| Peroral protein/peptide delivery system has been the persistent focus in drug delivery, and also represents one of the most challenging and frontier topics in the pharmaceutical world. To achieve effective oral deliver of such therapeutic agents, however, several barriers should be overcomed beforehand, such as instability, gastrointestinal enzymatic degradation, poor membrane permeability and so on. Various delivery strategies, especially those based on nanoscale delivery systems, for absorption enhancement have been explored using insulin as a model drug. However, the status of the art indicates that there seems to have reached a bottleneck. It is highly recommended to explore noval strategies to further enhance the performance of nanoscale drug delivery systems. In this paper, we aim to develop biotin-decorated liposomes to enhance the oral absorption of insulin by biotin receptor-mediated endocytosis targeted to the intestinal epithelia, and to explore and clarify the mechanisms involved in the gastrointestinal absorption thereof.The biotinylation of liposomes was accomplished by incorporation of biotin-DSPE into the lipid bilayers in liposomes preparation. Biotin-DSPE was synthesized via amidation between the carboxyl group of biotin and the amino group of DSPE. The structure of the final product was verified by TLC, FTIR and1H NMR.Iinsulin-loaded biotinylated liposomes were prepared by reverse evaporation in combination with sonication. The influencing factors, including drug/lipid ratio, lipid/cholesterol ratio, the ratio of organic to aqueous phase in preparing W/O emulsion and pH upon liposomes hydration, were optimized by monitoring entrapment efficiency and particle size. The resultant liposomes obtained under optimimum conditions were around150nm in diameter with insulin entrapment efficiencies of30%～40%. Circular dichroism (CD) analysis indicated that the secondary conformation of insulin was well preserved upon encapsulation into liposomes; the bioactivity was close to the insulin solution estimated by monitoring blood glucose level after subcutaneous injection to rats. It was indicated that the preparative stresses did not significantly alter the conformation and bioactivity of insulin. For stability investigation, we determined the phase transition temperature (Tt) of liposomes with different biotin-DSPE amount and assessed the ability of liposomes to resist acidic and enzymatic degradation when subjected to simulated physiological fluids. The results showed that the Tt of liposomes could be elevated to over40℃ when the content of biotin-DSPE in lipids was over20%. In comparison with conventional liposomes (CLP), biotinylated liposomes (BLP) exhibited improved protection of insulin from gastrointestinal enzymatic degradation.Following the preparation and characterization of liposomes, the animal trials including normal and diabetic rats were carried out to estimate the in vivo hypoglycemic effect and pharmacokinetics after oral administration of insulin biotin-liposomes. The relative pharmacological bioavailability (PA) based on pharmacological action and bioavailability (BA) based on insulin level in serum using diabetic rat as a pathological model were also evaluated. The results showed that biotinylated liposomes could achieve more significant blood glucose decline than conventional liposomes. The PA was up to11.04%, but to conventional liposomes it was just2.09%. In normal rats, it was found that liposomes with a particle size of-150nm produced the most significant hypoglycemic effect; when the ratio of biotin-DSPE in the lipids was more than20%, the hypoglycemic effects reached a plateau; at low doses, the hypoglycemic effect of biotinylated liposomes was dose-dependent, whereas nonlinearity was observed at high doses. The PA and BA of biotin-liposomes was observed to be12.09%and8.41%in diabetic rats, which was significantly higher than that of conventional liposomes. It could be concluded that biotinylation of liposomes could reinforce the enhancement of oral absorption of insulin on the basis of conventional liposomes.To explore the oral absorption mechanisms of biotinylated liposomes, a series of investigation using both in vitro and ex vivo models were carried out. Firstly, the expression of biotin receptors in intestinal epithelia was verified in Caco-2cell lines. At the cellular level, the transmembrane transport, uptake and internalization of insulin liposomes on Caco-2cell model were observed. The influence of liposomes on the tight junctions of Caco-2cell monolayer was also estimated. The results suggested that the transmembrane transport of insulin, mediated by liposomes, was significantly improved, especially for biotinylated liposomes, and the Papp was as much as1.87fold of conventional liposomes; the TEER of Caco-2cell monolayers was not influenced by the addition of liposomes, which indicated that tight junctions opening was not responsible for the enhanced oral absorption. The cellular uptake results showed that biotin decoration of liposomes increased the uptake amount of markers incorporated in liposomes and the degree of internalization. Moreover, the cellular uptake of biotinylated liposomes was temperature-dependent and biotin-competitive, indicating a different cellular uptake mechanism other than conventional liposomes. In the following studies of endocytotic pathways, we found that the internalization of biotinylated liposomes was by clathrin-mediated endocytosis. In addition, studies on fluorescent colocalization by CLSM showed that the internalization by cells of biotinylated liposomes was as intact nanoparticle form, and the intracellular trafficking thereof experienced early endosome, late endosomes and lysosomes that is the typical characteristics of receptor-mediated endocytosis. The real-time ex vivo imaging and transepithelial absorption immunofluorescence analysis were also performed. Ex vivo imaging studies suggested that biotinylated liposomes, relative to conventional liposomes, has a faster transportation rate in the gastrointestinal tract, and the fluorescent immunohistochemistry analysis also indicated that biotinylated liposomes has better membranous permeability to the absorptive epithelia. The evidences from absorption mechanism studies illustrated that the gastrointestinal absorption of biotin-modified liposomes was mainly achieved by the transcellular route by virtue of clathrin-mediated endocytosis. However, the overall gastrointestinal uptake of insulin loaded in biotin-liposomes was a synergetic process, the contribution of lymphatic transport by M-cell, out of the particulate nature of liposomes, could not be ignored.To assess the oral safty of liposomes, cell viability and cell apoptosis after incubation with liposomal preparations were tested. The results proved that there was no significant cytotoxicity in comparison with the control, indicating that biotinylated liposomes have desirable safety as oral delivery carriers.In conclusion, by incorporating biotin-DSPE into the lipid bilayers, we successfully developed a novel oral insulin delivery system, biotinylated liposomes. The oral bioavailability of insulin could be significantly improved by this system, which provided proof of concept of biotin-liposomes as potential oral insulin delivery carriers. Besides, such strategy of targeting to enterocytic receptors also provided a new solution to the research and development of novel oral protein/peptide drug delivery system. |
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