Abstract
Bilosomes are a novel colloidal delivery system similar to niosomes but incorporating bile salts (BS) into the vesicular lipid bilayer membrane. Although of proven value in the delivery of antigens and vaccines, they have not been evaluated for drug delivery. The aim of this thesis was to prepare and characterize bilosomes and assess their potential as an oral drug delivery system for the hydrophilic peptidomimetic drug cefotaxime (CTX). BS used to make bilosomes included sodium cholate (NaC), deoxycholate (NaDC), taurocholate (NaTC) and 12-monoketocholate (NaMKC). Vesicles were classified according to their images. Biorelevant and other media used to test the stability of bilosomes in vitro included SGF, SGFpep, FaSSIF, FeSSIF, pancreatin solution and solutions of NaDC and NaTC.
In the first part of the study, 5,6-carboxyfluorescein (CF) was used as a marker compound to evaluate vesicle stability. CF-loaded bilosomes were prepared using the rotary film evaporation method in which lipid film (150 μmol) consisting of glycerol monopalmitate:dicetyl phosphate:cholesterol (4.5:4.5:1 molar ratio) was hydrated with a solution (3 ml) of CF (100 mM) and BS to prepare vesicles that were then size-reduced to ≈ 200 nm. Encapsulation efficiency was 40–52% depending on the particular BS and its concentration in the hydration buffer. Vesicle stability after size reduction was assessed in terms of changes in vesicle size and leakage of CF during incubation in biorelevant and other media for 3 h. All vesicles types were stable in SGF, SGFpep and FaSSIF but vesicles made with 20 mM NaDC (bilosomes and transfersomes) were more stable than those made without NaDC (niosomes and liposomes) in FeSSIF and NaTC solution; that the stability of NaDC bilosomes in FeSSIF and NaTC solution increased with increasing concentration of NaDC (6.3-50 mM) used to make them; and that stability of bilosomes made with 20 mM NaDC in solution of NaDC and NaTC (1-20 mM) was greater in NaTC solution and decreased with increasing concentration of both. It was found that: the stability of bilosomes made with different BS (20 mM) in 15 mM NaTC solution was in the order NaTC>NaDC≈NaC>NaMKC.
In the second part of the study, the characteristics and stability of size-reduced vesicles of CTX-loaded bilosomes prepared in the same way as CF-loaded bilosomes but using 10.5 mM NaCTX solution were assessed in terms of encapsulation efficiency (EE) and drug loading (DL) of CTX and BS loading. For bilosomes made using different BS (20 mM), DL of CTX and BS loading were not significantly different at 10-20 and 18-29% respectively. For bilosomes made using 0-11.8 mM NaDC concentrations, EE and DL of CTX were unchanged and equal to 48.2±1.6% and 13.3±1.5% respectively. For bilosomes made using 6.3-50 mM NaDC concentrations, DL of CTX remained unchanged at 10.3-12.3% but NaDC loading increased from ≈8% at ≤12.5 mM to ≈28% at ≥25 mM. For bilosomes made using 25 mM NaDC and increased volumes of hydration buffer (3-10 ml), EE and DL of CTX of 75.1±6.5% and 12.0±1.9% respectively for 3 ml hydration buffer both decreased as the volume increased. A study of CTX stability in NaCTX solution (unencapsulated CTX), size-reduced vesicles of CTX-loaded niosomes and CTX-loaded bilosomes made with 25 mM NaDC on incubation in SGF at 37°C over 6 h showed that both niosomes and bilosomes enhanced CTX stability in the acidic media. In terms of CTX release, incubation of CTX-loaded niosomes and bilosomes made using 20 mM NaTC and NaMKC in FeSSIF and SGF showed that vesicle stability was in the order NaTC bilosomes>NaMKC bilosomes ≈ niosomes.
In the third part of the study, the pharmacokinetics of CTX administered in sizereduced bilosomes, niosomes and as a solution of NaCTX was determined in rat. Male Wistar rats were randomly allocated into 5 groups (n = 5-6 per group) and administered either a single i.v. dose of 20 mg/kg NaCTX in solution or a single oral doses of 80 mg/kg CTX in solution or encapsulated in niosomes, bilosomes made with NaTC and bilosomes made with NaMKC. Blood was collected for up to 12 h and analyzed for CTX. Encapsulation of CTX in niosomes and bilosomes made with NaTC and NaMKC produced CTX oral bioavailabilities of 11.1 ± 2.6, 16.9 ± 4.7 and 12.8 ± 4.8% respectively compared to 1.53 % for CTX administered as CTX solution. Bilosomes made with NaTC produced a significantly higher CTX Cmax but the corresponding AUC0-∞ and F values were not significantly different. In conclusion, this work demonstrates the potential of bilosomes as an oral delivery system for peptidomimetic drugs. The research confirms that the instability of lipid-based vesicular systems in the gut can to some extent be overcome by incorporating BS during their preparation.