Investigation Of Archaeosomes As Novel Carrier For Oral Delivery

Administering drugs orally is by far the most widely used route of administration, although it is generally not feasible for peptide and protein drugs. The main reasons for the low oral bioavailability of biologicals are presystemic enzymatic degradation. Various strategies have been pursued to overcome such barriers and to develop safe and effective oral delivery systems for biologicals.The potential usefulness of liposomes as drug carriers has attracted considerable interest due to their controlled size, charge and membrane fluidity.These phospholipid vesicles are capable of encapsulating both hydrophobic and hydrophilic drugs. The drugs encapsulated in liposomes are sufficiently protected from enzymatic attack to some extent. In this context, nevertheless, a major drawback of liposome formulations that have been studied extensively to date is their poor stability at low pH, susceptibility to attack by bile salts and GI tract enzymes. Therefore, it was desirable to develop more stable liposome formulations to protect peptide and protein drugs.Archaeosomes are liposomes which are made from structurally typical of archaeobacterial membrane lipids. Archaeobacterial lipids consist of archaeol (diether) and caldarchaeol (tetraether) core structures. wherein tetraether lipids regularly branched and usually fully saturated phytanyl chains (40 carbons in length), are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone. In contrast, conventional phospholipids have fatty acyl chains of variable length, which may be unsaturated, and which are attached via ester bonds to the sn-1,2 carbons of the glycerol backbone. Moreover, the lipid moieties were composed of membrane-spanning caldarchaeol lipids, known to form a monomolecular thick membrane by span the entire lamellar structure, and the unique lipid structures in general confer considerable stability to archaeosomes.In the present study, PLFE (polar lipid fraction E) was distilled from S. acidocaldarius, the stability of archaeosomes composed of PLFE has been examined in stimulated GI environment. The objective of our study was to evaluate the potential of archaeosomes to induce humoral and CD8+ T cell mediated immune responses to entrapped antigen via the oral immunization. As oral carrier of insulin, the hypoglycemic effect of the archaeosomes in diabetic mice was also evaluated. Finally, we examined the intestinal transit of archaeosomes through In Vivo Fluorescence Imaging. The detailed results were summarized as following:PLFE lipids were purified as a single fraction from the crude lipid extract of S. acidocaldarius. The isolated PLFE lipids were characterized through TLC, mass spectral, infrared spectroscopy and NMR, and these results provided conclusive support that the final product was PLFE. In order to investigate the shape and surface morphology of the archaeosomes, atomic force microscopy (AFM) was employed. The AFM images showed that the form of archaeosomes was nearly spherical, and that there was no aggregation or adhesion among the particles, and the AFM micrograph of the archaeosomes revealed that the mean diameter was about 210 nm, which was in good agreement with data obtained by PCS. In addition, the mean surface potential archaeosomes was measured to be -35.27 mV, which was responsible for the good poly.index. One of the requirements of a successful oral delivery system is reasonable stability in the environment of GI tract. In the present study, to determine the stability of carriers during passage through GI via oral route, our study was performed with the release of calcein from vesicles in mimic intestinal condition, and these results indicated that the archaeosomes composed of PLFE are essentially stable in these mimic intestinal conditions.Based on the unique structure from PLFE and stability of archaeosomes, Our studies performed in mice have indicated that archaeosomes are superior in eliciting humoral immune response, as well as CD8+ T cell mediated immune response to encapsulated antigen, compared with conventional liposomal formulation. In order to explore primarily the distribution and absorption of various liposomal formulation post vaccination orally, one hand, Rhodamine-DHPE was employed, and the results showed that Rhodamine-DHPE associated with intestine when administered archaeosomes increased and attained a higher level than that of conventional liposomes, especially in the lower ileum. In the other hand, Cy5.5-labeled OVA was used as optical tracer, and the corresponding results showed archaeosomes prolonged intestinal residence time of optical tracer in GI tract, which reversibly increased the permeability of the mucosal epithelium. Further optical imaging results from incised small intestinal suggested that archaeosomes containing optical tracer were preferentially taken up in the lower ileum. These results may also be corrected with enhanced immunity. However, the mechanisms of action require further study. Nevertheless, these available data from our study indicate that archaeosomes may hold great promise in developing oral vaccines for weak antigens.Subsequent, we investigated the possibility of archaeosomes as a tool for the oral delivery of insulin. Firstly, the results from stability in vitro showed the release of insulin from archaeosomes was markedly reduced not only in the acidly solution but also in the bile salts. Secondly, when insulin was orally administered to diabetic mice as either a solution or conventional liposomes, no hypoglycemic effect was observed. Whereas administration of insulin encapsulated in archaeosomes caused the rapid decrease in the plasma glucose level and the hypoglycemic effect lasted for much longer duration than that of conventional liposomes, which, we speculated, was contributed to the stability of various carriers. Further optical imaging results from in vivo showed that the slow release of insulin from archaeosomes achieved the longer duration in the small intestine, which was benefit for drug absorption. Thirdly, we established Caco-2 cell monolayer model, and evaluated the absorption characteristics of insulin as various formulations. The results showed the cumulative amount of insulin transported through Caco-2 cell monolayer for archaeosomes was lower than that of conventional liposomes, which, we speculated, was ascribed to composition prepared for liposomes. Despite, the mechanisms of action require further study, these results suggested that an ideal delivery system for oral administration should not only protect drug or antigen from the harsh environment in the GI, but also enhance the absorption of them by small intestine. Therefore, we expect to optimize the delivery system through combination the stable archaeosomes with target strategy to increase the uptake of drug and improve the bioavailability.