Development and evaluation of mucoadhesive nanoparticles based ...

J Nanopart Res (2015) 17:98 DOI 10.1007/s11051-015-2909-5

RESEARCH PAPER

Development and evaluation of mucoadhesive nanoparticles based on thiolated Eudragit for oral delivery of protein drugs Yan Zhang • Zhijie Yang • Xi Hu • Ling Zhang Feng Li • Meimei Li • Xing Tang • Wei Xiao

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Received: 4 December 2014 / Accepted: 4 February 2015 / Published online: 20 February 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract The objective of this study was to develop pH-sensitive Eudragit L100–cysteine/reduced glutathione (Eul–cys/GSH) nanoparticles (NPs), which provided the mucoadhesion and protection for protein drugs against enzymatic degradation. Insulin was chosen as a model biomolecule for testing this system. The Eul–cys conjugate, which was obtained by grafting cysteine onto the carboxy group of Eudragit L100, was analyzed by HNMR and SEM, and the swelling degree (SD), cation binding, and enzymatic inhibition were also determined. The results obtained showed that the Eul–cys conjugate represent a pHsensitive delivery system which effectively protected the insulin from being degraded by the proteases, and this is related to the mechanism of Ca2? binding. Insulin-loaded Eul–cys/GSH NPs were prepared by a diffusion method involving an electrostatic interaction between the network structure of the polymer and the embedded proteins, including insulin and GSH. TEM images indicated that Eul–cys/GSH existed as smooth Y. Zhang F. Li M. Li Normal College, Shenyang University, Shenyang 110044, China Y. Zhang Z. Yang X. Hu L. Zhang X. Tang Department of Pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China Y. Zhang W. Xiao (&) Jiangsu Kanion Pharmaceutical Co., Ltd, No.58, Yuankang Road, Lianyungang 222047, China e-mail: [email protected]

and spherical NPs in aqueous solution with particle sizes of 260 ± 20 nm. The X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) findings showed the presence of amorphous insulin in thiolated NPs and higher free thiol oxidation than the result obtained by Ellman’s reagent method. In addition, thiolated NPs showed excellent binding efficiency to the mucin in rat intestine, indicating that Eul–cys/GSH NPs have great potential to be applied as safe carriers for the oral administration of protein drugs. Keywords Eudragit L100-cysteine Nanoparticles pH sensitive Mucoadhesive Enzymatic inhibition Nanomedicine

Introduction Protein and peptide drugs are not usually given by oral administration because they are rapidly degraded when they come into contact with gastrointestinal (GI) fluids and they are also poorly absorbed through the GI epithelium. Therefore, the development of oral delivery system for protein drugs presents a major scientific challenge (Bakhru et al. 2013; Ponchel and Irache 1998; Torchilin 2008). Numerous studies have been carried out in attempts to deliver proteins and polypeptides by the oral route. At present, the most popular method is to encapsulate them in colloidal nanocarriers that can offer protein protection against

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enzymatic degradation and control the drug release in a satisfactory manner (Su et al. 2012; Ganta et al. 2008; Yamagata et al. 2006). Polymeric nanoparticles (NPs) prepared from natural and synthetic polymers have received great attention due to their stability and ease of surface modification (Vauthier et al. 2003; Herrero et al. 2005; Pawar et al. 2012). Several natural polymers, like chitosan, alginate, and dextran sulfate, have been used in the synthesis of polyectrolytic complexes, which are promising carriers for biomacromolecules (Felber et al. 2012; Wee and Gombotz 1998; Tiyaboonchai et al. 2003). This self-assembly technique of NP synthesis has some advantages by lowering the risk of damage to the entrapped drug molecule as it involves no organic reagent or any harsh conditions in the NP preparation method (Mao et al. 2006). In addition, this process leads to the formation of polyelectrolyte complexes by electrostatic interaction, which are optically homogeneous, stable nano-dispersions at a colloidal level (Mukhopadhyay et al. 2013). NPs have been proven to be potentially useful systems allowing drugs to target the site of action directly or get very close to it (Ponchel and Irache 1998; Kim and Kang 2008; Bernkop 2005; Jin et al. 2012). They can be used to provide targeted delivery of drugs, to maintain the drug in the target tissue, to reversibly increase the permeability of the mucosal epithelium to enhance the absorption of drug, to improve the stability of therapeutic agents against enzymatic degradation, and to improve oral bioavailability. In addition, this delivery system is also safe after oral administration. In this study, a polymeric vehicle with pH-sensitivity, mucoadhesion, and penetration-enhancing capacity for improved oral absorption of insulin was investigated. Thiolated Eudragit, a potential carrier for the oral delivery of insulin, was also evaluated and investigated based on our previous research findings. Initially, the synthesis of the Eul–cys conjugate was carried out by grafting the cysteine onto the backbone of Eudragit L100 mediated by EDC. Characterization techniques, including nuclear magnetic resonance spectroscopy (HNMR) and scanning electron microscopy (SEM), were performed to investigate the polymeric structure and morphology, and swelling behavior, cation binding, and enzymatic-inhibition ability were studied to examine the protein protection offered. Insulin-loaded Eul–cys/GSH NPs were

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prepared by diffusion using an electrostatic interaction between the network structure of the polymer and the embedded proteins. Transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were used to study the particle size of the manufactured NPs and insulin distribution. Also, a rat intestinal mucoadhesion experiment was conducted to confirm that Eul–cys/GSH NPs could be potential candidates for the oral delivery of insulin.

Materials and methods Materials Porcine insulin (27.8 IU/mg) was purchased from Xuzhou Wanbang Biochemical Pharmaceutical Co., Ltd., China. EudragitÒ L-100 was obtained from Ionic Degussa (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), trypsin and a-chymotrypsin (from bovine pancreas) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Reduced Glutathione (GSH) was purchased from the National Chemical Reagent Co., Ltd, China. Fluorescein isothiocyanate (FITC) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were purchased from Sigma, St. Louis, MO, USA. All other reagents were of analytic grade. The animal experiments in this work complied with the regulations of the Committee on Ethics for the Care and Use of Laboratory Animals at Shenyang Pharmaceutical University. Synthesis of Eul–cys conjugates The covalent attachment of cysteine to Eudragit L100 was achieved by the formation of amide bonds between the primary amino groups of cysteine and the carboxylic acid groups of the polymer mediated by EDC as described by our research group previously (Zhang et al. 2012). In this study, the amounts of Eudragit L100 and EDC were fixed at 0.5 g and 50 mM, respectively, and three different amounts of cysteine (2, 0.5, and 0.125 g) were used, which led to the formation of conjugates with high, medium, and low contents of sulfhydryl groups, named Formations 1, 2, and 3 (F1, F2, and F3, respectively).


Characterization of conjugates The Eul–cys and Eul were dissolved in DMSO4-d6, and HNMR spectra were obtained on a Bruker ACF300 (300 MHz) Fourier Transform Spectrometer (Bruker, Germany). A Hitachi (Japan) S-3400 N SEM instrument was used to examine the morphology of the particles. The degree of free thiol groups immobilized on the polymer was quantified by Ellman’s method (Bernkop and Freudl 1999). Swelling characteristics The swelling behavior of the conjugate was determined by a gravimetric method as described by Thierry Schmitz (Schmitz et al. 2008). Thirty milligrams of each of the lyophilized Eul–cys powders (F1, F2, and F3) were compressed into 7.0-mmdiameter flat-faced tablets. The compaction force of 4 kN was kept constant during the preparation of all tablets. The tablets were then immersed in a series of solutions of different pH values for 2 h. The pH was adjusted by the addition of HCl or NaOH, and the ionic strength was adjusted to 0.5 M by the addition of sodium chloride. The swelling ratio of the polymeric tablet was calculated according to the following equation: Swelling ratio ¼

Wt W0 100 % W0

where Wt and W0 are the weights of the swollen and the dried tablets, respectively. All experiments were carried out in triplicate, and the average values were reported. The influence of the ionic strength was also examined. The dried conjugate tablets were immersed in NaCl solution (pH 7.0) at concentrations of 0.01, 0.1, 0.5, and 1 NaCl, respectively. After 2-h immersion, tablets were taken out, and water was wiped off with filter paper. Then, the tablets were weighed, and the swelling ratio was calculated as above. Cation-binding studies The efficiency of cation binding was evaluated by a calcium-binding assay using parent conjugates according to a modified EDTA titration method (Bernkop and Krajicek 1998). In brief, 30 mg Eudragit

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L100 and Eul–cys conjugates (F1, F2, and F3) were added to 10 mL 20 mM CaCl2 and incubated for 30 min. The assay system was centrifuged at 10,000 rpm for 10 min to remove the polymer, and the supernatant was withdrawn and mixed with the same volume of ammonia–ammonium chloride buffer solution at pH 12.0. Trace amounts of calconcarboxylic acid were added, and the system was shaken before titration until the samples became red in color. Subsequently, 0.01 M EDTA was added until a color change from red to blue was observed, and this point was taken as the end of the titration. The volume of titrant used was recorded, and the amount of calciumbound polymer was calculated from the formula given below. The blank titration was carried out with DI water, and the experiments were performed at room temperature in triplicate. Calcium ion ðmg=gÞ ¼

½C1 V1 CEDTA ðV2 V0 Þ 40:08 mg

where C1: concentration of calcium ion standard solutions, M; V1: volume of supernatant, mL; CEDTA: concentration of EDTA titrant, M; V2, V0: titrant volume consumed by supernatant and blank solvent, respectively, mL; 40.08 is the molar mass of calcium ions; and mg: weight of polymer, g. Enzymatic inhibition The protective effect of Eul–cys NPs against insulin degradation was examined using a modified method described by Yin (Yin et al. 2010). Trypsin and achymotrypsin were added to pH 7.4 PBS to obtain concentrations of 6, 12, and 24 lg/mL for trypsin; and 8, 16, and 32 lg/mL for a-chymotrypsin, respectively. Then, 40 mg of blank Eul–cys NPs was added to the enzyme fluid followed by incubation at 37 °C for 2 h. Following this, 10 mL enzyme solution containing NPs was blended with the same volume of insulin solution (100 lg/mL) at 37 °C and 100 rpm. The sample solutions in the absence of NPs and enzyme were the positive and negative controls, respectively. At predetermined times, 100 lL aliquots were withdrawn and diluted with 150 lL TFA solution (pH 1.8) to stop the enzyme reactions. Insulin degradation was monitored by determining the residual amount of insulin using HPLC.

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Preparation and characterization of insulin-loaded Eul–cys/GSH NPs The NPs were prepared by diffusion partitioning by mixing the insulin and GSH solutions with the Eul–cys aqueous dispersion under mild stirring and adjusting the pH to the value where NP formation occurred according to a method described previously (Zhang et al. 2014). It is based on the electrostatic interactions between positively charged proteins (insulin and GSH) and the negatively charged polyanionic chain of the polymer. In brief, insulin predissolved in 0.01 M hydrochloric acid at a final concentration of 0.1 % (w/v) was added dropwise to the Eul–cys aqueous dispersion (pH 10.0, adjusted with 1 M NaOH) under gentle magnetic stirring, and then GSH (0.5 %, w/v) was added followed by thorough mixing. Subsequently, the pH of the mixture was gradually adjusted to pH 4.8 with 0.02 M hydrochloric acid, and an opalescent suspension was obtained and incubated for a further 15 min at room temperature under magnetic stirring (100 rpm). The Eul–cys/GSH NPs were washed, collected by centrifugation, and lyophilized. The Eul–cys NPs were prepared by the same process but omitting GSH. The particle size, zeta potential, and polydispersity index (PI) of the NPs were measured by DLS and electrophoretic light scattering using a Nicomp 380ZLS instrument (NICOMP Particle Sizing Systems, Santa Barbara, CA, U.S.A.). X-ray diffraction studies (XRD) XRD was used to investigate the crystalline (or amorphous) phases for NPs prepared using a series of different parameters. The patterns were obtained on a Magix Pro PW2440 X-ray diffractometer, with a tube of CuKa, a voltage of 40 kW, and a current of 60 mA. The samples including insulin, Eul–cys/GSH NPs, and the mixture of insulin and the blank NPs were scanned between 2h of 5°–50° at a scanning speed of 5°/min. X-ray photoelectron spectroscopy (XPS) XPS (ESCALAB 250, Thermo VG, U.S.A.) was used to examine the surface chemistry of the blank Eul–cys/ GSH and insulin-loaded NPs. The spectra were collected at analyzer pass energy of 50 eV. The spectrometer had a

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monochromatic Al KR X-ray source operating at 1486.6 eV, 10 mA (150 W) for all data acquisitions. Data were analyzed using XPSPEAK41 software. Intestinal mucoadhesion Sprague–Dawley rats (250 g) were provided with a laboratory diet ad libitum and fasted 20 h before the experiments. Animals were given a general anesthesia with an intra-peritoneal injection of sodium pentobarbital. Intestinal segments (duodenum, jejunum, and ileum) were obtained by tying both ends of the segment, into which 1 mL of FITC-insulin-loaded Eul–cys/GSH NPs (10 mg/mL) was injected, and FITC-insulin solution served as a control. After 1 h, the animals were sacrificed, samples of the intestine (duodenum, jejunum, and ileum with Peyer’s patches) were removed, and fixed in 4 % (v/v) formalin in PBS. Tissues were dehydrated using a series of ethanol solutions, embedded in paraffin, and sections of 5 lm were cut using a microtome (Frigocut Mod. 2700, Reichert Jung, Germany). The sections were stained with DAPI (1 lg/mL), and viewed under laser confocal microscopy (CLSM).

Results and discussion Characterization of Eul–cys conjugate The Eul–cys conjugate was obtained by the covalent attachment of cysteine to the carboxyl groups of Eudragit L100 via amide bonds. The chemical structures of Eudragit L100 and Eul–cys conjugate were confirmed using HNMR, as shown in Fig. 1. The characteristic peaks of Eudragit L100 appeared at 3.4 and 0.8–1.5 ppm, which were assigned to the methyl protons on the methyl ester (-OCH3) and the backbone of the polymer (-C(CH3)-). Successful introduction of cysteine onto Eudragit L100 was confirmed by the peaks of the amido bond, which appeared at 8.1 ppm. In addition, some new peaks were observed around 3.0–4.0 ppm in the spectrum of the Eul–cys conjugate presumably caused by the signals of methylene and methyne protons in cysteine, as a result of the reaction of cysteine with the carboxyl groups of Eudragit L100. The SEM images of the Eul–cys conjugate are shown in Fig. 2. In general, they had a size distribution of 180 ± 20 nm, which were uniform and spherical in


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shape. The content of sulfydryl groups in each Eul–cys conjugate is also shown in Table 1. The lyophilized polymer was a white, odorless powder with free thiol contents of 368, 215, and 113 lmol/g for F1, F2, and F3, respectively. It can be concluded that more cysteine (within 2 g) leads to a higher sulfydryl group content in the conjugate. Swelling behavior

Fig. 1 HNMR spectra of a Eudragit L100 and b Eul–cys

Fig. 2 SEM image of lyophilized Eul–cys conjugate

Table 1 Amount of thiol groups immobilized on the basic thiomer Eul–cys conjugates and evaluation of their calciumbinding ability in ammonia–ammonium chloride buffer (pH = 12) at 25 °C (mean ± SD, n = 3) Polymer

Thiol content (lmol/g polymer)

Bound Ca2? (mg/g polymer)

Eudragit L100

–

62.0 ± 1.5

Eul–cys (F1) Eul–cys (F2)

368 215

67.0 ± 2.2 72.3 ± 2.8

Eul–cys (F3)

113

69.5 ± 1.3

The swelling behavior of the polymer has a significant impact on bioadhesion. During the process of rapid swelling, diffusion between the polymer and mucus was completed, and a closer contact was achieved. Figure 3 shows the swelling behavior of Eul–cys conjugates in PBS with different pH values and an ion strength of 0.5 M at 37 °C. As shown in Fig. 3a, the particles exhibited lower swelling ratios in the acidic medium due to the hydrogen bond interactions between the protonated carboxylic acid groups of the hydrogel networks. Only a small amount of water was absorbed by the polymeric tablets when the pH value was below 4. There is an obvious difference between pH 5 and 8 where the variation in pH produces a clear swelling of the tablets. An increase in sulfydryl groups led to a high water absorption at the same pH value. This may be due to the deprotonated carboxylic acid groups, which increase the electrostatic repulsion and charge density of the polymer, leading to chain expansion and an increase in tablet size. The sulfydryl groups are oxidized to disulfide bonds above pH 5.0, and the matrix is strengthened by forming crosslinks from the disulfide bond, resulting in a decrease in corrosion and markedly higher swelling ratios. In order to examine the effect of ionic strength on the swelling of the conjugates, we immersed the tablets in 0.01, 0.1, 0.5, and 1 M NaCl solutions and measured their weights after 2 h. From Fig. 3b, we can see that, at higher salt concentrations, the weight of the tablets exhibits a smaller swelling effect, as a consequence of the screening of the electrostatic repulsions between the Eul–cys chains. This difference in swelling behavior can be explained by the fact that the decreased coulombic repulsions of the adjacent molecular chains result in a decrease in osmotic pressure inside the polymeric tablets, and cause a decrease in the swelling ratios. In addition, anions like Cl- cause polymer deswelling because they strongly compete for the water molecules hydrating the

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dependent with regard to the thiol amount. A comparable Ca2?-binding capacity was observed with Eul– cys F1, F2, and F3. It is known that carboxylic acid groups on polyacrylate structures provide the main adsorption and chelating sites for calcium ions. Although consumption of carboxylic groups by sulfhydrylation modification reduces the availability of these chelating sites, the presence of thiol groups are able to improve the chelation of metal cations, and the binding behavior observed was higher than that of the nonmodified polymer (Bravo et al. 2007). Enzymatic inhibition

Fig. 3 a pH-sensitive swelling ratios of Eul–cys tablets (F1, F2, and F3) with PBS at different pH values (mean ± SD, n = 3), b The swelling ratio of Eul–cys tablets (F1, F2, and F3) in NaCl solutions (pH = 7.0) with various ion strength (mean ± SD, n = 3)

polymer, and the matrix becomes hydrophobic. Thus, the swelling ratio decreased with an increase in the NaCl concentration. Cation-binding studies Eudragit L100 and its derivatives modified with a sulfydryl group with different thiol contents were investigated with regard to their binding capacity for calcium ions, in order to elucidate the mechanism of their potential to protect peptides from enzyme degradation and possible Ca2?-dependent protease inhibition. The effects of Eudragit L100 and Eul–cys (F1, F2, and F3) on the cation-binding capacity were compared, and the results are listed in Table 1. As shown, both Eudragit and its derivatives had calcium-binding ability, and a stronger effect was observed with the Eul–cys polymers, indicating that the presence of thiol groups improved the chelation of metal cations. These results agreed well with those published by BernkopSchnu¨rch (Bernkop et al. 2001). However, the increment in the chelating effect was not concentration

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The progress of insulin degradation as a function of time by trypsin and a-chymotrypsin is shown in Fig. 4. As can be seen, the unprotected insulin was degraded by both enzymes significantly, and only 7–20 % of the insulin remained after 2 h. In comparison, a significant increase in the undegraded insulin was observed in the presence of Eul–cys. The insulin was incubated in Eul– cys suspension including a series of concentrations of trypsin (6, 12, and 24 lg/mL) for 2 h, and the contents were reduced to 89.1 ± 3.7, 76.9 ± 4.8, and 60.8 ± 5.3 % of the initial values, respectively. Under similar conditions with a-chymotrypsin (8, 16, and 32 lg/mL), the insulin contents were reduced to 80.0 ± 4.6, 63.7 ± 4.7, and 38.8 ± 5.5 % of the initial values, respectively. The degradation profile revealed that the degradation rate of insulin was proportional to the concentration of enzyme. However, inhibition of insulin degradation at the highest concentration of enzyme could still be observed compared with positive controls, indicating that Eul–cys produced significant protease inhibition and protected insulin from degradation. Protease has an important effect on the stability and absorption of insulin. Similar to the Carbopol and polycarbophil (Luessen et al. 1995; Luessen et al. 1996), Eudragit L100 and its derivatives display their potential for inhibiting proteases from the GI tract and protecting peptides from proteolytic degradation. One possible mechanism is enzyme incorporation by polymer and Ca2? deprivation for enzyme inactivation. At a physiological pH, the polymer produces significant swelling, and opening of the network structure entraps the enzyme in the NPs, leading to the inactivation of the proteases by physical or chemical interaction. Moreover, the calcium-binding capacity is related directly to a significant ability to


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inhibit proteases like trypsin and a-chymotrypsin. Calcium ions play an important role in maintaining the structure and function of protease, and polymers possessing abundant carboxylic groups provide chelating sites for it and, thus, inhibit proteolytic activity. Furthermore, sulfhydrylation can promote the binding capacity of calcium ions for the polymer and, consequently, improve enzymatic inhibition. Preparation and characterization of NPs

Fig. 4 a Trypsin inhibition by Eul–cys at enzyme concentration of 6 lg/mL (filled circle), 12 lg/mL (filled diamond), 24 lg/mL (filled triangle), positive control (insulin solution) at trypsin concentration of 6 lg/mL (open circle), 12 lg/mL (open diamond), 24 lg/mL (open triangle) and negative control (filled square) (insulin solution in the absence of enzyme). b achymotrypsin inhibition by Eul–cys at enzyme concentration of 8 lg/mL (filled circle), 16 lg/mL (filled diamond), 32 lg/mL (filled triangle), positive control (insulin solution) at achymotrypsin concentration of 8 lg/mL(open circle), 16 lg/ mL (open diamond), 32 lg/mL (open triangle) and negative control (filled square) (insulin solution in the absence of enzyme). Each value represented mean ± SD (n = 3)

Insulin is a protein drug which has an isoelectric point of 5.3, and GSH is also a tripeptide with a isoelectric point of 5.9. When a pH of 4.8 was chosen for the preparation, both proteins have a positive charge, while Eul–cys is negatively charged due to the ionization of carboxyl. Insulin-loaded Eul–cys/GSH NPs were formed based on the electrostatic interaction between the network structure of the polymer and the proteins including insulin and GSH, as illustrated in Fig. 5. DLS measurement revealed that the particle sizes of Eul–cys/GSH NPs were 260 ± 20 nm, zeta potential of -3.1 ± 0.9 mV, and with a PI of 0.215, measured in 2 % trehalose at pH 4.8. The TEM micrographs of the NPs showed them to be distinct, uniform, and spherical in shape. Currently, there is some disagreement about the influence of the surface charge of particles on the particle uptake in the intestine. It is generally accepted that particles with a positive charge are favorable for mucoadhesion and, consequently, offer better drug absorption, due to the presence of opposite electric charges in the intestinal mucosa. However, other authors argue that a strong interaction between

Fig. 5 Putative schematic structure of Eul–cys/GSH nanoparticles and TEM image

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Fig. 6 X-ray diffraction patterns: a insulin; b Eul–cys NPs; c Eul–cys/GSH NPs; d Eul–cys ? insulin physical mixture; e Eul–cys/GSH ? insulin physical mixture

positively charged NPs and mucin can lead to decreases in their absorption and uptake (Andreani et al. 2014). Some studies have reported that the use of anionic polymers leads to better mucosal adhesion than cationic polymers or nonionic polymers (Chickering and Mathiowitz et al. 1995). It is believed that the presence of numerous surface carboxyl groups on anionic polymers generates strong biodhesive interactions by hydrogen bonds with the mucin, thereby, improving the protein absorption. Interaction between insulin and Eul–cys nanoparticles XRD was used to investigate the crystalline (or amorphous) phases of insulin in polymeric NPs prepared using different parameters. The XRD spectra were recorded for pure insulin, empty NPs, and drug-loaded NPs as shown in Fig. 6. Insulin exhibits a partial sharp crystalline peak at 2h \ 10°, which is the characteristic of a macromolecule with some crystallinity (Fig. 6a). For the mixture of NPs and insulin, the presence of typical peaks for both insulin and NPs at 2h \ 10° and 20° is shown in Fig. 6e, f while the intensities of these peaks are slightly reduced. For Eul–cys and Eul–cys/GSH NPs (Fig. 6b, c), only a broad peak centered at 20° is observed, indicating a degree of interaction between insulin and NPs. It is also suggested that insulin was loaded into the Eul–cys or Eul–cys/GSH NPs, and insulin in the NPs was present in an amorphous or molecular dispersed form.

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Fig. 7 XPS spectra of blank Eul–cys/GSH NPs (a); insulinloaded Eul–cys/GSH (b); and S2p narrow scans with the curve fit for Eul–cys/GSH NPs, (c)

Surface chemistry XPS can quantitatively determine the chemical composition of the particle surface at a depth of 5-10 nm. The atomic compositions of the blank Eul–cys/GSH NPs and the insulin-loaded Eul–cys/GSH NPs were determined by XPS, and the spectra are shown in Fig. 7. Compared with the XPS spectrum in Fig. 7a, the nitrogen (from 1.22 to 1.46 %) and sulfur (from 0.68 to


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Fig. 8 Histologic analyses of different parts of the small intestine: duodenum, jejunum, and ileum [Peyer’s patches (PP)] exposed to Eul–cys/GSH NPs in rats after 60 min. Tissue

sections were stained with DAPI (nuclei, blue), and insulin was stained with FITC. Green dots refer to the insulin-loaded Eul– cys/GSH NPs

0.90 %) contents increased slightly in Fig. 7b, which indicated that the insulin was mainly distributed inside the NPs. Only a small amount of drug was present on the surface of the NPs due to the electrostatic attraction between insulin and the NPs. To further estimate the amount of thiol groups on the NP surface, the S 2p peaks were decomposed into subcomponents using a Gaussian–Lorentzian curve-fitting program with a nonlinear background. Four signals of S 2p or S 2p3 at binding energies of 162.4, 163.2, 163.8, and 164.6 eV were observed, corresponding to -S2O3, SH, -SS-, and -SH, respectively, indicating the presence of different sulfurs that can be assigned to free thiol groups and disulfide. Moreover, oxidized species, like SO3- (169 eV) or -SS- (168.8 eV), are also present. By the fit obtained, we estimate that about 50 % of the free

thiol groups represent surface oxide, which is obviously higher than that determined by Ellman’s reagent method. The degree of oxidation here is a qualitative estimation based on the structure and the chemical composition of the NPs studied in the work presented here. However, it is true that the degree of surface oxidation of the thiol-modified colloidal particles will play a role in the formation of a crosslinked structure of the NPs and, consequently, their mucoadhesive properties in vivo. Intestinal mucoadhesion measurements To validate our in vitro mucoadhesion results as described before (Zhang et al. 2014), we determined the interaction of Eul–cys/GSH NPs in different parts

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of the small intestine after intra-intestine injection in rats. Cross sections of the jejunum, duodenum, and ileum were prepared and stained with DAPI. As shown in Fig. 8, green NPs spots can be easily distinguished from the blue nuclei in all parts of the intestine compared with the control group. A significant number of NPs were attached to jejunal epithelial cells and Peyer’s patches. These results confirm our previous research from in vitro mucoadhesion and cell culture models (Zhang et al. 2014). It seems that a large number of NPs were attached to the apical surface of the GIT, especially in the jejunum. The cyan fluorescence suggests that some NPs were taken up by epithelial cells and Peyer’s patches. These findings indicate comparable uptakes of NPs in rat enterocytes and a potential mechanism perhaps involving the paracellular pathway, endo- or transcytosis or phagocytosis via M-cells.


delivery. Furthermore, Eul–cys/GSH NPs exhibited excellent mucoadhesive ability in an in situ rat intestinal model, especially to jejunal and ileum epithelial cells. The combination of all the properties of Eul–cys/GSH NPs, i.e., pH-sensitive swelling and controlled insulin release, protection from enzyme degradation, and possible protease inhibition and bioadhesion, makes these polymeric NPs very promising materials of interest for oral delivery of peptides and proteins. Acknowledgments The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (No. 81202482). Conflict of interest The authors have no conflict of interest to declare.

References Conclusion pH-sensitive thiolated conjugates were synthesized by grafting cysteine onto the chain of polymethylacrylic acid in Eudragit L100, and their potential as drug carriers for an oral delivery system for protein and peptide drugs was investigated. Here, insulin was incorporated into the conjugate as a model drug. A series of properties and functions of the Eul–cys conjugate were studied and evaluated. The Eul–cys showed representative pH-sensitivity, and it was swollen when the pH was above 5 due to the deprotonated carboxylic acid groups and the crosslinked disulfide bond structure, but water absorption shrinkage occurred below this. Cation-binding, and enzymatic-inhibition studies revealed that the Eul– cys conjugate had the potential to protect peptides from enzyme degradation and possible Ca2?-dependent protease inhibition. Following the above assumption, insulin-loaded Eul–cys/GSH NPs were prepared by a diffusion method and an electrostatic interaction between polymer and proteins, in which GSH acted as a penetration enhancer for insulin in oral delivery. Interaction between insulin and Eul– cys NPs confirmed by XRD and XPS showed that insulin was loaded into the NPs with an amorphous or molecular dispersed form. These results also suggest that thiolated NPs structures may improve the integrity of fragile biomolecules during oral

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