Article pubs.acs.org/molecularpharmaceutics
Effective Enhancement of Hypoglycemic Effect of Insulin by LiverTargeted Nanoparticles Containing Cholic Acid-Modified Chitosan Derivative Zhe Zhang, Huanxin Cai, Zhijia Liu, and Ping Yao* State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Fudan University, Shanghai 200433, China S Supporting Information *
ABSTRACT: Liver is responsible for the balance of blood glucose level. In this study, cholic acid and N-(2-hydroxy)-propyl-3trimethylammonium chloride modified chitosan (HTCC-CA) was used as a liver-targeted vehicle for insulin delivery. A novel approach was developed to effectively load insulin by mixing insulin and HTCC-CA in 50% ethanol and water mixed solvent at pH 2 and then dialysis against pH 7.4 phosphate buffer subsequently against water. The insulin-loaded HTCC-CA nanoparticles have an average diameter of 86 nm and insulin loading efficiency of 98.7%. Due to random distribution of the hydrophobic cholic acid groups in HTCC-CA, some of the cholic acid groups located on the nanoparticle surface. Compared with free insulin, the nanoparticles increased in vitro cellular uptake of insulin to 466%, and the nanoparticles accumulated in liver for more time after subcutaneous injection into mice. The therapy for diabetic rats displayed that the nanoparticles increased the pharmacological bioavailability of insulin to 475% relative to free insulin, and the nanoparticles could maintain the hypoglycemic effect for more than 24 h. This study demonstrates that the nanoparticles with cholic acid groups on their surface possess liver-targeted property and biocompatible insulin-loaded HTCC-CA nanoparticles can effectively enhance the hypoglycemic effect of insulin. KEYWORDS: chitosan, cholic acid, diabetes mellitus, insulin, liver target, nanoparticle
■
INTRODUCTION
of insulin. Although many targeting delivery systems have been developed, few studies focused on liver-targeted delivery of insulin by now. Bile acids are produced in liver and circulate 10−20 times per day as a part of enterohepatic circulation.11,12 Kramer et al. demonstrated that a drug covalently linked to a bile acid could be selectively absorbed by the liver, and peptide drugs, which were easily cleaved by membrane-bound peptidases in the liver, could escape membrane-permeated proteolysis by covalent conjugation with bile acids sharing hepatic bile acid transport systems.13 Moreover, Xiao et al. demonstrated that liver uptake was very high for highly charged nanoparticles.14 In this study,
Diabetes mellitus is one of the main threats to human health, which affects more than 415 million adults around the world.1,2 By now, subcutaneous injection of insulin once or multiple times per day is still the most effective way to control blood glucose level (BGL) of diabetic patients.3,4 To alleviate the patients’ pain, local tissue necrosis, microbial contamination, and nerve damage, various insulin delivery systems have been fabricated for controlled and sustained release of insulin to overcome the limitations and drawbacks of conventional delivery.5−7 As we know, liver plays a major role in carbohydrate metabolism and takes the responsibility for the balance of BGL by means of glycogenogenesis and glycogenolysis.8,9 After being transported to liver cells, insulin stimulates the synthesis of glycogen in the liver to store glucose as glycogen.7,10 Therefore, it is possible that transporting insulin to liver is a strategy to improve the hypoglycemic effect © XXXX American Chemical Society
Received: March 3, 2016 Revised: May 12, 2016 Accepted: June 6, 2016
A
DOI: 10.1021/acs.molpharmaceut.6b00188 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
was characterized using ANS as a fluorescence probe.18 The two probes were used separately. The fluorescence emission spectra were measured on a fluorescence spectrophotometer (FLS-920, Edinburgh). Preparation and Characterization of INS/HTCC-CA Nanoparticles. HTCC-CA and insulin stock solutions were prepared by separately dissolving lyophilized HTCC-CA and insulin in 0.01 M HCl solutions. Equivoluminal ethanol was added into individual HTCC-CA and insulin solutions. Subsequently, the HTCC-CA ethanol solution was added into the insulin ethanol solution. The mixed solution was kept at 4 °C overnight and then dialyzed (cutoff molecular weight 3.5 kDa) against 1 mM pH 7.4 phosphate buffer and water in succession. Finally, the solution was adjusted to pH 7.4, and INS/HTCC-CA nanoparticles were obtained. The final insulin concentration was 1 mg/mL, and HTCC-CA concentration was also 1 mg/mL in INS/HTCC-CA solution. The free insulin in INS/HTCC-CA solution was separated by ultrafiltration using Amicon Ultra centrifugal filter (Ultracel 100 kDa, Millipore), and the insulin concentration in the filtrate was analyzed using BCA assay;19 the work curve was obtained by analysis of a series of insulin standard solutions. Insulin loading efficiency (LE) and insulin loading capacity of the HTCC-CA sample (LC) were calculated using the following equations:
we used cholic acid and N-(2-hydroxy)-propyl-3-trimethylammonium chloride modified chitosan (HTCC-CA) as a livertargeted delivery vehicle of insulin. Chitosan, the only cationic natural polysaccharide, is biocompatible, biodegradable, and modifiable.5 N-(2-Hydroxy)-propyl-3-trimethylammonium chitosan chloride (HTCC), which retains cationic charges in neutral solution, can improve the solubility of chitosan and enhance the binding ability with negatively charged insulin by electrostatic attraction as well as can increase the liver uptake. The cholic acid (CA) moiety in the chitosan derivative can increase the binding with insulin by hydrophobic interaction and also can enhance the liver uptake. In vitro, ex vivo, and in vivo investigations were performed to demonstrate that insulinloaded HTCC-CA (INS/HTCC-CA) nanoparticles can deliver insulin to liver and greatly improve the hypoglycemic effect of insulin.
■
EXPERIMENTAL SECTION Materials. Chitosan (50 kDa, deacetylation degree 95%) was from Jinan Haidebei Marine Bioengineering Co., Ltd. N(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), glycidyltrimethylammonium chloride (GTMAC), and alloxan were from SigmaAldrich. Cholic acid (CA) was from Bio Basic Inc. Porcine insulin (INS, 21 IU/mg) was from Dingguo Biotech Co., Ltd. Fluorescein isothiocyanate (FITC), pyrene, and 8-anilino-1naphthalenesulfonic acid (ANS) were from Tokyo Chemical Industry Co., Ltd. NHS-functionalized cyanine 7.5 (Cy7.5NHS) was from Lumiprobe. Bicinchoninic acid (BCA) protein assay kit was from Thermo Fisher Scientific Inc. DMEM cell culture medium and fetal bovine serum were from GIBCO BRL Life Technologies Inc. HEK293 cell line was from American Type Culture Collection. MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was from Promega Co. The other chemicals were from Sinopharm Chemical Reagent Co., Ltd. Synthesis and Characterization of HTCC-CA. The molecular weight distribution (Mw/Mn) of the chitosan we used is 2.8, which was measured by a gel permeation chromatography (Model 1525, Waters) equipped with TSK PW 2500 and 3000 columns and a refractive index detector (Optilab T-rEX, WYATT) using 0.2 M CH3COOH/0.1 M CH3COONa buffer as the mobile phase at a flow rate of 1.0 mL/min. HTCC-CA was synthesized and purified as reported previously.15 First, HTCC was synthesized by using chitosan (2 g) and GTMAC (5 mL). Second, HTCC-CA was synthesized by using purified HTCC (0.5 g) and CA (0.1, 0.2, or 0.5 g) with EDC and NHS as activator and coupled agent. FTIR spectra were recorded on a FTIR spectrometer (Nicolet 6700, Thermo Fisher) using ATR accessory. 1H NMR spectra were acquired on a NMR instrument (DMX500, Bruker) at 25 °C. NMR samples were prepared by separately dissolving chitosan in D2O containing 10 vol % DCl, HTCC in D2O, and HTCC-CA in a mixed solvent of D2O and DMSO-d6 (2:1, volume ratio). The quaternization degree of HTCC was characterized by analysis of the Cl− ion content using AgNO3 standard solution as titrant, and the conductivity was measured on a conductivity meter (DDS-11AD) during the titration.16 The CA grafting degree of HTCC-CA was calculated according to the NMR spectra as reported previously.15 Hydrophobic aggregation of the HTCC-CA samples in aqueous solutions was characterized using pyrene as a fluorescence probe as reported in the literature,17 and hydrophobic surface of the aggregates
LE(%, wt/wt) =
total insulin − free insulin × 100% total insulin
LC(%, wt/wt) =
total insulin − free insulin × 100% polymer
Z-Average hydrodynamic diameter (Dh), polydispersity index (PDI), and ζ-potential values of INS/HTCC-CA nanoparticles were measured on a laser light scattering instrument (Zetasizer Nano ZS90, Malvern) at 25 °C and 90° scattering angle. Before the measurement, NaCl was added to reach 5 mM NaCl concentration in the solution. Transmission electron microscopy (TEM) observations were conducted on an electron microscope (Tecnai G2 TWIN, FEI). In Vitro Release of Insulin from INS/HTCC-CA Nanoparticles. Insulin release was investigated using a dialysis method. INS/HTCC-CA solution of 1 mL was added into a dialysis tube (cutoff molecular weight 100 kDa) and dialyzed against 9 mL of release medium (PBS, 10 mM pH 7.4 phosphate buffer containing 0.15 M NaCl) at 37 °C with shaking. At predetermined intervals, 1 mL of the release medium was taken out, and the same volume of fresh PBS was added. The insulin concentration in the release medium was determined using BCA assay. In Vitro Cytotoxicity. Cell viabilities against HTCC, HTCC-CA, and INS/HTCC-CA were evaluated using MTS assay.20 After being seeded and then incubated in complete culture medium (DMEM medium containing 10 vol % fetal bovine serum, 100 IU/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate) for 24 h, the HEK293 cells were treated with the media containing different concentrations of HTCC, HTCC-CA, and INS/HTCC-CA. After 48 h incubation, the cells were analyzed by MTS assay. In Vitro Cellular Uptakes of Fluorescence-Labeled INS/ HTCC-CA Nanoparticles. FITC-labeled insulin (FITC-INS) was synthesized and purified as described in the literature.21 After being seeded in 12-well plates and incubated with the complete culture medium for 24 h, the HEK293 cells were B
DOI: 10.1021/acs.molpharmaceut.6b00188 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics Scheme 1. Synthetic Routes and Structures of HTCC and HTCC-CA
Table 1. I1/I3 Ratios of Pyrene Fluorescence, ANS Fluorescence Intensities, and ζ-Potentials of HTCC and HTCC-CA Samples with Various CA Grafting Degrees in pH 7.4 Aqueous Solutions (n = 3) sample
quaternization degree (mol %)a
CA grafting degree (mol %)b
HTCC HTCC-CA3 HTCC-CA7 HTCC-CA11
41.5 41.5 41.5 41.5
3.3 7.2 11.2
a
I1/I3 ratio of pyrene fluorescence 1.78 1.70 1.64 1.52
± ± ± ±
0.01 0.04 0.02 0.01
ANS fluorescence intensity (cps) 2.59 6.05 1.81 3.89
× × × ×
104 104 105 105
ζ-potential (mV) 37.1 36.0 31.0 28.6
± ± ± ±
0.2 0.5 0.1 0.1
Quaternization degree of the chitosan glycosyl residues. bCA grafting degree of the chitosan glycosyl residues.
CHEK Active, Roche). About 60% of the induced rats had fasting BGL higher than 13 mM, which was considered as diabetes. The diabetic rats were randomly assigned to various treatment groups with five in each group and were fasting overnight with freedom to water prior to administration. Physiological saline, insulin, INS/HTCC, and INS/HTCC-CA solutions at insulin dose of 1 IU/kg were separately injected into the rats via subcutaneous injection. At predetermined intervals blood samples were collected and their BGLs were measured. At 24 h postinjection, rat chow was provided after sampling and removed at 12 h before the next sampling time. Water was supplied at all times. For repeated administrations, male KM mice (20−22 g) were injected with alloxan to induce diabetes as described above. Insulin and INS/HTCC-CA solutions were separately injected subcutaneously at insulin dose of 2 IU/kg once daily. Rat chow was provided at 6 h postinjection and removed at 12 h before the next injection. Water was provided at all times. Histological Analysis. Male KM mice were separately injected with physiological saline and HTCC-CA solution subcutaneously at HTCC-CA dose of 0.5 mg/kg once daily for 7 and 15 days continuously. After the mice were sacrificed, the heart, liver, spleen, lung, and kidney were surgically taken out, fixed, dehydrated, and embedded in paraffin in succession. The specimens were cut into 5 μm thick sections, and the sections were stained with hematoxylin−eosin. The histological images of the sections were observed on a microscope (BX53, OLYMPUS). Statistical Analysis. The data were expressed as mean ± SD (standard deviation). Statistical analysis was performed using independent samples-t test (Origin Pro 8.0 software), and a P value
