Preparation and Characterization of Glycoprotein-Resistant Starch

Apr 11, 2015 - For effective oral delivery of polypeptide or protein and enhancement their oral bioavailability, a new resistant starch–glycoprotein...
0 downloads 9 Views 3MB Size
Download PDF
Article pubs.acs.org/JAFC

Preparation and Characterization of Glycoprotein-Resistant Starch Complex As a Coating Material for Oral Bioadhesive Microparticles for Colon-Targeted Polypeptide Delivery Wenbei Situ, Xiaoxi Li,* Jia Liu, and Ling Chen* Ministry of Education Engineering Research Centre of Starch & Protein Processing, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, China ABSTRACT: For effective oral delivery of polypeptide or protein and enhancement their oral bioavailability, a new resistant starch−glycoprotein complex bioadhesive carrier and an oral colon-targeted bioadhesive delivery microparticle system were developed. A glycoprotein, concanavalin A (Con A), was successfully conjugated to the molecules of resistant starch acetate (RSA), leading to the formation of resistant starch−glycoprotein complex. This Con A-conjugated RSA film as a coating material showed an excellent controlled-release property. In streptozotocin (STZ)-induced type II diabetic rats, the insulin-loaded microparticles coated with this Con A-conjugated RSA film exhibited good hypoglycemic response for keeping the plasma glucose level within the normal range for totally 44−52 h after oral administration with different insulin dosages. Oral glucose tolerance tests indicated that successive oral administration of these colon-targeted bioadhesive microparticles with insulin at a level of 50 IU/kg could achieve a hypoglycemic effect similar to that by injection of insulin at 35 IU/kg. Therefore, the potential of this new Con A-conjugated RSA film-coated microparticle system has been demonstrated to be capable of improving the oral bioavailability of bioactive proteins and peptides. KEYWORDS: oral colon-targeted bioadhesive delivery, resistant starch, resistant starch−glycoprotein complex, polypeptide controlled-release, hypoglycemic response

■

INTRODUCTION

time, which allows a high local concentration or improved absorption of poorly absorbable bioactive ingredients. The colon displays some distinctive features that might be beneficial to successful bioadhesion there, including the less motile and thinner unstirred water layer adjacent to the mucosa; lower mucus turnover rates, which are less affected by secretory stimuli; the thicker mucus layer; and the less compact glycocalyx layer.5,6 Hence, a longer retention in close proximity to the colonic epithelial surface would be possible for substrates that are entrapped in the mucus layer. The combination of the bioadhesion with a colon-specific delivery vehicle would contribute to a more efficient colonic targeting and improve the absorption of bioactive peptides and proteins. Although work has been carried out on the biomaterials for oral colon-specific bioadhesive delivery carriers, the colonic bacteria-degradable polysaccharides, including alginate/chitosan and guar gum/chitosan,7,8 have gained more attention. Resistant starch (RS) is another colonic bacteria-degradable polysaccharide,9 the most successful carrier material among different polysaccharides for oral colon-specific delivery.10 At present, RS is generally classified into five categories:11 RS1 is the physically inaccessible starch; RS2 is raw starch granules from potato, pea, or high-amylose maize starch, which has the B- or C-type polymorphs; RS3 is retrograded starch after cooking; RS4 is chemically modified starch; and RS5 is amylose−lipid complexed starch. RS plays an important role in

Controlled release of food ingredients at the right place and right time is a key approach to improve the effectiveness and bioavailability of food ingredients.1−4 Great progress has been made over recent years to encounter biochemical and biophysical barriers for effective oral delivery of bioactive peptides and proteins for their physiological effects of health promoting or disease preventing. An ideal delivery system for oral administration of bioactive peptides and proteins should have great stability against enzymatic and hydrolytic degradation in the gastrointestinal (GI) tract and could maximize the absorption of these bioactive components. Regarding this, oral colon-specific controlled-release delivery systems have recently gained increasing attention for the delivery of a variety of therapeutic agents. This is because this kind of delivery system can take advantage of the potentially favorable characteristics of the colon environment, offering obvious advantages over administration to the small intestine.5,6 However, the main challenge of this kind of system for bioactive peptides and proteins is about how to enhance their oral bioavailability. To increase the absorption of bioactive peptides and proteins in the colon, a variety of strategies have been attempted, ranging from the coadministration of protease inhibitors and permeation enhancers to formulations within purposely designed delivery systems using suitable biomaterials, such as bacteria-degradable, pH-sensitive, pressure-sensitive, and bioadhesive natural or synthetic biopolymers. Among the carriers for oral colon-specific delivery, bioadhesive carriers and delivery systems are perhaps the most prominent candidates because they remain in contact with particular organs, tissues, or cells for an extended period of © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4138

January 21, 2015 April 11, 2015 April 11, 2015 April 11, 2015 DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry digestive physiology as a functional dietary fiber, which escapes digestion before the colon but can be fermented by the colonic bacteria. Depending on this property, it has been reported from our group that RS312 and RSA (resistant starch acetate; one of RS4) can be used as potential carrier materials for oral colonspecific drug delivery.13−16 However, the bioadhesion of RS is nonspecific, which may limit the delivery efficacy (including targeting accuracy) and the bioavailability of bioactive peptides and proteins.17,18 It has been found that the GI mucosa epithelial cells are covered with specific glycan moieties in different sites of the GI tract.19,20 If RS is coupled by certain ligands that can bind to the surface of predetermined colonic mucosa epithelial cells, the specific bioadhesion of RS carriers could be achieved, with increased residence time and enhanced adsorption of bioactive peptides and proteins in the colon. Lectins are glycoproteins of nonimmune origins that are able to specifically recognize sugar molecules and bind to glycosylated membrane components. Lectin−sugar interactions play crucial roles in many cell recognition and adhesion processes.21−24 It has been reported that lectin-conjugated particles were of benefit to bioactive ingredients absorption because of the interactions between lectin and cells.25,26 Considering this, it is worth conjugating suitable lectin to RSA, which has proved to have good colonic targeting and controlled-release properties.13 Using this method, a colon-specific bioadhesive delivery system might be achieved that will improve the oral bioavailability of bioactive peptides and proteins. In this regard, the fluorescein-labeled lectins (wheat germ agglutinin, concanavalin-A, peanut agglutinin, Lens culinaris, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, Pisum sativum agglutinin, Phaseolus vulgaris agglutinin and Galanthus nivalis agglutinin) of different carbohydrate specificities were screened in our lab for binding to human colonic mucosa epithelial cells through three types of human GI mucosa epithelial carcinoma cells, namely, MGC803, Hic, and Caco-2 cell lines, which were derived from human stomach, small intestine, and colon carcinomas, respectively. In addition, the colon-bioadhesion of the screened lectins was confirmed further in vivo in the gastrointestinal epithelial tissues of rats by tissue immunocytochemistry. Our previous research indicated that concanavalin A (Con A), one of the most abundant lectins that could bind to the mannose of glycoprotein on the surface of cells,27 showed better colon bioadhesion. Thus, this lectin was conjugated to the surface of RSA-film-coated microparticles. The structure of the Con A− RSA complex was investigated using Raman spectra and X-ray diffraction. On the basis of this, the insulin-loaded microparticles coated with this Con A−RSA complex film were prepared, and their colonic targeting and bioadhesion of the decorated microparticles were determined by in vivo optical imaging and tissue section tests. Furthermore, the in vivo hypoglycemic response of this kind of insulin-loaded microparticles was evaluated in streptozotocin (STZ)-induced diabetic rats.

■

Medicinal Accessary Material Co., Ltd. (Huainan, Anhui, China). Insulin was purchased from Newprobe Bioscience & Technology Co., Ltd. (Beijing, China). Native corn starch (medicinal grade) used for excipients in the preparation of insulin-loaded microparticles cores was supplied by Defeng Starch Sugar Co., Ltd. (Shunde District, Foshan, Guangdong, China). Con A, molecular weight 102 000, was purchased from Medicago (Quebec, Canada). Fluorescein isothiocyanate (FITC), glycine, and STZ were purchased from Sigma-Aldrich Co. LLC (Santa Clara, CA, USA). Acetone, ethanol, phosphate buffer solution, isoflurance, ether, and glucose were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The remaining chemical reagents were of analytical grade. Synthesis of Con A-Conjugated RSA. Con A was introduced to the hydroxyl groups of RSA by the CDI activation method.28 RSA was suspended in acetone, followed by the addition of CDI, and stirred at 30 °C for 8−26 h. The concentration of RSA was 10−35%. The weight ratio between CDI and RSA was 0.08−0.5. After reaction, the mixture was filtered and washed with ethanol to remove unreacted CDI. The product (N-resistant starch acetate imidazolyl carbamate) was dried at 45 °C and sieved. The nitrogen content was determined by a Kjeldahl meter (BÜ CHI distillation unit B-324, Buchi, Flawil, Switzerland). Then 1−10 g of N-resistant starch acetate imidazolyl carbamate was suspended in phosphate buffer solution (10 mmol/L; pH 7.4), followed by addition of Con A (the weight ratio between Nresistant starch acetate imidazolyl carbamate and Con A was varied from 0.001 to 0.03) and kept at 37 °C for 8−28 h. After reaction, the products were filtered and washed several times in phosphate buffer solution to remove unreacted Con A and incubated for 1 h in 0.5% glycine (w/v) in phosphate buffer solution to quench activated sites to which Con-A was not bound. The filtrate and washing liquor after reaction were collected, and the leftover of Con A was analyzed using a Coomassie Brilliant Blue kit (G-250, Bio-Rad Corp., Hercules, California, America) at 595 nm by a UV spectrophotometer (Unico, UV-3802, Shanghai, China). The coupled Con A was the difference value of Con A between the amount added and that left without reaction. Structures of Con A-Conjugated RSA. Raman Spectrometry. The Raman spectra were acquired using a micro-Raman spectroscopy system (LabRAM Aramis, Horiba Jobia Yvon Corp., Villeneuve d’Ascq, France) equipped with a 532 nm laser source and grating of 1200 g/mm. Data were collected over a range of 100−4000 cm−1 at an interval of 1 cm−1 using 2 scans per sample with an exposure time of 5 s. X-ray Diffraction. Wide-angle X-ray powder diffraction analysis was performed using an X-ray diffractometer (X’Pert Prox, Panalytial, Eindhoven, Netherlands) operating at 40 kV and 40 mA with Cu−K radiation (λ = 0.1542 nm). The diffractograms of samples were acquired at an angular range of 2θ from 4° to 60° with a step size of 0.033° and a counting time of 4 s for each step. The samples were sieved through 0.180 mm and equilibrated at 25 °C for 24 h prior to the analysis. Preparation of Con A−RSA Film-Coated Microparticles. The preparation of insulin-loaded microparticle cores were obtained via extrusion−spheronization.13 The N-resistant starch acetate formate imidazole obtained above was dissolved in acetone at a concentration of 5% and used as the initial coating material. The 1% insulin-loaded microparticle cores (500 μm in diameter) were coated by a bottomspray method in a fluidized bed coater (Mini-XYT, Xinyite Co., Ltd., Shenzhen, Guangdong, China) until a proper film coating thickness was achieved, which was represented by the dry weight gain of the microparticles.13 The parameters for the film coating process were as followed: inlet temperature, 30−35 °C; product temperature, 23−27 °C; spray rate, 0.7−0.8 mL/min; atomization pressure, 0.125−0.2 MPa; and fluidization pressure at 0.1−0.15 MPa. Then the film-coated microparticles were conjugated with Con A according to the method mentioned above and dried at 26 °C. In Vivo Colonic Targeting and Bioadhesion of the Con A− RSA Film-Coated Microparticles. In Vivo Fluorescent Imaging. Six 5- or 6-week-old SPF grade nude mice (half males and half females, Vital River Laboratory Animal Technology Co., Ltd., Beijing, China)

MATERIALS AND METHODS

Materials. RSA was synthesized in our laboratory according to a previously published method.13 1,1′-Carbonyldiimidazole (CDI) was purchased from Alfa Aesar. (Shanghai, China). Commercial longacting insulin glargine injection was supplied by Sanofi-Aventis Deutschland GmbH (Frankfurt am Main, Hessen, Germany). Microcrystalline cellulose was purchased from Anhui Shanhe 4139

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effects of reaction time (a), concentration of RSA (b), weight ratio between 1,1′-carbonyldiimidazole and RSA (c) on the RSA activation by 1,1′-carbonyldiimidazole method; and the effects of reaction time (d), concentration of RSA (e), and weight ratio between Con A and RSA (f) on the reaction of Con A conjugated to RSA. weighing approximately 12−14 g were fasted for 24 h before the study. The FITC-loaded microparticles coated with Con A−RSA film were orally administrated to the stomach via polyethylene tubing under light ether anesthesia. The microparticles were dosed at 0.2 mg per gram of body weight. At 2, 8, 14, and 44 min and at 24, 48, and 72 h after oral administration, the fluorescence intensity and transmission of the microparticles in the nude mice were observed by a whole-body smallanimal in vivo imaging system (IVIS 200, Xenogen Corp., Alameda, CA, USA) at 445−490 nm of exciting light and 515−575 nm of emitted light with an exposure time of 5 s. The nude mice were anesthetized by isoflurance before being photographed. Tissue Slices and Fluorescence Distribution. Six 5- or 6-week-old SPF grade KM mice (half males and half females, Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) weighing approximately 18−22 g were fasted for 24 h before the study. The oral administration and dosage of FITC-loaded microparticles coated with Con A−RSA film was described in the previous section In Vivo Fluorescent Imaging”. At 2, 4, 12, 24, 48, 72, and 144 h after oral administration, the mice were sacrificed by cervical dislocation, and the tissues of the stomach, small intestine, and colon were obtained. After washing the mucosal surface, the different parts of the mouse GI tract tissues were embedded with Optimum Cutting Temperature Compound and then cut on a cryostat microtome (Shandon cryotoZFE, ThermoFisher Scientific Inc., Waltham, MA USA) into 5 μm sections, and the tissue slices were thaw-mounted onto surfacetreated glass slides. The FITC distribution in the different parts of the mouse GI tract and the fluorescence intensity were observed by a fluorescence microscope (BX51TRF, Olympus, Tokyo, Japan) at 445−490 nm of exciting light and 515−575 nm of emitted light. In Vivo Hypoglycemic Response of Insulin-Loaded Microparticles Coated with Con A−RSA Film. The in vivo hypoglycemic response of the microparticles was evaluated using STZ-induced diabetic male Sprague-Dawley (SD) rats (Guangdong Animal Experimental Centre, Foshan, Guangdong, China).12 The type II diabetic rats were randomly divided into five groups (n = 5 for each group, and the average plasma glucose concentrations of the different groups differed less than 2 mmol/L). The group that was subcutaneously injected with commercial long-acting insulin glargine injection at 35 IU/kg was the positive control group, and the diabetic group without insulin treatment was the negative control group. The

other three groups are experimental groups. Before the tests, all the rats were fasted for 12 h with free access to water. Effect of Con A Conjunction on the Surface of RSA Film on the Hypoglycemic Response after Single-Dose Oral Administration. The insulin-loaded microparticles coated by RSA film and Con A− RSA film were orally administered to the stomach via polyethylene tubing under light ether anesthesia. Insulin was dosed at different dosages: 60, 80, and 100 IU per kg body weight. After insulin administration, the rats in all the groups were allowed to diet freely. At predetermined time points, the plasma glucose concentrations of the rats were monitored by the glucose oxidase method. Hypoglycemic Response after Successive Oral Administration. Current clinical use of long-acting insulin injections is generally subcutaneously injected at 19:00−21:00 every day. To coincide with the clinical use, the insulin-loaded microparticles coated with Con A− RSA film were orally administered at 19:00 on each of 5 consecutive days. On the first day, the insulin was dosed at 100 IU/kg; and from the second to the fifth days, the dose was 25, 40, 50 IU/kg, respectively. According to the predetermined time points for monitoring the plasma glucose concentrations of people with diabetes, the plasma glucose concentrations of the rats were monitored immediately before and 2 h after breakfast (8:00), lunch (12:00) and dinner (17:00) every day by the glucose oxidase method. Oral Glucose Tolerance Test (OGTT). After oral administration for 5 successive days of the insulin-loaded microparticles coated with Con A−RSA film, all the rats from each group were fasted for 12 h with free access to water and orally administered with 20% glucose solution at 5.0 g/kg, and the rats of the negative control group received the same volume of distilled water. After glucose administration, the plasma glucose concentrations of the rats were measured at 30, 60, and 120 min. Statistical Analysis. All data were subjected to statistical analysis using the SPASS 10.0 statistical package and were presented as mean ± standard deviation (± SD). Differences between groups were estimated by analysis of t test, and P < 0.01 was considered to indicate a statistically significant difference between two groups.

■

RESULTS AND DISCUSSION Synthesis of Con A-Conjugated RSA. Con A was introduced to the hydroxyl groups of RSA by the conventional

4140

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry CDI activation method.28 The RSA with a DS of 1.94 and resistant starch content of ∼90% was selected in this study. Its hydroxyl groups were activated by CDI and formed an imidazolyl carbamate complex that may be displaced by binding to the free amino group of Con A. The reaction is an N-nucleophilic substitution and results in a stable Nalkylcarbamate linkage of the ligand to the molecules of RSA. To investigate how the activation time, the RSA concentration, and the weight ratio between CDI and RSA will influence the level of activation, different amounts of RSA, activator and time periods were applied (shown in Figure 1). The optimized parameters for activating RSA were as follows: RSA concentration, 25% (w/v); weight ratio between CDI and RSA, 0.25; reaction temperature, 30 °C; and reaction time, 16 h. At these optimized conditions, the nitrogen content of RSA was at the highest value of 2.7%. This CDI-activated RSA was further treated with Con A. It can be seen from Figure 1b−d that the coupled Con A was at 0.86−34 μg/mg. The amount of coupled Con A could be 32 μg/mg when the concentration of RSA was 5% (w/v), the weight ratio between Con A and RSA was 0.03, and the reaction took place at 30 °C for 24 h. The structural changes of RSA after conjugation with Con A were analyzed using Raman spectrometry and X-ray diffraction. The Raman spectrum of Con A had two bands at 620−640 cm−1 that indicated the stretching frequency associated with the C−S bond. The intense band at 1006 cm−1 presented in the spectra was the characteristic of the ring breathing of the benzene ring of phenylalanine. From Figure 2, the Raman

Raman spectrum indicated that Con A had been successfully conjugated to RSA. The XRD patterns of Con A and RSA coupled with different contents of Con A are shown in Figure 3. It can be seen that

Figure 3. X-ray diffraction pattern of RSA with different contents of Con A (a, Con A; b, RSA; the contents of Con A are 8 (c), 18 (d), 32 (e), and 34 μg/mg (f)).

RSA had two humps on the XRD pattern at 7.2−7.3° and 19.0−19.7° that were related to the V-type starch crystalline structure. Thus, RSA presented a V-type diffraction pattern.32 Con A had two humps on the XRD pattern at around 9° and 19° that were similar to that of RSA. After Con A was conjugated to RSA, the XRD pattern of Con A-conjugated RSA changed slightly. All the XRD patterns of Con A-conjugated RSA showed two humps on the diffraction pattern, suggesting that Con A-conjugated RSA was amorphous. The amorphous molecular chains of Con A-conjugated RSA could be easy to extend, which makes it possible to pass through mucosa and adhere to the surface of colonic epithelium cells. Oral Colon-Targeting and Bioadhesive Property of the Con A−RSA Film-Coated Microparticles. In Vivo Colonic Targeting in Mouse GI Tracts. The size of Con A− RSA film-coated microparticles was ∼500 μm in diameter (data not shown). Using in vivo fluorescent imaging observed by a whole-body small-animal in vivo imaging system, the oral colonic targeting capability of FITC-labeled microparticles coated with Con A−RSA film (the coupled Con A was 32 μg/ mg) was studied. The images showing the microparticles distribution in different parts of the GI tract of nude mice at different times after oral administration of Con A−RSA filmcoated microparticles are shown in Figure 4. Before administration, no fluorescence was shown in the nude mice (Figure 4a). With increased transit time after oral administration of Con A−RSA film-coated microparticles (from 2 to 44 min), the fluorescence spots moved from the stomach to the small intestine and then to the colon. The fluorescence intensity in the colon gradually increased. At 44 min after oral administration, all the fluorescence spots concentrated in the colon, indicating that the microparticles had been moved mainly to the colon (Figure 4b−e). As the transit time was prolonged further, the fluorescence intensity at the colon was gradually increased, and the fluorescence spots were enlarged, which suggested that FITC was released from the microparticles. When the transit time reached 72 h, the size of the fluorescence spot was smaller, but the intensity was still strong (Figure 4f−h). This indicated that Con A−RSA film-coated microparticles still existed in the colon. These results showed

Figure 2. Effect of the conjugation of Con A on the molecular structure of RSA (from bottom to top are the Raman spectrum of Con A, RSA, and the Con A−RSA complex).

spectrum of Con A−RSA possessed an absorption peak at 1006 cm−1, indicating that the phenylalanine residue had been conjugated to RSA after reaction. In general, the presence of proteins can be noticed by amide I and amide III bands at about 1300 and 1655 cm−1.29,30 In the Raman spectrum of Con A, the absorption peaks at 1240 cm−1 were attributed to the N−H bending vibration band and the C−N stretching vibration band of amide III. The v(CO) stretch vibration band and N−H bending vibration of amide I caused an intense band at 1670 cm−1.31 The Raman spectrum of Con Aconjugated RSA had two bands at 1240 and 1670 cm−1, which means the material has an OC−NH group. The results of the 4141

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

Figure 4. Microparticle transit after administration of fluorescein isothiocyanate loaded microparticles coated with Con A−RSA film in the gastrointestinal tract of nude mice at different time intervals (from left to right are the blank group and two Con A−RSA film-coated microparticle groups).

Figure 5. Fluorescence distribution after administration of fluorescein isothiocyanate-loaded microparticles coated with Con A−RSA film in the different regions of the gastrointestinal tract epithelial tissue of mice at different time intervals.

that Con A−RSA film-coated microparticles could not only target bioactive molecules to the colon but also stay in the colon for a longer time and continuously release bioactive molecules (FITC). This indicated good colonic targeting and bioadhesion abilities of Con A−RSA film-coated microparticles. FITC Distribution in the Mouse GI Tract Epithelial Tissue. At 2 and 4 h after oral administration of Con A−RSA filmcoated microparticles, no fluorescence was found in the GI tract epithelial tissue of mice because no FITC was released (the pictures are not shown). At 12 h after oral administration, very slight fluorescence was observed in the stomach and small intestine epithelial tissues (Figure 5a,b), meaning a very small amount of FITC was released in the stomach and small intestine. Meanwhile, minor green fluorescence was shown in

the colon epithelial tissue (Figure 5c), suggesting that some of the FITC began to be released from the microparticles. When the administration time was prolonged to 24 h, the fluorescence intensity concentrated in the colon, indicating a large amount of FITC released in the colon (Figure 5d). These results showed that Con A−RSA film-coated microparticles could target and release the bioactive molecules to the colon. To estimate the bioadhesion of Con A−RSA film-coated microparticles to the colon epithelial tissue, the difference in the FITC distribution in the mouse colon epithelial tissue was analyzed after prolonged administration of Con A−RSA filmcoated microparticles and RSA film-coated microparticles, respectively. Figure 6 showed the FITC distribution in the mouse colon epithelial tissue at 24, 48, 72, and 144 h after oral 4142

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

Figure 6. Fluorescence distribution after administration of fluorescein isothiocyanate-loaded microparticles coated with RSA film and Con A−RSA film in the colon epithelial tissue of mice at different time intervals (left, RSA film-coated microparticle group; right, Con A−RSA film-coated microparticles group).

slices after administration of RSA film-coated microparticles showed the highest value and then decreased as the time was further prolonged. This indicated that the microparticles could be easily eliminated from the body. When the time reached 144 h, the fluorescence had almost disappeared; however, for the colon epithelial tissue slices after administration of Con A−RSA film-coated microparticles, the fluorescence intensity still increased from 24 to 72 h and reached the highest value at 72 h. At 144 h, the fluorescence intensity was weaker but still

administration of these two types of microparticles. By comparing the fluorescence intensity between images, the release behaviors of these two types of microparticles were shown to be different. It can be seen from Figure 6 that at the same time, the fluorescence intensity of the colon epithelial tissue slices after administration of Con A−RSA film-coated microparticles was stronger than that of the slices after administration of RSA film-coated microparticles. Moreover, at 24 h, the fluorescence intensity of the colon epithelial tissue 4143

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

Table 1. Plasma Glucose Concentrations at Different Post-Administration Times in Diabetic Rats Following the Injected Insulin and Oral Administration of Rsa Film-Coated Microparticles with Different Insulin Dosagesa time, h 0 1 2 4 6 8 10 12 24 28 30 36 48 52 56 60 a

diabetic model group, (mmol/L) 17.9 22.4 25.1 19.3 16.4 22.6 17.9 20.7 17.9 21.5 18.7 19.4 18.0 20.0 25.0 23.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 2.6 4.6 3.7 2.8 3.0 2.7 3.4 3.9 4.4 3.1 4.0 2.6 4.0 3.3 4.0

insulin injected group, (mmol/L) 20.8 11.6 9.0 5.6 3.5 6.5 6.8 6.4 10.4 14.5 19.7 22.7 21.9 24.5 26.5 22.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.3 3.8b 3.9b 2.4b 0.6b 1.3b 0.9b 1.6b 2.9b 3.9b 3.9 4.7 3.6 4.6 3.7 4.0

low-dose microparticles group, (mmol/L) 17.7 29.7 21.6 13.0 16.6 11.3 7.7 9.7 7.0 6.3 6.9 7.8 15.1 17.6 17.7 29.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.3 4.3b,c 5.2c 2.3b,c 3.5c 2.9b,c 2.2b 3.1b 1.9b 2.0b,c 1.3b,c 2.4b,c 2.5c 4.3 3.5b,c 5.0

medium-dose microparticles group, (mmol/L) 19.5 30.3 18.7 9.7 11.6 7.5 6.0 5.2 6.1 6.0 6.8 6.9 16.3 19.5 19.5 30.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.2 3.9b,c 4.0c 2.5b 2.8d 2.3b 1.3b 1.5b,d 1.8b 2.1b,c 1.7b,c 1.8b,c 2.3c 3.7 4.5 4.2

high-dose microparticles group, (mmol/L) 19.0 32.5 19.0 11.1 12.5 5.9 4.3 5.0 5.7 6.2 7.0 8.3 15.7 19.5 19.0 32.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.9 4.4b,c 3.4c 3.1b,c 2.8c 2.0b,d 1.5b,d 1.2b,d 0.8b,c 1.6b,c 2.3b,c 1.9b,c 2.7c 4.4 4.7c 5.6b,c

n=5 for each group. bCompared with those of the diabetic model group (P < 0.01). cCompared with those of the insulin injected group (P < 0.01). Compared with those of the low-dose microparticles group (P < 0.01).

d

Table 2. Plasma Glucose Concentrations at Different Post-Administration Times in Diabetic Rats Following Injected Insulin and Oral Administration of Con A−RSA Film-Coated Microparticles with Different Insulin Dosagesa time, h 0 2 6 8 10 12 24 30 36 48 54 60 72 78

diabetic model group, (mmol/L) 19.9 25.4 23.3 28.2 26.6 22.3 18.7 20.2 22.6 21.5 18.5 21.2 19.4 21.8

± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.5 3.7 4.2 3.3 2.4 2.9 3.1 3.4 2.8 3.1 4.0 2.7 3.0 3.7

insulin injected group, (mmol/L) 18.7 10.6 5.0 3.6 5.5 6.5 7.8 12.4 20.4 19.5 19.7 22.7 18.9 24.5

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.4 3.1b 1.5b 1.0b 0.9b 1.6b 2.1b 2.6b 3.4 2.9 3.7 4.0 2.9 3.6

low-dose microparticles group, (mmol/L) 18.1 30.9 12.0 12.9 9.2 6.4 5.2 6.9 7.6 7.3 7.5 11.7 18.0 19.9

± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.4 3.6c 2.8b 2.4b,c 1.5b 1.1b 0.9b 1.4b,c 1.8b,c 1.9b,c 2.2b,c 2.4b,c 2.9 3.4

medium-dose microparticles group, (mmol/L) 17.8 30.0 11.5 12.1 6.6 4.7 5.4 6.5 7.1 7.1 6.5 10.5 18.2 19.3

± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.0 2.9c 2.2b 2.1b,c 1.4b 1.0b 1.4b 0.7b,c 1.4b,c 2.1b,c 2.4b,c 2.8b,c 3.4 4.0

high-dose microparticles group, (mmol/L) 21.6 28.1 10.1 9.4 3.8 4.2 5.4 6.3 6.7 6.2 6.4 9.7 18.5 18.7

± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.0 3.4c 2.7b 2.4b 1.8b,d 1.4b 1.6b 0.8b,c 1.3b,c 2.0b,c 1.6b,c 2.1b,c 3.1 3.2

a

n = 5 for each group. bCompared with those of the diabetic model group (P < 0.01). cCompared with those of the insulin injected group (P < 0.01). dCompared with those of the low-dose microparticles group (P < 0.01).

abnormally low plasma glucose level of 3.5 ± 0.6 mmol/L) occurred at 6 h after injection. On the other hand, after RSA film-coated microparticles with different insulin dosages were orally administered, the hypoglycemic response was slowly but steadily maintained for a long time, and no glycopenia was observed between 8 and 36 h (total 28 h) (seen from Table 1). The hypoglycemic response was increased by an increase in the dosage of oral administration. It can be seen that the plasma glucose concentration declined into a lower range (6.3−9.7 mmol/L) at 10 h after oral administration of the low-dose microparticles and stayed within the lower range for 26 h. The medium-dose and the high-dose groups experienced a steady decrease in the plasma glucose concentration and stayed within the lower range for a total of 28 h, which was a little more rapid than that of the low-dose group. These results indicated that the plasma glucose concentration of the diabetic model rats was decreased steadily by using the RSA film-coated microparticles for the delivery of insulin; however, the plasma glucose

stronger compared with that of slices after administration of RSA film-coated microparticles, indicating that Con A conjugated RSA film-coated microparticles can extend the residence time of the microparticles in the colon. These results showed that Con A conjugation to the surface of the RSA film could increase the microparticle residence time in the colon and enhance the bioactive molecules’ (FITC) absorption effectively through bioadhesion to the surface of colonic mucosa cells. In Vivo Hypoglycemic Response of Insulin-Loaded Microparticles Coated with Con A−RSA Film. Hypoglycemic Response Improvement after Con A Conjunction on the Surface of RSA Film-Coated Microparticles. After administration of the commercial long-acting insulin solution subcutaneously, the plasma glucose concentration decreased significantly within 1 h. A strong hypoglycemic response was observed between 4 and 12 h after injection; subsequently, the plasma glucose concentration increased with time and returned to the basal level at 30 h (Table 1). However, glycopenia (an 4144

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

Table 3. Plasma Glucose Concentrations at Different Post-Administration Times in Diabetic Rats Following the Injected Insulin and Continuous Oral Administration of Con A−RSA Film-Coated Microparticles with Different Insulin Dosagesa time before administration first day 8:00 10:00 12:00 14:00 17:00 19:00 second 8:00 day 10:00 12:00 14:00 17:00 19:00 third day 8:00 10:00 12:00 14:00 17:00 19:00 fourth 8:00 day 10:00 12:00 14:00 17:00 19:00 fifth day 8:00 10:00 12:00 14:00 17:00 19:00

diabetic model group, (mmol/L)

insulin injected group, (mmol/L)

19.9 ± 2.5

20.0 ± 2.0

18.4 23.3 22.2 26.6 20.3 27.7 20.2 26.6 23.5 29.5 22.2 27.4 18.8 25.2 22.4 28.3 21.1 26.8 21.0 26.5 23.1 27.2 22.1 26.9 19.5 23.9 20.6 25.7 21.1 26.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.7 3.2 3.4 2.8 2.9 3.1 2.9 2.7 3.1 3.2 2.7 3.0 2.6 3.1 3.1 2.6 2.7 3.0 3.5 2.5 2.6 2.9 3.0 3.1 2.8 3.0 3.2 3.1 3.8 3.3

6.7 9.5 6.8 10.7 7.1 11.4 6.1 10.7 5.6 11.1 6.0 11.6 6.0 10.7 5.9 10.6 6.0 11.3 6.2 10.4 6.1 10.3 5.8 10.2 5.5 10.1 6.1 10.2 6.0 10.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

low-dose microparticles group, (mmol/L)

1.5b 1.1b 1.2b 1.9b 1.1b 1.5b 1.8b 0.9b 1.2b 1.5b 1.0b 2.1b 1.3b 2.2b 1.4b 1.5b 1.8b 1.3b 1.2b 2.1b 1.5b 1.4b 1.0b 1.8b 1.3b 2.0b 0.9b 1.4b 1.5b 2.0b

18.2 ± 2.1 4.3 9.2 5.7 10.9 5.1 9.2 6.6 12.4 7.0 12.8 7.8 14.2 7.7 13.3 9.5 13.8 9.6 14.5 7.9 13.4 9.8 14.4 9.8 16.3 13.0 16.8 13.4 17.5 13.3 16.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2b 1.7b 1.2b 1.3b 0.5b 0.9b 1.5b 1.6b 1.3b 1.5b 2.0b 1.1b 1.2b 1.7b 1.3b 2.0b 1.5b 2.1b 1.4b 1.8b 1.5b 0.9b 1.3b 2.1b,c 1.7b,c 1.5b,c 2.5b,c 1.4b,c 2.5b,c 2.5b,c

medium-dose microparticles group, (mmol/L) 17.9 ± 1.9 4.3 8.6 5.4 11.0 5.6 10.4 5.6 9.7 6.1 11.2 6.3 11.4 5.7 11.1 6.2 11.3 6.7 11.4 5.7 11.2 6.6 11.1 6.7 11.8 6.8 11.6 7.0 12.9 7.0 12.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.3b 1.2b 1.8b 1.5b 1.1b 1.1b 1.4b 1.0b 0.6b 1.3b 1.7b 1.0b 0.8b 1.1b 0.9b 1.2b 1.4b 1.3b 0.7b 0.5b 0.8b 1.2b 0.3b 1.8b 1.5b,d 2.0b 1.3b,d 1.5b 1.4b,c 1.1b

high-dose microparticles group, (mmol/L) 19.0 ± 2.5 4.1 8.6 6.2 9.9 5.7 10.3 5.3 10.9 5.8 10.9 5.9 10.8 5.4 10.2 5.7 10.8 6.1 10.9 5.3 10.4 6.0 10.8 6.0 10.2 5.5 10.3 6.1 10.6 5.8 10.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.7b 0.9b 1.5b 1.2b 1.1b 1.4b 1.5b 0.5b 0.8b 0.9b 0.6b 1.1b 1.2b 1.4b 0.8b 1.0b 0.5b 1.5b 1.6b 1.2b 0.8b 1.0b 0.8b 1.6b,d 1.5b,d 1.2b,d 0.7b,d 1.5b,d 0.5b,c 1.5b,c

a

n = 5 for each group. bCompared with those of the diabetic model group (P < 0.01). cCompared with those of the insulin injected group (P < 0.01). dCompared with those of the low-dose microparticles group (P < 0.01).

glucose concentration within the normal range could be maintained for a prolonged time (18−24 h with different insulin dosages), showing improved bioavailability of insulin. Hypoglycemic Response after Successive Oral Administration. For investigating the continuous bioavailability of insulin delivered through Con A−RSA film-coated microparticles, the insulin-loaded microparticles were orally administered at 19:00 on each of 5 consecutive days. The plasma glucose concentration was assayed at 2 h before and after meals every day. Table 3 shows the blood glucose change by continuous administration of insulin-loaded Con A−RSA filmcoated microparticles. After administration of microparticles, the plasma glucose concentration of experimental groups on the first day was at a low range, which was consistent with the results above. The differences among groups existed the following day. For the low-dose microparticles group, the plasma glucose concentration was at the upper level of the normal range on the second day. Then the plasma glucose concentration was out of the normal range, indicating that insulin for the low-dose microparticles group was not enough to decrease the plasma glucose. The plasma glucose concentration of the medium-dose microparticles group was within the normal range until the final day, but in the end, the plasma glucose concentration was higher than the normal level.

concentration increased with time and returned to the basal level from 48 h after administration, suggesting that the RSA film-coated microparticles could be easily eliminated from the body. The hypoglycemic response with different insulin dosages is shown in Table 2 for after Con A was conjugated to the surface of RSA film-coated microparticles. Comparing the data between Table 2 and Table 1, it is concluded that the time of the hypoglycemic response (when the plasma glucose concentration kept within a low range) was prolonged after the Con A−RSA film-coated microparticles with the same insulin dosage were orally administered. It can be seen that the plasma glucose concentration declined to a low range (5.2−9.2 mmol/L) from 10 to 54 h after administration of the low-dose and mediumdose microparticles and was kept within the lower range for 44 h. The high-dose group also showed the longest time of staying within a lower plasma glucose concentration from 8 to 60 h (for a total of 52 h). There was no glycopenia due to the steady release of insulin from the microparticles. In the treatment of diabetes mellitus, steadily decreasing the plasma glucose concentration is important to the patients.33 Con A−RSA film-coated microparticles could adhere to the surface of colonic mucosa cells, increase the residence time in the colon, and steadily and sustainably release insulin. Thus, the plasma 4145

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry *E-mail: [email protected].

The plasma glucose concentration of the high-dose microparticles was steady during all the 5 days, which showed a hypoglycemic response similar to that of the group subcutaneously injected with commercial long-acting insulin. OGTT mimics the physiological conditions of the glucose/ insulin system closely and evaluates the insulin sensitivity and the β-cell function.34,35 After 5 days of continuous administration with insulin-loaded Con A−RSA film-coated microparticles, the OGTTs were conducted; the results of different groups are shown in Figure 7. It can be seen from Figure 7 that

Funding

We acknowledge the National Natural Science Funds of China (No. 31271824); the National Science and Technology Supporting Program Projects (2012BAD33B04); the Ministry of Education Special R&D Funds for the Doctoral Disciplinary Stations in Universities (20120172110014); the Office of Education of Guangdong Province Science and Technology Innovation (Key) Projects in Universities (2012CXZD0006); the Ministry of Education Program for Supporting New Century Excellent Talents (NCET-12-0193); and the Fundamental Research Funds for the Central Universities, SCUT (2013ZG0009). Notes

The authors declare no competing financial interest.

■ Figure 7. Results of oral glucose tolerance test in different rat groups.

the glucose tolerance of the type II diabetic rats after administration of the microparticles was gradually enhanced, with greater enhancement seen with increased insulin dosage. Compared with the diabetic model group, the high-dose microparticles group had a glucose tolerance similar to that of the insulin-injected group. In conclusion, Con A−RSA filmcoated microparticles could adhere to the surface of colonic mucosa cells, prolonging the insulin release time and enhancing the hypoglycemic response. Thus, this study demonstrates the potential of Con A−RSA film-coated microparticles for oral colon-targeted delivery of polypeptide or protein ingredients with enhanced oral bioavailability. Although the use of lectins as targeting ligands remains problematic because of their cytotoxic effects, which would preclude their use in targeting delivery systems,36,37 the toxic effects of lectins are varied with different varieties. We did the toxicity tests when we screened the lectins of different carbohydrate specificities for binding to human colonic mucosa epithelial cells. The relative growth rate of Caco-2 cells was high (80%) when Caco-2 cells were exposed to Con A solutions in the microgram range (7.8−50 μg/cell) for 60 h (data not shown). This result indicated that the toxic effects of Con A would not be aggravated because the amounts of Con A conjugated to the microparticle surface were in the microgram range. Furthermore, the approach to overcome this limitation is to produce recombinant lectins with modified properties and to design and synthesize peptides or other organic molecules that mimic the function of the lectins. 38−40 Thus, better biocompatibility may result from the lectin-sugar interactions, which is applicable to the colon-specific bioadhesive delivery system.

■

REFERENCES

(1) Day, L.; Seymour, R. B.; Pitts, K. F.; Konczak, I.; Lundin, L. Incorporation of functional ingredients into foods. Trends Food Sci. Technol. 2009, 20, 388−395. (2) Mills, S.; Stanton, C.; Hill, C.; Ross, R. New developments and applications of bacteriocins and peptides in foods. Annu. Rev. Food Sci. Technol. 2011, 2, 299−329. (3) Hartmann, R.; Meisel, H. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 2007, 18, 163−169. (4) McClements, D. J.; Decker, E. A.; Park, Y.; Weiss, J. Structural design principles for delivery of bioactive components in nutraceuticals and functional foods. Crit. Rev. Food Sci. Nutr. 2009, 49, 577−606. (5) Maroni, A.; Zema, L.; Del Curto, M. D.; Foppoli, A.; Gazzaniga, A. Oral colon delivery of insulin with the aid of functional adjuvants. Adv. Drug Delivery Rev. 2012, 64, 540−56. (6) Varum, F. J. O.; McConnell, E. L.; Sousa, J. J. S.; Veiga, F.; Basit, A. W. Mucoadhesion and the Gastrointestinal Tract. Crit. Rev. Ther. Drug Carrier Syst. 2008, 25, 207−258. (7) Wittaya-areekul, S.; Kruenate, J.; Prahsarn, C. Preparation and in vitro evaluation of mucoadhesive properties of alginate/chitosan microparticles containing prednisolone. Int. J. Pharm. 2006, 312, 113− 118. (8) Haupt, S.; Zioni, T.; Gati, I.; Kleinstern, J.; Rubinstein, A. Luminal delivery and dosing considerations of local celecoxib administration to colorectal cancer. Eur. J. Pharm. Sci. 2006, 28, 204−211. (9) Sharma, A.; Yadav, B. S.; Ritika. Resistant starch: Physiological roles and food applications. Food Rev. Int. 2008, 24, 193−234. (10) McConnell, E. L.; Short, M. D.; Basit, A. W. An in vivo comparison of intestinal pH and bacteria as physiological trigger mechanisms for colonic targeting in man. J. Controlled Release 2008, 130, 154−60. (11) Thompson, D. B. Strategies for the manufacture of resistant starch. Trends Food Sci. Technol. 2000, 11, 245−253. (12) Situ, W.; Chen, L.; Wang, X.; Li, X. Resistant starch film-coated microparticles for an oral colon-specific polypeptide delivery system and its release behaviors. J. Agric. Food Chem. 2014, 62, 3599−609. (13) Pu, H. Y.; Chen, L.; Li, X. X.; Xie, F. W.; Yu, L.; Li, L. An Oral Colon-Targeting Controlled Release System Based on Resistant Starch Acetate: Synthetization, Characterization, and Preparation of FilmCoating Pellets. J. Agric. Food Chem. 2011, 59, 5738−5745. (14) Li, X.; Liu, P.; Chen, L.; Yu, L. Effect of resistant starch film properties on the colon-targeting release of drug from coated pellets. J. Controlled Release 2011, 152, e5−e7. (15) Chen, L.; Pu, H. Y.; Li, X. X.; Yu, L. A novel oral colon-targeting drug delivery system based on resistant starch acetate. J. Controlled Release 2011, 152, E51−E52. (16) Chen, L.; Li, X.; Li, L.; Guo, S. Acetylated starch-based biodegradable materials with potential biomedical applications as drug delivery systems. Curr. Appl. Phys. 2007, 7, e90−e93.

AUTHOR INFORMATION

Corresponding Authors

*Phone: +86 20 8711 3252. Fax: +86 20 8711 3252. E-mail: [email protected]. 4146

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147

Article

Journal of Agricultural and Food Chemistry

mimetics from combinatorial libraries. Pharm. Res. 2003, 20, 1258− 1266. (40) des Rieux, A.; Pourcelle, V.; Cani, P. D.; Marchand-Brynaert, J.; Preat, V. Targeted nanoparticles with novel non-peptidic ligands for oral delivery. Adv. Drug Delivery Rev. 2013, 65, 833−844.

(17) Vasir, J. K.; Tambwekar, K.; Garg, S. Bioadhesive microspheres as a controlled drug delivery system. Int. J. Pharm. 2003, 255, 13−32. (18) Smart, J. D. The basics and underlying mechanisms of mucoadhesion. Adv. Drug Delivery Rev. 2005, 57, 1556−1568. (19) Gabius, H. J.; Andre, S.; Kaltner, H.; Siebert, H. C. The sugar code: functional lectinomics. Biochim. Biophys. Acta, Gen. Subj. 2002, 1572, 165−177. (20) Gabius, H. J. The sugar code in drug delivery. Adv. Drug Delivery Rev. 2004, 56, 421−424. (21) Ensign, L. M.; Cone, R.; Hanes, J. Oral drug delivery with polymeric nanoparticles: The gastrointestinal mucus barriers. Adv. Drug Delivery Rev. 2012, 64, 557−570. (22) Gabor, F.; Bogner, E.; Weissenboeck, A.; Wirth, M. The lectincell interaction and its implications to intestinal lectin-mediated drug delivery. Adv. Drug Delivery Rev. 2004, 56, 459−480. (23) Kilpatrick, D. C. Animal lectins: a historical introduction and overview. Biochim. Biophys. Acta, Gen. Subj. 2002, 1572, 187−197. (24) Gajbhiye, V.; Gong, S. Lectin functionalized nanocarriers for gene delivery. Biotechnol. Adv. 2013, 31, 552−562. (25) Jepson, M. A.; Clark, M. A.; Foster, N.; Mason, C. M.; Bennett, M. K.; Simmons, N. L.; Hirst, B. H. Targeting to intestinal M cells. J. Anat. 1996, 189 (Pt 3), 507−16. (26) Pusztai, A.; Ewen, S. W.; Grant, G.; Peumans, W. J.; Van Damme, E. J.; Coates, M. E.; Bardocz, S. Lectins and also bacteria modify the glycosylation of gut surface receptors in the rat. Glycoconjugate J. 1995, 12, 22−35. (27) Rü d iger, H.; Gabius, H.-J. Plant lectins: occurrence, biochemistry, functions and applications. Glycoconjugate J. 2001, 18, 589−613. (28) Stollner, D.; Scheller, F. W.; Warsinke, A. Activation of cellulose membranes with 1,1′-carbonyldiimidazole or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as a basis for the development of immunosensors. Anal. Biochem. 2002, 304, 157−65. (29) De Gelder, J.; De Gussem, K.; Vandenabeele, P.; Moens, L. Reference database of Raman spectra of biological molecules. J. Raman Spectrosc. 2007, 38, 1133−1147. (30) Zhu, G.; Zhu, X.; Fan, Q.; Wan, X. Raman spectra of amino acids and their aqueous solutions. Spectrochim. Acta, Part A 2011, 78, 1187−1195. (31) Jaaskelainen, A.-S.; Holopainen-Mantila, U.; Tamminen, T.; Vuorinen, T. Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy. J. Cereal Sci. 2013, 57, 543−550. (32) Rindlava, A.; Hulleman, S. H. D.; Gatenholm, P. Formation of starch films with varying crystallinity. Carbohydr. Polym. 1997, 34, 25− 30. (33) Monnier, L.; Mas, E.; Ginet, C.; Michel, F.; Villon, L.; Cristol, J. P.; Colette, C. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA-J. Am. Med. Assoc. 2006, 295, 1681−1687. (34) Bartoli, E.; Fra, G. P.; Schianca, G. P. C. The oral glucose tolerance test (OGTT) revisited. Eur. J. Intern. Med. 2011, 22, 8−12. (35) Palumbo, P.; Ditlevsen, S.; Bertuzzi, A.; De Gaetano, A. Mathematical modeling of the glucose-insulin system: A review. Math. Biosci. 2013, 244, 69−81. (36) Smart, J. D. Lectin-mediated drug delivery in the oral cavity. Adv. Drug Delivery Rev. 2004, 56, 481−489. (37) Yun, Y.; Cho, Y. W.; Park, K. Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery. Adv. Drug Delivery Rev. 2013, 65, 822−832. (38) Eck, J.; Langer, M.; Möckel, B.; Witthohn, K.; Zinke, H.; Lentzen, H. Characterization of recombinant and plant-derived mistletoe lectin and their B-chains. Eur. J. Biochem. 1999, 265, 788− 797. (39) Lambkin, I.; Pinilla, C.; Hamashin, C.; Spindler, L.; Russell, S.; Schink, A.; Moya-Castro, R.; Allicotti, G.; Higgins, L.; Smith, M. Toward targeted oral vaccine delivery systems: selection of lectin 4147

DOI: 10.1021/acs.jafc.5b00393 J. Agric. Food Chem. 2015, 63, 4138−4147