Present study prepared anionic powdered-nanoliposomes loaded with algal oil using phosphatidylserine and different wall materials (maltodextrin and hydroxypropyl β-cyclodextrin). All the powdered liposomes were nanosized (238.97 ± 4.67–256.30 ± 0.85 nm) with negative zeta potential (-71.80 ± 7.84 – -51.46 ± 1.27 mv). We have investigated and compared the effects of different wall materials on the oxidative stability, in-vitro digestibility and cellular uptake of the spray-dried powders were investigated. Results showed MD was able to provide higher oxidative stability to anionic-powdered liposomes loaded with algal oil. In addition, powdered liposomes produced from MD demonstrated better in-vitro digestibility with increased in SLC27A4, FABP2 and CD36 gene expression indicating good cellular uptake of the powdered liposomes.
Research Article
Anionic powdered-nanoliposome loaded with algal oil: Effects of carrier agents on storage stability, in-vitro digestibility and cellular uptake
https://doi.org/10.21203/rs.3.rs-1840648/v1
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DHA
Nanoliposome
Spray drying
Stability
Digestion
Lipid uptake
Docosahexaenoic acid (DHA, C22:6 ω-3) is the primary structural component of brain and retina. DHA has many healthful properties including promote brain development (Echeverría et al. 2017), reduce blood lipid and risk of cardiovascular disease and involve in anti-inflammatory processes (Calder 2015). Biosynthesis of DHA in human body is very limited; thus, it is very important to incorporate DHA into our diet (Ahonen et al. 2022). Due to its high degree of unsaturation, DHA is prone to oxidative rancidity (Wang et al. 2021) resulted in unpleasant odor, decreased in nutritional value and formation of compounds that maybe detrimental to human health (Jamshidi et al. 2020). Therefore, it is very important to develop a suitable delivery system capable of improving DHA storage stability without compromising its bioavailability.
Liposomes are nano-to micron-sized vesicles composed of amphiphilic substances such as phospholipid bilayer that surrounds an aqueous core (Jiang et al. 2020). The core of liposomes can be used to encapsulate water-soluble compounds (Liu et al. 2020); meanwhile, the hydrophobic bilayer can be used to entrap lipid-soluble compounds. Anionic liposomes are structures that are made of negatively charged lipids such as phosphatidylserine (PS) and phosphatidylglycerol. Anionic liposomes show greater storage stability with lower degree of aggregation in solution as compared to neutrally charged liposomes. For example, PEGylated anionic liposomes were prepared and used to sequester amitriptyline in human serum. The anionic liposomes remained stable for extended periods of time at 2–8°C with little effect on drug absorption properties (Howell and Chauhan 2008). In addition, anionic liposomes demonstrated enhanced endocytosis and bioavailability than cationic liposomes. Anionic liposomes prepared from 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), Egg phosphatidylglycerol (EPG) and 1,2-dimiristoyl-sn-glycero-3-phosphoglycerol (DMPG) demonstrated a higher rate of uptake by J774.A1 macrophages (Sahebkar et al. 2014).
Spray drying of liposomes can increase its storage stability and ease of transportation (Sultana et al. 2021). Nevertheless, high temperature during spray drying process will destroy liposome membranes (Sepúlveda et al. 2021). Maltodextrin and hydroxypropyl β-cyclodextrin (β-CD) are some of the commonly used carrier agents/wall materials in spray-dried liposomes. For example, maltodextrin was used as a carrier to produce powdered liposomes containing black carrot extract (BCE) which have high retential rate of 88% after 21 days of storage. (Guldiken et al. 2019). In another study, β-CD was used as a wall material for preparation of powdered PEGylated liposomes loaded with prednisolone disodium phosphate (PLP). β-CD can protect the liposome membrane and ensure its structure remained intact during spray drying (van den Hoven et al. 2012).
Present study aims to prepare anionic powdered-nanoliposomes loaded with algal oil using PS and different carrier agents (maltodextrin and/or β-CD). We postulated that powdered liposomes can improve the storage stability of liposomes. Firstly, we have optimized the liposome preparation process (weight ratio of PS to algal oil, volume and concentration of PBS buffer, ultrasonic time and evaporation temperature) to obtain high algal oil encapsulation rate. The anionic nanoliposomes loaded with algal oil were spray dried using different carrier agents. The effects of different carrier agents on physicochemical, oxidative properties (particle size, zeta potential, phase transition temperature, and morphology), in-vitro digestibility and cellular uptake of the spray-dried powdered nanoliposomes were compared and elucidated (Fig. 1). Findings from present work will be useful for development of powdered liposome loaded with algal oil with increased storage stability and bioavailability.
Materials
Phosphatidylserine (purity ≥ 50%) was provided by Enzymecode Biotechnology Co. Ltd. (Tianjin, China) and algal oil (DHA ≥ 60%) was obtained from Cabio Biotechnology Co., Ltd. (Wuhan, China). (2-Hydroxypropyl)-β-cyclodextrin (C63H112O42, ≥ 97%) and maltodextrin (C6H12O6) were purchased from Yuan Ye Bio-Technology Co, Ltd. (Shanghai, China). Fatty acid methyl esters (FAMEs) standards (USP Reference Standard), mucin (1500 μ/mg), pepsin (≥ 250 μ/mg), trypsin (1000 -2000 u/mg), porcine pancreatic lipase (100 - 500 μ/mg) and α-amylase were purchased from Aladdin Chemicals (Shanghai, China). Caco-2 (CW2042S) cells was purchased from CoWin Biosciences (Jiangsu, China), DMEM high glucose liquid medium (SH30243.01B) and penicillin-streptomycin solution (SV30010) were purchased from Hyclone (Utah, USA). Porcine bile salts, sodium chloride, calcium chloride, and dihydrogen phosphate were purchased from Solarbio or Sinopharm (Shanghai, China). Chemical reagents such as ethanol and n-hexane were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals and reagents used were of analytical grade.
Fatty acid composition of the algal oil determined by gas chromatography triple quadrupole mass spectrometry (GC- QQQ- MS)
Fatty acid composition of the algal oil was determined using GC-QQQ-MS according to AOCS method Ce-1b 89 (7890B-7000C, Agilent Technologies Inc., America). Firstly, algal oil was methylated according to our previous study (Wu et al. 2020). Briefly, 200 μL of the algal oil was dissolved in 4 mL of n-hexane. One milliliter of methanol-sodium hydroxide solution (0.5 M) was added, and the mixture was vortexed for 2 min at room temperature. Anhydrous sodium sulfate was added to the mixture, and the mixture was shaken and allowed to stand for 2 min. The upper organic phase was diluted 10 times with n-hexane, filtered and transferred into a GC vial for fatty acid composition analysis.
FAMEs were separated using a DB-2560 capillary column (100 m × 250 μm × 0.2 μm). Nitrogen was used as carrier gas at a flow rate of 1.5 mL/min and a split ratio of 30:1. The injector and detector temperatures were 250 and 300 °C, respectively. The oven temperature was held at 140 °C for 5 min, increased to 240 °C at a rate of 4 °C/min, and finally held at 240 °C for 25 min. Fatty acid peaks were identified by matching retention times to FAME standards (Wu et al. 2020). Relative content of the fatty acids is calculated as ratio of peak area of fatty acids to total peak area.
Preparation and encapsulation efficiency of the anionic liposomes loaded with algal oil
Algal oil loaded liposome was prepared using PS by thin film evaporation and ultrasonic dispersion. Firstly, PS and algal oil were fully dissolved in n-hexane. The solution was heated, and rotary evaporated to form a thin film. The thin film was then hydrated with PBS buffer (0.01 M, pH 7.2) for two hours and then subjected to ultrasonic treatment (JY92-IID, Ningbo Xinzhi Biotechnology Co., Ltd.) at 3 sec ON / OFF for 10 min (power 300 w) to obtain algal oil loaded-liposome with uniform dispersion.
In order to obtain algal oil liposome with high encapsulation rate, the following preparation conditions were optimized: weight ratio of algal oil to PS (5:1, 2.5:1, 1:1, 1:2.5, 1:5); pH of the PBS buffer (6.8, 7.0, 7.2, 7.4, 7.6); volume of PBS buffer (10, 15, 20, 25, 30 mL); concentration of PBS buffer (0.005, 0.01, 0.02, 0.04, 0.06 mol/L); ultrasound time (10, 15, 20, 25, 30 min); evaporation temperature (30, 35, 40, 45, 50 °C).
The prepared anionic liposomes were centrifuged at 25 °C for 30 min at 10000 g (H1850R, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.) to obtain supernatant containing non-encapsulated algal oil. Two milliliter of the supernatant was added to 10 mL of petroleum ether and their absorbance was measured at 274 nm. Concentration of the algal oil was calculated using a standard curve (Regression equation: y = 0.3269x + 0.0262，R2=0.9575). Encapsulation efficiency (EE) of the anionic liposomes is calculated as follows:
Preparation and encapsulation efficiency of the anionic powdered-liposomes loaded with algal oil
Carrier agents (Hydroxypropyl-β-cyclodextrin, maltodextrin and a mixture of hydroxypropyl-β-cyclodextrin and maltodextrin (1:1, w/w) was dissolved in PBS buffer (5%, 0.01 M, pH 7.2). The carrier solution was mixed with liposome dispersion (2:1, v/v) for 1 h. The resulting solution was pumped into spray dryer (YC-500, Shanghai Yacheng Instrument Equipment Co., Ltd., China) at a flow rate of 1.8 m3/min by a peristaltic positive displacement pump. The inlet and outlet air temperatures were set at 150 and 50 °C, respectively. The collected anionic powdered-liposomes loaded with algal oil was stored in a closed container at 4 °C. Spray-dried yield was calculated as mass ratio of spray-dried powder to the total solid in the feed solution.
Encapsulation efficiency of the anionic powdered-liposomes loaded with algal oil was determined according to a previously published method with minor modifications (Jafari et al. 2008a). Liposome powder (3 g) was gently mixed with 15 mL of n-hexane (15 mL) to wash off the surface oil of the powder. Hexane containing the extracted surface oil was collected and evaporated for 3 h at 60 ± 2 °C and weighed. Encapsulation efficiency (EE) is calculated as follows:
Average particle size, size distribution and zeta potential of the anionic liposomes and powdered liposomes loaded with algal oil
Average particle size, particle size distribution (PDI) and zeta potential of liposomes and powdered liposomes were measured using dynamic light scattering particle size analyzer (Malvern Instruments Co., Ltd, United Kingdom). Powdered- liposomes were dissolved in PBS (pH 7.2) buffer prior to analysis.
Thermal properties of the anionic liposomes loaded with algal oil
Phase transition temperature of the anionic liposomes was analyzed using a differential scanning calorimeter (DSC 200F3，Netzsch，Deutschland). Fifty microliter of the liposome suspension was placed in a hermetically sealed aluminum pan. The sample was firstly heated to 25 °C and held at this temperature for 5 min; and cooled to −40 °C at a cooling rate of 5 °C/min and held at this temperature for 10 min. Finally, the sample was heated to 90 °C at a heating rate of 5 °C/min.
Morphology of the anionic powdered liposomes loaded with algal oil
Morphology of the powdered-liposomes was observed using scanning electron microscope (Sigma 300, Zeiss, Germany). Powdered-liposomes (10 mg) were placed in an aluminium sample holder and treated with gold–palladium immediately prior to analysis and images were obtained at 3.0 kv.
Oxidative stability of anionic powdered liposome loaded with algal oil
Anionic powdered-liposomes loaded with algal oil were stored at 45 °C for 15 days. Oxidative stability of anionic powdered liposome loaded with algal oil were determined by measuring peroxide value (China 2016b) and p-anisidine value (China 2009) at 0, 3, 6, 9, 12, and 15 days, respectively.
Powdered liposome (0.5 g) was mixed with acetic acid-chloroform (3:2; v/v). Saturated potassium iodide solution (1 mL) was added to the mixture and shake for 30 s and place in dark for 3 min. The solution was then mixed with 50 mL of distilled water and 1 mL of starch indicator; and titrated with Na2S2O3 (0.01 M) solution until the blue color disappeared. Peroxide value was calculated according to the following equation:
Peroxide value (mmol/ kg) = S×M×1000 / 2m (3)
where S is the volume of Na2S2O3; M is the molar concentration of Na2S2O3; m is the sample mass.
Anisidine value (p-AV) of the powdered liposomes was determined according to the following method. Powdered liposome (0.5 g) was dissolved in isooctane (25 mL). The test solution (5 mL) and solvent (5 mL) of the sample were added to different test tubes. p-anisidine reagent (1 mL) was added to each test tube under shaking condition and kept at 25 °C for 8 min in complete darkness. Absorbance of the solution (A0) and blank solution (A) at 350 nm was measured with UV−vis absorption spectroscopy (UV-2600 spectrophotometer, Shimadzu, Japan). p-AV was calculated according to the following equation:
Where V is the volume of dissolved sample, in ml; Q is determining the concentration of the sample in the solution, in g/ml; m is the mass of the sample, in g. A is absorbance of the reaction solution; A0 is absorbance of solution in blank group.
In-vitro stimulated lipolysis of the anionic powdered liposomes loaded with algal oil
In-vitro stimulated lipolysis of the powdered liposomes were determined according to previously published method (Mou et al. 2021). Simulated digestive fluid was prepared according to Table S1 of Supplementary material.
Oral Phase: Powdered liposome (1 g) was mixed with 10 mL of simulated oral digestive fluid (pH adjusted to 6.8) and amylase (50 mg). The mixture was incubated at 37 °C for 10 min with continuous agitation at 100 rpm to simulate oral digestion.
Stomach Phase: Sample obtained from mouth digestion was mixed with simulated gastric fluid (1:1 v/v), pepsin (32 mg) and pH adjusted to 2.0. The solution was incubated for 2 hours at 37 °C under continuous stirring at 100 rpm.
Small Intestinal Phase: The gastric digested liposomes were pH adjusted to 7.0. Simulated intestinal solution (2.6 mL), bile salt solution (2.6 mL) and pancreatin solution from porcine pancreas (2.6 mL) were added and incubated at 37 °C for 2 h at 100 rpm. The solution was titrated using NaOH solution (1 M) and the volume of 1 M NaOH solution required to maintain the system at pH 7.0 was recorded.
Unhydrolyzed lipids were extracted from the digested samples according to Folch method with slight modification (Mou et al. 2021). Five milliliter of digested sample was mixed with 50 mL of chloroform/methanol (2:1, v/v) and vortexed for 20 min. Five milliliter of NaCl solution (0.9%) was then added to the mixture and mixed for 30 min. Finally, the mixture was centrifuged for 10 min at 8000 rpm. The organic phase was transferred to a new tube and washed with 5 mL of NaCl solution (0.9%). Finally, the chloroform layer was evaporated to dryness under nitrogen flow. Fatty acid composition of the extracted lipid was determined according to method described in section 2.2.
Caco-2 cellular uptake of the digested anionic powdered-liposomes
Caco-2 human colorectal cancer cells were grown in Dulbecco's modified Eagles medium (DMEM) containing 20% FBS at 37 °C in a humidified environment with 5% CO2 and expanded in tissue culture flasks (75 cm2, BD Bioscences, USA) ,changing the medium every two days , Caco-2 cells were grown up to 80% confluency in 6-well cell culture plates (Calvello et al. 2016).According to Section 2.2 method, lipids were extracted from different liposome samples after intestinal digestion. Thirty milligrams of the extracted lipids were dissolved in 1 mL of ethanol. Two hundred microliter of this solution was added to 10 mL of 0.33 M bovine albumin (0.01 M PBS, pH = 7.4), mixed with DMEM (1:4, v/v) and incubated with cells in 6-well cell culture plates at 37 °C and 5% CO2. The control was basal medium containing only bovine albumin. After 24 hours of incubation, Caco-2 cells were washed twice with 1 M PBS and harvested for extract genes extraction (Mou et al. 2021).
Changes in gene expression following lipid uptake in Caco-2 cells were measured using quantitative real-time PCR. RNA was extracted using the TransZol Up (ER501,Beijing Quanshijin Biotechnology Co., Ltd.), RNA concentration was measured, and RNA samples with an A260/280 ratio between 1.7 and 2.1 had better purity(Vincent et al. 2020). cDNA was synthesized using the TransScript One-Step kit (AT311, Beijing Quanshijin Biotechnology Co., Ltd.) and analyzed by real-time PCR on the PrefectStart Green qPCR superMix (AQ601,Beijing Quanshijin Biotechnology Co., Ltd.)(Maares et al. 2022), PCR primers are listed in Table S2 of Supplementary material. Relative changes in mRNA levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001).
Statistical analysis
All experiments were performed in triplicate and expressed as mean ± standard deviation (SD). Data were shown as means ± standard deviations (SD). Statistical analyses were performed using a one-way analysis of variance (ANOVA), followed by a Duncan post hoc test using SPSS software (Version 25.0, SPSS Inc., Chicago, IL, USA). Significant differences were set at P < 0.05.
Optimization of the preparation conditions of anionic liposomes loaded with algal oil
Figure 2 shows the effects of processing parameters on encapsulation efficiency of anionic liposomes loaded with algal oil. Encapsulation efficiency firstly increased and then gradually decreased with ratio of algal oil to PS. Algal oil encapsulation efficiency was highest (74.39%) at the optimal algal oil to PS ratio of 1:2.5 (Fig. 2A).
Increased in PS content resulted in formation of more liposomes for encapsulation of algal oil. However, there is a tendency for the liposomes to aggregate and fuse at high PS content resulting in a decrease in overall encapsulation efficiency (Jin et al. 2016).
In terms of pH of PBS, our results showed a maximum encapsulation efficiency of 74.39% at pH 7.2 (Fig. 2B). Phospholipids can be easily hydrolyzed to form lysophospholipids under both acidic and alkaline conditions resulting in decreased encapsulation efficiencies at these conditions (Rasti et al. 2012).
Encapsulation efficiency increased firstly and then decreased with concentration of PBS. Highest encapsulation efficiency (74.39%) of algal oil was obtained at a concentration of 0.01 mol/L algal oil (Fig. 2C). High ionic strength will reduce the surface charge of the membrane causing aggregation, fusion or floating of liposomes which decrease encapsulation efficiency (Molina et al. 2004).
The volume of PBS used in preparation of liposomes affects its encapsulation efficiency. Encapsulation efficiency of algal oil liposomes decreased with increase of PBS volume. Highest encapsulation efficiency of (74.39%) algal oil was obtained at PBS volume of 10 mL (Fig. 2D). PBS can fully extend the hydrophilic head group of phospholipids for formation of liposomes (Odeh et al. 2019). However, excess amount of PBS negatively effects the encapsulation efficiency of algal oil.
In terms of ultrasonic time, encapsulation efficiency increased firstly and then decreased with ultrasonic time. Highest encapsulation efficiency of algal oil (88.03%) at ultrasonic time of 15 min (Fig. 2E). Ultrasonic vibration was used to prepare liposomes, but prolonged ultrasonication may lead to damage of the liposomes structure and leakage of the encapsulated algal oil (Vergara and Shene 2019).
Figure 2F shows the effects of evaporation temperature on encapsulation efficiency. At evaporation temperature of 45 °C, encapsulation efficiency of algal oil was highest at 88.03% (Fig. 2F). Our results showed low temperature was not conducive for formation of thin film. Meanwhile, high evaporation temperature might cause leakage of algal oil from the interior of liposome and reduced encapsulation efficiency (Ickenstein et al. 2006).
The optimal preparation conditions for DHA liposomes with high encapsulation were as follows: algal oil/ PS1:2.5 (w/w), PBS buffer (pH 7.2, 0.1 M, 10 mL), ultrasonic time 15 min, evaporation temperature 45 ℃. At optimal preparation conditions, the encapsulation efficiency was 88.03%.
Average particle size, size distribution, zeta potential and thermal properties of the anionic liposomes loaded with algal oil
Table 1 shows average particle size, size distribution and zeta potential of the anionic liposomes loaded with algal oil. Particle size and PDI are two important parameters affecting the stability and of liposome systems. Anionic liposomes loaded with algal oil prepared at optimal conditions were nanosized (188.53 ± 1.50) with a narrow size distribution (PDI = 0.238 ± 0.01). They had a zeta potential values of -80.07 ± 1.01 mV indicating that liposomes have high electrostatic repulsion and good stability (Sahari et al. 2017). In terms of thermal properties, the anionic liposomes demonstrated an exothermic peak at -9.39 °C (switching of the phospholipid bilayer from liquid crystal state to gel state) and an endothermic peak at 5.72 °C (switching of the phospholipid bilayer from gel state to liquid crystal state) (Fig.4). This indicated that the anionic liposomes loaded with algal oil will have good physical stability when stored at temperature below 5 °C.
Morphology, encapsulation efficiency and drying yield of the anionic powdered-liposomes loaded with algal oil
Figure 3 shows SEM images of the anionic powdered liposomes loaded with algal oil. All the powdered liposomes were spherical shape with concavities (2 to 10 μm) which are typical characteristics of spray-dried powders. It is interesting to note that β-CD powdered liposomes showed a smoother surface than MD and β-CD-MD powdered liposomes which is beneficial in ensuring high encapsulation efficiency. There was no significant difference (P < 0.05) in terms of algal oil encapsulation efficiencies between β-CD powdered liposomes and liposomes dispersion indicating β-CD is able to protect the liposomes structures during high temperature spray drying (Table 1).
On the other hand, MD and β-CD-MD powdered liposomes demonstrated tiny cracks on the spray-dried powders which might cause leakage of the encapsulated algal oil. Both MD and β-CD-MD powdered liposomes demonstrated significantly lower encapsulation efficiencies as compared to liposomes dispersion. MD forms less elastic wall matrix which are unable to resist structural collapse of droplets during spray drying (Jafari et al. 2008b).
Drying yield of the powdered-liposomes was shown in Table 1. All the powdered liposomes have a drying yield between 59.33% and 64.41% (Table 1). This yield was noticeably higher than the previously reported on spray-dried liposomes using maltodextrin as wall material (52%) (Sarabandi et al. 2019).
Average particle size, size distribution, zeta potential and physical properties of the reconstituted powdered liposomes
The reconstituted spray-dried powders liposomes have increased average particle sizes as compared to liposomes dispersion. β-CD-MD powdered liposomes demonstrated highest average particle sizes of 256.30 ± 0.85 nm followed by β-CD and MD powdered liposomes which demonstrated average particle sizes of 244.57 ± 2.61 and 238.97 ± 4.67 nm, respectively. Spray drying process damage the liposomes structures and rehydration process might cause flocculation and aggregation of the liposomes which increase the average particle sizes of the reconstituted spray-dried powders liposomes (Sarabandi et al. 2019). In addition, β-CD form a emulsion with higher viscosity which cause formation of larger droplets during atomization and subsequently resulting in larger powder particles. (Turchiuli et al. 2014). All the reconstituted spray-dried powders liposomes demonstrated a low PDI value of < 0.5 indicating narrow particle size distribution. This shows the flocculation and aggregation of the liposomes were not serious (Ramezanzade et al. 2017).
In terms of zeta potential, all three powdered liposomes demonstrated less negative zeta potential than liposomes dispersion. Among the three powdered liposomes, β-CD powdered liposomes exhibited highest negative zeta potential of -71.80 mV indicating highest stability of β-CD powdered liposomes. A zeta potential higher than ± 30 mV indicated excellent stability as repulsion charges prevent aggregation due to collisions between adjacent particles (Mohan et al. 2016).
Oxidative stability of the anionic powdered-liposomes loaded with algal oil
PV is an important indicator of lipid oxidation which determines the concentration of primary oxidative products such as hydroperoxide and etc. (Sahari et al. 2017). All the powdered liposomes meet the requirement of fish oil microcapsules with a PV < 5 mmol/kg (China 2016a). Figure 5A showed all the powdered liposomes had increased PV during the accelerated oxidation test (P < 0.05). After 15 days, MD powdered liposomes demonstrated lowest increased in PV (from 3.62 mmol/kg to 6.58 mmol/kg). On the other hand, β-CD-MD powdered liposomes demonstrated the most significant increase increased in PV (from 3.65 mmol/kg to 18.07 mmol/kg) (P < 0.05).
During accelerated storage test, hydroperoxide was further oxidized into secondary oxidation products (aldehydes, ketones, quinones, and volatile compounds). These secondary oxidation products can be measured by AV (Fu et al. 2020). p-AV of the powdered-liposomes increased gradually with MD powdered liposomes demonstrated lowest increased in p-AV (from 0.93 to 1.90) and β-CD powdered liposomes demonstrated highest increased in p-AV (from 0.57 to 1.94) (Fig. 5B). At the end of the accelerated oxidation test, p-AV of all the powdered liposomes meet the requirement of first-class fish oil microcapsules (China 2016a) indicating minor oxidation of the powdered liposomes.
It is interesting to note that β-CD-powdered liposomes which demonstrated smooth spherical structures had lowest oxidative stability. We postulated that the liposomes loaded with algal oil was not fully entrapped within the β-CD structures. The ability of MD to induce higher oxidative stability is in agreement with previously reported study. MD was used as a carrier to produce powdered liposomes loaded with soybean oil bodies (OBs) demonstrated higher oxidative stability after storage at high temperature of 60 °C for 7 days (Zhu et al. 2022).
In-vitro digestibility of the anionic powdered-liposomes loaded with algal oil
Figure 6A shows the in-vitro digestibility of the anionic powdered-liposomes loaded with algal oil. All powdered liposomes demonstrated fast release of titratable acids within the first 20 min before reaching constant at 120 min. Initial lipolysis rate (20 min) of the anionic powdered-liposomes increased in the following order: MD powdered liposomes > β-CD-MD powdered liposomes > β-CD powdered liposomes (P < 0.05). MD powdered liposomes demonstrated minor more cracks and pores or their structures. It is possible that the algal oil can be released rapidly through these cracks and pores.
Final lipolysis rate following 120 min gastrointestinal digestion increased in the following order: β-CD-MD powdered liposomes > MD powdered liposomes > β-CD powdered liposomes (P < 0.05). β-CDs has been previously reported to be resistant towards gastric acid and pancreatic enzyme digestion (Stella and He 2008). Therefore, the final release of titratable acid from β-CD powdered liposomes was the lowest.
We collected and analyzed the fatty acid composition of algal oil and liposome powder after 120 min in-vitro digestion. Figure 6B shows the fatty acid compositions of the unhydrolyzed lipid fraction. The algal oil used for encapsulation in this study contained 62.52% DHA, 16.60% EPA and 16.45% oleic acid. The algal oil used for encapsulation in this study contained stearic acid (C18:0, 1.33%), oleic acid (C18:1, 16.45%), linoleic acid (C18:2, 2.06%), EPA (C20: 4, 1.04%), DPA (C22:5, 16.60%) and DHA (C22:6, 62.52%). All powdered liposomes exhibited higher levels of stearic, oleic, and linoleic acids in the unhydrolyzed lipid fraction compared to algal oil, suggesting that these fatty acids were less likely to be hydrolyzed and hydrolyzed during in-vitro digestion. This was consistent with previous findings showing that kink structures in unsaturated fatty acids reduce binding to gastric and pancreatic lipases, thereby reducing their digestibility (Chen et al. 2019). There was no significant difference in the DHA retention of the three liposome powders after digestion (P > 0.05), indicating that the type of wall material has little effect on the digestion and hydrolysis of DHA.
Gene expression
Figure 6C shows the expression of genes (SLC27A4, FABP2, CD36) involved in fatty acid transport after incubation with powdered liposomes with Caco-2 cells. SLC27A4 and CD36 enhances extracellular fatty acid uptake by increasing intracellular esterification efficiency. Meanwhile, FABP2 participates in and regulates the processes related to lipid metabolism in the body. Upregulation of the aforementioned genes indicate reduced cellular lipid uptake.
In comparison to control group, all powdered liposomes demonstrated increased in SLC27A4 (1.19 fold for β-CD-MD powdered liposomes, 1.61 fold for β-CD powdered liposomes, 1.30 fold for MD powdered liposomes), FABP2 (1.30 fold for β-CD-MD powdered liposomes, 3.05 fold for MD powdered liposomes) and CD36 (3.01 fold for β-CD-MD powdered liposomes, 1.91 fold for β-CD powdered liposomes, 1.15 fold for MD powdered liposomes) gene expression indicating good cellular uptake of powdered liposomes (Ding et al. 2021).
Anionic powdered liposomes loaded with algal oil were successfully prepared using phosphatidylserine, MD and β-CD. The effects of different dry wall materials on physical properties, storage stability, in-vitro digestibility and cellular uptake of the powdered liposomes were studied. All the powdered liposomes were nanosized (238.97 ± 4.67–256.30 ± 0.85 nm) with negative zeta potential (-71.80 ± 7.84 – -51.46 ± 1.27 mv). MD was found to induce higher oxidative stability and better in-vitro digestibility in anionic-powdered liposomes loaded with algal oil. Powdered liposomes produced from MD demonstrated increased in SLC27A4, FABP2 and CD36 gene expression indicating good cellular uptake of the powdered liposomes.
Author contributions
Yuanyuan Liu: Methodology, Investigation, Writing - original draft. Xiaoxiao Feng: Investigation, Formal analysis. Jiahe Zhao: Investigation, Formal analysis. Jiale Lin: Investigation, Formal analysis. Cai Shen: Writing - review & editing. Oi-Ming Lai: Conceptualization, Writing - review &editing. Chin-Ping Tan: Conceptualization, Writing - review & editing. Ling-Zhi Cheong: Conceptualization, Writing - review & editing, Funding acquisition.
Conflicts of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding
This work was sponsored by Zhejiang “Jianbing Lingyan” Technology Corporation Fund (2022C04009), Natural Science Foundation of Zhejiang Province (LGJ20C200001). Ling-Zhi Cheong acknowledged funding support from Ningbo “3315” Talent Program.
Availability of Data and Materials Data will be available on request.
Conflicts of interest The authors declare no competing interests.
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Table 1. Physical properties of the liposomes and spray-dried liposomes loaded with algal oil.
|
Sample |
Size (nm) |
PDI |
Zeta potential (mV) |
Encapsulation efficiency (%) |
Drying yield (%) |
|
Algal oil liposome |
188.53±1.50d |
0.238±0.01c |
-80.07±1.01d |
88.03±0.07a |
|
|
β-CD powder |
244.57±2.61c |
0.257±0.01bc |
-71.80±1.84c |
87.42±0.88a |
59.76±2.12b |
|
MD powder |
238.97±4.67b |
0.370±0.02a |
-57.53±1.59b |
84.32±0.50b |
64.41±1.76a |
|
β-CD-MD powder |
256.30±0.85a |
0.262±0.01b |
-51.46±1.27a |
81.60±1.03c |
59.33±0.91b |
Values are expressed as mean ± standard deviations.
Means with different superscript lowercase letters within the same column differ significantly (P < 0.05).
No competing interests reported.