Artificial Oil Bodies Research Articles

Novel method to improve oxidative stability of safflower oil: An artificial oil body loaded with curcumin coupled rosmarinic acid

Safflower oil is easily oxidized; therefore, its oxidative stability can be improved using artificial oil bodies (AOBs). AOBs were assembled using safflower oil, phospholipids, and a conjugate comprising safflower caleosin (Ctcaleosin) and rosmarinic acid. AOBs coupled with rosmarinic acid exhibited better physical and thermal stability. The conjugate comprising safflower caleosin and rosmarinic acid was obtained by forming stable C-N covalent bonds. Circular dichroism spectroscopy analysis revealed that α-helix levels increased in safflower caleosin in the conjugate, which could be ascribed to the improvement in the stability and flexibility of safflower caleosin anchored by rosmarinic acid. Rosmarinic acid covering the surface of the AOBs and curcumin (Cur) located in the core of the artificial oil bodies significantly improved the physical and lipid oxidation stabilities of the artificial oil bodies. In vitro, release assay confirmed that curcumin in safflower caleosin-rosmarinic acid conjugate-modified AOBs loaded with curcumin (RAOBC) had a slower release efficiency than the free curcumin solution. The hemolysis assay confirmed the biological safety of RAOBC. The assembly of RAOBC can prevent the oxidation and deterioration of safflower oil, broadening its application in industry.

Exploring The Relative Astringency of Tea Catechins and Distinct Astringent Sensation of Catechins and Flavonol Glycosides via an In Vitro Assay Composed of Artificial Oil Bodies.

Artificial oil bodies covered by a recombinant surface protein, caleosin fused with histatin 3 (a major human salivary peptide), were employed to explore the relative astringency of eight tea catechins. The results showed that gallate-type catechins were more astringent than non-gallate-type catechins, with an astringency order of epicatechin gallate > epigallocatechin gallate > gallocatechin gallate > catechin gallate > epigallocatechin > epicatechin > gallocatechin > catechin. As expected, the extension of brewing time led to an increase in catechin content in the tea infusion, thus elevating tea astringency. Detailed analysis showed that the enhanced proportion of gallate-type catechins was significantly higher than that of non-gallate-type catechins, indicating that tea astringency was elevated exponentially, rather than proportionally, when brewing time was extended. Rough surfaces were observed on artificial oil bodies when they were complexed with epigallocatechin gallate (a catechin), while a smooth surface was observed on those complexed with rutin (a flavonol glycoside) under an atomic force microscope and a scanning electron microscope. The results indicate that catechins and flavonol glycosides induce the sensation of rough (puckering) and smooth (velvety) astringency in tea, respectively.

Investigation of structure–stability correlations of reconstructed oil bodies

To overcome the challenges of complex synthesis methods and the thermodynamic instability associated with conventional emulsions, the artificial oil bodies (AOBs) containing oil body proteins (OBPs) and phospholipids (PLs) were fabricated using ultrasonication and the correlations between their reconstituted structures and characteristics were investigated. As the PL concentration was increased, the concentrations of α-helix and β-turns in interfacial proteins decreased, whereas those of β-sheets and random coils increased. Moreover, the number of the intermolecular hydrogen bond first increased and then decreased. At an OBP/PL ratio of 0.5, the AOBs exhibited the lowest mean particle size (unimodal distribution), highest creaming index, and lowest KE. The oxidation stabilities of the AOBs with OBP/PL ratios of 0.05 and 0.015 were higher than those of other AOBs. Furthermore, statistical analysis indicated that the secondary structure and tyrosine residues of the interface protein were significantly correlated with the stability of the AOBs (p < 0.05), but the hydrogen bond of the interface protein has a low correlation with the stability of the oil body.

Economic purification of recombinant uricase by artificial oil bodies

Rasburicase is an expensive treatment used to control hyperuricemia caused by tumour lysis syndrome (TLS). In this study, a non-chromatographic method was designed based on nano-oil bodies for convenient and economical purification of the recombinant uricase. For this purpose, two chimaeras were synthesized with a different arrangement of the uricase, caleosin and intein fragments. After confirming the protein expression by measuring the uricase activity at 293 nm, purification was conducted through oil-body construction. The results were resolved on the 12% SDS-PAGE gel. Finally, the stability of the oil bodies was examined against different salts, surfactants, temperatures, and pH values. According to our results, the overexpression of uricase–caleosin chimaera under the T7 promoter in Escherichia coli led to the production of soluble protein, which was successfully purified by artificial oil bodies. The active uricase was subsequently released through the self-splicing of intein. Further investigations highlighted the importance of the free C-terminus of caleosin in constructing artificial oil bodies. Moreover, surfactants and low temperature, in contrast to salts, improved the stability of oil bodies. In conclusion, caleosins are an efficient purification tag reducing the cost of purification compared to conventional chromatography methods.Graphical

Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

In vitro assay to estimate tea astringency via observing flotation of artificial oil bodies sheltered by caleosin fused with histatin 3

Astringency, a sensory characteristic of food and beverages rich in polyphenols, mainly results from the formation of complexes between polyphenols and salivary proteins, causing a reduction of the lubricating properties of saliva. To develop an in vitro assay to estimate the astringency of oolong tea infusion, artificial oil bodies were constituted with sesame oil sheltered by a modified caleosin fused with histatin 3, one of the human salivary small peptides. Aggregation of artificial oil bodies was induced when they were mixed with oolong tea infusion or its major polyphenolic compound, (−)-epigallocatechin gallate (EGCG) of 100μM as observed in light microscopy. The aggregated artificial oil bodies gradually floated on top of the solution and formed a visible milky layer whose thickness was in proportion to the concentrations of tea infusion. This assay system was applied to test four different oolong tea infusions with sensory astringency corresponding to their EGCG contents. The result showed that relative astringency of the four tea infusions was correlated to the thickness of floated artificial oil bodies, and could be estimated according to the standard curve generated by simultaneously observing a serial dilution of the tea infusion with the highest astringency.

Enhanced Nuclear Localization of Photosensitizer Using Artificial Oil Bodies for Photodynamic Therapy

Photodynamic therapy (PDT) is a light-activated photochemical reaction that induces localized tissue damage for the treatments of cancers and other nonmalignant conditions through the generation of reactive oxygen species. In the clinic, patients treated with PDT reveal strong skin photosensitivity and thus should be kept away from direct sunlight. In our previous study, we demonstrated that the skin phototoxicity of meta-tetra(hydroxyphenyl)chlorin (m-THPC), a photosensitizer used in the clinic, can be significantly reduced after micellar encapsulation. In this present work, m-THPC was loaded into artificial oil bodies (AOBs) to improve its biocompatibility with self-aggregation. Our results show that the m-THPC-loaded oil bodies with particle size around 100 nm significantly enhanced the nuclear localization of m-THPC, whereas m-THPC-loaded Pluronic® F68 micelles were only observed in the cytosol. In addition, m-THPC-loaded oil bodies had better PDT efficacy than those m-THPC-loaded polymeric micelles. It is speculated that the nuclear accumulation of m-THPC using AOBs as drug carriers significantly enhanced the PDT effects in cancer cells. Thus, m-THPC-loaded oil bodies with improved anticancer efficacy have a great potential for developing nanotechnology-based PDT.

Identification of steroleosin in oil bodies of pine megagametophytes

Three classes of integral proteins termed oleosin, caleosin and steroleosin have been identified in seed oil bodies of diverse angiosperm species. Recently, two oleosin isoforms and one caleosin were identified in megagametophyte oil bodies of pine (Pinus massoniana), a representative gymnosperm species. In this study, a putative steroleosin of approximately 41 kDa was observed in isolated oil bodies of pine megagametophytes, and its corresponding cDNA fragment was obtained by PCR cloning and further confirmed by mass spectrometric analysis. Phylogenetic tree analysis showed that pine steroleosin was evolutionarily more closely-related to steroleosin-B than steroleosin-A found in angiosperm seed oil bodies. As expected, artificial oil bodies constituted with recombinant steroleosin over-expressed in Escherichia coli were less stable and larger than native pine oil bodies. Filipin staining of artificial oil bodies sheltered by recombinant steroleosin with or without its sterol binding domain showed that the sterol binding domain was responsible for the sterol binding capability of steroleosin. Sterol-coupling dehydrogenase activity was demonstrated in artificial oil bodies constituted with recombinant steroleosin as well as in purified pine oil bodies.

Artificial oil body as a potential oral administration system in zebrafish

Zebrafish are well recognized as an excellent vertebrate model which, in particular, facilitates the rapid investigation of drug effects on cancers. A prerequisite for this anti-cancer study appears to have a non-invasive and effecicve method for admininstration of drugs. In this study, we explored the potential of artificial oil bodies (AOBs) for the oral administration of a hydrophobic agent in adult zebrafish. A hydrophobic dye was employed for illustration, and AOBs were assembled with phospholipids, a storage protein, and an equal volume of olive oil and brine shrimp oil. Consequently, more than 90% of the dye was encapsulated in AOBs which exhibited a prolonged release profile for the dye cargo. The dye-loaded AOBs collected as an oil cake remained integral within 30 min in the aquatic solution while it was completely consumed by one zebrafish in a few seconds. As revealed by the fluorescence microscopy and frozen section, the fluorescence signal was detected in the zebrafish intestinal tract 2 h after the oral administration. The dye cargo was completely absorbed in 12 h and almost no trace was left in zebrafish feces. Overall, the AOBs system is biocompatible and shows promise in the oral administration of chemical entities for zebrafish.

The characteristics and potential applications of structural lipid droplet proteins in plants

Plant cytosolic lipid droplets are storage organelles that accumulate hydrophobic molecules. They are found in many tissues and their general structure includes an outer lipid monolayer with integral and associated proteins surrounding a hydrophobic core. Two distinct types can be distinguished, which we define here as oleosin-based lipid droplets (OLDs) and non-oleosin-based lipid droplets (NOLDs). OLDs are the best characterized lipid droplets in plants. They are primarily restricted to seeds and other germinative tissues, their surface is covered with oleosin-family proteins to maintain stability, they store triacylglycerols (TAGs) and they are used as a source of energy (and possibly signaling molecules) during the germination of seeds and pollen. Less is known about NOLDs. They are more abundant than OLDs and are distributed in many tissues, they accumulate not only TAGs but also other hydrophobic molecules such as natural rubber, and the structural proteins that stabilize them are unrelated to oleosins. In many species these proteins are members of the rubber elongation factor superfamily. NOLDs are not typically used for energy storage but instead accumulate hydrophobic compounds required for environmental interactions such as pathogen defense. There are many potential applications of NOLDs including the engineering of lipid production in plants and the generation of artificial oil bodies.

Immobilization of Clostridium cellulolyticum D-psicose 3-epimerase on artificial oil bodies.

The rare sugar D-psicose possesses several fundamental biological functions. D-Psicose 3-epimerase from Clostridium cellulolyticum (CC-DPEase) has considerable potential for use in D-psicose production. In this study, CC-DPEase was fused to the N terminus of oleosin, a unique structural protein of seed oil bodies and was overexpressed in Escherichia coli as a CC-DPEase-oleosin fusion protein. After reconstitution into artificial oil bodies (AOBs), refolding, purification, and immobilization of the active CC-DPEase were simultaneously accomplished. Immobilization of CC-DPEase on AOB increased the optimal temperature but decreased the optimal pH of the enzyme activity. Furthermore, the AOB-immobilized CC-DPEase had a thermal stability and a bioconversion rate similar to those of the free-form enzyme and retained >50% of its initial activity after five cycles of enzyme use. Thus, AOB-immobilized CC-DPEase has potential application in the production of d-psicose at a lower cost than the free-form enzyme.

Elevating Bioavailability of Curcumin via Encapsulation with a Novel Formulation of Artificial Oil Bodies

Utilization of curcumin has been limited due to its poor oral bioavailability. Oral bioavailability of hydrophobic compounds might be elevated via encapsulation in artificial seed oil bodies. This study aimed to improve oral bioavailability of curcumin via this encapsulation. Unfortunately, curcumin was indissoluble in various seed oils. A mixed dissolvent formula was used to dissolve curcumin, and the admixture was successfully encapsulated in artificial oil bodies stabilized by recombinant sesame caleosin. The artificial oil bodies of relatively small sizes (150 nm) were stably solidified in the forms of powder and tablet. Oral bioavailability of curcumin with or without encapsulation in artificial oil bodies was assessed in Sprague-Dawley male rats. The results showed that encapsulation of curcumin significantly elevated its bioavailability and provided the highest maximum whole blood concentration (Cmax), 37 ± 28 ng/mL, in the experimental animals 45 ± 17 min (t(max)) after oral administration. Relative bioavailability calculated on the basis of the area under the plasma concentration-time curve (AUC) was increased by 47.7 times when curcumin was encapsulated in the artificial oil bodies. This novel formulation of artificial oil bodies seems to possess great potential to encapsulate hydrophobic drugs for oral administration.

A useful method integrating production and immobilization of recombinant cellulase

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and β-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose-hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum

Endoglucanase CelA from Clostridium thermocellum (CtCelA) is a thermophilic endo-β-1,4-glucanase and has a low solubility when expressed in Escherichia coli. To make industrial application of CtCeA more appealing, artificial oil bodies (AOBs) was implemented for one-step renaturation and immobilization of recombinant CtCelA. CtCelA was first fused with oleosin (Ole-CtCelA), a structural protein of plant seed oils. Ole-CtCelA was overexpressed in E. coli, and its insoluble form was recovered and mixed with plant oils to assemble AOBs. Moreover, the Box–Behnken design and the central composite design were employed to optimize the condition for assembly of AOBs and the enzymatic reaction condition, respectively. Consequently, the approach led to the resumption of active CtCelA on AOBs. CtCelA-bound AOBs exhibited an optimum activity at 69°C and pH 6.3 while the immobilized protein remained stable for several hours at 70°C and after 5 repeated uses. Overall, it indicates a promise of this novel approach for direct processing and immobilization of recombinant CtCelA.

Stabilization of Fish Oil‐in‐Water Emulsions with Oleosin Extracted from Canola Meal

International dietary guidelines advocate replacement of saturated and trans fat in food with unsaturated oils. Also, there is growing interest in incorporating highly unsaturated omega-3 oils in to food products due to beneficial health effects. A major obstacle to incorporating highly unsaturated oils in to food products is the extreme susceptibility to oxidative deterioration. Oil bodies were prepared from tuna oil, oleosin, and phospholipid mimicking natural oil bodies within oilseed. Oleosin was extracted from canola (Brassica napus) meal by solubilization in aqueous sodium hydroxide (pH 12) and subsequent precipitation at its isoelectric point of pH 6.5. The tuna oil artificial oil bodies (AOBs) readily dispersed in water to produce oil-in-water (o/w) emulsions, which did not coalesce on storage and were amenable to pasteurization using standard conditions. Accelerated oxidation studies showed that these AOB emulsions were substantially more resistant to lipid oxidation than o/w emulsions prepared from tuna oil using Tween40, sodium caseinate, and commercial canola protein isolate, respectively. There is potential to use commercial canola meal, which is cheap and abundant, as a natural source of oleosin for the preparation of physically and oxidatively stable food emulsions containing highly unsaturated oils.

Integral Proteins in Plant Oil Bodies

Hydrophobic storage neutral lipids are stably preserved in specialized organelles termed oil bodies in the aqueous cytosolic compartment of plant cells via encapsulation with surfactant molecules including phospholipids and integral proteins. To date, three classes of integral proteins, termed oleosin, caleosin, and steroleosin, have been identified in oil bodies of angiosperm seeds. Proposed structures, targeting traffic routes, and biological functions of these three integral oil-body proteins were summarized and discussed. In the viewpoint of evolution, isoforms of oleosin and caleosin are found in oil bodies of pollens as well as those of more primitive species; moreover, caleosin- and steroleosin-like proteins are also present in other subcellular locations besides oil bodies. Technically, artificial oil bodies of structural stability similar to native ones were successfully constituted and seemed to serve as a useful tool for both basic research studies and biotechnological applications.

Development of a protocol to solidify native and artificial oil bodies for long‐term storage at room temperature

Oil bodies isolated from sesame seeds coalesced to form large oil drops when they were solidified in a drying process commonly used for food products. The aim of this study was to develop a protocol to solidify oil bodies for long-term storage at room temperature. On the basis of testing several excipients, the coalescence of oil bodies could be effectively prevented when they were combined with mannitol. Sizes of oil bodies appeared similar under a light microscope before and after powderisation in combination with 70% or more mannitol. Artificial oil bodies were successfully generated with sesame oil, phospholipid and recombinant sesame caleosin. Following the developed protocol, native and artificial oil bodies were stably solidified in tablets. Both native and artificial oil bodies dissolved from the tablets remained stable after an accelerated stress test under a condition of 75% humidity at 40 °C for 4 months. A protocol was successfully developed for the solidification of native and artificial oil bodies in stable powder and tablet forms. This successful protocol is very likely to expedite the utilisation of artificial oil bodies in their potential applications.

Influence of the spinning rate in the HR-MAS pattern of mobile lipids in C6 glioma cells and in artificial oil bodies

To evaluate how spinning rate affects mobile lipid (ML) resonances visibility in HR-MAS spectra of C6 glioma cells and artificial oil bodies (AOB), as models of cytosolic lipid droplets. Using C6 cells and AOB of two different sizes, 780 ± 580 and 240 ± 293 nm, as models, we acquired HR-MAS pulse and acquire spectra at different spinning rates between 500 and 15,000 Hz, all at 37 °C. Sample spinning at 15,000 Hz increased by 2.3 and 4.6-fold with respect to 500 Hz spinning the area of resonances corresponding to ML at 0.88 and 1.28 ppm, respectively, for log phase C6 cells. Furthermore, postconfluent C6 cells displayed an increase of 2.5-fold at 0.88 ppm and 4.2-fold at 1.28 ppm. These changes were reversible upon low speed spinning. AOBs did show much lower ML area increases (1.4-1.5-fold) upon high-speed HR-MAS. ML can be reversibly mobilized in C6 glioma cells by high-speed HR-MAS, partially unveiling the NMR "invisible" ML pool. A small part of the ML pool also shows reduced visibility in freely tumbling AOBs.

Caleosin-assembled oil bodies as a potential delivery nanocarrier

Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we have sought to use nanoscale artificial oil bodies (NOBs) as an alternative delivery carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu affibody with a high affinity towards the HER2/neu receptor. After overproduction in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in one step. Consequently, resulting NOBs had a zeta potential around -49mV and ranged in size from 150 to 200nm. Upon loading with a hydrophobic fluorescence dye, NOBs were found to be selectively internalized into HER2/neu-positive tumor cells. Further analyses showed that more than 90% cells were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. In addition, the in vitro assay revealed the release of the dye from NOBs in a slow and prolong manner. Overall, these results indicate the potential of Cal-based NOBs as a delivery vehicle.

Caleosin-based nanoscale oil bodies for targeted delivery of hydrophobic anticancer drugs

Nanoscale artificial oil bodies (NOBs) could be assembled from plant oil, phospholipids (PLs), and oleosin (Ole) as previously reported. NOBs have a lipid-based structure that contains a central oil space enclosed by a monolayer of Ole-bound PLs. As an oil structural protein, Ole functions to maintain the integrity of NOBs. Like Ole, caleosin (Cal) is a plant oil-associated protein. In this study, we investigated the feasibility of NOBs assembled by Cal for targeted delivery of drugs. Cal was first fused with anti-HER2/neu affibody (ZH2), and the resulting fusion gene (Cal–ZH2) was then expressed in Escherichia coli. Consequently, NOBs assembled with the fusion protein were selectively internalized by HER2/neu-positive tumor cells. The internalization efficiency could reach as high as 90%. Furthermore, a hydrophobic anticancer drug, Camptothecin (CPT), was encapsulated into Cal-based NOBs. These CPT-loaded NOBs had a size around 200 nm and were resistant to hemolysis. Release of CPT from NOBs at the non-permissive condition followed a sustained and prolonged profile. After administration of the CPT formulation, Cal–ZH2-displayed NOBs exhibited a strong antitumor activity toward HER2/neu-positive cells both in vitro and in vivo. The result indicates the potential of Cal-based NOBs for targeted delivery of hydrophobic drugs.