Enhanced hypoglycemic effect of biotin-modified liposomes loading insulin: effect of formulation variables, intracellular trafficking, and cytotoxicity
© Zhang et al.; licensee Springer. 2014
Received: 6 January 2014
Accepted: 3 April 2014
Published: 16 April 2014
Peroral protein/peptide delivery has been one of the most challenging, but encouraging topics in pharmaceutics. This article was intended to explore the potential of biotin-modified liposomes (BLPs) as oral insulin delivery carriers. By incorporating biotin-DSPE into the lipid bilayer, we prepared BLPs using reverse evaporation/sonication method. We investigated hypoglycemic effects in normal rats after oral administration of BLPs, and the possible absorption mechanism by a series of in vitro tests. The relative pharmacological bioavailability of BLPs was up to 11.04% that was as much as 5.28 folds of conventional liposomes (CLPs). The results showed that the enhanced oral absorption of insulin mainly attributed to biotin ligand-mediated endocytosis. The results provided proof of BLPs as effective carriers for oral insulin delivery.
With the advent of biotech epoch, more and more proteins and peptides become available for clinical treatment, such as growth hormone , calcitonin , and octreotide . Nevertheless, due to short half-life in the blood circulation, it is inevitable to take the medications subjected to multi-dosage over a long time for chronic diseases. Insulin, a protein secreted by the β cells of the pancreas, is one of the most important therapeutic agents for insulin-dependent (type I) and deteriorative insulin-independent (type II) diabetes mellitus , and commonly administered subcutaneously; however, besides pain, which may bring about unwanted complications, e.g. allergic reactions, hyperinsulinemia, insulin lipodystrophy around the injection site . Problems encountered with insulin injection vitalize the demands to develop alternative delivery systems.
However, to achieve effective oral delivery of insulin, several barriers like instability, gastrointestinal enzymatic degradation, and poor membrane permeability, etc., should be overcome beforehand . Various delivery strategies, especially those based on nanoscaled delivery systems, have been explored to enhance the oral delivery of insulin, including microemulsions , nanospheres , polymeric nanoparticles [9, 10], niosomes , and liposomes [12–14]. However, the state of the art indicates that there seems to have reached a bottleneck in terms of oral bioavailability enhancement of insulin. It is highly recommended to explore novel strategies to ameliorate the performance of nanoscaled drug delivery systems. As known, receptor-mediated endocytosis, a process of internalization of extracellular molecules during which a binding occurs between the molecules and the receptors, is an important absorption mechanism for substances like proteins, hormones, growth factors, and fatty nutrients . In the intestinal cavity, there are a variety of receptors expressing on the membrane of enterocytes such as vitamins, transferrin, amino acids, sugars, and bile salt receptors . Targeted drug delivery to absorptive epithelia by receptor-mediated endocytosis has emerged as a prominent means to improve oral delivery of drugs . Vitamins as ligands, which can specifically bind to enterocytic receptors, have been extensively studied for the oral delivery of poorly permeable molecules [18–22].
Biotin receptors that distribute in the small intestine and partially in the colon are responsible for the essential absorption of biotin by nonspecific receptor-mediated endocytosis . Additionally, biotin plays an important role in maintaining the homeostasis of blood glucose . Improved cellular permeability and higher hypoglycemic effect after oral administration of biotin-conjugated glucagon-like peptide-1 has been observed . Biotin-modified vehicles have been investigated for nonparenteral delivery of active ingredients [26–29]. Our previous report has also proved that biotin-modified liposomes (BLPs) have ability to improve the oral delivery of insulin, and studied the uptake and transport mechanisms in the gastrointestinal tract . However, particular enhanced absorption mechanisms and cytotoxicity of BLPs are not clear enough.
Herein, we performed several experiments to further probe the oral absorption mechanism of BLPs based on previous studies  as well as the cytotoxicity thereof. We evaluated hypoglycemic effects of BLPs of various particles, or with different amounts of biotin-DSPE using normal rats. Meanwhile, the influence of BLPs on tight junctions and internalization process was further investigated by Caco-2 cells.
Porcine insulin (29 IU/mg) was provided by Jiangsu Wanbang Biochemical Pharmaceutical Co, Ltd (Xuzhou, China). Soybean phosphatidylcholine (SPC, Lipoid S75), cholesterol (CH), and 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE) were supplied by Lipoid (Ludwigshafen, Germany). Fluorescein isothiocyanate (FITC) and biotin were purchased from Sigma (Shanghai, China). Sephadex G-50 was obtained from Pharmacia (Shanghai, China). Deionized water was prepared by a Milli-Q purification system (Molsheim, France). HPLC-grade acetonitrile was provided by Merck (Darmstadt, Germany). All other chemicals were of analytical grade and used as received.
Preparation of BLPs
SPC, biotin-DSPE (synthesized according to previous report ), and cholesterol were dissolved in absolute ether to prepare the organic phase, into which the aqueous phase, insulin citric acid-Na2HPO4 buffer solution (pH 4.0, if not specified otherwise), was added dropwise following ultra-sonication to prepare the W/O emulsion. The organic solvent in the emulsion was then evaporated toward a rota-evaporator under 0.05- to 0.06-MPa pressure at a rotating speed of 50 rpm at 30°C until glutinous gel formed. Afterwards, citric acid-Na2HPO4 buffer with pH 3.8 was instilled to hydrate the lipidic gel until a homogeneous dispersion was formed. Finally, the dispersion was homogenized through an ultrasound cell cracker (SCIENTZ IID, Scientz Biotechnology, Ningbo, China) to obtain insulin-loaded BLPs. To prepare insulin-loaded conventional liposomes (CLPs) and blank liposomes, same procedures were followed as described above.
The particle size of liposomes was measured by dynamic light scattering using Zetasizer Nano ZS (Malvern, Worcestershire, UK) at 25°C. The morphology of the liposomes was characterized by transmission electron microscopy (TEM). Briefly, liposomes were dripped onto a piece of copper grid and negatively stained with 1% (w/v) phosphotungstic acid for 1 min at room temperature. The stained nanoparticles allowed to dry at ambient condition and then were observed with TEM (JEM-1230, Tokyo, Japan) at an acceleration voltage of 120 kV.
Entrapment efficiency of insulin-loaded liposomes was determined by molecular exclusion chromatography using Sephadex G50 column to separate the free insulin from liposomes . Briefly, liposome samples were added into the top of column and eluted with the same buffer to liposomes hydration. The eluted fraction of insulin-enveloped liposomes was analyzed by HPLC according to the reported procedure . The entrapment efficiency (EE) was defined as the ratio of liposome-enveloped insulin (insulinenv) to total insulin (insulintot), namely EE (%) = Insulinenv/Insulintot × 100%.
Hypoglycemic effect in normal rats
where AAC was the overall area above the plasma glucose levels vs. time curve calculated by the trapezoidal method using a reference line obtained from the base control.
Absorption mechanism studies
In order to continue to investigate the absorption mechanism, the changes of the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers after incubation with insulin-loaded liposomes and the intracellular trafficking of BLPs were determined. The methods of cell culture, Caco-2 cell monolayer construction and the synthesis of fluorescent probes were the same as the previous report .
To determine the TEER, the well-cultured Caco-2 cell monolayers were incubated with insulin preparations, and the TEERs of Caco-2 cell monolayers were determined at different times by a Millicell Electrical Resistance System equipped with STX-2 electrodes (Millipore, Bedford, MA, USA).
To study the intracellular trafficking of BLPs, cells were cultured on coverslips for 5 days prior to testing. For endosome investigation, the cells were treated with rhodamine-labeled BLPs for 2 h. Then, the cells were continued to be incubated with Rabbit polyclonal antibody Rab5 (ab18211, Abcam, UK) and Mouse monoclonal Rab7 (ab50533, Abcam, UK) overnight at 37°C followed by the addition of a secondary antibody FITC-goat anti-rabbit IgG to identify the early and later endosomes. For lysosome investigation, the medium containing LysoTracker® Red DND-99 g was added into the cells beforehand to label the lysosomes for 2 h. Subsequently, the cells washed with PBS were incubated with FITC-labeled insulin (FITC-ins) loaded BLPs for another 2 h. Finally, the media were removed from the cells and the co-localizations of BLPs with cytoplasmic vesicles were observed by confocal laser scanning microscopy (CLSM).
In vitro cytotoxicity evaluation of liposomes
The cytotoxicity of the liposomes was examined by assessing the viability and apoptosis of Caco-2 cells in the presence of different concentrations of liposomes. The viability of the cells was measured using the MTT assay. Caco-2 cells were cultured for 48 h and rinsed with PBS three times, into which liposomes with various lipid levels were introduced. After incubation for 5 h at 37°C, the MTT solution (20 μL, 5 mg/mL) was added to each well holding cells and continued to incubate for 4 h. DMSO (200 μL) was added to each well to dissolve completely the internalized purple formazan crystals when the medium and excess MTT were removed. UV absorbance of each well was tested at a wavelength of 490 nm. Cell viability was calculated from the ratio between the number of cells treated with the liposomes and that of the control (blank) following the equation: Cell viability (%) = (Atri/Acon) × 100%, where Atri was the absorbance intensity of the cells treated with liposomes, and Acon was the value treated with PBS. The cells treated with culture medium served as 100% cell viability.
To assess the effect of liposomes on cell apoptosis, liposomes with different lipid concentrations were added into the cells and incubated for 4 h. The state of apoptosis were analyzed by detecting the phosphatidylserine (PS) translocation of cell membranes using annexin V-FITC and PI double staining in order to differentiate apoptotic cells from necrotic cells. The analysis was conducted by flow cytometry with an annexin V-FITC/PI apoptosis detection kit (Keygen Biotech, Nanjing, China). Each sample was repeated three times using 105 cells per test. The cells treated with PBS were set as the control.
Results and discussion
Preparation and characterization of BLPs
Formulation variables influence the physicochemical properties of insulin-loaded liposomes such as entrapment efficiency and particle size [32, 33]. It is of high importance to effectively entrap insulin into liposomes so as to reduce the bulk dosage and avoid waste of drug. The main variables including lipid/cholesterol ratio, drug/lipid ratio, the buffer pH upon hydration, and phase ratio in preparing W/O emulsion were optimized to obtain liposomes with high insulin entrapment efficiency and suitable particle size.
Factors influencing hypoglycemic effect of BLPs
The blood glucose profiles of rats after oral administration of liposomes with different biotin-DSPE levels are shown in Figure 3B. Liposomes with 10% biotin-DSPE, to some extent, produced the decline of blood glucose of rats after dosing, whereas more obvious downtrends occurred in the rats those were given of liposomes with biotin-DSPE above 20%. However, there was no significant difference between the liposomes composed of 20% and 30% biotin-DSPE in terms of hypoglycemic effect. The hypoglycemic effect produced by liposomes with 10% biotin-DSPE weaker than that of liposomes with more biotin-DSPE may be as a consequence of relatively weak stability rather than the insufficiency of ligand amount, because DSPE that possesses a high phase transition temperature could reinforce the rigidity of liposomes if more biotin-DSPE was incorporated into lipid bilayer. However, since there was not much difference in the stability of liposomes, increasing the amount of biotin-DSPE is not helpful to the absorption of liposomes, which could also be deduced from the results of hypoglycemic effect using liposomes containing 20% and 30% biotin-DSPE that presented similar profiles of blood glucose decline.
The changes in the blood glucose level of rats after oral administration of different doses of BLPs are displayed in Figure 3C. Below the dose of 20 IU/kg, the hypoglycemic effect of BLPs increased with the increase of oral dose, presenting a dose dependency. At high doses above 20 IU/kg, however, the in vivo hypoglycemic effects of BLPs were maintained in the analogous level and seemingly arrived to a plateau. The phenomenon that the hypoglycemic effect of BLPs linearly correlated with the dose given at low doses and expressed nonlinearity at high doses may be ascribed to the saturability of biotin receptors on enterocytes.
Enhanced hypoglycemic effect of insulin via BLPs
Potential absorption mechanism
In previous studies, enhanced cellular uptake and internalization by specific clathrin-mediated endocytosis was found in terms of BLPs, and the enhanced performance had nothing to do with the opening of intercellular tight junctions . To further interpret the absorption mechanism of BLPs, we executed another several cell experiments to deepen the prior results.
Cytotoxicity of BLPs
This research provided insight into the potential of biotinylated liposomes as novel nanocarriers for oral insulin delivery. Liposomes prepared under optimal conditions can effectively entrap insulin into the inner aqueous cavity and improve the stability of transportation through the GI tract. By biotinylation, the GI absorptive feature of liposomes was notably enhanced. Significant hypoglycemic effect was observed in rats in comparison with CLPs after oral administration of BLPs, especially using liposomes with a particle size about 150 nm. The enhanced oral delivery of insulin was mainly ascribed to ligand-mediated endocytosis by targeting to biotin receptor on enterocytes.
XZ, XH, and WH are Ph.D students at Fudan University. JQ holds a lecturer position at Fudan University. YL and WW hold associate professor and professor position at Fudan University, respectively.
This work was supported by the National Key Basic Research Program of China (2009CB930300) and the Ministry of Education (NCET-11-0114). The authors should also be thankful for the financial assistance from the Shanghai Commission of Education (10SG05).
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