Oral Delivery of Peptide Drugs Using Nanoparticles Self-Assembled

Jun 3, 2008 - Nanoparticles (NPs) self-assembled by the synthesized TMC and poly(γ-glutamic acid) (γ-PGA, TMC/γ-PGA NPs) were prepared for oral del...
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Bioconjugate Chem. 2008, 19, 1248–1255

Oral Delivery of Peptide Drugs Using Nanoparticles Self-Assembled by Poly(γ-glutamic acid) and a Chitosan Derivative Functionalized by Trimethylation Fwu-Long Mi,† Yong-Yi Wu,‡ Yu-Hsin Lin,‡,§ Kiran Sonaje,‡ Yi-Cheng Ho,† Chiung-Tong Chen,| Jyuhn-Huarng Juang,⊥ and Hsing-Wen Sung*,‡ Department of Biotechnology, Vanung University, Chungli, Taoyuan, Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC, Department of Biological Science and Technology, China Medical University, Taichung, Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli, and Division of Endocrinology and Metabolism, Chung Gung University and Memorial Hospital, Kweishan, Taoyuan, Taiwan, ROC. Received February 26, 2008; Revised Manuscript Received May 3, 2008

In the study, chitosan (CS) was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) polymers with different degrees of quaternization. Nanoparticles (NPs) self-assembled by the synthesized TMC and poly(γ-glutamic acid) (γ-PGA, TMC/γ-PGA NPs) were prepared for oral delivery of insulin. The loading efficiency and loading content of insulin in TMC/γ-PGA NPs were 73.8 ( 2.9% and 23.5 ( 2.1%, respectively. TMC/γ-PGA NPs had superior stability in a broader pH range to CS/γ-PGA NPs; the in Vitro release profiles of insulin from both test NPs were significantly affected by their stability at distinct pH environments. At pH 7.0, CS/γ-PGA NPs became disintegrated, resulting in a rapid release of insulin, which failed to provide an adequate retention of loaded insulin, while the cumulative amount of insulin released from TMC/γ-PGA NPs was significantly reduced. At pH 7.4, TMC/γ-PGA NPs were significantly swelled and a sustained release profile of insulin was observed. Confocal microscopy confirmed that TMC40/γ-PGA NPs opened the tight junctions of Caco-2 cells to allow the transport of insulin along the paracellular pathway. Transepithelial-electrical-resistance measurements and transport studies implied that CS/γ-PGA NPs can be effective as an insulin carrier only in a limited area of the intestinal lumen where the pH values are close to the pKa of CS. In contrast, TMC40/γ-PGA NPs may be a suitable carrier for transmucosal delivery of insulin within the entire intestinal tract.

INTRODUCTION The oral route is the most convenient and comfortable means of administering protein/peptide drugs such as insulin, because the pain caused by injection could be avoided, leading to a higher patient compliance (1, 2). However, protein/peptide drugs are readily degraded by the low pH of gastric medium in the stomach. Additionally, different digestive enzymes in the stomach and small intestine may lead to degradation of protein/ peptide drugs (3–5). Therefore, a pH-responsive drug delivery system that is stable in the gastrointestinal (GI) tract and can swell significantly or disintegrate and subsequently release the loaded drug when adhering and infiltrating into the mucus of the intestinal tract is desired. Chitosan (CS) and poly(γ-glutamic acid) (γ-PGA) have been used for the purpose of gene or drug delivery (6–11). In our recent study, a nanoparticle (NP) system composed of CS and γ-PGA for oral delivery of insulin via the paracellular transport was developed (12, 13). We found that the prepared NPs remained intact in the pH range 2.5-6.6; however, outside this range, they became unstable and disintegrated, thus limiting the efficacy of insulin delivery. * Correspondence to Hsing-Wen Sung, PhD, Professor, Department of Chemical Engineering/Bioengineering Program, National Tsing Hua University, Hsinchu, Taiwan 30013. Phone: 886-3-574-2504, Fax: 8863-572-6832. E-mail: [email protected]. † Vanung University. ‡ National Tsing Hua University. § China Medical University. | National Health Research Institutes. ⊥ Chung Gung University and Memorial Hospital.

It is known that the natural pH environment in the GI tract varies from acidic in the stomach to slightly alkaline in the small intestine (14). CS lacks the advantage of good solubility at neutral pH values. It aggregates in solutions at pH values above 6.5, and only protonated CS (i.e., in its uncoiled configuration) can trigger the opening of tight junctions, thereby facilitating the paracellular transport of hydrophilic compounds (15). To overcome these problems, CS was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) polymers with different degrees of quaternization. The partially quaternized TMC, which shows higher aqueous solubility than CS in a much broader pH range (15), was used in this study to prepare NPs. The synthesized TMC polymers were analyzed by the proton nuclear magnetic resonance (1H NMR) and Fourier transformed infrared (FT-IR) spectroscopy. Preparation of NPs self-assembled by TMC and γ-PGA (TMC/γ-PGA NPs) was reported and their mean particle sizes as well as zeta potential values in response to simulated GI media were examined by dynamic light scattering. Molecular dynamic (MD) simulations were used to investigate the structural changes of the self-assembled TMC/ γ-PGA complex at distinct pH environments. Evaluation of test NPs in enhancing the intestinal paracellular transport was investigated in Vitro in Caco-2 cell monolayers. The change of transepithelial electrical resistance (TEER) for the tightness of cell monolayers was measured and the paracellular transport of insulin was visualized using confocal laser scanning microscopy (CLSM) and quantified by enzyme-linked immunosorbent

10.1021/bc800076n CCC: $40.75  2008 American Chemical Society Published on Web 06/03/2008

TMC/γ-PGA Nanoparticles

assay (ELISA). Additionally, the release profiles of insulin from test NPs were studied in simulated GI media.

MATERIALS AND METHODS Synthesis and Characterization of TMC. CS (MW 60 kDa) with a degree of deacetylation of approximately 85% was acquired from Koyo Chemical Co. Ltd. (Japan). TMC55 with a degree of quaternization of about 55% was synthesized from CS using a two-step methylation procedure described by Sieval et al. (16). By varying the number of reaction steps and the amount of base used, TMC25 and TMC40 with degrees of quaternization of approximately 25% and 40%, respectively, were synthesized by a method reported by Polnok et al. (17). The obtained TMC was purified by dialysis against water for 3 days and then freeze-dried. The purified TMC was analyzed by 1H NMR (Varian Unityionva 500 NMR Spectrometer, Missouri) and FT-IR (Perkin-Elmer Spectrum RX1 FT-IR System, Buckinghamshire, England). Degrees of quaternization of the synthesized TMC polymers were estimated using 1H NMR spectra (17). Preparation and Characterization of NPs. TMC/γ-PGA NPs were prepared using a simple ionic-gelation method under magnetic stirring at room temperature. In brief, an aqueous γ-PGA (Vedan, Taichung, Taiwan, MW 60 kDa, 1.0 mg/mL, 2 mL) was added by flush mixing with a pipet tip into an aqueous TMC [1.2 mg/mL, 10 mL, in deionized (DI) water, pH 6.0]. The self-assembled NPs were collected by centrifugation at 15 000 rpm for 50 min. Supernatants were discarded and NPs were resuspended in DI water for further studies. The mean particle sizes and zeta potential values of NPs were measured using a Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK). The characteristics of NPs were investigated at distinct pH values (simulating the pH environments in the GI tract) (18–20). MD Simulation of NPs at Distinct pH Environments. MD simulations were performed with the program NAMD (21) using parameters adapted from the CHARMM (22) force field. TMC and γ-PGA used in simulations were oligomers consisting of 10 monomers. The models were minimized to remove unfavorable contacts, brought to 310 K by velocity rescaling and equilibrated for 1 ns. Before any MD trajectory was run, 40 ps of energy minimization was performed to relax conformational and structural tensions. This minimum structure was the starting point for MD simulations. For this purpose, the molecule was embedded into a cubic simulation box of 80 Å. A cutoff distance of 12 Å was employed for the nonbonded and electrostatic interactions. The heating process was performed from 0 to 310 K through Langevin damping with a coefficient of 10 ps-1. A time step of 2 fs was employed for rescaling the temperature. After 20 ps heating to 310 K, equilibration trajectories of 1 ns were recorded, which provided the data for the structural and thermodynamic evaluations. The equations of motion were integrated with the Shake algorithm with a time step of 1 fs. Figures displaying atomistic pictures of molecules were generated using VMD (23). Preparation of Insulin-Containing NPs. A sample of 100 mg of insulin (from bovine pancreas, 27.4 IU/mg, SigmaAldrich, St. Louis, MO) was dissolved in 10 mL of 0.01 N HCl, and this solution was neutralized with 0.1 N NaOH (24). The insulin solution was then diluted with DI water to make a 3 mg/mL insulin stock solution. The insulin stock solution (1 mL) was premixed with an aqueous γ-PGA (3 mg/mL, 1 mL). Subsequently, MgSO4 (6 mg/mL, 1 mL) was blended into the mixture and thoroughly stirred for 30 min. The mixed solution was added into an aqueous TMC (1.2 mg/mL, 10 mL) under magnetic stirring at room temperature as described before. To determine their insulin loading content and loading efficiency, NPs were collected by centrifugation at 15 000 rpm,

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4 °C for 50 min, and the insulin concentration in the supernatant was assayed by high-performance liquid chromatography (HPLC). The drug loading content and loading efficiency of NPs were determined as follows (12, 13). Loading Content (%) ) total amount of insulin - free insulin × 100% weight of NPs Loading Efficiency (%) ) total amount of insulin - free insulin × 100% total amount of insulin The release profiles of insulin from test NPs were investigated in distinct dissolution media (pH 2.5, 6.4, 7.0, and 7.4, simulating the pH environments in the GI tract) at 37 °C under agitation (100 rpm, DISTEK-2230A, North Brunswick, NJ). At particular time intervals, samples were taken out and centrifuged and supernatants were used for the HPLC analysis. The amount of insulin released was expressed as a percentage of the total insulin associated with NPs as calculated from the loading efficiency (25). The stability of the released insulin was determined by analyzing its conformation using an Aviv 202 spectropolarimeter (Aviv Associates, Lakewood, NJ) and comparing the spectrum with that of standard insulin (26). TEER Measurements and Transport Studies. Caco-2 cell monolayers were cultured on the tissue-culture-treated polycarbonate filter (diameter 24.5 mm, growth area 4.7 cm2) in Costar Transwell 6 wells/plates (Corning Costar Corp., NY) and were used for transport experiments approximately 21 days after seeding (TEER values in the range 600-800 Ω cm2). Evaluation of test NPs (0.2 mg/mL, 1 mL) in enhancing the intestinal paracellular transport at pH 6.6, 7.0, and 7.4 (in the donor compartment; at pH 7.4 in the receiver compartment) was investigated in Caco-2 cell monolayers (27). The change of TEER for the tightness of cell monolayers was measured with a Millicell-Electrical Resistance System (Millipore Corp., Bedford, MA). Simultaneously, samples (50 µL) were collected from the receiver compartment at distinct time intervals and the insulin concentrations were measured using ELISA (bovine insulin kit, Mercodia AB, Uppsala, Sweden) (28). The transport of insulin through Caco-2 cell monolayers was expressed as the cumulative insulin transport (29). Fluorescent NP Preparation and CLSM Visualization. Cy3-labeled insulin (Cy3-insulin) was synthesized by a method described in the literature (30). To remove the unconjugated Cy3, the synthesized Cy3-insulin was dialyzed in the dark against 5 L of 0.01 N HCl and replaced on a daily basis until no fluorescence was detected in the supernatant. The resultant Cy3-insulin was lyophilized in a freeze-dryer. Fluorescent NPs loaded with Cy3-insulin were then prepared for the CLSM study as per the procedure described above. The transport medium (pH 7.0) containing fluorescent NPs (0.2 mg/mL, 1 mL) was introduced into the donor compartment of Caco-2 cell monolayers, while the medium in the receiver compartment was maintained at pH 7.4 (31). After incubation for specific time intervals at 37 °C, test samples were aspirated. Cells were then washed twice with prewarmed phosphate buffered saline (PBS) before they were fixed in 3.7% paraformaldehyde (32). Subsequently, the fixed cells were examined under a CLSM (TCS SL, Leica, Germany). Visualization of Opening Tight Junctions. Caco-2 cell monolayers were incubated with test NPs (0.2 mg/mL, 1 mL). After 120 min, the incubated NPs were removed and cells were fixed in 3.7% paraformaldehyde. Cells were washed three times with PBS and permeabilized with 0.2% Triton X-100 and RNase (100 µg/mL) for 15 min at 37 °C. The wash was repeated and cells were blocked with 5% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for

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Mi et al. Table 1. Mean Particle Sizes, Zeta Potential Values, and Polydispersity Indices of Nanoparticles (NPs) Self-Assembled by TMC Polymers with Different Degrees of Quaternization and γ-PGA (n ) 5 batches)a mean zeta polydispersity particle size (nm) potential (mV) index CS/γ-PGA NPs TMC25/γ-PGA NPs TMC40/γ-PGA NPs TMC55/γ-PGA NPs

104.1 ( 1.2 101.3 ( 3.1 106.3 ( 2.3 114.6 ( 2.3

36.2 ( 2.5 30.9 ( 2.1 32.3 ( 2.1 30.6 ( 3.8

0.11 ( 0.02 0.13 ( 0.04 0.15 ( 0.14 0.12 ( 0.03

a TMC: N-trimethyl chitosan. CS: Chitosan. γ-PGA: Poly(γ-glutamic acid).

Figure 1. (a) 1H NMR spectra of chitosan (CS) and N-trimethyl chitosan (TMC) polymers with different degrees of quaternization; (b) FT-IR spectra of CS and TMC40.

60 min at 37 °C. Subsequently, cells were treated with the rabbit anti-ZO-1 mAb (Zymed Laboraties, San Francisco, CA) at 1:50 dilution for 60 min at 37 °C. After washing three times with PBS, cells were incubated in the Cy-5 conjugated goat antirabbit IgG at 1:100 dilution (Jackson ImmunoResearch Laboratories). Additionally, cells were costained to visualize nuclei by propidium iodide (PI, P4864, Sigma-Aldrich, St. Louis, MO). The stained cells were evenly mounted on slides and examined under a CLSM. Superimposed images were performed with LCS Lite software (version 2.0). Statistical Analysis. Comparison between two groups was analyzed by the one-tailed Student’s t-test and multiple group comparison was performed by one-way ANOVA followed by Fisher’s LSD using statistical software (SPSS, Chicago, IL). All data are presented as a mean value with its standard deviation indicated (mean ( SD). Differences were considered to be statistically significant when p values were less than 0.05.

RESULTS AND DISCUSSION Characterization of TMC. Figure 1a,b shows the 1H NMR and FT-IR spectra of CS and the synthesized TMC, respectively. As shown in Figure 1a, TMC showed a new signal at 3.82 ppm [-N(CH3)3+], while those observed at 3.82-4.04 ppm corresponded to -OCH3 (13, 33). In the FT-IR spectra, the intensity of the characteristic peak (2890 cm-1, C-H stretch) increased and a new peak at 1489 cm-1 (-CH3 antisym deformation) was observed on TMC, while the characteristic peak (1580 cm-1) of -NH2 on CS decreased (33). These results indicated

that TMC was successfully synthesized. Degrees of quaternization of the synthesized TMC polymers, estimated from the obtained 1H NMR spectra, were 24.8% (TMC25), 42.8% (TMC40), and 54.5% (TMC55), respectively. Preparation of NPs. In the study, NPs were self-assembled instantaneously upon addition of an aqueous γ-PGA into an aqueous TMC (with a TMC/γ-PGA weight ratio of 6:1) under magnetic stirring at room temperature. The amount of positively charged TMC significantly exceeded that of negatively charged γ-PGA; some of excessive TMC molecules were entangled onto the surfaces of NPs, thus displaying a positive surface charge (Table 1). The degree of quaternization on TMC had little effects on the mean particle size and zeta potential of NPs. pH-Responsive Characteristics of NPs. The stomach pH is about 1.0-2.0 in the presence of food, while the fasting pH of the stomach is 2.5-3.7 (18–20). The pH values in the duodenum and the jejunum and proximal ileum are 6.0-6.6 and 6.6-7.0, respectively, while the mean pH in the distal ileum and in the body fluid at intercellular spaces between enterocytes is about 7.4 (14, 19, 20). Therefore, characterization of test NPs in response to distinct pH environments must be investigated. Peppas et al. prepared poly(methacrylic acid-g-ethylene glycol) (PMAA-g-EG) NPs for oral administration of insulin (24, 34, 35). It was shown that the prepared NPs were pH-responsive and mucoadhesive. The results of TEER measurements and transport studies implied that PMAA-g-EG NPs were effective as a transmucosal carrier and able to open the tight junctions between Caco-2 cells. In contrast to PMAA, TMC is a positively charged polymer and has been used as an intestinal permeation enhancer (29), and its mechanism of opening tight junctions is similar to that of protonated CS (15). Additionally, TMC showed no indication of epithelial damage or cytotoxicity (15). The mucoadhesive characteristics of the CS-coated NPs (FITC-labeled) in vivo were previously reported by our group (36). In this study, the FITClabeled CS NPs were orally administered in a rat model and the intestinal segments were retrieved 3 h afterwards. It was found that the administered CS NPs were colocalized with ZO-1 proteins at the epithelial junctions. Also, it has been reported that TMC-containing formulations exceeded the potency of CS in mucoadhesion (29). The mucoadhesive properties of CS and TMC have also been reported in large animals such as pigs (37). The basic concept of the study is that the orally administered NPs with excessive mucoadhesive TMC (29) on their surfaces may adhere and infiltrate into the mucus of the intestinal tract, and then mediate transiently opening the tight junctions between enterocytes (Figure 2). It is known that the tight junctions opened by absorption enhancers are less than 20 nm in width (38–40). Consequently, the NPs infiltrated into the mucus must become unstable (swelling or disintegration); thus, their loaded insulin can be released and permeated through the paracellular pathway to the bloodstream. It is known that the pKa values of CS (amine groups) and γ-PGA (carboxylic groups) are 6.5 and 2.9, respectively (12).

TMC/γ-PGA Nanoparticles

Bioconjugate Chem., Vol. 19, No. 6, 2008 1251 Table 2. Mean Particle Sizes, Zeta Potential Values, and Polydispersity Indices of Nanoparticles (NPs) Self-Assembled by TMC Polymers with Different Degrees of Quaternization and γ-PGA at Distinct pH Environments (n ) 5 batches)a mean particle size (nm)

zeta potential (mV)

polydispersity index

CS/γ-PGA NPs pH pH pH pH pH pH pH

1.2 2.0 2.5 6.0 6.6 7.0 7.4

N/A N/A 113.3 ( 1.6 104.1 ( 1.2 245.6 ( 4.5 N/A N/A

N/A N/A 38.6 ( 0.8 36.2 ( 2.5 12.9 ( 0.4 N/A N/A

1 1 0.14 ( 0.01 0.11 ( 0.02 0.17 ( 0.11 1 1

TMC25/γ-PGA NPs pH pH pH pH pH pH pH

1.2 2.0 2.5 6.0 6.6 7.0 7.4

N/A N/A 396.4 ( 4.7 101.3 ( 3.1 N/A N/A N/A

N/A N/A 32.1 ( 1.6 30.9 ( 2.1 N/A N/A N/A

1 1 0.32 ( 0.11 0.13 ( 0.04 1 1 1

TMC40/γ-PGA NPs pH pH pH pH pH pH pH pH

1.2 2.0 2.3 2.5 6.0 6.6 7.0 7.4

N/A N/A 272.2 ( 2.3 252.4 ( 3.5 106.3 ( 2.3 238.3 ( 3.1 296.7 ( 4.7 498.4 ( 6.8

pH pH pH pH pH pH pH

1.2 2.0 2.5 6.0 6.6 7.0 7.4

N/A 252.5 ( 4.1 221.4 ( 3.5 114.6 ( 2.3 141.2 ( 1.6 144.6 ( 4.8 141.2 ( 0.9

N/A N/A 38.6 ( 2.7 35.4 ( 1.1 32.3 ( 2.1 24.3 ( 1.4 20.4 ( 0.3 17.3 ( 0.6

1 1 0.25 ( 0.23 0.21 ( 0.04 0.15 ( 0.14 0.09 ( 0.03 0.18 ( 0.11 0.38 ( 0.21

TMC55/γ-PGA NPs

Figure 2. Schematic illustrations of the presumed mechanism of opening tight junctions by test nanoparticles and transporting of their encapsulated drug (using duodenum as an example).

In the study, NPs were prepared in DI water (pH 6.0). At pH 6.0, CS (TMC25) and γ-PGA were ionized. The ionized CS (TMC25) and γ-PGA could form polyelectrolyte complexes, which resulted in a matrix structure with a spherical shape. At pH 1.2-2.0, most carboxylic groups on γ-PGA were in the form of -COOH. Hence, there was little electrostatic interaction between CS (TMC25) and γ-PGA; thus, NPs became disintegrated (Table 2). Similarly, at pH values above 6.6, the free amine groups on CS (TMC25) were deprotonated, thus leading to the disintegration of NPs. This might limit the efficacy of drug delivery and absorption in the small intestine. With increasing the degree of quaternization on TMC (TMC40 and TMC55), the stability of NPs in the pH range 6.6-7.4 increased significantly. However, the swelling of TMC55/γ-PGA NPs at pH 7.4 was minimal (due to the highly quaternized TMC55), which might limit the release of loaded drugs. In contrast, TMC40/γ-PGA NPs swelled significantly with increasing pH value (Figure 3). Therefore, TMC40/γ-PGA NPs were chosen to load insulin and used for the rest of the study. As shown in Table 2, TMC40/γ-PGA NPs still retained a positive surface charge with a zeta potential value of 17.3 mV at pH 7.4. The results of MD simulations showed that the molecular chains of TMC40 (in blue) and γ-PGA (in red) in their selfassembled complex were tightly entangled to each other at pH 6.0 (Figure 4). The surface of the complex was dominated by TMC40 molecules. Relaxations of TMC40 and γ-PGA molecular chains at pH 2.5 (pH 7.4) resulted in a moderate (significant) swelling of the TMC40/γ-PGA complex, while its surface was still dominated by the positively charged TMC molecules, thus retaining a positive surface charge.

N/A 35.6 ( 4.2 32.5 ( 3.4 30.6 ( 3.8 24.8 ( 3.4 20.4 ( 1.7 18.9 ( 4.1

1 0.16 ( 0.08 0.15 ( 0.02 0.12 ( 0.03 0.15 ( 0.02 0.18 ( 0.14 0.11 ( 0.11

a N/A: Precipitation of aggregates was observed; TMC: N-trimethyl chitosan; CS: chitosan; γ-PGA: Poly(γ-glutamic acid).

Figure 3. Schematic illustrations of the pH-responsive characteristics of TMC40/γ-PGA NPs at distinct pH environments. TMC: N-trimethyl chitosan. γ-PGA: poly(γ-glutamic acid).

Characteristics of Insulin-Containing NPs and Their Release Profiles. In the study, we found that the loading efficiency and loading content of insulin in TMC40/γ-PGA NPs were only 20.8 ( 1.9% and 4.2 ( 1.6% (n ) 5 batches), respectively. To increase the amount of insulin loaded, Mg2+ was blended in the premixed aqueous insulin/γ-PGA in the

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Mi et al. Table 3. Mean Particle Sizes, Zeta Potential Values, and Polydispersity Indices of Insulin-Containing TMC40/γ-PGA Nanoparticles at Distinct pH Environments (n ) 5 batches)a

pH pH pH pH pH pH pH pH

Figure 4. Structural changes of the self-assembled TMC40/γ-PGA complex at distinct pH environments obtained by molecular dynamic simulations. TMC: N-trimethyl chitosan (in blue). γ-PGA: poly(γglutamic acid) (in red).

1.2 2.0 2.3 2.5 6.0 6.6 7.0 7.4

mean particle size (nm)

zeta potential (mV)

polydispersity index

N/A N/A 312.2 ( 2.2 292.5 ( 2.5 148.6 ( 2.1 262.2 ( 3.2 336.7 ( 2.3 522.4 ( 5.9

N/A N/A 36.6 ( 1.7 32.1 ( 1.4 30.1 ( 1.8 25.5 ( 2.4 18.4 ( 1.3 14.2 ( 0.6

1 1 0.23( 0.21 0.18( 0.13 0.12( 0.01 0.11( 0.15 0.23( 0.13 0.44( 0.36

a TMC: N-trimethyl chitosan. γ-PGA: Poly(γ-glutamic acid). N/A: Precipitation of aggregates was observed.

Figure 5. TEM micrographs of the insulin-containing TMC40/γ-PGA nanoparticles at distinct pH environments. TMC: N-trimethyl chitosan. γ-PGA: poly(γ-glutamic acid).

preparation of NPs. It was reported in our previous study that insulin could conjugate with γ-PGA via divalent metal ions such as Zn2+ (13, 36), thus increasing the drug loading efficiency and loading content in NPs. In the present study, we found that the NPs prepared with Mg2+ had a better stability than their counterparts with Zn2+ (data not shown). Mg2+ is one of the most abundant ions present in living cells. It was reported that daily Mg2+ administration in diabetic patients improves insulinmediated glucose uptake (41). In the presence of Mg2+, the insulin loading efficiency (73.8 ( 2.9%) and loading content (23.5 ( 2.1%, n ) 5 batches) in TMC40/γ-PGA NPs increased significantly. The morphology, mean particle sizes, zeta potential values, and polydispersity indices of the insulin-containing TMC40/γPGA NPs at distinct pH values are shown in Figure 5 and Table 3, respectively. As shown, the pH-responsive characteristics of the insulin-containing TMC40/γ-PGA NPs were similar to their empty counterparts (Table 2). Figure 6a shows the release profiles of insulin from the insulin-containing CS/γ-PGA and TMC40/γ-PGA NPs at pH 2.5, 6.4, 7.0, and 7.4, simulating the pH environments in the stomach before a meal (similar to the conventional injectable insulin therapy), the small intestine, and the body fluid at intercellular spaces between enterocytes, respectively. As mentioned already, the insulin-containing TMC40/γ-PGA NPs were prepared at pH 6.0, which was close to the pI of native insulin. It has been reported that at this pH value, insulin associated with CS/TPP NPs primarily through hydrophobic interactions and the associated insulin released rapidly and

Figure 6. (a) Release profiles of insulin from CS/γ-PGA and TMC40/ γ-PGA nanoparticles (NPs) at pH 2.5, 6.4, 7.0, and 7.4, simulating the pH environments in the fasting stomach, the small intestine, and the body fluid at intercellular spaces between enterocytes, respectively. (b) Circular dichroism spectra of the insulin released from test nanoparticles at pH 7.4 and the standard insulin. CS: chitosan. TMC: N-trimethyl chitosan. γ-PGA: poly(γ-glutamic acid).

completely in aqueous media at pH 2.0 to 7.4 (25). Therefore, the release of insulin from the prepared NPs should be mainly influenced by their pH-dependent swelling property. At pH 2.5 (the stomach before meal) and 6.4 (the duodenum), the release profiles of insulin from both test NPs were similar. At pH 7.0 (the jejunum and the proximal ileum), CS/γ-PGA NPs became disintegrated (Table 2), resulting in a rapid release of insulin, which failed to provide an adequate retention of loaded insulin (approximately 85% of the loaded insulin was released). In contrast, for TMC/γ-PGA NPs, the cumulative amount of insulin released was significantly reduced to about

TMC/γ-PGA Nanoparticles

Figure 7. (a) Effects of CS/γ-PGA and TMC40/γ-PGA nanoparticles (NPs) on TEER values of Caco-2 cell monolayers at distinct pH values. (b) Cumulative amounts of insulin transported through Caco-2 cell monolayers incubated with test nanoparticles as a function of time at distinct pH values. CS: chitosan. TMC: N-trimethyl chitosan. γ-PGA: poly(γ-glutamic acid).

30%, due to their comparative stability at pH 7.0. At pH 7.4 (the distal ileum and the body fluid at intercellular spaces), TMC/ γ-PGA NPs were significantly swelled and a sustained release profile of insulin was observed. As indicated by the circular dichroism spectra (Figure 6b), no significant conformation change was observed for the insulin released from TMC/γ-PGA NPs at pH 7.4 as compared to the standard insulin. TEER Measurements and Insulin Transport Studies. Effects of CS/γ-PGA and TMC40/γ-PGA NPs on the TEER and the amount of insulin transported across Caco-2 cell monolayers as a function of time at distinct intestinal pH environments are shown in Figure 7a,b. As shown, incubation of CS/γ-PGA NPs on the apical side of Caco-2 cell monolayers at pH 6.4 led to an immediate reduction in TEER and a significant amount of insulin transported. However, at pH 7.0 and 7.4, the reduction in TEER and the amount of insulin transported through Caco-2 cells were significantly limited. These results implied that CS/γ-PGA NPs can be effective as an insulin carrier only in a limited area of the intestinal lumen (the duodenum) where the pH values are close to the pKa of CS. In contrast, TMC40/γ-PGA NPs significantly decreased the TEER and increased the amount of insulin transported across Caco-2 cell monolayers at all test intestinal pH environments,

Bioconjugate Chem., Vol. 19, No. 6, 2008 1253

Figure 8. (a) DIC and fluorescence images (taken by an inverted confocal laser scanning microscope) of four optical sections (at different depths) of Caco-2 cell monolayers 120 min after incubation with the Cy3-insulin containing TMC40/γ-PGA NPs. (b) Fluorescence images of Caco-2 cell monolayers immunofluorescently stained for ZO-1 proteins after incubation with TMC40/γ-PGA NPs for 120 min. TMC: N-trimethyl chitosan. γ-PGA: poly(γ-glutamic acid).

indicating a specific interaction of the cationic NPs (Table 3) with components of the tight junctions at neutral pH values. These findings suggested that TMC40/γ-PGA NPs may be a suitable carrier for transmucosal delivery of protein/peptide drugs within the entire intestinal lumen (the duodenum, jejunum, and ileum). After removal of the incubated test particles, a gradual increase in TEER was observed, indicating that the effect of TMC40/γ-PGA NPs on the tight junction’s regulation is transient and reversible. CLSM Visualization. Figure 8a shows the differential interference contrast (DIC) and fluorescence images of 4 optical sections of Caco-2 cell monolayers 120 min after incubation with the Cy3-insulin-containing TMC40/γ-PGA NPs using CLSM. This noninvasive method allows for optical sectioning and imaging of the transport of insulin across Caco-2 cell monolayers, without disrupting their structure (42). As shown, fluorescent images were observed at intercellular spaces between adjacent cells, suggesting that TMC40/γ-PGA NPs opened the tight junctions of Caco-2 cells to allow the transport of insulin by passive diffusion along the paracellular pathway. These

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findings confirmed the results observed in our TEER measurements and insulin-transport experiments (Figure 7a,b). The opening of the tight junctions of cell monolayers by TMC40/γ-PGA NPs was shown in Figure 8b. As shown, the staining for ZO-1 tight junction proteins showed a continuous ring appearance between adjacent cells before incubation with test NPs (control). After incubation with test NPs, the staining for ZO-1 proteins appeared discontinuous (indicated by white arrows), indicating the opening of cell tight junctions. ZO-1 proteins are thought to be a linkage molecule between occludin and actin cytoskeleton and play important roles in the rearrangement of cell-cell contacts (43, 44). After removal of NPs, the discontinuous staining for ZO-1 proteins appeared again, indicating the restoration of cell tight junctions.

CONCLUSIONS A pH-responsive NP system self-assembled by TMC and γ-PGA for oral delivery of insulin was successfully prepared in the study. TEER experiments showed that TMC/γ-PGA NPs were able to open the tight junctions between Caco-2 cells, and their effect on the tight junction’s integrity appeared to be reversible. CLSM confirmed that TMC/γ-PGA NPs opened the tight junctions of cell monolayers to allow the transport of insulin along the paracellular pathway at all test intestinal pH environments. These findings suggested that TMC40/γ-PGA NPs may be a suitable carrier for transmucosal delivery of protein/peptide drugs within the entire intestinal lumen.

ACKNOWLEDGMENT This work was supported by a grant from the National Science Council (NSC 96-2120-M-007-004), Taiwan, Republic of China.

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