Preparation, Characterization, and Oral Delivery of Insulin Loaded

Mar 17, 2009 - State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai...
0 downloads 5 Views 5MB Size
Download PDF
Biomacromolecules 2009, 10, 1253–1258

1253

Preparation, Characterization, and Oral Delivery of Insulin Loaded Carboxylated Chitosan Grafted Poly(methyl methacrylate) Nanoparticles Fuying Cui,†,‡ Feng Qian,†,‡ Ziming Zhao,†,‡ Lichen Yin,† Cui Tang,† and Chunhua Yin*,†,‡ State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China, and Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China Received January 8, 2009; Revised Manuscript Received February 15, 2009

To improve the efficiency of insulin via oral administration, pH-sensitive carboxylated chitosan grafted poly(methyl methacrylate) nanoparticles (CCGN) were prepared. CCGN were characterized by 1H NMR, dynamic light scattering, zeta potential, and transmission electron microscopy, and the hypoglycemic effect of insulin loaded CCGN via the oral route was evaluated in normal and diabetic rats. CCGN exhibited a homogeneous morphology and a spherical shape with core-shell structure. They were aggregated in simulated gastric fluid while separated in simulated intestinal fluid. Insulin was mainly located in the shell of the CCGN via hydrogen bonding, electrostatic interaction, and Van der Waals force. Insulin release from the CCGN exhibited a pH-sensitive property in that it had a slow release rate at pH 2.0 and a fast release rate at pH 6.8 and 7.4. The pharmacological bioavailability after oral administration of insulin loaded CCGN at a dose of 25 IU/kg was found to be 9.7%. Besides, CCGN showed desirable tissue and blood compatibility. Therefore, the CCGN would be a promising delivery carrier for protein drugs via the oral route.

Introduction The oral route seems to be the most convenient and comfortable way for insulin administration. However, insulin is readily degraded under the low pH of the gastric medium and by various proteolytic enzymes in the gastric intestinal (GI) tract. Therefore, it needs to be protected from the harsh environment when given orally.1 To overcome these problems, delivery systems including nanoparticles, microcapsules, liposomes, and emulsions are used.2 Some investigators have observed that the number of nanoparticles that pass the intestinal epithelium is larger than the number of microspheres and that not only the M cells but also the normal enterocytes are involved in the transport.3,4 Despite great progress of the knowledge in this field, there are still some limitations considering nanoparticles as transmucosal protein delivery systems, including their instability in the GI fluids, limited interaction, and transport across the mucosal barrier, use of organic solvent during preparation,5 and poor absorption of many drugs as a result of their high hydrophilicity. Shinji Sakuma’s research group has developed a method for preparing nanoparticles composed of novel graft copolymer which possesses a hydrophobic backbone and hydrophilic branches. For example, they developed nanoparticles having pH sensitive anionic, cationic, thermosensitive nonionic PNIPAAm, and poly(N-vinylisobetyramide) on the surfaces,6 which lacked use of organic solvent during preparation. Meanwhile, stability of the formed nanoparticles was improved. However, the reaction process was complex, which limited its effective application as a carrier for hydrophilic protein drugs. * To whom correspondence should be addressed. Tel.: +86-21-65643797. Fax: +86-21-55522771. E-mail address: [email protected]. † Department of Pharmaceutical Sciences. ‡ Department of Biochemistry.

Presently, chitosan as mucoadhesive polymer is widely used.7-9 However, chitosan is a polycation with an intrinsic pK (pKo) of 5.5, which will thus lose its charge and precipitate in neutral and basic environment. With carboxyl groups on the glucosamine units of the chitosan structure, carboxylated chitosan has successfully overcome the limited solubility at neutral and weakly alkaline pH values. So, in this investigation, carboxylated chitosan was used as hydrophilic branches and poly(methyl methacrylate) (PMMA) was used as hydrophobic backbone. They can easily react with each other through graft polymerization10-12 and, thus, spontaneously form nanoparticles in aqueous solution. This method involves simple reaction process without use of organic solvents, and existence of carboxylated chitosan in the nanoparticles greatly contributed to their high encapsulation efficiency for hydrophilic proteins, enhancement of drug absorption, prolonged drug residence in the gut, and favorable enzymatic inhibition.13-15 With insulin as a model protein, drug loading, in vitro release, in vivo hypoglycemic effect, and its absorption mechanism in the GI tract were also evaluated.

Experimental Section Materials. Carboxylated chitosan (Mη: 8.5 × 104 Da and the pKo value about 3.0) with the substitution degree (carboxyl groups) of more than 60% was supplied by Yuhuan Ocean Biochemistry Co. Ltd. (Zhejiang, China). Methyl methacrylate (MMA, Mw: 100.12 Da) and ammonium persulfate (APS, Mw: 228.20 Da) were purchased from Lingfeng Chemical Co. (Shanghai, China) and Anjian Chemical Co. (Shanghai, China), respectively. Insulin (28 IU/mg) was purchased from Xuzhou biochemical plant (Jiangsu, China). Phycocyanin was extracted from spiral algae in our laboratory. 6-Coumarin (content above 99%) wasobtainedfromSigma-Aldrich(St.Louis,U.S.A.).MaleSprague-Dawley (SD) rats (body weight 200-250 g) were provided by the Animal Care Center, Fudan University. The study protocol was reviewed and

10.1021/bm900035u CCC: $40.75  2009 American Chemical Society Published on Web 03/17/2009

1254

Biomacromolecules, Vol. 10, No. 5, 2009

approved by the Institutional Animal Care and Use Committee, Fudan University, China. Preparation of Insulin Loaded CCGN and 6-Coumarin Labeled CCGN. CCGN were prepared by graft polymerization according to our previous work.16 Briefly, carboxylated chitosan was dissolved in water under magnetic stirring. Methyl methacrylate was dissolved in the above mixture at 75 °C. Thereafter, APS solution was added. The reaction was completed after 24 h. PMMA was prepared when carboxylated chitosan was not added. Insulin loaded CCGN and 6-coumarin labeled CCGN were also prepared according to our previous work.16 In brief, insulin was dissolved in 0.01 N HCl and mixed with CCGN colloid. The mixture was centrifuged 1 h later at 15000 rpm for 40 min at 4 °C and was washed three times with water to remove the free drug in the supernatant. Insulin concentration in the supernatant was determined by the Lowry method.17 The drug encapsulation efficiency and loading capacity was defined as follows

encapsulation efficiency(%) ) (total amount of insulin insulin in the supernatant) × 100/total amount of insulin loading capacity(%) ) (total amount of insulin insulin in the supernatant) × 100/weight of nanoparticles 6-Coumarin was dissolved in ethanol and mixed with CCGN colloid. The labeled nanoparticles were collected 1 h later by centrifugation at 12000 rpm for 40 min, washed with water three times to remove excessive 6-coumarin, and then lyophilized. Characterization of CCGN. The chemical structure of carboxylated chitosan and CCGN was confirmed by 1H NMR (D2O or CF3COOD; AVANCE DMX500, Bruker, German). Morphology of the nanoparticles was observed using transmission electronic microscopy (TEM, Hitachi, Japan). Samples were immobilized on copper grids and negatively stained with phosphothungstic acid (PTA). After drying at room temperature, morphology of the particles was examined. The particle size, zeta potential, and polydispersity index of the nanoparticles were measured with dynamic light scattering and electrophoretic light scattering technique using Nicomp 380ZLS (NICOMP Particle Sizing Systems, Santa Barbara, CA, U.S.A.). The fixed aqueous layer thickness (FALT) of particles was determined by zeta potential measurement with various concentrations of NaCl. The calculation of FALT (L) was based on the linear correlation between ln(§) (zeta potential) and κ (Debye-Huckel-parameter): ln(§) ) ln A - κL, where A was a constant and κ could be expressed in nm-1 unit as κ ) c/ 0.3, where c was the concentration of NaCl solution. Then ln(§) was plotted against κ and a straight line was obtained. The slope gave L, FALT in nm unit. Stability of CCGN. CCGN were incubated at 37 °C for 15 min in Na2SO4 solution with concentrations ranging from 0 to 1 mol/L. The turbidity was measured using a UV spectrophotometer at 400 nm. CCGN and 6-coumarin labeled CCGN were incubated in simulated gastric fluids (pH 1.2 HCl solution with pepsin), simulated small intestinal fluids (pH 6.8 PBS with trypsin), and simulated large intestinal fluids (pH 7.4 PBS with trypsin), respectively. Their morphology was observed by TEM and fluorescence microscopy, respectively. Their diameters were determined using Nicomp 380ZLS. Characterization of Insulin Loaded Nanoparticles. Endogenous Fluorescence Spectroscopy. Insulin solution, CCGN colloid, and insulin loaded CCGN colloid was scanned by fluorescence spectroscopy (Hitachi, Japan). Excitation wavelength was 282 nm for emission spectra, and the scanning range was 200∼400 nm. Extrinsic Fluorescence Spectroscopy. Fluorescein isothiocyanate (FITC) labeled insulin (FITC-insulin), CCGN, and FITC-insulin loaded CCGN colloids were also scanned by fluorescence spectroscopy. Excitation wavelength was 488 nm for emission spectra, and the

Cui et al. scanning range was 470∼600 nm. Circular Dichroism (CD) Spectroscopy. CD spectra of free insulin and insulin loaded CCGN colloid were recorded on a CD spectrometer (Jasco, Japan) using a 1 cm cell length at a speed of 200 nm/min, a response time of 0.25 s, and a bandwidth of 1 nm. Double Labeled Fluorescence. Phycocyanin was dissolved in water in a foil-covered flask. Thereafter, 6-coumarin labeled CCGN colloid was added, stirred under light exclusion for 1 h, and centrifuged at 12000 rpm for 30 min. In Vitro Release. The mixture of insulin and CCGN (1 mL) was centrifuged at 12000 rpm for 30 min to separate the free drug and the settlement was redispersed in water three times, and the settlement that was insulin loaded CCGN were incubated in 1 mL release media of pH 7.4 PBS, pH 6.8 PBS, and pH 2.0 HCl, respectively. In vitro release was carried out at 37 °C and 100 rpm. At each time interval, the supernatant was withdrawn after centrifugation at 12000 rpm for 30 min, and replaced with fresh release media.18 The amount of insulin released was determined by the Lowry method. Hypoglycemic Effects Following Subcutaneous Administration of Insulin Loaded CCGN Colloid in Normal Rats. To verify the bioactivity of insulin after drug loading, insulin loaded CCGN colloid and free insulin solution were s.c. injected to normal rats at a dose of 1 IU/kg with four rats per group. Blood samples were collected from the tail vein at predetermined time intervals. The plasma glucose levels were determined using a glucose-oxidase kit (Shanghai Kexin Biochemical Reagent Industry, Shanghai, China).19 Hypoglycemic Effects Following Oral Administration of Insulin Loaded CCGN Colloid in Normal Rats. The following formulations were administered intragastrically to rats (four rats per group): (1) insulin solution (100 IU/kg), (2) insulin loaded CCGN colloid (100 IU/kg), (3) insulin loaded CCGN colloid (50 IU/kg), (4) insulin loaded CCGN colloid (25 IU/kg), and (5) insulin loaded CCGN colloid (15 IU/kg). Blood sampling and determination of serum glucose level were the same as above. The percentage of glucose reduction at each time after oral administration was calculated. The extent of hypoglycemic response was calculated using the trapezoidal method as the area above the curve (AAC) for 0∼12 h. The relative pharmacological availability (PA (%)) of the orally administered insulin was calculated according to the following equation

PA(%) ) ([AACoral(0 ∼ 12h)]/doseoral) × 100/([AACs.c.(0 ∼ 12h)]/doses.c.)

where [AACoral (0∼12 h)] was the area determined after oral administration of insulin loaded CCGN and [AACs.c. (0∼12 h)] was the area determined after subcutaneous injection of 1.0 IU/kg insulin solution. Hypoglycemic Effects Following Oral Administration of Insulin Loaded CCGN in Diabetic Rats. Type 1 diabetes was induced in normal rats through injection of 40 mg/kg alloxan via the tail vein. Rats with blood glucose level higher than 16.67 mmol/L were used for further studies. The following formulations were administered intragastrically to diabetic rats: (1) insulin solution (50 IU/kg), (2) insulin loaded CCGN colloid (50 IU/kg), and (3) insulin loaded CCGN colloid (25 IU/kg). Blood sampling and determination of serum glucose level were the same as above. Biocompatibility of CCGN. Biochemical EValuation of Intestinal Damage. Intestinal damage induced by CCGN was evaluated in the ileum of normal rats as described previously.16 Briefly, 0.5 mL of the CCGN colloid (1%) was administered to rat ileum and incubated in the 10 cm segment for 1 h. Then, the ileal loop was washed with 1.0 mL of PBS, and the concentration of lactate dehydrogenase (LDH) in it was determined using a LDH-UV kit (Xinchang Company, China). PBS served as a negative control, while 1% sodium tauroglycocholate served as a positive control.

Nanoparticles for Oral Insulin Delivery

Biomacromolecules, Vol. 10, No. 5, 2009

1255

Hemolysis Assay. Hemolysis induced by CCGN was evaluated according to the work of Cenni et al.20 A total of 100, 200 and 300 µL of CCGN colloid (1%) were added to the erythrocyte suspension and was incubated at 37 °C for 1 h. Samples were centrifuged at 3000 rpm for 5 min and the absorbance of the supernatant was determinted. Water served as a positive control and normal saline served as a negative control. Statistical Analysis. Data were expressed as mean ( standard deviation (S.D.). Significant differences in mean values were evaluated by a Student’s t-test. A statistical difference was considered when the p value was less than 0.05, while a significant statistical difference was considered when the p value was less than 0.01.

Results and Discussion Characterization of CCGN. As a chitosan derivative, carboxylated chitosan with moieties bearing carboxylic acid groups could also be grafted by methyl methacrylate.12 The amine (-NH2) groups in carboxylated chitosan and the initiator of APS together formed a redox system and initiated the copolymerization by generating free radicals in the macromolecules.21 The reaction could be divided into a two-stage process. First, the ionic initiator (S2O82-) decomposed into HSO3• radicals at a higher reaction temperature. Then, the HSO3• radicals attacked the saccharide unit of carboxylated chitosan. If more carboxylated chitosan macroradicals were generated, more active sites could react with methyl methacrylate, and thus initiated the propagation reaction of methyl methacrylate. In present study, carboxylated chitosan reacted with methyl methacrylate through graft polymerization. Therefore, their nanostructures were relatively stable compared with other chitosan nanoparticls such as chitosan coated nanoparticles or nanoparticles produced through ionic gelation of tripolyphosphate (TPP) and chitosan.22 The 1H NMR spectrum of carboxylated chitosan dissolved in D2O was given in Figure 1A. The multiplet signals at 3.0-4.0 ppm corresponded to the ring methenyl protons of carboxylated chitosan backbones. The 1H NMR spectrum of CCGN dispersed in D2O was given in Figure 1B and only the proton signals of carboxylated chitosan were observed. All the characteristic peaks including CH3 (0.67-0.99 ppm), methylene-methine envelope (1-2.5 ppm), and 3.0-4.0 ppm were observed for both MMA and carboxylated chitosan when CCGN was dissolved in CF3COOD (Figure 1C). The hydrophobic segments in CCGN were limited in D2O, thus causing shielding of the proton signal in CCGN. As CCGN was dissolved, the dissociation of CCGN led to orientation of the hydrophobic chains to the aqueous phase, and the alkyl groups appeared accordingly. Therefore, the hydrophilic carboxylated chitosan and the hydrophobic acrylate polymer portions spontaneously formed nanoparticles with core-shell structures in aqueous solution. TEM images showed that CCGN were spherical and separated from each other. CCGN demonstrated a unimodal size distribution within 200-300 nm and the polydispersity index was 0.014, indicating that the nanoparticles were a relatively homogeneous dispersion.23 The zeta potential ranged from -57 to -41 mv. The FALT of CCGN was 2.36 nm, which was larger than that of PMMA (0.9 nm). The highly hydrophilic brush of CCGN could prolong its circulation time.24 Figure 2 showed the morphology of CCGN in the GI tract. CCGN aggregated in simulated gastric fluid, reaching 1200 nm in diameter, which led to the zeta potential not to be measured. In simulated small intestinal and large intestinal fluid, CCGN separated from each other, reaching 200-300 nm in diameter, and the zeta potentials were -21.4 and -8.0 mv, respectivly.

Figure 1. 1H NMR spectra of carboxylated chitosan dissolved in D2O (A), CCGN dispersed in D2O (B), and CCGN dissolved in CF3COOD (C).

The stability of CCGN in the GI tract might be affected by pH value and ionic strength. No change in the turbidity was observed as the ionic strength increased from 0 to 1 mol/L, indicating that an increase in salt concentration would not shield the electrostatic forces and ionic strength did not bring about significant influence on the behavior of CCGN. In simulated gastric fluid, most of carboxylic groups of carboxylated chitosan were in the protonated form, thus leading to loss of the electrostatic repulsion between particles and correspondingly aggregation of them. In contrast, almost all the carboxylic groups of carboxylated chitosan were in the ionized form of -COO- in simulated intestinal fluid. The electrostatic repulsive forces increased, thereby resulting in the separation of the nanoparticles. Characterization of Insulin Loaded CCGN. The loading capacity and encapsulation efficiency of CCGN for insulin was 11.9 and 85%, respectively, while the encapsulation efficiency

1256

Biomacromolecules, Vol. 10, No. 5, 2009

Cui et al.

Figure 2. Morphology of 6-coumarin labeled CCGN in simulated gastric (A), small intestinal (B), and large intestinal fluid (C) by fluorescence microscopy. Bar represented 3.9 µm. Morphologies of CCGN in simulated gastric (D), small intestinal (E), and large intestinal fluid (F) by TEM, bar represents 200 nm.

Figure 3. Effect of CCGN on insulin as monitored by circular dichroism in the far (A) and near (B) UV region.

of PMMA was only 10%. Diameters of insulin loaded CCGN with theoretical loading efficiencies of 5, 10, and 15% were 251.6 ( 85.5, 319.6 ( 46.9, and 256.3 ( 86.1 nm, respectively. Zeta potentials of them were -26.4, -24.3, and -22.0 mv, respectively, which showed a slight decrease in their absolute values. Such results demonstrated that insulin might be loaded into the hydrophilic shell of CCGN through interaction with the amide, hydroxyl, and carboxyl groups. The fluorescent characteristics of tyrosine residue in insulin were sensitive to the microenvironment. Free insulin gave the fluorescence emission spectra with a maximum at 308 nm, and the fluorescence disappeared after addition of CCGN. The maximum fluorescence wavelength of insulin at 308 nm was due to the tryptophan residue (Tyr-A14, -A19), and it was likely that, through interaction between CCGN and insulin, the surface positional tyrosine residue was concealed within the CCGN, which led to a disappearance of the strong fluorescence intensity. With the application of FITC-insulin, such presumption was confirmed. The fluorescence of FITC-insulin at 520 nm disap-

peared after being loaded in CCGN, which was due to the shielding effect of CCGN (data not shown). Figure 3 showed the CD spectra of insulin, CCGN, and insulin loaded CCGN at near and far UV region. In the far-UV region, two peaks at 208 and 222 nm were observed for insulin, which corresponded to the R-helix and β-folding. For CCGN, two peaks at 210 and 215 nm were observed, while for insulin loaded CCGN, three peaks at 212, 217, and 222 nm appeared. In the near UV region, the peak at 275 nm for free insulin represented the aromatic side-chains. Three peaks at 275, 320, and 480 nm were detected for CCGN, so as for insulin loaded CCGN. In insulin loaded CCGN, the CD spectra of both insulin and CCGN greatly changed, indicating that insulin might be distributed in the hydrophilic shell of CCGN via hydrogen bonding, electrostatic interaction, Van der Waals force, and chain entanglement between insulin and nanoparticles. Insulin molecules tend to aggregate and form dimer or hexamer due to its hydrophobicity between nonpolar groups. It

Nanoparticles for Oral Insulin Delivery

Biomacromolecules, Vol. 10, No. 5, 2009

1257

Figure 4. Double labeled fluorescence images of 6-coumarin labeled CCGN (A), phycocyanin loaded 6-coumarin CCGN (B), and phycocyanin loaded CCGN (C). Bar represents 6.3 µm.

Figure 5. Insulin release from insulin loaded CCGN in pH 2.0 HCl (9), pH 6.8 PBS (b), and pH 7.4 PBS (0). *p < 0.05, **p < 0.01 vs pH 2.0 HCl group according to Student’s t-test.

usually exists in the form of hexamer and dissociates into monomer when it is absorbed into the body. The interaction between CCGN and insulin could avoid the intramolecular interaction of insulin and allow the insulin to maintain the monomer configuration, which was important for its oral absorption. The insulin distribution in CCGN could be directly simulated through different fluorescence labeling. 6-Coumarin, a lipophilic green fluoresent dye, was used to label the hydrophobic core of CCGN. Phycocyanin, a hydrophilic blue fluorescent pigmentprotein, was used as a model of biological protein for studying the interaction with nanoparticles. Figure 4 showed that the green fluorescent point coming from the core of 6-coumarin labeled CCGN overlapped with the blue fluorescent point of phycocyanin, which directly showed that phycocyanin was distributed in the CCGN. In Vitro Release. The in vitro release profiles of CCGN at pH 2.0, 6.8, and 7.4 were shown in Figure 5. A slower release rate of insulin was detected at pH 2.0 when compared to that at pH 6.8 and 7.4. However, a burst release profile within the first 15 min was observed at all of the above pH values. The burst release of the CCGN was due to the drug dissociating from the nanoparticles surface. Under strong acidic conditions, the carboxylic groups of the CCGN were in the protonated form, leading to aggregation of the particles. Therefore, insulin release from the nanoparticles was significantly limited. In comparison, the carboxylic groups of the CCGN were ionized at pH 7.4 and 6.8. The increased electrostatic repulsion between the carboxylic groups on CCGN and insulin that were both negatively charged resulted in a relatively faster release rate of insulin. Such sensitivity of insulin release toward pH would be favorable for the protection of orally delivered insulin when the nanoparticles passed through the acid environment of the stomach. Hypoglycemic Effects. After subcutaneous injection of insulin loaded CCGN, maximal decrease in blood glucose level

Figure 6. Hypoglycemic effect following s.c. administration of insulin (9) and insulin loaded CCGN colloid (b) at a dose of 1 IU/kg.

Figure 7. Hypoglycemic effect following oral administration of insulin loaded CCGN at a dose of 100 IU/kg (b), 50 IU/kg (2), 25 IU/kg (3), 15 IU/kg ()), and insulin solution at a dose of 100 IU/kg (9) in normal rats. *p < 0.05, **p < 0.01 vs control group (insulin solution) according to Student’s t-test.

of 41% was noted after 1.5 h and maintained up to 12 h (Figure 6). The hypoglycemic effect of insulin loaded CCGN showed no significant difference (p > 0.05) to insulin solution, which suggested that the bioactivity of insulin was preserved in CCGN and it would not be affected by the encapsulation process. Figure 7 showed that oral administration of insulin solution induced no significant change in blood glucose levels in normal rats over 24 h postadministration, while a marked and sustained hypoglycemic effect was observed after oral administration of insulin loaded CCGN at 100 IU/kg. A maximal decrease to 67% in blood glucose was noted after 12 h. Similar hypoglycemic effect was observed after oral administration of insulin loaded CCGN at 50 and 25 IU/kg, respectively, while inappreciable lowering of blood glucose level was found for insulin loaded CCGN at 15 IU/kg. The pharmacological bioavailability of insulin loaded CCGN at 25 IU/kg was found to be 9.7%, which was superior to the previously reported chitosan-insulin nanoparticles, which exhibited pharmacological bioavailablity of 4.4 and 3.2% at 50 and 100 IU/kg in fasted diabetic rats,25 respectively. Such notable hypoglycemic effect of insulin loaded CCGN might be attributed to the following reasons. CCGN with

1258

Biomacromolecules, Vol. 10, No. 5, 2009

Cui et al.

with the pharmacological bioavailability of 9.7% at a dose of 25 IU/kg in normal rats. Furthermore, CCGN induced minimal impairment to rat intestine and erythrocyte, which confirmed its desired tissue and blood compatibility. Therefore, the CCGN would be a promising peroral delivery carrier for protein drugs, especially insulin. Acknowledgment. This work was supported by a grant from the National Natural Science Foundation of China (No. 30772656). Figure 8. Hypoglycemic effect following oral administration of insulin loaded CCGN at a dose of 50 IU/kg (b), 25 IU/kg (2), and insulin solution at a dose of 50 IU/kg (9) in diabetic rats. *p < 0.05, **p < 0.01 vs control group (insulin solution) according to Student’s t-test.

diameters of 200-300 nm were preferentially taken up by the M cells in Peyer’s patches of gut associated lymphoid tissue, and therefore, insulin could be released into circulation systems.26 Carboxylic groups of the CCGN could provide a strong protective effect toward pancreatic serine proteases due to deprivation of Ca2+ and Zn2+.27 Besides, CCGN could increase the drug concentration gradient and control the release of insulin due to its pH sensitivity properties as mentioned above. As illustrated in Figure 8, blood glucose level of diabetic rats decreased to below 80% following oral administration of insulin loaded CCGN within 6-16 h, and reached the nadir at 16 h (35% of the initial level). Because the rats were fasted over the whole experiment, an appreciable decrease in blood glucose level was observed in control rats. The pharmacological action in diabetic rats was different from that in normal rats, which was attributed to the interference of the autoregulation system after alloxan treatment. In normal rats, the autoregulation system could distinguish the content of blood glucose and autoregulate in vivo insulin secretion. In diabetic rats, the islet cell was disturbed by alloxan and could not secrete insulin and the regulation system was inactivated. Therefore, only the exogenous insulin could regulate blood glucose. Biocompatibility of CCGN. LDH leakage in rat ileum 1 h following administration of the PBS, STG, and CCGN was 0.838 ( 0.047, 3.144 ( 0.669, and 0.894 ( 0.485 [U], respectively. Negligible leakage of LDH into the ileal loop was observed after CCGN treatment (p > 0.05 vs control), suggesting that the epithelial integrity and cellular tight junctions remained mostly unchanged. In contrast, notable LDN leakage after administration of commonly used permeation enhancer STG confirmed validity of the study. In addition, the CCGN induced no significant hemolysis at different test volumes when compared with blood incubated with normal saline. Such result indicated that CCGN had desirable blood compatibility (data not shown).

Conclusions Oral administration of insulin loaded CCGN led to a significant hypoglycemic effect in both normal and diabetic rats,

References and Notes (1) Borchard, G.; Lueβen, H. L.; de Boer, A. G.; Verhoef, J. C.; Lehr, C. M.; Junginger, H. E. J. Controlled Release 1996, 39, 131–138. (2) Graf, A.; Jack, K. S.; Whittaker, A. K.; Hook, S. M.; Rades, T. Eur. J. Pharm. Sci. 2008, 33, 434–444. (3) Ponchel, G.; Irache, J. M. AdV. Drug DeliVery ReV. 1998, 34, 191– 219. (4) Win, K. Y.; Feng, S. S. Biomaterials 2005, 26, 2713–2722. (5) Vila, A.; Sa´nchez, A.; Tobı´o, M.; Calvo, P.; Alonso, M. J. J. Controlled Release 2002, 78, 15–24. (6) Sakuma, S.; Hayashi, M.; Akashi, M. AdV. Drug DeliVery ReV. 2001, 47, 21–37. (7) Dodane, V.; Khan, M. A.; Merwin, J. R. Int. J. Pharm. 1999, 182, 21–32. (8) Thanou, M.; Nihot, M. T.; Jansen, M.; Verhoef, J. C.; Junginger, H. E. J. Pharm. Sci. 2001, 90, 38–46. (9) Chen, F.; Zhang, Z. R.; Yuan, F.; Qin, X.; Wang, M.; Huang, Y. Int. J. Pharm. 2008, 349, 226–233. (10) Harish Prashanth, K. V.; Tharanathan, R. N. Carbohydr. Polym. 2003, 54, 343–351. (11) Zhang, Z. X.; Jiao, Y. P.; Li, L. H.; Zhou, C. R. Shangdong J. Biomed. Eng. 2002, 21, 14–17. (12) Chen, Y.; Lu, M.; Tang, Y.; Tan, H. M. Petrochem. Technol. 2004, 33, 1137–1141. (13) Schipper, N. G. M.; Olsson, S.; Hoogstraate, J. A.; de Boer, A. G.; Varum, K. M.; Artursson, P. Pharm. Res. 1997, 14, 923–929. (14) Van der Lubben, I. M.; Verhoef, J. C.; Borchard, G.; Junginger, H. E. Eur. J. Pharm. Sci. 2001, 14, 201–207. (15) Thanou, M.; Nihot, M. T.; Jansen, M.; Verhoef, J. C.; Junginger, H. E. J. Pharm. Sci. 2001, 90, 38–46. (16) Cui, F. Y.; He, C. B.; Yin, L. C.; Qian, F.; He, M.; Tang, C.; Yin, C. H. Biomacromolecules 2007, 8, 2845–2850. (17) Peterson, P. L. Anal. Biochem. 1977, 83, 346–356. (18) Merodio, M.; Arnedo, A.; Renedo, M. J.; Irache, J. M. Eur. J. Pharm. Sci. 2001, 12, 251–259. (19) Pan, Y.; Li, Y. J.; Zhao, H. Y.; Zheng, J. M.; Xu, H.; Wei, G. Int. J. Pharm. 2002, 249, 139–147. (20) Cenni, E.; Granchi, D.; Avnet, S.; Fotia, C.; Salerno, M.; Micieli, D. Biomaterials 2008, 29, 1400–1411. (21) Sun, T.; Xu, P. X.; Liu, Q.; Xue, J.; Xie, W. M. Eur. Polym. J. 2003, 39, 189–192. (22) Xu, Y. M.; Du, Y. M. Int. J. Pharm. 2003, 250, 215–226. (23) Haidar, Z. S.; Hamdy, R. C.; Tabrizian, M. Biomaterials 2008, 29, 1207–1215. (24) Sadzuka, Y.; Nakade, A.; Tsuruda, T. J. Controlled Release 2003, 91, 271–280. (25) Ma, Z.; Lim, T.; Lim, L. Int. J. Pharm. 2005, 293, 271–280. (26) Khafagy, E. S.; Morishita, M.; Onuki, Y.; Takayama, K. AdV. Drug DeliVery ReV. 2007, 59, 1521–1546. (27) Yin, L. C.; Ding, J. Y.; Fei, L. K.; He, M.; Cui, F. Y.; Tang, C.; Yin, C. H. Int. J. Pharm. 2008, 350, 220–229.

BM900035U