestingly, the presence of cyclodextrins in the nanoparticle formulations slightly decreased the release rate of RBITC. 3.4. Gastrointestinal transit studies Fig. 4 shows the distribution of the adhered fractions of nanoparticles, in the different regions of the gut, as a function of time

236

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

Fig. 3. RBITC release profiles from poly(anhydride) nanoparticles and cyclodextrin–poly(anhydride) nanoparticles (CD-NP, HPCD-NP and NHCDNP) after the incubation in simulated gastric fluid (0–1 h) and simulated intestinal fluid (1–24 h) at 37 ± 1 ◦ C. Data represented as mean ± SD (n = 3).

after the oral administration of 10 mg RBITC-labelled nanoparticles. Thirty minutes after the oral administration of a single dose of RBITC-labelled nanoparticles, all the cyclodextrin-nanoparticle formulations displayed a higher ability to adhere to the stomach and the upper regions of the small intestine (I1 and I2 segments) than

conventional ones (NP). This phenomenon was particularly intense for HPCD-NP and NHCD-NP. About 12–20% of the given dose of cyclodextrin nanoparticles was adhered to the stomach and about 14–22% in the small intestine. These values were up to two times higher than the values found for control nanoparticles. In fact, for NP, less than 10% and no more than 12% of the given dose was found adhered to the stomach and small intestine, respectively. On the other hand, 1 h after the administration, the adhered fraction of the cyclodextrin nanoparticles in the gastrointestinal mucosa significantly decreased and appeared to move to the distal part of the gastrointestinal tract. This fact was also observed 3 h and 8 h post-administration. In all cases, all the cyclodextrin–poly(anhydride) formulations displayed a homogeneous and similar distribution within the gut than the control nanoparticles. In addition, none of the formulations tested showed specificity for a particular gut region. Fig. 5 shows, for the different formulations tested, the evolution of the cumulative amount of adhered particles (expressed in mg) on the whole gastrointestinal tract over time. Analysis of these curves of bioadhesion revealed that, for all the formulations tested, the profiles of the curves of bioadhesion were quite similar and characterized by a maximum of adhesion 30 min post-administration, followed by a decrease of the amount of adhered nanoparticles versus time. Table 3 summarizes the parameters of bioadhesion, derived from curves in Fig. 5. For HPCD-NP and NHCD-NP, both the maximum amount (Qmax ) and the total amount (AUCadh ) of adhered

Fig. 4. Distribution of HPCD-NP (A), CD-NP (B), NHCD-NP (C) and NP (D) in the gastrointestinal tract of rats after the oral administration of 10 mg RBITC-loaded nanoparticles. The x-axis represents the different gut segments: stomach (Sto.), intestine portions (I1, I2, I3, I4) and caecum. The y-axis represents the adhered fraction of the nanoparticles in the mucosa, expressed in mg. The z-axis represents the time post-oral administration. Each value was represented by the mean (n = 3; SD was less than 20% of the mean).

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

237

hour, the 99m Tc-HPCD-NP remained in the stomach, while the activity slowly moved to distal parts of the gut at later times. On the other hand, free 99m TcO4 − was visualized in the stomach with no detection in the small intestine. After 3 h, the radioactive tracer was detected in the bladder and no presence was observed in other organs of the animals.

4. Discussion

Fig. 5. Kinetics of bioadhesion of cyclodextrin–poly(anhydride) nanoparticles through the whole gastrointestinal tract versus time: () HPCD-NP, (䊉) CD-NP, () NHCD-NP and () NP. Each value was represented by the mean ± SD (n = 3). Dose: 10 mg nanoparticles or 45 mg nanoparticles/kg.

nanoparticles were found to be significantly higher than for the CD-NP or control nanoparticles (p < 0.05). On the other hand, the elimination rate (Kel ) of the adhered fraction of nanoparticles was significantly lower for the cyclodextrin–poly(anhydride) nanoparticles than for NP (p < 0.01; Table 2). Similarly, the mean residence time of the adhered fraction of cyclodextrin nanoparticles was about 40 min longer than for NP. 3.5. Tissue fluorescence microscopy Fig. 6 shows fluorescence microscopy images of ileum samples, 2 h after the oral administration of 10 mg RBITClabelled nanoparticles to laboratory animals. In all cases, cyclodextrin–poly(anhydride) nanoparticles can be visualised as red fluorescent spots. Control nanoparticles (NP) displayed a low affinity to the normal mucosal tissue (M), and were found mainly in the outer layer (mucus layer) of the ileum (Fig. 6A). On the contrary, cyclodextrin nanoparticles were found broadly and homogeneously distributed along the ileum mucosa (Fig. 6B–D). Particularly, HPCD-NP appeared to show the highest ability to establish bioadhesive interactions with the ileum mucosa. 3.6. In vivo biodistribution studies Fig. 7 shows the comparison of the biodistribution of oral administered 99m Tc-HPCD-NP and free 99m technetium. During the first Table 3 Parameters of bioadhesion for the formulations tested. Data expressed as mean ± SD (n = 3). Qmax (mg) NP CD-NP HPCD-NP NHCD-NP

2.1 2.3 3.5 3.2

± ± ± ±

0.2 0.3 0.5** 0.5**

AUCadh (mg h) 10.49 13.86 18.16 18.04

± ± ± ±

2.10 1.03 4,47* 3.49*

Kel (h−1 ) 0.292 0.077 0.098 0.097

± ± ± ±

MRT (h) 0.03 0.029** 0.084** 0.063**

2.7 3.5 3.4 3.4

± ± ± ±

0.23 0.10* 0.41* 0.28*

Qmax (mg): maximum amount of nanoparticles adhered; AUCadh (mg h): area under the curve of the adhered nanoparticles; Kel (h−1 ): terminal elimination rate of the adhered fraction; MRTadh (h): mean residence time of the adhered fraction of the nanoparticles. * p < 0.05 HPCD-NP, CD-NP and NHCD-NP versus NP. (Mann–Whitney U-test). ** p < 0.01 HPCD-NP, CD-NP and NHCD-NP versus NP. (Mann–Whitney U-test).

In the last years, poly(anhydride) nanoparticles have demonstrated an interesting potential to develop adhesive interactions within the gut mucosa (Arbos et al., 2004; Ochoa et al., 2007; Gomez et al., 2007). However, the drug loading capacity of these nanoparticles, expressed as the amount of drug associated to a unit mass of polymer, is often limited, especially when drugs are very weakly soluble in water. This fact has been also described for other types of polymer nanoparticles, such as poly (alkylcyanoacrylate) (Boudad et al., 2001; Monza Da Silveira et al., 1998) and poly(ethylene oxide)[PEO]-b-poly(lactic acid) nanoparticles (Choi and Kim, 2002; Kim et al., 2001). One possibility to overcome this drawback may be the association between the poly(anhydride) and cyclodextrins. Cyclodextrins and their derivatives are considered biocompatible and, in general, they do not elicit immune responses and have low toxicities (Stella and He, 2008). In addition, they are currently used as solubilizers, stabilizers, and to increase the loading capacity of nanoparticles (Duchene et al., 1999a). This increased loading capacity has been reported to be of two different mechanisms. The former would be as a consequence of the increase of the drug concentration in the medium in which the nanoparticles are formed, due to the addition of the drug:cyclodextrin complex. The latter, would be related with the presence of immobilyzed cyclodextrins into the structure of the nanoparticles. This fact would create numerous lipophilic sites available for the complexation of a lipophilic drug (Monza Da Silveira et al., 1998; Duchene et al., 1999a). Besides, oral administration of ␤-cyclodextrin based systems raises minimal safety concerns since they are poorly absorbed from gastrointestinal tract (Sajeesh and Sharma, 2006). Thus, these cyclodextrin–poly(anhydride) nanoparticles may combine the advantages of the bioadhesive nanoparticles in terms of increasing the residence time at the site of absorption, with the solubilizing properties of these oligosaccharides. Preliminary studies were intended to determine the range of conditions suitable for the preparation of poly(anhydride) nanoparticles associated to different cyclodextrins. Firstly, the oligosaccharide/polymer ratio was optimized in order to obtain the highest encapsulation efficiency. This ratio was found to be 0.25 (w/w). However, the amount of the cyclodextrin associated to the nanoparticles was found to be dependent on the type of oligosaccharide used. It is remarkable that ␤-cyclodextrin was associated in a more effective way to the poly(anhydride) nanoparticles than its hydroxyl or amino derivatives. This fact would be probably due to the relatively lower aqueous solubility of ␤-cyclodextrin (1.85 mg/ml) compared with the hydroxypropyl and amino derivatives (higher than 40 mg/ml). Surprisingly, a maximum of efficiency was observed when the oligosaccharide and the copolymer where incubated in acetone for 30 min (Fig. 1); although, as expected, by increasing this time of incubation the encapsulation efficiencies of the three cyclodextrins decreased. Probably, a long period of incubation between both compounds would modify the aggregative properties of the poly(anhydride) and negatively affect the formation of the nanoparticles during the incorporation of ethanol and water. Under these conditions (30 min of incubation), the cyclodextrin loading in the resulting nanoparticles was calculated to be

238

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

Fig. 6. Fluorescence microscopic visualization of the nanoparticles in the ileum of animals 3-h post-administration. (A) NP; (B) HPCD-NP; (C) CD-NP and (D) NHCD-NP. M: mucosal intestinal villi. E: enterocytes.

around 84 ␮g for CD and 70 ␮g for HPCD and NHCD per mg nanoparticle. Under the experimental conditions, all the cyclodextrin nanoparticles displayed a similar and homogeneous size (see Table 1 and Fig. 2), being slightly smaller for than control nanoparticles (140 versus 180 nm). This decrease of the mean size of poly(anhydride) nanoparticles in the presence of cyclodextrins is in agreement with previous results described by Maestrelli et al. (2006) and Monza Da Silveira et al. (1998) who demonstrated that the presence of HPCD significantly decreased the size of chitosan and poly(isobutylcyanoacrylate) nanoparticles, respectively. In order to evaluate the ability of cyclodextrin–poly(anhydride) nanoparticles to develop bioadhesive interactions within the gut, a quantitative study was performed by measuring the adhered fraction of the nanoparticles in different segments of the gastrointestinal tract. Previously, in vitro RBITC release studies confirmed the stability of the association between the fluorescent marker and the nanoparticles. In addition, as the in vitro release profiles of RBITC were similar for all the formulations, differences observed in their in vivo behaviour can only be ascribed to their different bioadhesive properties. On the other hand, it has been previously described that free RBITC is rapidly removed from the gut. Thus, 3 h post-oral administration of a single dose of RBITC in water, less than 2% of the given fluorescent marker can be found adhered to the gut mucosa (Salman et al., 2005). Overall, all the cyclodextrin–poly(anhydride) nanoparticles displayed a higher ability to develop bioadhesive interactions with the gut mucosa than the control nanoparticles. In addition, in spite of no evidences of a clear ability of these nanoparticles to target any specific regions within the gut were found, these nanoparticles showed a certain potential to concentrate in the stomach mucosa at the first time-point (30 min) post-administration was obtained (see Fig. 4) This homogeneous distribution appears to be quite

similar to that obtained previously with pegylated nanoparticles (Yoncheva et al., 2007) or dextran-coated nanoparticles (unpublished data). Concerning the curves of bioadhesion (Fig. 5), all the nanoparticles formulations displayed a similar profile, characterized by an initial maximum of bioadhesion followed by a reduction of the total adhered amount of nanoparticles versus time. Nevertheless, for control nanoparticles, this reduction was found to be progressive and constant with time whereas for cyclodextrin nanoparticles a plateau of adhesion was obtained between 1 and 3 h post-administration. All of these profiles are different to those obtained for pegylated nanoparticles (Yoncheva et al., 2007) or other ligand-coated nanoparticles (i.e. mannosamine or vitamin B12 derivatives), in which the maximum of bioadhesion was obtained after 1 or 3 h post-administration (Salman et al., 2006, 2007, 2008). In any case, the capacity to develop adhesive interactions (expressed as Qmax ) and the intensity of the bioadhesion phenomenon (expressed as AUCadh ) (see Table 3) were found to be 1.8-times higher for HPCD-NP and NHCD-NP than for NP (p < 0.01 and <0.05, respectively). For CD-NP, these parameters were only slightly higher than for conventional nanoparticles. These results may me related with the morphological differences observed in the surface of nanoparticles (Fig. 2). In fact, HPCD-NP and NHCD-NP displayed a rough surface whereas CD-NP showed a smooth aspect. The rough surface would provide a larger surface area to establish bioadhesive interactions with the gut mucosa and consequently rougher nanoparticles should interact in a larger extent than smooth ones. On the other hand, the higher content of cyclodextrin nanoparticles in hydroxyl groups (as revealed by elemental analysis in Table 2), would increase the possibilities of these carriers to establish hydrogen bonds with components of the gut mucosa and, then, improve their bioadhesive potential. This fact appears to be corroborated by both the significantly lower elimination rate of the adhered fraction (p < 0.01) and the higher

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

239

Fig. 7. Comparison of the biodistribution of 99m Tc HPCD-NP (panels A–C) and a solution of 99m TcO4 − used as control (panels D–F). Panels A–C show gammacamera images after oral administration of 10 mg of 99m Tc-labelled – HPCD NP 75, 210 and 480 min post-injection, respectively (arrow: stomach; arrow head: gut). Panels D–F show gammacamera images after oral administration of 1 mCi of 99m TcO4 − solution (arrow: stomach; arrow head: bladder). A 1 mm diameter tube loaded with a diluted 99m TcO4 − solution was used to outline the contour of the animal to help proper organ localisation.

MRTadh (about 30%) of cyclodextrin nanoparticles compared with conventional carriers (Table 3). In comparison with pegylated nanoparticles or thiamine-coated nanoparticles, cyclodextrin nanoparticles appear to show a hypothetical lower ability to cross the mucus layer (as observed in Fig. 6) and, thus, reach the surface or the enterocytes. In any case, from in vivo imaging biodistribution studies, it appears that the possibility of cyclodextrin–poly(anhydride) nanoparticles to be “translocated” and/or absorbed is negligible (Fig. 7) because no distribution to other organs different that the gut was observed. In addition, from these results it is clear that the association of 99m Tc to poly(anhydride) nanoparticles dramatically modified the absorption and distribution of this radioactive marker. In fact, during the 8 h of the experiment, no absorption of the marker and accumulation in the bladder was observed.

Notwithstanding the possible reduction of the gut motility in anesthetized animals (Torjman et al., 2005; Freye et al., 1998), these in vivo imaging studies put in evidence a similar biodistribution of HPCD-NP in the gut of animals than that obtained from the bioadhesion studies (Fig. 4). In summary, the incorporation of cyclodextrins in Gantrez® AN nanoparticles increase the bioadhesive capacity of poly(anhydride) nanoparticles. This fact may be related with high content of hydroxyl groups in the resulting nanoparticles which can increase the possibilities of these carriers to interact with components of the mucosa by means of hydrogend bonds. In addition, the high bioadhesive capacity for HPCD-NP and NHCD-NP could be related with their rough morphology and, thus, a higher specific surface than for smooth nanoparticles. Finally, from in vivo imaging studies, HPCD-NP remained in the gut with no evidence of

240

M. Agüeros et al. / European Journal of Pharmaceutical Sciences 37 (2009) 231–240

translocation of distribution to other organs of the body of animals. Acknowledgements This research was supported by “Ministerio de Ciencia y Tecnología” (SAF2008-02538), “Gobierno de Navarra”, “Fundación Universitaria de Navarra”, and ISP Corp. Authors were also thankful to Dr. Rafael Jordana (Dep. Zoología y Ecología, University of Navarra) for SEM photographs. References Agueros, M., Campanero, M.A., Lrache, J.M., 2005. Simultaneous quantification of different cyclodextrins and Gantrez by HPLC with evaporative light scattering detection. J. Pharm. Biomed. Anal. 39, 495–502. Arangoa, M.A., Ponchel, G., Orecchioni, A.M., Renedo, M.J., Duchene, D., Irache, J.M., 2000. Bioadhesive potential of gliadin nanoparticulate systems. Eur. J. Pharm. Sci. 11, 333–341. Arangoa, M.A., Campanero, M.A., Renedo, M.J., Ponchel, G., Irache, J.M., 2001. Gliadin nanoparticles as carriers for the oral administration of lipophilic drugs. Relationships between bioadhesion and pharmacokinetics. Pharm. Res. 18, 1521–1527. Arbos, P., Arangoa, M.A., Campanero, M.A., Irache, J.M., 2002a. Quantification of the bioadhesive properties of protein-coated PVM/MA nanoparticles. Int. J. Pharm. 242, 129–136. Arbos, P., Wirth, M., Arangoa, M.A., Gabor, F., Irache, J.M., Gantrez, 2002b. AN as a new polymer for the preparation of ligand-nanoparticle conjugates. J. Control. Release 83, 321–330. Arbos, P., Campanero, M.A., Arangoa, M.A., Renedo, M.J., Irache, J.M., 2003. Influence of the surface characteristics of PVM/MA nanoparticles on their bioadhesive properties. J. Control. Release 89, 19–30. Arbos, P., Campanero, M.A., Arangoa, M.A., Irache, J.M., 2004. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J. Control. Release 96, 55–65. Babu, V.R., Patel, P., Mundargi, R.C., Rangaswamy, V., Aminabhavi, T.M., 2008. Developments in polymeric devices for oral insulin delivery. Expert Opin. Drug Deliv. 5, 403–415. Boudad, H., Legrand, P., Lebas, G., Cheron, M., Duchene, D., Ponchel, G., 2001. Combined hydroxypropyl-beta-cyclodextrin and poly(alkylcyanoacrylate) nanoparticles intended for oral administration of saquinavir. Int. J. Pharm. 218, 113–124. Choi, S.K., Kim, D., 2002. Drug-releasing behavior of MPEG/PLA block copolymer micelles and solid particles controlled by component block length. J. Appl. Polym. Sci. 83, 435–445. Damge, C., Michel, C., Aprahamian, M., Couvreur, P., 1988. New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier. Diabetes 37, 246–251. Daoud-Mahammed, S., Ringard-Lefebvre, C., Razzouq, N., Rosilio, V., Gillet, B., Couvreur, P., Amiel, C., Gref, R., 2007. Spontaneous association of hydrophobized dextran and poly-beta-cyclodextrin into nanoassemblies. Formation and interaction with a hydrophobic drug. J. Colloid Interface Sci. 307, 83–93. Dass, C.R., Jessup, W., 2000. Apolipoprotein A-I, cyclodextrins and liposomes as potential drugs for the reversal of atherosclerosis. A review. J. Pharm. Pharmacol. 52, 731–761. Des Rieux, A., Fievez, V., Garinot, M., Schneider, Y.J., Preat, V., 2006. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J. Control. Release 116, 1–27. Duchêne, D., Vauthion, F.G.C., 1987. Cyclodextrins. Their industrial uses. Editions de Santé, 211. Duchene, D., Ponchel, G., Wouessidjewe, D., 1999a. Cyclodextrins in targeting. Application to nanoparticles. Adv. Drug Deliv. Rev. 36, 29–40. Duchene, D., Wouessidjewe, D., Ponchel, G., 1999b. Cyclodextrins and carrier systems. J. Control. Release 62, 263–268. El-Shabouri, M.H., 2002. Positively charged nanoparticles for improving the oral bioavailability of cyclosporin-A. Int. J. Pharm. 249, 101–108. Freye, E., Sundermann, S., Wilder-Smith, O.H., 1998. No inhibition of gastro-intestinal propulsion after propofol- or propofol/ketamine-N2 O/O2 anaesthesia. A comparison of gastro-caecal transit after isoflurane anaesthesia. Acta Anaesthesiol. Scand. 42, 664–669.

Gomez, S., Gamazo, C., Roman, B.S., Ferrer, M., Sanz, M.L., Irache, J.M., 2007. Gantrez AN nanoparticles as an adjuvant for oral immunotherapy with allergens. Vaccine 25, 5263–5271. Gu, J., Yuasa, H., Hayashi, Y., Watanabe, J., 1998. First-pass metabolism of 5fluorouracil in the perfused rat small intestine. Biol. Pharm. Bull. 21, 871–873. Italia, J.L., Bhatt, D.K., Bhardwaj, V., Tikoo, K., Kumar, M.N., 2007. PLGA nanoparticles for oral delivery of cyclosporine: nephrotoxicity and pharmacokinetic studies in comparison to Sandimmune Neoral. J. Control. Release 119, 197–206. Kihara, F., Arima, H., Tsutsumi, T., Hirayama, F., Uekama, K., 2003. In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer. Bioconjug. Chem. 14, 342–350. Kim, S.Y., Kim, J.H., Kim, D., An, J.H., Lee, D.S., Kim, S.C., 2001. Drug-releasing kinetics of MPEG/PLLA block copolymer micelles with different PLLA block lengths. J. Appl. Polym. Sci. 83, 2599–2605. Loftsson, T., Brewster, M.E., 1996. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J. Pharm. Sci. 85, 1017–1025. Longer, M.A., Ch’ng, H.S., Robinson, J.R., 1985. Bioadhesive polymers as platforms for oral controlled drug delivery III: oral delivery of chlorothiazide using a bioadhesive polymer. J. Pharm. Sci. 74, 406–411. Maestrelli, F., Garcia-Fuentes, M., Mura, P., Alonso, M.J., 2006. A new drug nanocarrier consisting of chitosan and hydoxypropylcyclodextrin. Eur. J. Pharm. Biopharm. 63, 79–86. Matsuda, H., Arima, H., 1999. Cyclodextrins in transdermal and rectal delivery. Adv. Drug Deliv. Rev. 36, 81–99. Mckinnon, R.A., Burgess, W.M., Hall, P.M., Roberts-Thomson, S.J., Gonzalez, F.J., Mcmanus, M.E., 1995. Characterisation of CYP3A gene subfamily expression in human gastrointestinal tissues. Gut 36, 259–267. Monza Da Silveira, A., Ponchel, G., Puisieux, F., Duchene, D., 1998. Combined poly(isobutylcyanoacrylate) and cyclodextrins nanoparticles for enhancing the encapsulation of lipophilic drugs. Pharm. Res. 15, 1051–1055. Ochoa, J., Irache, J.M., Tamayo, I., Walz, A., Delvecchio, V.G., Gamazo, C., 2007. Protective immunity of biodegradable nanoparticle-based vaccine against an experimental challenge with Salmonella enteritidis in mice. Vaccine 25, 4410–4419. Ponchel, G., Irache, J., 1998. Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract. Adv. Drug Deliv. Rev. 34, 191–219. Reddy, L.H., Couvreur, P., 2008. Novel approaches to deliver gemcitabine to cancers. Curr. Pharm. Des. 14, 1124–1137. Robertson, J.M., Mckenzie, N.H., Duncan, M., Allen-Vercoe, E., Woodward, M.J., Flint, H.J., Grant, G., 2003. Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. J. Med. Microbiol. 52, 91–99. Sajeesh, S., Sharma, C.P., 2006. Cyclodextrin-insulin complex encapsulated polymethacrylic acid based nanoparticles for oral insulin delivery. Int. J. Pharm. 325, 147–154. Sakuma, S., Sudo, R., Suzuki, N., Kikuchi, H., Akashi, M., Hayashi, M., 1999. Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int. J. Pharm. 177, 161–172. Salman, H.H., Gamazo, C., Campanero, M.A., Irache, J.M., 2005. Salmonella-like bioadhesive nanoparticles. J. Control. Release 106, 1–13. Salman, H.H., Gamazo, C., Campanero, M.A., Irache, J.M., 2006. Bioadhesive mannosylated nanoparticles for oral drug delivery. J. Nanosci. Nanotechnol. 6, 3203–3209. Salman, H.H., Gamazo, C., Agueros, M., Irache, J.M., 2007. Bioadhesive capacity and immunoadjuvant properties of thiamine-coated nanoparticles. Vaccine 25, 8123–8132. Salman, H.H., Gamazo, C., De Smidt, P.C., Russell-Jones, G., Irache, J.M., 2008. Evaluation of bioadhesive capacity and immunoadjuvant properties of vitamin b(12)-Gantrez nanoparticles. Pharm. Res. 25, 2859–2868. Shen, D.D., Kunze, K.L., Thummel, K.E., 1997. Enzyme-catalyzed processes of firstpass hepatic and intestinal drug extraction. Adv. Drug Deliv. Rev. 27, 99–127. Stella, V.J., He, Q., 2008. Cyclodextrins. Toxicol. Pathol. 36, 30–42. Szejtli, J., 1991. Cylodextrin in drug formulations: Part I. Pharm. Technol. Int. 3, 15–23. Szejtli, J., 1994. Medicinal applications of cyclodextrins. Med. Res. Rev. 14, 353–386. Torjman, M.C., Joseph, J.I., Munsick, C., Morishita, M., Grunwald, Z., 2005. Effects of isoflurane on gastrointestinal motility after brief exposure in rats. Int. J. Pharm. 294, 65–71. Uekama, K., Hirayama, F., Irie, T., 1998. Cyclodextrin drug carrier systems. Chem. Rev. 98, 2045–2076. Woodley, J., 2001. Bioadhesion: new possibilities for drug administration? Clin. Pharmacokinet. 40, 77–84. Yoncheva, K., Guembe, L., Campanero, M.A., Irache, J.M., 2007. Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles. Int. J. Pharm. 334, 156–165.