Liposomes Containing Bile Salts As Potential Oral Insulin Delivery Systems

Liposomes, being among the most studied particulate carrier systems, have shown appealing potential in enhancing oral bioavailability of proteins and peptides. However, the efficacy of conventional liposomes, mainly composed of phospholipids and cholestoral, has been compromised due to the instability of liposomes in the GI tact and poor permeability across the epithelial membrane. Recentaly, an novel liposomes containing a bile salt, including sodium glycocholate, sodium taurocholate and sodium deoxycholate, which is also called ’bilosomes’, showed promising poteintial in oral immuzation due to improved stability in GI tract. In this study, insulin was selected as a model drug, drug loading, in vitro characterization, in vivo hypoglycemic effect and absorption mechanism in the GI tract, as well as primary safety of bile salt liposomes were investigated.As a biomacromolecular drug, insulin suffers from low entrapment efficiency (EE) in liposomes. To achieve high EE, bile salt liposomes were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and EE of rhINS-loaded SGC-liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid/SGC ratio, insulin/phospholipid ratio, the water/ether volume ratio, interior water phase pH and the hydration buffer pH. The optimal formulation showed an insulin entrapment efficiency of 30±2% and a particle size of 154±18 nm. The conformational study by circular dichroism spectroscopy and bioactivity study confirmed the preserved integrity of rhINS against the preparative stress, the percentage ofα-helix andθ208/θ223 were close to insulin solution; reduction of blood glucose level after s.c. broken bile salt liposomes was also close to s.c. insulin solution (46%).Based on the optimal preparative conditions, three bile salt liposomes including sodium glycocholate liposomes (SGC-liposomes), sodium taurocholate liposomes (STC-liposomes) and sodium deoxycholate (SDC-liposomes), were prepared and characterized in vitro and in vivo. The particle size of three bile salt liposomes were between 130～160 nm, smaller than conventional liposomes 187 nm. No significant difference was found as for PI or EE. Transmission electron micrographs revealed a near spherical and deformed structure with discernable lamellar for SGC-liposomes. Leakage of insulin study indicated that leakage of rhINS from SGC-liposomes was slow in pH 2.0,5.6 and 6.8 buffers within 6 h. The conformational study by circular dichroism spectroscopy and bioactivity study confirmed the preserved integrity of rhINS in three types of bile salt liposomesAfter oral administration by gavage, all bile salt liposomes elicited a certain degree of hypoglycemic effect in parallel with increase of blood insulin level. However, a series of controls including insulin solution, the blank liposomes vehicles, a mixture of rhINS with blank liposomes and a mixture of rhINS with sodium glycocholate showed no hypoglycemic activities and increase in blood insulin level. The most significant blood glucose reduction resulted from insulin-loaded SGC-liposomes, which produced maximum blood glucose reduction of 63% at 10 h after oral administration and returned to normal at around 20 h, with relative pharmacological bioavailability (PA) and relative bioavailability (F) value of 6.9% and 6.7%, respectively. Liposomes with smaller particle size exhibited a rapid reduction and quick return in blood glucose level. On the contrary, liposomes with larger particle size exhibited slower and sustained hypoglycemic effect. Good linear correlation could be observed between administered doses of 2,5,10 and 20 IU/kg and the actual hypoglycemic activity and serum insulin level increase. Same results were found in diabetic rats, except the hypoglycemic effect was more obvious:the maximum blood glucose reduction was 55%, with PA and F value of 10.2% and 9.9 % for SGC-liposomes.Bile salt liposomes showed better protection of insulin against enzymatic degradation by enzyme than conventional liposomes. SGC-liposomes provided the best protection, preserving 48%,67% and 81% of rhINS at 4 h for pepsin, trypsin and a-chymotrypsin. This may be attributable to the enzyme-inhibiting ability of SGC. However, as the amount of SGC increased further, less protection of liposomal rhINS was observed.In vivo imaging of bile salt liposomes loaded with IR 783-rhINS, after oral administeration to rat showed that, the residence time of liposomes in GI tract is dependent on enzyme inhibition ability. SGC-liposomes with the most enzyme inhibition exhibited increased residence time in GI tract and more rhINS amount in blood.Cellular uptake of rhINS was greatly enhanced by bile salt liposomes compared with conventional liposomes. rhINS cellular uptake was relative to the particle size and concentration of liposomes:lipsomes with smaller size and higher concentration had more rhINS uptake by Caco-2 cells. The incubation of bile salt liposomes on the apical side of Caco-2 cell monolayers led to an immediate reduction in TEER and increase amount of rhINS transported through caco-2 cell monolayers. The apparent permeability (Papp) value of rhINS for SGC-liposomes (4.79±0.55×10-6) was nearly 3 times higher than that for conventional liposomes. The cumulative amounts of insulin increased as the increase of liposomal concentration and decrease of particle size. After incubation with bile salt liposomes, shortening of filaments and aggregates of F-actin were identified; ZO-1 proteins appeared discontinuous, indicating the opening of tight junctions.Three types of bile salt liposomes with phospholipids concentration 1～25 mM showed little inhibition of the Caco-2 viability within 4 h by MTT method. Apoptosis study indicated bile salt liposomes inducing little apopotosis in Caco-2 cells within 24 h. Overal, the toxicity of bile salt liposomes was low.On a whole, enhanced pharmacological bioavailability (PA) and relative bioavailability (F) of 10.2% and 9.9% respectively were achieved after oral administration of rhINS loaded SGC-liposomes in diabetic rats. Protection of rhINS from enzymatic degradation and increased residence time in GI were founded in bile salt liposomes.Besides, increased rhINS cellular uptake and transport across Caco-2 cell monolayers were achieved with little toxicity to cells. On bases of cellular uptake and transport results, the bile salt liposomes are considered as potential oral delivery systems for rhINS.