Article pubs.acs.org/JAFC
Resistant Starch Film-Coated Microparticles for an Oral ColonSpecific Polypeptide Delivery System and Its Release Behaviors Wenbei Situ, Ling Chen,* Xueyu Wang, and Xiaoxi Li* Ministry of Education Engineering Research Centre of Starch and Protein Processing, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, China S Supporting Information *
ABSTRACT: For the delivery of bioactive components to the colon, an oral colon-specific controlled release system coated with a resistant starch-based film through aqueous dispersion coating process was developed. Starch was modified by a hightemperature−pressure reaction, enzymatic debranching, and retrogradation, resulting in a dramatic increase in the resistibility against enzymatic digestion (meaning the formation of resistant starch, specifically RS3). This increase could be associated with an increase in the relative crystallinity, a greater amount of starch molecular aggregation structure, and the formation of a compact mass fractal structure, resulting from the treatment. The microparticles coated with this RS3 film showed an excellent controlled release property. In streptozotocin (STZ)-induced type II diabetic rats, the RS3 film-coated insulin-loaded microparticles exhibited the ability to steadily decrease the plasma glucose level initially and then maintain the plasma glucose level within the normal range for total 14−22 h with different insulin dosages after oral administration; no glycopenia or glycemic fluctuation was observed. Therefore, the potential of this new RS3 film-coated microparticle system has been demonstrated for the accurate delivery of bioactive polypeptides or protein to the colon. KEYWORDS: oral colon-specific delivery, resistant starch, aqueous dispersion film coating, controlled release, polypeptide delivery, hypoglycemic effect
■
INTRODUCTION For health promotion or disease prevention, the challenge in the field of functional foods is how to add a bioactive ingredient and keep its stability not only during food processing and storage but also in the gastrointestinal (GI) system.1−3 It is very important to facilitate the bioavailability of these ingredients by controlled release at the appropriate GI target.4,5 Nevertheless, the physiological barriers to the oral delivery of bioactive components have not thoroughly been overcome and the bioavailability is generally still lower and far from being reliable in terms of consistency. Bioactive peptides and proteins have recently gained increasing attention for their physiological effects. Increased progress has been made over the years against the biochemical and biophysical barriers to effective oral delivery of peptide or protein biomolecules. Oral colon-specific controlled release delivery systems have been proven useful for systemic action of bioactive polypeptides such as insulin, calcitonin, and metenkaphalin.6−8 However, technical difficulties, resulting from the variation in the pH condition, the digestion enzymes, and the long transit time,9 are involved in the effective oral delivery of these biomolecules to the colon. To overcome these obstacles, recent developments in biomaterials for oral colon delivery carriers, including bacteria-degradable, pH-sensitive, pressure-sensitive, and time-dependent polymer coating films,8,10,11 have provided renewed hope for the effective targeting of polypeptides or proteins to the colon. Particularly, because of the nontoxicity, safety, and good biocompatibility, growing attention has been focused on many polysaccharides, such as amylose,12 guar gum,13 pectin,14,15 chitosan,16 hydroxypropyl methylcellulose,17 and inulin,18 as carrier © 2014 American Chemical Society
materials to be used for oral colon-specific controlled-release delivery systems. For the same purpose, starch, another polysaccharide, is also interesting, as it is renewable and biodegradable and has already been widely used in foods.19,20 Starch can be modified easily to overcome its native hydrophilicity and limitations against the acid and enzymes in the GI tract.21,22 Starch that is not hydrolyzed in the small intestine but can be degraded by colon microorganisms is considered resistant starch (RS).23 At present, RS in foods is generally classified into five categories:24 RS1, RS2, RS3, RS4, and RS5. RS can escape digestion before the colon and be fermented by the colonic bacteria,25 which plays an important role in digestive physiology.26 Depending on these properties, it has been reported from our group that resistant starch acetate (RS4) can be used as a potential carrier for oral colon-specific delivery.5,21,27−29 Meanwhile, it has also been reported elsewhere that the retrogradation of gelled starch molecules upon drying or dehydration could result in the formation of RS3.23,30 From this point, if microparticles loaded with bioactive components are coated with an RS3 film by an aqueous dispersion coating process where the gelled starch molecules with suitable molecular molar mass aggregate upon dehydration and drying and then an RS3 film coating layer subsequently forms in situ on the surface of the microparticles, a colon-specific controlled-release system might be achieved. Received: Revised: Accepted: Published: 3599
January 27, 2014 March 31, 2014 March 31, 2014 March 31, 2014 dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Fifty milligrams of starch was dispersed in 10 mL of dimethyl sulfoxide (DMSO) containing LiBr (50 mM) with heating in a boiling water bath for 1 h, followed by the shaking of the sample at 60 °C in the water bath for another 12 h. Before injection, the starch sample solutions were filtered using a membrane filter (5.0 μm LS, Millipore Corp., Bedford, MA, USA). The mobile phase used for GPC was DMSO (HPLC grade) containing LiBr (50 mM), which had been filtered through a 0.2 μm membrane filter (0.2 μm FG, Millipore Corp.) and degassed by ultrasound. The flow rate of the mobile phase was 0.7 mL/min; the column was maintained at 45 °C, and the detectors were maintained at 25 °C. The specific RI increment value (dn/dc) of starch in DMSO was found to be 0.074 mL/g.32 The data were analyzed by the Astra 3.4 software program (Wyatt Technology). X-ray Diffraction (XRD). Wide-angle X-ray powder diffraction analysis was performed using an X-ray diffractometer (X’Pert Prox, PANalytical, Eindhoven, The Netherlands) operating at 40 kV and 40 mA with Cu Kα radiation (λ = 0.1542 nm). The diffractograms of the RS3 samples were acquired at an angular range of 2θ from 4° to 40° with the step size of 0.033° and the 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. The relative crystallinity of the samples was quantitatively estimated according to the method of Nara and Komiya.33 A smooth curve connected with the constant peak baseline was computer-plotted on the diffraction. The ratio of the upper area (crystalline portion) to the total diffraction area (based on a linear baseline) was taken as the relative crystallinity with the software MDI Jade 6.0. Small Angle X-ray Scatting (SAXS). SAXS measurements were performed on a SAXSess camera (Anton-Paar, Graz, Austria) using Cu Kα radiation (0.1542 nm wavelength) at 40 kV and 50 mA according to our previous studies.31,34 The starch slurries with similar moisture content (60%, w/w) were prepared for this experiment and equilibrated at 26 °C for 24 h before the analysis. The samples were filled into a paste sample cell and fixed and placed in a TCS 120 temperature-controlled unit (Anton Paar) along the line-shaped X-ray beam in the evacuated camera housing. Each measurement was collected for 10 min. The 2D data were integrated into the 1D scattering function I(q) as a function of the magnitude of the scattering vector q and normalized. The background and smeared intensity were removed using the SAXSquant 3.0 software program for further analysis. If Bragg’s law is applied, q is proportional to the inverse of the apparent periodic length d, as d = 2π/q. The surface/mass fractal structure can be obtained from the slope of the log−log SAXS graph according to the method of Suzuki, Chiba, and Yarno.35 The scattering objects with a surface fractal dimension (Ds) are more compact than those with a mass fractal dimension (Dm). Preparation of the RS3 Film-Coated Microparticles. Bioactive component-loaded microparticle cores (containing microcrystalline cellulose and starch in the ratio of 3:1 and the bioactive component in varied content) were obtained via extrusion−spheronization.5 5-ASA and insulin were chosen as model bioactive components, respectively. During the extrusion−spheronization, the temperature was kept at 5− 10 °C to maintain the activity of the bioactive components. Purified water was added into the retrograded starch pastes until starch-based aqueous dispersions (8%, w/w) were obtained. After homogenization by a high-pressure homogenizer (Panda Plus2000, Gea Niro Soavi, Italy), 1,2-propanediol was added into the dispersions, which were then stirred for 8 h by a magnetic stirrer. Thus, the RS3based aqueous coating dispersions were obtained. The bioactive component-loaded microparticle cores were coated using a bottom spray fluid bed coater (Mini-XYT; Xinyite Technology Co., Shenzhen, China) until the coated-film thickness of 5−30% (w/ w) was achieved, which was representative of the dry weight gain of the microparticles.5 The process parameters were as follows: inlet temperature at 45 ± 1 °C; temperature of the bioactive componentloaded microparticles at 30 ± 1 °C; spray rate of coating dispersion at 0.7−0.8 mL/min; atomization pressure at 0.15 MPa; and fluidization pressure at 0.15 MPa. After coating, the coated microparticles were further fluidized dried at 35 °C for different times.
The present investigation attempts to develop an oral colontargeted controlled-release system by an aqueous dispersion film coating process at low temperature. Starch was treated by a high-temperature−pressure reaction, enzymatic debranching, and retrogradation. The structural changes and the digestion resistibility were investigated. With the aqueous dispersion film coating technique, the formation of RS3 and the film coating could be achieved simultaneously. Thus, in this study, insulinloaded microparticles coated with the in situ formed RS3 film were prepared, following optimization of the colon-targeting controlled-release property of the RS3 film-coated microparticles by in vitro experiments (i.e., under the conditions of simulated human GI tract). Furthermore, the in vivo effectiveness of the RS3 film-coated insulin-loaded microparticles was evaluated by tissue immunocytochemistry and in vivo insulin bioactivity studies in streptozotocin (STZ)-induced diabetic rats.
■
MATERIALS AND METHODS
Materials. High-amylose corn starch (Gelose 50, G50) was obtained from Penford (Australia). Pullulanase was of analytical grade and supplied by Yulibao Biology and Technology Co., Ltd. (Guangzhou, China). Termamyl α-amylase (120 KNU/g) from Bacillus licheniformis and Amyloglucosidase (300 AGU/g) from Aspergillus niger were purchased from Novo Nordisk Bioindustrials (Guangzhou, China). Microcrystalline cellulose was purchased from Anhui Shanhe Medicinal Accessary Material Co., Ltd. (Huainan, China). 5-Aminosalicylic acid (5-ASA) was supplied by Yuancheng Technology Development Co., Ltd. (Wuhan, China). Commercial long-acting insulin was purchased from Newprobe Bioscience and Technology Co., Ltd. (Beijing, China). Medicinal starch was supplied by Defeng Starch Sugar Co., Ltd. (Shunde District, Foshan, China). Pepsin, pancreatin, and STZ were purchased from Sigma-Aldrich Co. LLC (Santa Clara, CA, USA) and were of pharmaceutical grade. The rest of the chemical reagents were of analytical grade. Preparation of RS. The preparation procedure used in this work was according to our earlier method of high-temperature−pressure and debranching treatment, which can increase the RS content of starch.31 About 25 g of dry high-amylose corn starch was dispersed in 225 mL of water and cooked in a sealed pressure reactor (Parr 4545, Parr Instrument Co., USA) at 110 °C under the pressure of 12.4 MPa with stirring at 200 rpm for 30 min. Then, the gelatinized starch dispersion was cooled to 60 °C, and its pH value was adjusted to 5.0 using 10% (w/v) citric acid. Subsequently, different amounts of pullulanase were added to the reactor for debranching the starch molecules at 60 °C under atmospheric pressure with stirring at 20 rpm for 6 h. The debranched starch was cooked at 100 °C for 10 min to stop the enzymatic hydrolysis and then cooled and stored at 4 °C for 24 h (during which retrogradation happened). Then, parts of the retrograded starch pastes were spray-dried for the analysis of characteristics, and the other parts were diluted by purified water for the preparation of the RS3-based aqueous dispersions. Digestion Resistibility and Structural Changes of the RS Samples. Digestion Resistibility in the Upper GI Tract. The RS content of the RS3 samples was determined by using method 991.43, total dietary fiber (TDF), of the Association of Official Analytical Chemists (AOAC) in in vitro model with Termamyl α-amylase and Amyloglucosidase. Molecular Molar Mass (Mw) Analysis. The molecular molar mass (M w ) analysis was carried out by using a gel permeation chromatography (GPC) system coupled with a multiangle light scattering (MALS) detector and a refractive index (RI) detector. The GPC system consisted of a pump (1515, Waters, Milford, MA, USA), an autoinjector with a 0.1 mL loop (717, Waters), and a column (Sytyragel HMW7 GPC column, 7.8 × 300 mm, Waters). The MALS detector (632.8 nm, DAWN HELEOS, Wyatt Technology, Santa Barbara, CA, USA) and the RI detector (Optilab rex, Wyatt Technology) were connected to the column. 3600
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Table 1. RS3 Samples and Their GPC-MALLS Parameters, Crystallinity, and SAXS Parameters pullulanase activity (U/g)
RS contenta (%)
0 5 10 50 100 200
± ± ± ± ± ±
a
10.4 10.8 15.6 20.1 34.3 41.8
A
0.4 0.7A 0.6B 0.7C 0.7D 0.8E
Rga (nm)
Mw (g/mol) 3.501 1.463 7.315 3.271 1.692 3.067
× × × × × ×
6
10 106 105 105 105 104
b A
(2% ) (5%)B (2%)C (1%)D (3%)E (1%)F
85.0 56.2 47.5 36.2 33.8 29.4
(1%)A (1%)B (2%)C (3%)D (9%)D (5%)E
conformation
crystallinity (%)
α
Ds
spherical spherical spherical spherical spherical spherical
34.4 37.2 41.4 45.6 59.5 62.7
−3.82 −1.71 −1.81 −1.87 −2.06 −2.06
2.18
Dm
Aq
1.71 1.87 1.87 2.06 2.06
0.1077 0.7190 0.7637 0.7681 0.8395 0.8420
Values followed by the different upper case letters within a column differ significantly (n = 3, P < 0.05). bPrecision of global fit.
In Vitro Release Testing and Related Morphological Observation. The bioactive component release tests were carried out using a dissolution rate test apparatus according to the USP23 dissolution method. The RS3 film coated microparticles were incubated in simulated gastric fluid (SGF) for the first 2 h, then in simulated intestinal fluid (SIF) for another 6 h, and afterward in simulated colonic fluid (SCF) for an additional 22 h, in sequence, all at 37 °C.5,28,29 When the simulated digestive fluid was changed, the microparticles were filtered by filter paper under vacuum, washed with distilled water two times, and then put into the following simulated digestive fluid. At each of the predetermined time points, 5 mL of the tested sample was withdrawn and analyzed for the 5-ASA or insulin content by a UV spectrophotometer (Unico, UV-3802, Shanghai, China). The morphological change of the RS3 film-coated microparticles during the release process in the simulated fluids was monitored by a scanning electronic microscope Hitachi S3700 (Tokyo, Japan). In Vivo Release of Insulin from Microparticles after Oral Administration. The in vivo insulin bioactivity of the microparticles was evaluated using a STZ-induced diabetic animal model. STZ (40 mg/kg of rat) in 0.1 M citrate buffer (pH 4.5) was injected intraperitoneally into male Sprague−Dawley (SD) rats, each weighing 273 ± 12 g (Guangdong Animal Experimental Centre, Foshan, China). Two weeks after the STZ treatment, the fasting plasma glucose levels of the rats were monitored at 24 h after fasting. If two successive glucose levels of the rats were >7.8 mmol/L, the rats were diagnosed as having type II diabetes and were used for the in vivo insulin release studies. Tissue Immunocytochemistry. The rats with type II diabetes were fasted with free access to water for 24 h before the study. The RS3 film-coated insulin-loaded microparticles were orally administered to the stomach via polyethylene tubing under light ether anesthesia. Insulin was dosed at 25 U/kg body weight. Dose selection was based on the expected pharmacodynamics of insulin. At certain times after their oral administration, the rats were sacrificed by cervical dislocation; the tissues of the stomach, small intestine, and colon were obtained, and the insulin distribution in the different parts of the rat GI tract were detected using the hematoxylin−eosin and immunohistochemical staining method. Formalin-fixed, paraffinembedded tissue sections of the stomach, small intestine, and colon of the rats were used for immunohistochemical studies according to the protocol specified in the ImmunoCruz rat ABC staining kit (Santa Cruz Biotechnology, Inc.). The section slides were washed in xylene and hydrated in different concentrations of alcohol and then were pretreated with 3% H2O2 for 10 min and 5% nonimmune bovine serum albumin for 15 min to seal the compounds, which can result in nonspecific background brown color. The slides were incubated with the primary antibody against insulin at 4 °C for 12 h. A horseradish peroxidase-conjugated secondary antibody was applied to locate the primary antibody. The specimens were stained with diaminobenzidine chromogen and counterstained with hematoxylin. The presence of brown staining was considered to be a positive identification of insulin. An optical microscope was used to observe the images on the slides, and the images were analyzed by the Image-Pro Plus (IPP) software program. Insulin Bioactivity. The type II diabetic rats were randomly divided into five groups (the average plasma glucose concentrations of the different groups differed by ≤2 mmol/L). Before the test, all of the rats
were fasted for 12 h with free access to water. The insulin-loaded microparticles coated by RS3 film were orally administered to the stomach via polyethylene tubing under light ether anesthesia. Insulin was dosed at different dosages, that is, 15, 25, and 35 U/kg body weight, respectively. The group that was subcutaneously injected with 25 U/kg of commercial long-acting insulin was used as the positive control group and the diabetic group without insulin treatment was used as the negative control group. After insulin administration, the rats in all of the groups were allowed to eat freely. At predetermined time points, the plasma glucose concentrations of the rats were monitored by the glucose oxidase method. The relative pharmacodynamic bioactivity of insulin (Frel%) was calculated using the equations
Frel =
[AUCT ] dose T [AUCiv ] dose iv
× 100
8
AUC =
∑ (tn+ 1 − tn) × n=0
Pn% =
(100 − Pn) + (100 − Pn + 1) 2
Cn × 100 C0
where Cn and tn are the plasma glucose concentration (mmol/L) and monitored time (h) at the nth blood collection, respectively; Pn % is the relative plasma glucose level (% of initial plasma glucose concentration); AUC is the total area of all the trapeziums between every two neighboring points and the 100% horizontal line in the relative plasma glucose concentration−time profile; dose is the intake dosage of insulin; and the subscripts T and iv represent oral administration and subcutaneous injection, respectively. For the analysis of plasma insulin concentrations, blood samples were centrifuged and subsequently determined by an enzyme immunoassay kit (BG Co., USA), and the relative insulin bioavailability (Brel%) of the insulin-loaded microparticles coated with RS3 film after oral administration was calculated.36 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 Preparation and Characteristics of the RS3 Samples. Preparation of the RS3 Samples. As the delivery carrier for colon-specific controlled release system, the starch-based film coated to the surface of the microparticles loaded with bioactive components must have suitable digestion resistibility to realize the delivery of the microparticles targeting the colon. Thus, the digestion resistibility of the RS3 films with different molecular molar masses, formed by the aggregation of gelled starch molecules upon dehydration and drying during the aqueous dispersion coating process, was investigated. The gelled starch molecules with different molecular molar masses were obtained by the high-temperature−pressure and debranching treatment and the RS contents of the treated starch samples with different 3601
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Figure 1. XRD patterns (a) and SAXS patterns of native G50 starch and the RS3 samples with different RS contents: (b) double-logarithmic SAXS patterns; (c) I × q2 − q SAXS patterns; (d) double-logarithmic SAXS patterns in the range of 0.08 < q < 1. The real lines show the relationship I ∼ qα). All of the intensities are shifted along the ordinate to avoid overlapping of the symbols.
amounts of the debranching enzyme, pullulanase, are shown in Table 1. A significant increase in the RS content, reaching a maximum value of 41.8%, was observed when the pullulanase content used in the treatment was increased to 200 U/g. Nevertheless, when the amount of pullulanase was increased to 300 U/g, the RS content was reduced to 33.7%, which indicated that a further increase in the amount of pullulanase reduced the RS content (P < 0.05). The high-temperature−pressure and debranching treatment with a suitable amount of pullulanase, followed by subsequent retrogradation and drying, could result in the degradation of starch molecules, and some of the degraded molecules of suitable molar mass could reassociate to form tightly packed ordered aggregations stabilized by hydrogen bonding, which were more difficult for starch enzymes to hydrolyze, leading to the formation of a higher amount of RS3. However, with a further increase in the amount of pullulanase, the starch molecules could be degraded further, and these molecules could not preferably form tightly packed ordered aggregations; thus, a decrease in the RS content was observed. The results here demonstrate that the digestion resistibility of starch could be improved by the hightemperature−pressure and debranching treatment and the following retrogradation and drying processes. Thus, the RS3 film with suitable digestion resistibility can be obtained through controlling the starch molecular molar mass during the preparation of starch-based aqueous coating dispersion and
the subsequent aqueous dispersion coating process (where retrogradation and drying occur). Weight-Average Molecular Molar Mass (Mw). After the high-temperature−pressure and debranching treatment, the Mw of native G50 starch was reduced from 3.501 × 106 to 1.463 × 106 g/mol when the RS content was 10.8% (Table 1). The Mw was further reduced to 3.271 × 105 g/mol when the RS content was 20.1% and to 3.067 × 104 g/mol when the RS content was 41.8%. This shows that the high-temperature−pressure and debranching treatment could result in a significant decrease in the Mw of starch (P < 0.05). The decrease in the Rg value with an increase in the RS content suggests that the starch molecular size became smaller after the high-temperature−pressure and debranching treatment, which is consistent with the results regarding the Mw. In addition, as seen from the results of the logMw/log Rg, the conformation of molecules of the RS3 samples was spherical and was not changed with the varied RS content. Along with the RS contents of the samples after the treatment with pullulanase, it seems that the degraded starch molecules with Mw = 3.067 × 104 g/mol should preferably form RS3 and the starch molecules with Mw below or above 3.067 × 104 g/mol could be easily digested by the enzyme. Crystalline Structure. Figure 1a shows the wide-angle XRD spectra of native G50 starch and the RS3 samples with different RS contents, and the relative crystallinity values are shown in Table 1. It can be seen that the native G50 starch gives the strongest diffraction peak at around 2θ = 17.0°and a few small 3602
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
increase in the RS content from 34.3 to 41.8% would contribute to the formation of a mass fractal structure in the larger length scale of 20.3 < d < 59.3 nm with the Dm of 2.06. This suggests that the treatment contributed to an increase in the compactness of the scattering objects and the formation of the mass fractal structure in a larger scale range. On the basis of the fractal geometry, the Dm of the linear arrangement, the surface-like arrangement, and the regular arrangement (such as a cube or sphere) are 1, 2, and 3, respectively.35 Thus, the arrangement of the scattering objects in these RS3 samples changed from the linear form to the surface-like form. Considering the RS contents, it is evident that the mass fractal structure in the larger length scale of 20.3 < d < 59.3 nm with a higher Dm value contributed to a higher RS content. The reason could be that some glucosidic bonds of the starch molecules were masked by the ordered aggregations of starch molecular chains in the scattering objects. Meanwhile, when the scattering objects became more compact with an increasing Dm value, it could be more difficult for the starch enzymes to attack the active sites of starch molecules of these scattering objects with a mass fractal structure. In Vitro Insulin Release from the RS3 Film-Coated Microparticles. In Vitro Release Behavior. To optimize the colon-targeting release property of RS3 film, the RS3 filmcoated microparticle delivery system was obtained using 5-ASA as the model bioactive compound and the effects of RS content, plasticizer content, film coating thickness, and heat treatment time after coating on the 5-ASA release behavior from the RS3 film-coated microparticles in the simulated human GI tract were investigated, and the results were presented in Figure 1S of the Supporting Information. On the basis of the optimization of the coating process for colon-targeting obtained from Figure 1S, the RS3 film-coated microparticles, in which insulin was loaded, were prepared using the RS3 film with the molecular molar mass of 3.067 × 104 g/mol (of which the RS content was 41.8%) as the coating material, with the 1,2-propanediol content being 10%, the coating thickness being 30%, and the time of heat treatment after coating being 8 h; the insulin release profiles are shown in Figure 2. It can be seen from the results that the RS3 film-coated microparticles showed a good colon targeting and release property. The cumulative release percentage of insulin was up to 25.31% within the first 8 h in the upper GI tract and then reached 80.66% at 30 h, suggesting that about 55% of insulin could be delivered to the SCF and
peaks at around 2θ values of 5.6°, 15.0°, 22.0°, 23.4°, and 26.0°, which are indicative of the B-type diffraction pattern, and one peak at 19.8°, which was related to the V-type diffraction pattern. Thus, native G50 starch presented a B+V-type diffraction pattern.37,38 Comparaed with the XRD pattern of native G50 starch, RS3 samples with RS content between 10.8 and 20.1% showed a new diffraction peak at around 2θ = 13.0°, and this peak changed to about 14.0° as the RS content increased. These two peaks are also related to the B-type diffraction pattern. For the RS3 samples with RS content of 34.3 and 41.8% the peak at around 2θ = 26.0° became distinct as the RS content increased. Although the RS3 samples exhibit a similar B+V-type crystal form, most of the peak intensity was gradually strengthened with an increase in the RS content, which indicated that the starch molecules in RS3 samples can form better-crystallized B- and V-type crystals after the hightemperature−pressure and debranching treatment. The relative crystallinity sharply increased with an increase in the RS content. Particularly, when the RS content was 41.8%, the relative crystallinity reached 62.7%. Combined with the Mw data, it is concluded that the starch molecules with Mw = 3.067 × 104 g/mol tended to arrange and form the crystalline structure. These results indicate that the total amount of crystallinity should be one of the reasons for the enhanced starch resistibility against enzymatic digestion. Lamellar Structure. Figure 1b shows the double-logarithmic SAXS patterns of native G50 starch and the RS3 samples. It can be seen from Figure 1b that native G50 starch had an apparent scattering peak at q = 0.65 nm−1, which corresponds to a Bragg distance of about 9.7 nm and is considered due to the longrange periodicity occurring from the alternating crystalline and amorphous lamellar structure of the starch granule.34,39−41 After the high-temperature−pressure and debranching treatment with the following retrogradation and drying processes, the peak at q = 0.65 nm−1 disappeared in the scattering patterns of the RS3 samples. Instead, there was a shoulder-like peak at q = 0.42 nm−1, corresponding to a Bragg distance of about 14.9 nm, suggesting the formation of a new repeat aggregation structure with the repeat distance of about 14.9 nm. The difference in the aggregation structure can cause a difference in the electron density, which influences the scattering intensity. The ordering of starch molecules can be 2 quantified by calculating the area under the curve (∫ ∞ 0 I(q)q dq) (Figure 1c). As the RS content increased, the integrated area (Aq) under the SAXS curve also increased (Table 1). During the high-temperature−pressure and debranching treatment with following retrogradation and drying processes, the starch molecules rearranged and aggregated. Thus, the molecular ordering in the aggregation structure of RS3 samples increased. These results indicate that the higher amount of starch molecular aggregation orders as a result of the newly formed repeat aggregation structure with the repeat thickness of about 14.9 nm contributed to the higher resistibility against enzymatic digestion of the RS3 starch samples. Fractal Structure. As presented in the double-logarithmic SAXS patterns (Figure 1d), the solid lines show the relationship of I ∼ q−α and the fractal dimensions are shown in Table 1. It can be seen from Figure 1d and Table 1 that native G50 starch showed a surface fractal structure in the length scale of 36.5 < d < 59.3 nm and the Ds was close to 2.18. For the RS3 samples, a mass fractal structure in the length scale of 31.7 < d < 59.3 nm was shown, with the Dm increasing from 1.71 to 1.87 with an increase in the RS content from 10.8 to 20.1%. A further
Figure 2. Insulin release from RS3 film-coated microparticles using the RS3 film with RS content of 41.8% as the coating material, with the 1,2-propanediol content being 10%, the coating thickness being 30%, and the time of heat treatment after coating being 8 h. 3603
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Figure 3. Surface change of RS3 film-coated microparticles during drug release: (a) microparticles, before release; (b) surface of microparticles, before release; (c) microparticles, after release for 2 h; (d) surface of microparticles, after release for 2 h; (e) microparticles, after release for 8 h; (f) surface of microparticles, after release for 8 h; (g) microparticles, after release for 24 h; (h) surface of microparticles, after release for 24 h; (i) microparticles, after release for 30 h; (j) surface of microparticles, after release for 30 h.
could be released sustainably from the RS3 film-coated microparticles in SCF. Morphological Change of the RS3 Film-Coated Microparticles. To investigate the insulin release mechanism from RS3 film-coated microparticles in the simulated human GI tract, SEM was used to observe the coating film surface on the microparticles after incubation in SGF, SIF, and SCF for the specific periods. The insulin-loaded microparticles coated by the RS3 film under the optimization coating process were incubated in SGF, SIF, and SCF for the specific periods, and the results are shown in Figure 3. It is revealed that the surface morphology of the RS3 film-coated microparticles could be affected by the incubation time. From Figure 3a,b, it can be seen that the microparticles were rounded and the surface of the coated RS3 film showed no obvious change after 2 h in SGF (Figure 3c,d), suggesting that the film could resist the acid in SGF. Transferred to and immersed in SIF (pH 6.8) for another 6 h, the microparticles displayed a few small holes on their coated film. After 2 h in SGF and 6 h in SIF (Figure 3e,f), although a few small holes appeared, the film still kept its integrity, which showed the ability to resist the enzyme in SIF. The next immersion of the microparticles in SCF (pH 7.0) for another 16 h resulted in an increased amount of holes on the surface. After 24 h in these digestive fluids (2 h in SGF, 6 h in SIF, and 16 h in SCF, Figure 3g,h), more holes and cracks emerged and the integrity of the film was impaired. With the prolongation of soaking time (Figure 3i,j), the sizes of holes on the microparticle surfaces were even larger and cracks appeared, deepening toward the interior, suggesting that the coated RS3 film was almost disintegrated. It is shown that the RS3 film has good resistance to degradation by the acid and enzymes in the simulated upper GI tract. Therefore, the RS3 film exhibits a good colon-targeting property.
In Vivo Insulin Release from RS3-Film Coated Microparticles. Distribution of Insulin in the GT Epithelial Tissues of Rats. After testing the in vitro colon targeting behavior of the RS3-film coated microparticles, it was worthwhile to assess the in vivo behavior of formulation in terms of the residence time of formulation in different parts of the GI tract. By tissue slices and immunohistochemistry, the insulin release property of the RS3 film-coated microparticles in the type II diabetic model rats was studied. The images showing the insulin distribution in the GI epithelial tissues of rats after administration of the RS3 film-coated microparticles for different times are shown in Figure 4. Before administration, the immunohistochemical images displayed a blue background, indicating no positive insulin sign in the stomach, small intestine, and colon of the rats (Figure 4a−c). At 2 h after intragastric administration, there was a slight insulin positive sign (the brown part) in the stomach of rats (Figure 4d), which means a very small amount of insulin released in the stomach. Meanwhile an insulin positive sign was also observed in the small intestine (Figure 4e), which was due to the fact that part of the insulin released in the stomach had reached the small intestine and some of the insulin released from the microparticles reached the small intestine, although no positive sign was observed in the colon at this moment (Figure 4f). At 8 h after intragastric administration, although there was no insulin positive sign in the stomach, a strong insulin positive sign was shown in the small intestine (Figure 4g,h) and a slight insulin positive sign was reflected in the colon (Figure 4i). From 24 h after intragastric administration, the insulin positive sign concentrated in the colon, suggesting that the microparticles had reached the colon, where the insulin had been released from the microparticles (Figure 4l,o). 3604
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Figure 4. Insulin distribution in the gastrointestinal epithelial tissues of rats after administration of the RS3 film-coated microparticles for different times: (a) stomach, 0 h; (b) small intestine, 0 h; (c) colon, 0 h; (d) stomach, 2 h; (e) small intestine, 2 h; (f) colon, 2 h; (g) stomach, 8 h; (h) small intestine, 8 h; (i) colon, 8 h; (j) stomach, 24 h; (k) small intestine, 24 h; (l) colon, 24 h; (m) stomach, 30 h; (n) small intestine, 30 h; (o) colon, 30 h.
By the color image analytic system IPP, the optical density of insulin could be determined, and the result is shown in Table 2. Before administration, no insulin was determined in the stomach, small intestine, and colon of the rats. At 2 h after administration, a small amount of insulin was released in the stomach and small intestine. Then, the microparticles reached the small intestine and released insulin at 8 h, as shown by a high optical density of insulin determined in the small intestine and a slight optical density of insulin in the colon. At 24 h after administration, the optical density of insulin in the colon reached the highest value (0.57 ± 0.02). When the administration time reached 30 h, a low optical density of insulin was determined in the small intestine, but the optical density of insulin was maintained at a high level, indicating a
Table 2. Integrated Optical Density of Insulin in the Digestive Tract Tissues of Rats at Different Times after Administration time (h) 0 2 8 24 30
stomacha 0.21 ± 0.02
small intestinea
colona
± ± ± ±
0.16 ± 0.02A 0.57 ± 0.02C 0.40 ± 0.02B
0.12 0.49 0.13 0.11
0.01A 0.03B 0.01A 0.01A
Values are means ± SD of three determinations (n = 3). Values followed by different upper case letters within a column differ significantly (P < 0.05). a
3605
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Table 3. Plasma Glucose, Corresponding Plasma Insulin Concentrations at Different Postadministration Times, and Pharmacokinetic Parameters of Insulin in Diabetic Rats Following the Administration of Different Insulin Formulations (n = 6 for Each Group)a control group plasma glucose (mmol/L) C (mIU/L)
0 2 6 8 10 12 24 26 30
Cmax(mIU/L) Tmax(h) AUC (mIU·h/L) Brel (%)
25.4 26.4 28.0 22.8 19.7 20.9 24.6 28.0 27.1
± ± ± ± ± ± ± ± ±
1.1 1.4 1.2 1.3 0.8 1.0 0.9 0.8 0.3
insulin injected group (25 U/kg) insulin (mIU/L) 18.52 20.11 19.81 17.09 15.35 20.12 29.90 27.03 25.73
± ± ± ± ± ± ± ± ±
2.62 1.78 2.20 3.54 4.77 2.23 3.78 5.90 4.69
plasma glucose (mmol/L) 24.0 19.5 6.2 5.3 6.3 3.4 4.2 17.0 21.6 667.79 12 13052.2 100
± ± ± ± ± ± ± ± ± ±
1.5 1.5* 0.5* 0.4* 0.4* 0.4* 0.5* 2.3* 0.6* 68.34
± 20.5
insulin (mIU/L) 20.65 39.06 493.25 524.17 490.40 667.79 635.53 67.72 39.56
± ± ± ± ± ± ± ± ±
3.22 4.32* 34.54* 22.98* 56.34* 68.34* 34.44* 7.23* 4.32
RS3 film-coated microparticles group (25 U/kg) plasma glucose (mmol/L) 23.0 18.7 11.8 7.2 6.5 5.6 5.0 6.9 15.7 601.17 24 11508.1 88.2
± ± ± ± ± ± ± ± ± ±
0.9 0.6* 0.7*# 0.4*# 0.5* 0.6* 0.6* 0.3*# 0.6*# 29.79
insulin (mIU/L) 21.90 40.60 196.80 343.76 500.14 530.23 601.17 302.91 128.81
± ± ± ± ± ± ± ± ±
3.80 7.34* 19.10*# 18.73*# 27.89* 23.90* 29.79* 11.8*#1 10.23*#
± 18.6 ± 1.2
a
Control, diabetic model control group without any treatment; C, plasma glucose concentration at different postadministration times; Cmax, maximum plasma insulin concentration; Tmax, time at which Cmax is attained; AUC, area under the plasma concentration−time curve. *, means compared with those of the diabetic model control group (P < 0.01); #, means compared with those of the insulin injected group (P < 0.01).
Figure 5. Relative plasma glucose concentration of the diabetic rats following (a) the administration of different insulin formulations and (b) the oral administration of different doses of insulin.
Although theoretically endogenous insulin could still be produced in the type II diabetic rats, the plasma insulin concentration was too low to control the plasma glucose level. Therefore, the plasma glucose concentration of this group remained higher during the whole monitoring period (30 h). For the insulin-injected (positive control) group, from 2 to 6 h after injection, there was a sharp increase (from 39.06 ± 4.32 to 493.25 ± 34.54 mU/L) in the plasma insulin concentration, accompanied by a sharp decrease (from 19.5 ± 1.5 to 6.2 ± 0.5 mmol/L) in the plasma glucose concentration. The percentage of decrease in the plasma glucose concentration was 74.2 ± 0.8% from the beginning (0 h) to 6 h (Figure 5a). Although a strong hypoglycemic effect was observed between 6 and 24 h after injection in the insulin-injected group rats (Figure 5a), a glycopenia (an abnormally low plasma glucose level of 3.4 ± 0.4 mmol/L) occurred at 12 h after injection when a maximum plasma insulin concentration of 667.79 ± 68.34 mU/L was reached. After that, the plasma glucose concentration experienced a rapid increase. After the RS3 film-coated microparticles containing 25 U/kg insulin were orally administered, a high level of the plasma
large amount of insulin released in the colon. These results showed that the RS3 film-coated microparticles could target insulin to the colon. In Vivo Bioactivity of Insulin-Loaded RS3 Film-Coated Microparticles. In the treatment of diabetes mellitus, steadily decreasing the plasma glucose concentration is important to the patients. The fluctuation of plasma glucose concentration in the therapeutic process could make the patients suffer from glycopenia and glycemic excursion, which might lead to diabetic complications.42 In this study, the plasma glucose concentration and corresponding plasma insulin concentration of the different type II diabetic rat groups with different treatments at different postadministration times were investigated, and the results are presented in Table 3. It can be seen from Table 3 that the plasma glucose concentration of the type II diabetic rats (negative control group) without insulin treatment was higher (18.6−29.7 mmol/L) than those of the positive control group, subcutaneously injected with commercial long-acting insulin, and the test groups which were fed by the RS3 film-coated microparticles with the same insulin load through an oral route. 3606
dx.doi.org/10.1021/jf500472b | J. Agric. Food Chem. 2014, 62, 3599−3609
Journal of Agricultural and Food Chemistry
Article
Table 4. Plasma Glucose Concentrations and Relative Bioavailability in the Different Groups with Different Dosages of Insulin in the RS3 Film-Coated Microparticles (n = 6 for Each Group) insulin dose* 15 U/kg plasma glucose concentration (mmol/L) at different postadministration times (h)
0 2 6 8 10 12 24 26 30
AUC Frel(%)
23.7 20.1 16.6 9.1 7.6 6.4 5.7 11.8 17.2 1644.7 88.2
± ± ± ± ± ± ± ± ± ± ±
1.4 1.6 0.8 2.0 2.3 0.8 0.3 0.8 0.2 74.0 4.0
25 U/kg 23.0 18.7 11.8 7.2 6.5 5.6 5.0 6.9 15.7 1817.9 97.5
± ± ± ± ± ± ± ± ± ± ±
0.9 0.6 0.7* 0.4* 0.5 0.6 0.6 0.3* 0.6* 30.5 1.6
35 U/kg 25.9 15.7 9.1 5.3 4.5 4.9 4.5 6.9 7.6 2145.4 115.1
± ± ± ± ± ± ± ± ± ± ±
0.8 0.4*# 1.2*# 1.1*# 0.6*# 0.8 0.2 0.3* 0.7*# 23.8 1.3
*
*, means compared with those of the low-dose group (P < 0.01); #, means compared with those of the medial-dose group (P < 0.01).
range for another 14 h (seen from Figure 5b). The medial-dose group experienced a steady decrease in the plasma glucose concentration from 8 h after oral administration, which was a little more rapid than that of the low-dose group. In contrast to those of these two groups, the plasma glucose concentration of the high-dose group decreased more rapidly within 2−10 h after oral administration (P < 0.01) and kept within the lower range from 8 to 30 h (for a total of 22 h) (seen from Figure 5b). The maximum percentages of decrease in the plasma glucose concentration of the low-dose microparticles group, medial-dose group, and high-dose group were 76.0 ± 1.1, 78.0 ± 2.0, and 82.8 ± 0.6%, respectively (Figure 5b). These results indicate that the insulin plasma concentration was related to the dosage of administration. An increase in the dosage of oral administration of insulin could lead to a remarkable decrease in the plasma glucose level. Compared with the insulin-injected group, the relative pharmacodynamic bioactivities (Frel%) of insulin in the high-dose microparticles group, medial-dose microparticles group, and low-dose microparticles group were 115.1 ± 1.3, 97.5 ± 1.6, and 88.2 ± 4.0%, respectively (Table 4). In conclusion, the RS3 film-coated microparticles can protect the insulin from enzymatic degradation in the upper digestive tract, prolong the insulin release time, and enhance the hypoglycemic effect. The plasma glucose concentration of the diabetic model rats was decreased steadily by using the RS3 film-coated microparticles for the delivery of insulin. Thus, this study demonstrates the potential of the RS3 film-coated microparticles for the colon-targeting delivery of insulin and other polypeptide or protein ingredients.
insulin concentration could be maintained from 6 h after oral administration as well; furthermore, for a longer period of time (up to 30 h) compared with the trend shown by the insulininjected (positive control) group. A moderate hypoglycemic effect was observed at 6 h after oral administration but the plasma glucose concentration was still higher than that of the insulin-injected group at the same time. There was a 47.8 ± 1.6% decrease in the plasma glucose concentration from 0 to 6 h when the plasma insulin concentration was increased to 196.80 ± 19.10 mU/L (Figure 5a), indicating that some insulin released from the RS3 film-coated microparticles had entered the colon. At 8 h after oral administration, the plasma glucose concentration was decreased to 7.2 ± 0.4 mmol/L, and there was a 68.3 ± 1.3% decrease from the beginning (0 h) (Figure 5a). A significant hypoglycemic effect was shown from 8 to 26 h after oral administration of the RS3 film-coated insulin-loaded microparticles (Figure 5a). Although the plasma glucose concentration increased to 15.7 ± 0.6 mmol/L at 30 h after oral administration, it was still much lower than that of the insulin-injected group rats (21.6 ± 0.6 mmol/L). Furthermore, no glycopenia was observed between 8 and 26 h (total 18h) after oral administration and the hypoglycemic effect was slowly but steadily maintained for a long time (Figure 5a), suggesting that insulin could be well protected by the RS3 film-coated microparticles from the degradation by the various proteolytic enzymes in the upper digestive tract and therefore the colontargeting controlled release of insulin was achieved. The results here show that the RS3 film-coated microparticles could target insulin to the colon and could maintain a sustainable release of insulin, which would not lead to hypoglycemia and glycemic excursion. Compared with that of the insulin-injected group, the relative insulin bioavailability (Brel%) of the RS3 film-coated microparticles containing the same insulin dosage after oral administration was 88.2 ± 1.2% (Table 3), and the relative pharmacodynamic bioavailability (Frel%) was as high as 97.5 ± 1.6% (Table 4). Experiments were further carried out to investigate the effect of dosage of oral administration of the RS3 film-coated microparticles received by the type II diabetic model rats on the change in the plasma glucose concentration, and the results are shown in Table 4. The hypoglycemic effect was further increased by an increase in the dosage of oral administration. It can be seen that the plasma glucose concentration declined to a lower range (
