The Polar Lipid Fraction E from Sulfolobus acidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents to Reduce the Leakage of the Antivascular Drug Combretastatin A4 Disodium Phosphate from Tetraether/Diester Hybrid Archaeosomes

Liposomes have many advantages as therapeutic capsules over free drugs such as small molecule drugs and nucleic acids. Cholesterol is commonly used as a membrane stabilizing agent in liposomal drugs (e.g., mRNA-lipid nanoparticle COVID-19 vaccines). However, due to the vulnerability of cholesterol to oxidation and the etiological role of cholesterol in many disorders, it is desirable to find an alternative means to stabilize liposomal membranes for drug delivery. In this study, we demonstrated that the polar lipid fraction E (PLFE), which contains exclusively bipolar tetraether macrocyclic lipids, isolated from the thermoacidophilic archaeon S. acidocaldarius can greatly stabilize the liposomal formulation of the anti-vascular drug, combretastatin A4 disodium phosphate (CA4P). Stability was assessed by determining the leakage rate constant k of entrapped CA4P fluorometrically. We found that, at 37 °C, PLFE decreases the k value monotonically from 1.54 × 10−2 s−1 for 100% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes to 3.4 × 10−5 s−1 for 100% PLFE archaeosomes, a change of k by two orders of magnitude. The changes in k of CA4P leakage are correlated well with the changes in liposomal CA4P’s cytotoxicity against MCF-7 breast cancer cells. We further showed that the reduction in spontaneous leakage of entrapped CA4P by PLFE can be attributed to the increased membrane surface charge and the increased membrane order and packing tightness in liposomes, as reflected by the zeta potential (−6.83 to −41.1 mV from 0 to 100 mol% PLFE) and diphenylhexatriene (DPH) fluorescence polarization (0.13 to 0.4 from 0 to 100 mol% PLFE) measurements. Moreover, we showed that PLFE slows down CA4P leakage more than cholesterol in POPC liposomes. These results together suggest that PLFE lipids can serve as an effective stabilizing agent for liposomal drugs and could potentially be useful for the optimization of liposomal CA4P for cancer treatment.

Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from −48 mV at 37 °C to −16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid ‘flip-flop’. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell’s cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42–44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions.

Separation and Purification of Wax from Nyamplung (Calophyllum inophyllum) Seed Oil

Nyamplung (Calophyllum inophyllum) is a multi-functional plant which is spread widely over the coast of Indonesia. Its seed produces a high content of oil, but its utilization is still limited. It is because C. inophyllum seed oil contains toxic compounds. Therefore, C. inophyllum seed oil has been used as a biodiesel raw material for many years. It was reported that C. inophyllum seed oil contains wax, but its percentage remains unknown. Wax has been used in cosmetics, pharmaceuticals, foods, and coatings industries as oil binder, water repellent, scratch resistance, and dispersion medium. In this work, wax was separated from C. inophyllum seed oil by solvent crystallization with and without separating non-polar lipid fraction (NPLF) from crude oil. Non-polar lipid fraction was separated by batch-wise solvent extraction using petroleum ether to methanol mass ratio of 3:1 (w/w) for eight stages. After eight stages, non-polar lipid fraction was collected for further separation by solvent crystallization method. The ratios of non-polar lipid fraction to acetone were 1:10, 1:20, and 1:40 (w/v). Then, the isolated wax was analyzed by gas chromatography. It was found that wax (purity of 40% and yield of 0.35%) was successfully isolated by separating non-polar lipid fraction from crude oil (batch wise solvent extraction for eight stages) and followed by solvent crystallization (non-polar lipid fraction to acetone ratio of 1:40 (w/v)).

Seasonal Variations of Lipid Content and Composition in Starfish Asterias amurensis Lütken

The total lipid content, lipid classes and fatty acid composition in the internal organs of starfish (Asterias amurensis) were analyzed to determine the effects of seasons (winter and spring). The non-polar and polar lipid fractions obtained from starfish internal organs were analyzed through two seasons using thin layer chromatography (TLC) and gas liquid chromatography. Total lipid content of internal organs was 10.18% in spring and 8.21% in winter as wet weight basis. The predominant lipid class in spring was triglyceride whereas free fatty acids were the main lipid class in winter. The most abundant fatty acid of non-polar lipid fraction was eicosamonoenoic acid (C20:1) in spring having the highest proportion (29.2% of total fatty acid) as compared to the winter. Eicosapentaenoic acid (EPA) was also found significantly (P < 0.05) higher in spring compared to winter in the non-polar lipid fraction. On the other hand, comparatively lower amount of EPA was observed in spring than winter in the polar lipid fraction. Proportions of other fatty acids in non-polar and polar lipid fractions were also varied seasonally. This result might be useful for commercial production of lipid from internal organ of starfish with a view to potential use in food, pharmaceutical, cosmetics and other non-food industries.