Penetratin Derivative-Based Nanocomplexes for Enhanced Intestinal

Nov 20, 2013 - The relative pharmacological availability and bioavailability of P-bis-CD nanocomplexes were 10.6% and 7.1%, which were 3.0-fold and ...
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Penetratin Derivative-Based Nanocomplexes for Enhanced Intestinal Insulin Delivery Xi Zhu, Wei Shan, Peiwen Zhang, Yun Jin, Shan Guan, Tingting Fan, Yang Yang, Zhou Zhou, and Yuan Huang* Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P. R. China S Supporting Information *

ABSTRACT: Sufficient mucosal permeability is the bottleneck problem in developing an efficient intestinal delivery system of insulin. Cell-penetrating peptide-based nanocomplexes for the enhanced mucosal permeation of insulin were developed in this study. Penetratin, a cell-penetrating peptide was site-specifically modified with a bis-β-cyclodextrin group. Insulin-loaded nanocomplexes were prepared by selfassembly using penetratin or its bis-β-cyclodextrin modified derivative (P-bis-CD). A stronger intermolecular interaction and higher complex stability were observed for P-bis-CD nanocomplexes than the penetratin nanocomplexes. P-bis-CD nanocomplexes were significantly more efficient for the permeation of insulin as compared to the penetratin nanocomplexes both in vitro and in situ. Interestingly, different cellular internalization mechanisms were observed for the two nanocomplexes. In diabetic rats, intestinal administration of P-bis-CD nanocomplexes resulted in a prominent hypoglycemic effect which lasted for 6 h with maximum inhibitory rate at 60%. The relative pharmacological availability and bioavailability of P-bis-CD nanocomplexes were 10.6% and 7.1%, which were 3.0-fold and 2.3-fold higher than that of penetratin nanocomplexes, respectively. In addition, no sign of toxicity was observed after 7 consecutive days of administration of P-bis-CD nanocomplexes with endotoxin. These results demonstrated that P-bis-CD was a promising epithelium permeation enhancer for insulin and suggested that the chemical modification of cell penetration peptides was a feasible strategy to enhance their potential. KEYWORDS: cell-penetrating peptides, nanocomplexes, insulin, intestinal absorption, diabetes

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epithelial uptake.3 Yang and co-workers reported that, by conjugating insulin with Tat peptide, the amount of insulin transported across Caco-2 cell monolayer was dramatically increased.7 However, the chemical conjugation approach could affect the pharmacological activity of the biotherapeutic agents, and thus tailor-made synthetic processes should be established for individual drugs.3,8,9 More recently, Morishita and coworkers reported that CPPs, such as penetratin (PEN), were capable of enhancing the permeation of negatively charged biomacromolecules across intestinal mucosa through forming complexes with the biomacromolecules.1,9 While this noncovalent electrostatic interaction strategy does not require chemical conjugation, the complexes may be unstable due to the relative high ionic strength and plenty of charged contents in the gastrointestinal environment.10,11 The biomolecules that are electrostatically associated with the CPPs are likely to be substituted by other biological contents.8 Furthermore, the

INTRODUCTION The gastrointestinal route is acknowledged as the most convenient way of drug delivery due to its noninvasive and patient-friendly properties. Nevertheless, the oral delivery of biomacromolecular agents, such as insulin, has shown limited therapeutic efficacy mainly due to their low enzymatic stability and poor mucosal permeability.1,2 It has been realized that strategies directed at overcoming the enzymatic barrier alone can only moderately enhance the efficiency of gastrointestinal delivery of macromolecules.3 Sufficient mucosal permeability is necessary for achieving satisfactory bioavailability of therapeutic biomacromolecules. In the past few decades, different carrier or receptormediated transport systems have been explored to enhance the permeation of biomacromolecules through the intestinal mucosa, such as Vitamin B12, transferrin, and so forth.4,5 However, the efficiency of these systems is often limited by their low expression level of receptors on enterocytes and their confined uptake capacity.6 Cell-penetrating peptides (CPPs) have recently been utilized for the systemic delivery of biomolecules across intestinal mucosa, due to their capability of facilitates the permeability of biomolecules via efficient © 2013 American Chemical Society

Received: Revised: Accepted: Published: 317

August 17, 2013 November 12, 2013 November 20, 2013 November 20, 2013 dx.doi.org/10.1021/mp400493b | Mol. Pharmaceutics 2014, 11, 317−328

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Figure 1. (a) Synthetic route of PEN-bis-β-cyclodextrin conjugates (P-bis-CD, compound 5). (b) Schematic of the self-assembly of insulin-loaded PEN and P-bis-CD nanocomplexes. (c) Transmission electron microscope of PEN NC and P-bis-CD NC. (d) Release profile of PEN NC and P-bisCD NC (pH = 6.8, mean ± SD, n = 3).

that the P-bis-CD nanocomplexes will not induce the absorption of unwanted toxins presenting in the small intestine.

electrostatic interactions between CPPs and the negatively charged glycosaminoglycans on epithelial cell surface may result in competitive dissociation of CPPs and the cargo.12,13 Herein, we have developed a PEN derivative-based nanocomplex system for the effective intestinal delivery of insulin by enhancing the complex stability and mucosal permeability. The PEN derivative (P-bis-CD) was synthesized by site-specifically conjugating PEN to a bis-β-cyclodextrin (bis-CD) group (Figure 1). β-cyclodextrin (β-CD) is a cyclic oligosaccharide which possesses an hydrophobic cavity and was reported to interact with specific surface residues of insulin through hydrophobic force.14,15 Unlike electrostatic interactions, hydrophobic interactions are less affected by other charged molecules.16 The P-bis-CD can thus self-assemble with insulin to form nanocomplexes through both electrostatic and hydrophobic interactions. The effectiveness of the nanocomplexes for intestinal delivery of insulin was evaluated both in vitro and in vivo. The mechanism of improved permeation enhancing effect of the P-bis-CD, as compared with PEN, was investigated in terms of intermolecular interaction, cellular uptake pathway, and in vivo absorption status in intestinal villi. The hypoglycemic activity and the pharmacokinetic behavior of the nanocomplexes were evaluated on diabetic rats. In addition, long-term toxicity investigation was also performed to confirm

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EXPERIMENTAL SECTION Materials. Porcine insulin (INS, 30 IU/mg) was purchased from Wanbang Bio-Chemical Co., Ltd. (Jiangsu, China). Penetratin peptide was chemically synthesized by Kaijie Biopharmaceuticals Co., Ltd. (Sichuan, China). Fluorescein isothiocyanate (FITC), rhodamine-conjugated ulex europaeus agglutinin I lectin (Rho-UEA-I), 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), streptozotocin, and lipopolysaccharide (LPS) (Escherichia coli, serotype 0111:B4) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Acetyl-L-cysteine was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). BCA assay kit was obtained from KeyGen Biotech Co. (Nanjing, China). FM4-64 was purchased from Invitrogen (Carlsbad, CA). All other chemicals were commercial products of analytical or reagent grade and were used without further purification. Caco-2 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). The HT29-MTX cell line was a kind gift from Dr. Thecla Lesuffleur (INSERM, Paris, France). Synthesis. The synthesis procedure was schemed in Figure 1a. Mono-6-deoxy-6-amino-β-cyclodextrin (β-CD-NH2; degree of substitution in one β-cyclodextrin molecule = 1.0) was 318

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Investigation. The effect of ionic strength on the integrity of the nanocomplexes was evaluated by incubating nanocomplexes with different concentrations of NaCl. The nanocomplex integrity was determined by measuring the turbidity at 450 nm. Hydrophobic domains in the nanocomplexes were detected with Coomassie Brilliant Blue (CBB) G-250 dye as reported.19 Briefly, 1 mL of the nanocomplexes solutions was mixed with 1 mL of CBB solution (0.1 mM), and the mixture was vortexed for 10 min at room temperature. The absorption spectrum of CBB was monitored between 400 and 800 nm to measure the maximum absorption peak with a UV−vis spectrophotometer (Cary100-EL05033098, Zhejiang, China). For the intrinsic fluorescence spectroscopic investigation, insulin solution (0.5 mg/mL) was mixed with bis-cyclodextrin-glutamic acid derivative (compound 3) at molar ratios of 0, 1, 2, and 4 and stirred for 20 min. The fluorescence emission spectrum of the insulin solution from 290 to 390 nm was recorded at the excitation of 280 nm (Shimadzu RF-5301 fluorescence spectrophotometer, Japan). Enzymatic Stability Study. A sample of 100 μL of trypsin solution (3000BAEE IU/mL, pH 7.4) was added to 900 μL of free insulin solution (control) or different nanocomplex solution containing 250 μg/mL of insulin, thus to get the concentrations of insulin and trypsin at 225 μg/mL and 300BAEE IU/mL, respectively. Aliquots (100 μL) were withdrawn at predetermined time points. Disintegration of nanocomplexes and halt of enzymatic reaction were achieved by the addition of 400 μL of ice cold water containing 0.1% trifluoroacetic acid. The insulin concentration was quantified by RP-HPLC (Agilent 1200 series, CA, USA). Experiments were performed in triplicate. In Vitro Cellular Studies. Caco-2 and HT29-MTX cells were cultivated separately in tissue culture flasks using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% L-glutamine. Both cultures were maintained at 37 °C under 5% CO2. An intracellular uptake investigation was performed on the Caco-2 cell model and the Caco-2/HT29-MTX cocultured cell model. The Caco-2 cells and Caco-2/HT29-MTX cells (mixed at 1:1) were seeded onto 24-well plates (Corning, NY, USA) at a density of 5 × 104 cells/well and cultured for 7 days. The culture medium was replaced with 500 μL of free F-insulin or nanocomplexes in Hank’s balanced salt solution (HBSS). The concentration of F-insulin was 0.2 mg/mL for all tested samples. After incubation at 37 °C for 2 h, the test media were removed, and the cells were washed with ice-cold phosphatebuffered saline (PBS) and heparin (0.5 mg/mL, 3 min) to remove extracellular bound nanocomplexes. Then cell lysis buffer was added, and the cell-associated fluorescence and protein were determined by Varioskan Flash (Thermo Fisher Scientific, MA, USA) and BCA assay kit (KeyGen Biotech Co., Ltd., Nanjing, China), respectively. The amounts of uptake were expressed as the quantity of F-insulin associated with 1 mg of cellular protein. Tests were performed in quadruplicate for each sample. To study the influence of mucus on the uptake of nanocomplexes, the cellular internalization studies were also performed on Caco-2/HT29-MTX cell cocultures after the removal of mucus, which was achieved by incubation of the cocultured cells with 10 mM N-acetyl-L-cysteine dissolved in HBSS under agitation for 60 min at 37 °C before study.18 The cellular uptake on Caco-2 cells was also visualized using confocal laser scanning microscopy. The uptake of the

prepared by the reported procedure via tosylation, nucleophilic substitution with sodium azide, and reduction with triphenyl phosphine. The bis-CD-glutamic acid derivative (2-(tert-butylloxycarbonylamino)-N1,N5-bis(6-mono-6-deoxy-βcyclodextrin)pentanediamide) was obtained by dicoupling one molecule of N-protected glutamic acid (N-Boc-L-glutamic acid) with two moieties of β-CD-NH2. Compound 3 was obtained by removing the N-protecting Boc group using TFA. Compound 4 was obtained by coupling compound 3 with 2-maleimido acetic acid using dicyclohexylcarbodiimide. The PEN peptide was synthesized with an additional cysteine residue at its amino terminus in order to facilitate unambiguous conjugation (CRQIKIWFQNRRMKWKK). The PEN-bis-β-CD conjugate (compound 5, P-bis-CD) was synthesized by attaching PEN peptide to bis-β-CD using chemo-selective maleimide−thiol chemistry. A detailed description of synthesis and purification procedure, qualification, and spectrum can be found in Supporting Information. Preparation and Characterization of Nanocomplexes. Insulin was dissolved in hydrochloric acid (HCl) solution (0.01N, pH 2.0) at a concentration of 1 mg/mL (0.172 mM), and the pH was adjusted to 7.0 using 1 M NaOH solution. PEN and P-bis-CD were dissolved in water at concentration of 0.689 mM. For the preparation of insulin loaded PEN nanocomplexes (PEN NC) or P-bis-CD nanocomplexes (Pbis-CD NC), the insulin solution was added dropwise (two drops per second) to an equal volume of PEN or P-bis-CD solution under stirring. The molar ratio of insulin to CPP was 1:4 for both nanocomplexes. The mixture was stirred at room temperature for another 20 min, yielding an opalescent suspension. Fluorescent labeled nanocomplexes were also prepared with the same method using FITC-labeled insulin (F-insulin). F-insulin was synthesized according to the previous report.17 The prepared nanocomplexes were characterized for particle size and zeta potential with a Malvern Zetasize NanoZS90 (Malvern Instruments Ltd., UK). All measurements were performed in triplicate. The morphology of the PEN NC and P-bis-CD NC was examined by transmission electron microscope (TEM, H-600, Hitachi, Japan). For the evaluation of encapsulation efficiency, freshly prepared nanocomplexes were centrifuged at 16 000 rpm for 15 min at 4 °C. After the ultracentrifugation, the amount of insulin in the supernatant was measured by a reverse-phase high-performance liquid chromatography (RP-HPLC) method (Agilent 1200 series, CA, USA) according to literature.18 The entrapment efficiency (EE %) were calculated as the following equation: EE(%) = (total amount of insulin − insulin in supernatant)/(total amount of insulin) × 100; the drug loading efficiency (LE%) were calculated as following equation: LE(%) = (amount of loaded insulin)/(load amount of insulin + the amount of PEN or Pbis-CD) × 100. The in vitro release profile of F-insulin from the nanocomplexes was investigated by the dialysis method. Freshly prepared F-insulin loaded nanocomplexes (0.5 mL) or free Finsulin were sealed within a dialysis bag and immersed in 20 mL of phosphate buffer solution (pH 6.8). At predetermined time point, samples were withdrawn, and the concentration of Finsulin was determined by fluorescence spectrometry (excitation at 488 nm and emission at 518 nm). Experiments were performed in triplicate, and the accumulative release is expressed as the total percentage of drug released over time. Effect of Ionic Strength, Detection of the Hydrophobic Interaction, and Fluorescence Spectroscopic 319

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rhodamine-conjugated ulex europaeus agglutinin I lectin (Rho-UEA-I) and DAPI to visualize the mucus and nuclei, respectively. The tissue-sections were observed by inverted fluorescence microscope (Sunny Instrument Co., Ltd., Ningbo, China). In Vivo Evaluation on Diabetic Animals. Male Sprague− Dawley rats weighing between 180 and 220 g were used. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) dissolved in a 10 mM citrate buffer (pH 4.5) as reported.18 The blood glucose level was determined using a glucose meter (JPS-6, Yicheng Biotech. Co. Ltd., Beijing, China). Rats were considered to be diabetic with the fasting blood glucose higher than 16.0 mM one week after treatment. Rats were fasted for 12 h prior to drug administration with water available ad libitum. Then the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). A midline laparotomy was done to expose the small intestine. Physiological saline, free insulin solution (30 IU/kg), PEN NC solution (30 IU/kg, 1:4), and P-bis-CD NC solution (30 IU/ kg, 1:4) were administered by intestinal injection. Another group of rats which underwent the same surgery was administered with 1 IU/kg of free insulin solution by subcutaneous (s.c.) injection. Blood samples were collected from the tail veins of rats prior to drug administration and at different time intervals, and then the blood glucose levels were determined using a glucose meter. For the analysis of plasma insulin levels, blood samples were centrifuged (3000 rpm, 5 min) and then quantified using porcine insulin ELISA kit (R&D System, Inc., MN, USA). The extent of glycemia reduction response was calculated using the trapezoidal method to determine the area above the curve (AAC) of the blood glucose level time curves for 0−8 h.20 The area under the curve (AUC) of plasma insulin concentration curve was calculated from the pharmacokinetic study. The pharmacological availability (PA %) and bioavailability (BA %) relative to subcutaneous injection were calculated. Toxicity Studies. The cytotoxicity of PEN and the P-bisCD was evaluated on Caco-2 cells using the MTT assay. The cells were seeded in 96 well plates at a density of 1 × 104 cells/ well and cultured for 7 days. The cells were incubated with 100 μL of PEN or P-bis-CD solution at the concentration of 1, 2, and 5 mg/mL in HBSS for 24 h at 37 °C and then submitted to an MTT assay. HBSS was used as reference for 100% cell viability. Tests were performed in triplicate, and the data were expressed as the percentage of viable cells by comparing with the control. To investigate whether long-term administration of the P-bisCD NC can also promote the absorption of unwanted toxins presented in the small intestine, an in vivo toxicity study was performed.21 Male KM mice weighing between 18.0 and 22.0 g were randomly divided into four groups (five mice for each group). The mice were administrated with 0.1 mL of NaHCO3 solution (3%) intragastrically to neutralize the gastric acid 15 min prior to the administration tested samples.22 Then the experimental groups received once-daily oral doses of LPS (5 mg/kg) with or without P-bis-CD NC solution (50 IU/kg of INS) for 7 consecutive days. One group without any treatment was used as a control, and another group receiving intraperitoneal injection of LPS (5 mg/kg) served as a reference for the toxicity produced by the systemic LPS. After 7 days of treatment, animals were anaesthetized with pentobarbital sodium, and then the blood samples were collected via cardiac

nanocomplexes was performed as described. Then the cells were treated with 10 μM endocytotic fluorescent marker FM464 for 15 min at 37 °C. The living cells were viewed using a Zeiss LSM 510 confocal laser scanning system (Carl Zeiss, Germany). To study the mechanism of cellular internalization, the cellular uptake was performed with the treatment of specific endocytosis inhibitors or in different temperatures. Caco-2 cells were seeded and cultured as described above. In the specific inhibition studies, the cells were preincubated with different endocytosis inhibitors including chlorpromazine (Chl, 10 μg/ mL), filipin (Fil, 1 μg/mL), sodium azide (NaN3, 1 mg/mL), and amiloride (Ami, 0.3 mg/mL) in HBSS for 1 h at 37 °C. Then the medium was replaced by nanocomplex solution with the presence of the same inhibitors. In the temperaturedependent uptake studies, the cells were incubated with test solution at 37, 16, and 4 °C. The uptake amount was measured as described above, and the results were presented as the percentage of control conducted at 37 °C without any treatment. The effectiveness of the nanocomplexes on improving the permeation of the insulin across the Caco-2 cell monolayers was also investigated. Caco-2 cells were seeded at a density of 1 × 104 cells/well on a polycarbonate filter (diameter 6.5 mm, growth area 0.33 cm2) in Costar Transwell 24 wells/plate (Corning Costar Corp., NY) and were cultured for 14−18 days. The TEER values were measured using a Millicell-ERS system (Millipore, USA), and monolayers with TEER values higher than 500 Ω·cm2 were used. Prior to the studies, the mediums in the apical and basolateral chambers were replaced with prewarmed HBSS and the cells were allowed to equilibrate at 37 °C for 30 min. Then the medium in apical chambers was replaced with 100 μL of free F-insulin or nanocomplexes solution at the concentration of 0.2 mg/mL for F-insulin. At the determined time points (0.5, 1, 1.5, 2, and 3 h), an aliquot of sample (0.1 mL) were taken from the basolateral chamber and equal volumes of HBSS were supplemented. The amount of transported F-insulin was determined using Varioskan Flash (Thermo Fisher Scientific, USA). The apparent permeability coefficient (Papp, cm/s) of insulin was calculated using the following equation: Papp = (dQ/dt) × (1/A·C0), where dQ/dt is the permeability rate, A is the surface area of the membrane filter, and C0 is the initial concentration in the apical chamber. In Situ Absorption Studies in Intestinal Loop. In situ absorption studies in rats were performed with F-insulin to visualize its absorption status in the intestinal villi. All experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University. Male Sprague− Dawley rats weighing between 180 and 220 g were fasted overnight before experiments with free access to water. The rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg), and then a midline laparotomy was done to expose the ileum. A 10 cm section of intestinal loop was made and ligated at both ends. Free F-insulin solution or nanocomplexes were administered directly into the loop (1 mg/kg for F-INS). At 60 min after administration, the rats were sacrificed by cervical dislocation. The loops were removed, mildly washed with 10 mL of PBS, fixed with 4% paraformaldehyde (PFA) for 2 h, and then dehydrated in 30% sucrose at 4 °C overnight. Samples were frozen quickly in liquid nitrogen-cooled OCT-compound (optimal cutting temperature-compound, Miles Laboratories Inc., Indiana, USA). The tissue was cross-sectioned and stained with 320

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Table 1. Characterizations of the PEN NC and P-bis-CD NCa

a

nanoparticles

size (nm)

PDI

zeta potential (mv)

encapsulation efficiency (%)

drug loading (%)

PEN NC P-bis-CD NC

101.2 ± 19.2 119.7 ± 21.3

0.22 0.34

18.7 ± 4.7 15.6 ± 6.7

96.2 ± 2.1 95.8 ± 2.4

36.7% 22.0%

Data were presented as mean ± standard deviation, n = 3.

Figure 2. (a) Percentage of turbidity of PEN NC or P-bis-CD NC in different concentrations of sodium chloride (mean ± SD, n = 3). (b) The absorption spectrum of Coomassie Brilliant Blue G-250 dye (CBB) with insulin, PEN NC, or P-bis-CD NC. The peak wavelengths of free CBB, CBB/insulin, CBB/PEN NC, and CBB/P-bis-CD NC were 589 nm, 594 nm, 598 nm, and 606 nm, respectively. (c) Intrinsic fluorescence spectrum of insulin with different mole ratio of bis-cyclodextrin-glutamic acid derivative (1:0, 1:1, 1:2, 1:4). (d) Percentage of residual amount of insulin in free insulin solution, PEN NC or P-bis-CD NC after certain times of incubation with trypsin (mean ± SD, n = 3).

PEN is a highly positively charged peptide, and insulin molecules possess negative charges in the neutral environment. Nanocomplexes could be easily formed by self-assembly in the form of polyelectrolyte complex (PEC). To form a nanosized complex rather than precipitation, pH, and concentration of insulin solution, the mixing speed and the ratio between insulin and PEN or its derivative were adjusted in our preliminary study. Insulin solution was added dropwise into the PEN or Pbis-CD solution at the molar ratio of 1:4. The TEM image of the insulin-PEN nanocomplexes (PEN NC) and insulin-P-bisCD nanocomplexes (P-bis-CD NC) are shown in Figure 1c. The particle size, zeta potential, encapsulation efficiency, and drug loading of both nanocomplexes are listed in Table 1. PEN NC and P-bis-CD NC exhibited average diameters of 101.2 nm and 119.7 nm, respectively. Both nanocomplexes had encapsulation efficiencies above 95%. Drug loadings of PEN NC and P-bis-CD NC were 36.7% and 22.0%, respectively. The lower loading of P-bis-CD NC was due to the higher molecular weight of P-bis-CD, since all nanocomplexes were prepared at the same molar ratio of insulin and cell-penetrating peptide. In vitro release profiles of the two prepared nanocomplexes are shown in Figure 1d. Much shower release profiles were observed for both nanocomplexes compared with free F-insulin. P-bis-CD complexes exhibited sustained release within 8 h. However, the drug release of PEN complexes reaches the equilibrium within 4 h.

puncture. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with an ALT and AST assay kit (Jiancheng Biotech. Co. Ltd., Nanjing, China). After being sacrificed, the livers of each animal were harvested for histological examinations. Specimens of liver were fixed in 10% paraformaldehyde, dehydrated with ethanol, embedded and frozen in OCT-compound, and then sectioned and stained with hematoxylin and eosin (H&E).

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RESULTS

Chemical Synthesis and Nanocomplexes Preparation. PEN-bis-β-cyclodextrin conjugates (P-bis-CD, compound 5) were synthesized by attaching a bis-β-CD group to the Cterminus of PEN, as outlined in Figure 1a. β-cyclodextrin (βCD) was first monoaminalized (mono-6-deoxy-6-amino-βcyclodextrin, β-CD-NH2) to render only one reactive functional group for further conjugation. Bis-CD-glutamic acid derivative (Compound 3) was then synthesized by dicoupling one molecule of glutamic acid with two moieties of β-CD-NH2. Cysteine was introduced as an N-terminal residue of the penetratin peptides to facilitate unambiguous conjugation. Penetratin was attached to bis-β-CD via a succinimidopropionyl-sulfide linker, using chemoselective maleimide−thiol chemistry. A detailed description of synthesis, purification, qualification, and spectrum can be found in the Supporting Information. 321

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Figure 3. (a) Cellular uptake of free F-insulin or fluorescent labeled nanocomplexes on the Caco-2 cell model (mean ± SD, n = 4, *: P < 0.05 vs free F-insulin). (b) Cellular uptake of free F-insulin or fluorescent labeled nanocomplexes on cocultured Caco-2/HT29-MTX cell model with or without mucus (mean ± SD, n = 4, *: P < 0.05). (c) Caco-2 cells uptake of F-insulin with presence of various endocytosis inhibitors or in different temperatures. The results were presented as the percentage of the control at 37 °C without any inhibitor (mean ± SD, n = 4, *: P < 0.05 vs control). (d) Confocal microscopic images of intracellular uptake of PEN NC and P-bis-CD NC on Caco-2 cells. F-insulin exhibited a green signal, and FM 4−64 (red) was used as an endosome marker.

Effect of Ionic Strength, Detection of the Hydrophobic Interaction, and Fluorescence Spectroscopic Investigation. To determine the role of electrostatic attraction in the formation of nanocomplexes, the integrity of nanocomplexes were investigated with different concentrations of sodium chloride, which can shield the electrostatic interaction. As shown in Figure 2a, the turbidity of both PEN NC and Pbis-CD NC decreased with the increase of salt concentration. This phenomenon indicated the disintegration of the nanocomplexes in high salinity, which is a typical characteristic of PEC.23 However, the turbidity of P-bis-CD NC became steady when it reached around 20% of the initial turbidity when no sodium chloride was added, while PEN NC totally disintegrated at the salt concentration of 20 mg/mL. This phenomenon demonstrated that P-bis-CD NC presented a better stability

than PEN NC in high ionic strength, suggesting different association mechanisms of the two nanocomplexes. Electrostatic attraction might be the only driving force for the selfassembly of PEN NC, while other interactions that were independent of ionic strength existed in P-bis-CD NC. The hydrophobic domains in the PEN NC and P-bis-CD NC were detected with Coomassie Brilliant Blue (CBB) G-250 dye. CBB is a highly water-soluble dye which also possesses a domain with a high hydrophobicity. The wavelength shift of the maximum absorption of CBB could be observed in hydrophobic media; thus it is a sensitive probe for microenvironmental polarity.19 Figure 2b shows the absorption spectrum of CBB with different samples, and the peak wavelengths of free CBB, CBB/insulin, CBB/PEN NC, and CBB/P-bis-CD NC were 589 nm, 594 nm, 598 nm, and 606 322

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permeation of the insulin across the cell monolayers was investigated. The Papp value was calculated from the cumulative amount of F-insulin transported from the apical side to the basolateral side of the monolayers and is listed in Table 2. The Papp values of PEN NC and P-bis-CD NC were 3.0-fold and 25.6-fold higher than that of free F-insulin solution, respectively.

nm, respectively. The peak wavelength shift of CBB/insulin (5 nm) may be due to the interaction between CBB and the hydrophobic residues of protein.24 A higher wavelength shift for P-bis-CD NC (17 nm) compared to PEN NC (9 nm) suggested the existence of a more hydrophobic microenvironment in the former, which may due to the bis-β-cyclodextrin modification. To further investigate the complexation of insulin with the bis-β-cyclodextrin group, the intrinsic fluorescence spectrum of insulin with different mole ratios of biscyclodextrin-glutamic acid derivative was studied. Intrinsic fluorescence spectroscopy of insulin is mainly due to the presence of tyrosine and tryptophan residues and considered to be a sensitive indicator of conformational variations resulted from the interaction with the other component.20 The fluorescence emission intensity of insulin with different molar ratios of a bis-cyclodextrin-glutamic acid derivative was shown in Figure 2c. The change of fluorescence intensity with the increase of bis-β-cyclodextrin concentration indicated the microenvironmental variation of the two hydrophobic residues. This result indicated the occurrence of interaction between insulin and the bis-β-cyclodextrin group. Enzymatic Degradation Study. Rapid enzymatic degradation of insulin in gastrointestinal tract is the one of the two main causes of its poor oral bioavailability.1 The stability of free insulin and nanocomplexes was investigated with the presence of trypsin, one of the major proteases in intestinal tract.25 The addition of trifluoroacetic acid not only quenched the enzymatic reaction but also disintegrated the nanocomplex. The pH of sample was tested below 4 after the addition of the trifluoroacetic acid, and insulin would possess positive charges in this environment. All intact insulin, no matter encapsulated or released, was determined by the HPLC method. Figure 2d depicts the residual amount of insulin after certain times of incubation in enzymatic environment. The stability of insulin was significantly improved in both nanocomplexes compared with the free insulin solution. The degradation rates of free insulin, PEN NC, and P-bis-CD NC were calculated to be 4.36 μg/mL·min, 2.56 μg/mL·min, and 1.99 μg/mL·min, respectively. The degradation rate of P-bis-CD NC was 2.2-fold and 1.3-fold slower than free insulin and PEN NC, respectively. In Vitro Cell Uptake and Transport Studies. Intracellular uptake of free FITC-labeled insulin (F-insulin) solution and Finsulin loaded nanocomplexes were evaluated on the Caco-2 cell model (Figure 3a) and the cocultured Caco-2/HT29-MTX cell model (Figure 3b). The uptake of PEN NC and P-bis-CD NC was 3.6-fold and 8.6-fold higher than that of free F-insulin solution in Caco-2 cells, respectively. In the Caco-2/HT29MTX cell model (with mucus), uptake of PEN NC and P-bisCD NC was 6.6-fold and 24.6-fold higher than that of the free F-insulin solution, respectively. P-bis-CD NC exhibited higher uptake than PEN NC in both cellular models with a 2.4-fold and 3.7-fold increase, respectively. HT29-MTX cells are mucussecreting cells which could mimic the mucus-producing goblet cells of intestinal epithelium. To evaluate the influence of mucus on the intracellular uptake of the nanocomplexes, the study was also performed on the cocultured Caco-2/HT29MTX cell model after the removal of the mucus.26 No significant difference was observed for P-bis-CD NC with or without mucus. However, interestingly, the cellular uptake of PEN NC was increased after mucus was removed. A Caco-2 cell monolayer cultured on permeable membrane was used as in vitro model to mimic the intestinal epithelium. The effectiveness of the nanocomplexes on improving

Table 2. Papp Values for F-Insulin Solution, PEN NC, and Pbis-CD NC across Caco-2 Cell Monolayers in Transport Studya sample

Papp (cm/s)

F-insulin solution PEN NC P-bis-CD NC

2.32 ± 0.77 × 10−7 7.04 ± 4.78 × 10−7* 5.95 ± 0.31 × 10−6*

Mean ± standard deviation, n = 4; *: Significant difference with Finsulin solution (p < 0.05)).

a

Mechanism of Cellular Uptake. To investigate the uptake mechanism of the prepared nanocomplexes, specific inhibition studies were performed. Sodium azide (NaN3) was used for energy depletion as a comprehensive active transport inhibitor. Chlorpromazine, filipin, and amiloride were used as specific endocytosis inhibitors for clathrin-mediated endocytosis, caveloae-mediated endocytosis, and macropinocytosis, respectively.18,27 Results were shown in Figure 3c. For PEN NC, NaN3 presented no effect on the uptake, and chlorpromazine, filipin, and amiloride even upgraded the amount of internalization (p < 0.05). However, for P-bis-CD NC, the cellular uptake was inhibited by 53.3% with the presence of sodium azide. Filipin and amiloride also significantly reduced the amount of uptake (p < 0.05). This result suggested that different pathways of internalization existed for the two nanocomplexes. Since energy depletion has almost no effect on the internalization of PEN NC, their uptake might occur via energy-independent pathway. For P-bis-CD NC, the significant inhibition by sodium azide, filipin, and amiloride suggested the existence of energy-dependent pathways in which macropinocytosis and caveloae-mediated endocytosis were involved. A temperature-dependent uptake study was performed at 37, 16, and 4 °C to further verify the uptake mechanism. Energydependent endocytosis and energy-independent direct transduction could be distinguished at 16 °C, in which the former would be inhibited due to the interference of intracellular vesicular fusion events, while the latter is not affected.28 As shown in Figure 3c, the uptake amount of PEN NC was not affected at 16 °C but reduced to 59.7% at 4 °C (p < 0.05) compared with the control at 37 °C. However, the uptake amounts of P-bis-CD NC were decreased to 30.1% at 16 °C and 18.3% at 4 °C (p < 0.05). These results were concordant with the specific inhibition study and indicated that the energydependent endocytosis was involved in P-bis-CD NC rather than PEN NC. The uptake inhibition at 4 °C for PEN NC may be due to the altered membrane fluidity which was highly related to the direct transduction.29 Confocal microscopic images of intracellular uptake on Caco2 cells were shown in Figure 3d. Much stronger signals of Finsulin (green) were observed for P-bis-CD NC than PEN NC. Interestingly, a different subcellular distribution of green signals was observed for the two nanocomplexes. Diffused green fluorescence in cytoplasm was observed for PEN NC, while a punctate cytoplasmic distribution (white arrow) was observed 323

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Figure 4. (a) Fluorescence microscopy of rat intestinal villi after administration of free F-insulin solution, PEN NC, or P-bis-CD NC (F-insulin exhibited green fluorescence). The tissue section was stained with Rho-UEA-I (red) and DAPI (blue) to visualize the mucus and nucleus. (b) Hypoglycemic effect in the in vivo pharmacodynamic study on diabetic rats. Blood glucose level was presented as the percentage of the blood glucose concentration before administration. Dosages were 1 IU/kg for s.c. injection of insulin solution and 30 IU/kg for intestinal administration of insulin solution, PEN NC, and P-bis-CD NC. (*: P < 0.05 vs insulin solution group, n = 7−8). (c) Plasma insulin concentration in the in vivo pharmacokinetic study on diabetic rats (*: P < 0.05 vs insulin solution group, n = 7−8).

within the villi. Obvious absorption of F-insulin was observed in the epithelial cells for both PEN NC and P-bis-CD NC. Interestingly, strong green signals were observed in the mucus layer (overlapped with red) for PEN NC. However, P-bis-CD NC group exhibited a much stronger signal than PEN NC within the villi, and much of the signals were observed in the interior of villi where the villus capillaries should be located (white arrows). An in vivo pharmacodynamic and pharmacokinetic investigation was then performed on diabetic rats. Free insulin solution, PEN NC, and P-bis-CD NC were administered directly to the small intestine. Figure 4b and c show the variation of blood glucose concentration and plasma insulin concentration after their intestinal administration (30 IU/kg) or subcutaneous injection (1 IU/kg) of insulin solution. No evident hypoglycemic effect and increase of plasma insulin

for P-bis-CD NC. FM4-64 (red signal) is a marker of endocytosis which indicates the punctate cytoplasmic vesicles. Co-localization of the endosome marker with F-insulin (yellow signal) could only be observed in P-bis-CD NC. This result also demonstrated the existence of endocytosis in P-bis-CD NC rather than PEN NC. In Situ Absorption and in Vivo Pharmacodynamics and Pharmacokinetics. The purpose of intestinal delivery is to convey the therapeutics across the epithelium on intestinal villi and enter the villus capillary. Therefore, in situ absorption of F-insulin (green) in intestinal villi was visualized by fluorescent microscopy and is shown in Figure 4a. 4',6Diamidino-2-phenylindole (DAPI) (blue) and Rho-UEA-I (red) were used to visualize the nuclei of epithelial cells and the mucus layer that surrounded the villi, respectively. For free F-insulin solution, hardly any green signal could be observed 324

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Toxicity Study. The cytotoxicity of PEN and P-bis-CD was evaluated on Caco-2 cells by an MTT assay at the concentration of 1, 2, and 5 mg/mL. As shown in Figure 5a, the relative cell viability for both PEN and P-bis-CD at 1−2 mg/mL exhibited no significant difference compared with the control. At 5 mg/mL, a significant reduction of cell viability was observed for PEN (p < 0.05), while P-bis-CD still showed no significant difference compared with the control. To investigate whether long-term administration of the P-bisCD NC causes the absorption of unwanted toxins that exists in the small intestine, a 7-day in vivo toxicity study was performed. Lipopolysaccharide (LPS) secreted by Gram-negative bacteria is the major bacterial toxin in the GI tract.30 If delivered into the systemic circulation, LPS triggers systemic inflammatory response which may progress to hepatocyte necrosis.31,32 After 7 days of consecutive administration, two of the liver function indicators (ALT and AST) were evaluated (Figure 5b). Intraperitoneal injection of LPS resulted in a remarkable increase of both ALT and AST compared with the control (p < 0.05). However, oral administration of LPS with or without Pbis-CD NC did not show significant changes in levels of ALT and AST compared with the control. Histological examination

concentration was observed after the intestinal administration of insulin solution. The administration of PEN NC or P-bis-CD NC induced noticeable hypoglycemic responses and increase of plasma insulin concentration. P-bis-CD NC exhibited the strongest hypoglycemic effect and highest plasma insulin concentration which lasted for 6 h with a maximal reduction of 60%. The AUC value, pharmacological availability (PA), and relative bioavailability (BA) are shown in Table 3. P-bis-CD NC exhibited a PA of 10.6% and BA of 7.1%, which were 3.0fold and 2.3-fold higher than that of PEN NC, respectively. Table 3. Pharmacological Availability and Bioavailability of Insulin in Diabetic Rats after Ileal Administration of PEN NC or P-bis-CD NCa sample

dose (IU/kg)

AUC (mIU h/L)

PA (%)

BA (%)

S.C. INS PEN NC P-bis-CD NC

1 30 30

132.0 ± 26.6 122.8 ± 23.9 279.3 ± 47.7

3.5 ± 0.7 10.6 ± 2.7

3.1 ± 0.6 7.1 ± 1.2

a

AUC: area under the plasma concentration−time curve; PA: pharmacological availability; BA: bioavailability. (mean ± standard deviation, n = 7−8).

Figure 5. (a) Cytotoxicity of PEN or P-bis-CD on Caco-2 cells at 1, 2, and 5 mg/mL.*: P < 0.05 vs control). (b) Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) in mice after 7 days of consecutive oral administration of LPS (Oral LPS) and oral administration of LPS in combination of P-bis-CD NC (oral LPS + P-bis-CD NC) or once intraperitoneal administration of LPS (IP LPS). The group without receiving any treatment was used as a control (mean ± SD, n = 5, *: P < 0.05 vs control). (c) Photomicrographs of the liver sections obtained in the toxicity study. 325

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ization. It was suggested by some researchers that the drug− CPPs interaction might be dissociated by competitive interaction between CPPs and GAG.12,13 Since intestinal mucus lay that covering the epithelium contains a large amount of GAGs,36 competitive dissociation of the cargo is also likely to occur within the mucus layer. The influence of mucus was evaluated on the cocultured Caco-2/HT29-MTX cell model with or without mucus. Interestingly, the cellular uptake of PEN NC was increased when mucus was removed, while that of P-bis-CD NC was not significantly affected (Figure 3b). This phenomenon may suggest that less competitive dissociation happened in P-bis-CD NC than PEN NC as they went through the mucus. Different researchers have proved that multiple mechanisms were involved in cellular uptake of CPPs, and the mechanisms can be categorized into energy-dependent endocytosis and energy-independent direct transduction across the lipid bilayer. It has become evident that the pathway of CPP-mediated internalization was cell type- or cargo type-specific and was significantly affected by other factors such as concentration and the linkage of cargo.37,38 In the mechanism study, different internalization mechanisms were observed for PEN NC and Pbis-CD NC (Figure 3c). Both specific inhibition and a temperature-dependent uptake study demonstrated that the uptake of PEN NC mainly occurred via energy-independent pathway (direct transduction). Interestingly, specific endocytosis inhibitors even increased the uptake of PEN NC. This phenomenon was also observed by other researchers for Tat and R9 peptides, and they suggested that perturbation of endocytotic mechanisms promoted accumulation of peptide on the membrane for cellular import via direct transduction.38 However, energy-dependent pathways were involved in the uptake of P-bis-CD NC, including at least macropinocytosis and caveloae-mediated endocytosis. The confocal microscopic images of P-bis-CD NC exhibited the typical modality of endocytosis, and the colocalization of F-insulin with the endosome marker was observed (Figure 3d). Some previous studies proved that CPPs can harness alternate mechanisms of uptake according to the environmental conditions.37 As demonstrated in our study, stronger intermolecular association might result in less CPP-cargo dissociation and better nanocomplex integrity. The difference of the internalization mechanism of the two nanocomplexes may be due to the variation of the intermolecular interaction and the complex stability. The different uptake mechanism may also be the reason for the improved internalization of the P-bis-CD NC compared with PEN NC. In situ absorption studies demonstrated that P-bis-CD NC were more effective in conveying the drug into the villus capillaries. Meanwhile, it was noticed that the colocalization between the drug (green) and mucus (red) was more obvious for PEN NC than P-bis-CD NC. This phenomenon was consisted with the mucus influencing study and suggested that higher CPP−drug dissociation might happen in PEN NC. An in vivo study in diabetic rats demonstrated a noticeable hypoglycemic effect of P-bis-CD NC that lasted for 6 h. The pharmacological availability (PA) and relative bioavailability (BA) of P-bis-CD NC were 3.0-fold and 2.3-fold higher than that of PEN NC. The improved absorption could be the comprehensive result of stronger intermolecular interaction, improved nanocomplex integrity, better enzymatic stability, less CPP-cargo dissociation, and altered internalization pathway.

of liver sections showed broad hemorrhagic necrosis and hepatocyte degeneration (black arrow) in the group with intraperitoneal injection of LPS (Figure 5c). However, no changes in the liver sections were observed in other groups compared to the control. These results indicated that P-bis-CD NC did not promote the unwanted intestinal absorption of LPS.

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DISCUSSION β-cyclodextrin is a cyclic oligosaccharide which possesses an hydrophobic cavity and hydrophilic surface and is able to form inclusion complexes with drugs of adequate size.33 The cyclodextrin family exerted effects on the intestinal barrier function and modulated flux of small molecular drug through intestinal barrier via different mechanisms.34 Class II or class III drugs in the Biopharmaceutics Classification System usually formed inclusion complexes with cyclodextrin for a subsequent permeation enhancing effect. Macromolecular drugs, such as insulin, which represented too large molecules for total inclusion, could partially associate with β-CD via their hydrophobic side chains.14 It had been previously demonstrated that interaction between β-CD and insulin occurs on specific amino acids via hydrophobic interactions.14,15 Unlike ionic interactions, the strength of hydrophobic interactions is less affected with the existence of other charged molecules.16 In our preliminary study, the derivative of PEN with just one βcyclodextrin group attached exhibited a slight increase in the permeation-enhancing effect compared with PEN (data not shown). P-bis-CD, in which a bis-β-cyclodextrin group was attached and possessed two hydrophobic cavities, was expected to form a stronger intermolecular binding with the insulin. Both nanocomplexes exhibited the typical characteristic of PEC such as disintegration in high salinity.23 However, the different response of P-bis-CD NC compared to PEN NC in the sodium chloride solution (Figure 2a) suggested that electrostatic attraction might be the only driving force for the assembly of the latter, while other interaction was involved in the formation of P-bis-CD NC. As shown in Figure 2b and c, microenvironments with higher hydrophobicity existed in the P-bis-CD NC compared with PEN NC, and interaction occurred between hydrophobic residues of insulin and the bis-β-cyclodextrin group. Therefore, the improved stability of P-bis-CD NC could be the result of the hydrophobic interaction between the bis-βcyclodextrin group and the hydrophobic residues of the insulin. In addition, better protection of insulin against protease was observed for P-bis-CD NC compared with PEN NC (Figure 2d). This phenomenon may also due to the association between the bis-β-cyclodextrin group and insulin, since the cavities could sequester some of the moieties of insulin that are sensitive to protease degradation.35 Significantly improved cellular internalization was observed for both nanocomplexes compared with free insulin solution in vitro, suggesting the effectiveness of CPPs-based nanocomplexes for enhancing epithelial uptake. P-bis-CD NC exhibited 2.4-fold and 3.7-fold higher uptake than PEN NC in the two cell models, respectively, and the Papp value of P-bisCD NC in the transport study was 8.5-fold higher than the latter. This indicated the enhanced capability of the peptide for the delivery of insulin, which may due to the enhanced intermolecular binding and better nanocomplex stability. The electrostatic interaction between CPPs and the negatively charged glycosaminoglycans (GAGs) on epithelial cell surface plays an essential role for the subsequent cellular internal326

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ACKNOWLEDGMENTS The research described above was supported by the National Natural Science Foundation of China (81173010) and the Open Project Program of State Key Laboratory of Long-acting and Targeting Drug Delivery System.

Cell-penetrating peptides were generally reported to be nontoxic and not to induce the leakage of lactate dehydrogenase of intestinal epithelium in the working concentration.39,40 The observed toxicity in higher concentrations was considered to be associated with the positive charge.41 The results of cytotoxicity study suggested that PEN or P-bis-CD presented no cytotoxicity at 2 mg/mL, which was highly above the concentration that utilized. P-bis-CD exhibited less cytotoxicity than PEN at 5 mg/mL, which may due to the reduction of charge density by the bis-β-cyclodextrin modification. Lipopolysaccharide (LPS), or bacterial endotoxin, presents in the gastrointestinal tract at all times. Since CPPs could nonspecifically enhance the permeation of oppositely charged macromolecules,40,42 a CPP-based delivery system could only be claimed as safe when no absorption of the negatively charge LPS was observed. An in vivo toxicity study showed that administration of LPS with P-bis-CD NC for 7 consecutive days did not induce change in liver function or histopathology, indicating that administration of P-bis-CD NC could not promote the intestinal absorption of unwanted toxins.

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CONCLUSION In this study, derivatives of PEN were synthesized in which a bis-β-cyclodextrin group was site-specifically attached to the PEN. Insulin-loaded nanocomplexes were prepared by selfassembly using PEN or P-bis-CD. Due to the existence of the hydrophobic interaction between the bis-β-cyclodextrin group and insulin, stronger CPP-cargo association, better complex stability, and higher enzymatic stability were observed for P-bisCD NC compared with PEN NC. Greatly improved cellular uptake and trans-monolayer transport were observed for P-bisCD NC compared with PEN NC both in vitro and in situ. Interestingly, different cellular internalization mechanisms were observed for the two nanocomplexes. After intestinal administration in diabetic rats, P-bis-CD NC exhibited a relative pharmacological availability of 10.6% and relative bioavailability of 7.1%, which were 3.0-fold and 2.3-fold higher than that of PEN NC, respectively. In addition, no absorption of coadministrated endotoxin was observed after 7 days of consecutive treatment. These results demonstrated that the permeation enhancing effect of PEN can be improved by strengthening the CPP-cargo intermolecular binding and the Pbis-CD have good potential to be utilized for gastrointestinal insulin delivery. The effectiveness of bis-β-cyclodextrin modification also suggested that chemical modification was a feasible strategy to tap the latent potential of CPPs by strengthening the cargo-CPP noncovalent binding. ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details, including description of synthesis and purification procedure, qualification, and spectrum. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel./fax: +86-28-85501617. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 327

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