Docosahexaenoic acid (DHA) is an essential n-3 polyunsaturated fatty acid for brain development, which can promote nerve and vision development[1] , improve lipid metabolism disorders[2] , and prevent and alleviate neurodegenerative diseases[3] . However, since the body cannot synthesize DHA to meet its own nutritional needs, DHA must be consumed through dietary intake. Compared with ethyl ester-type and triacylglycerol-type DHA, which are commonly found in commercial products, phospholipid-type DHA is an ideal form of DHA supplementation, as it helps to enrich DHA in the brain[4-5] . DHA-enriched phos phatidylcholine (DHA-PC) is one of the major classes of phospholipid DHA and is widely found in marine organisms. Due to the hydrophilic head group and hydrophobic fatty acid tail, DHA-PC is capable of self-assembling into liposomes in the aqueous environment for active substance transportation. However, DHA-PC liposomes are susceptible to lipid peroxidation due to the presence of polyunsaturated fatty acids, which leads to the destabilization of the liposome phospholipid membrane structure and the production of harmful oxygenated products, such as aldehydes and epoxide groups[6] . H amadou et al[7] encapsulated the antioxidant β-carotene into marine phospholipid liposomes, and found that β-carotene could inhibit the lipid peroxidation reaction, and the inhibition rate could reach about 40%. Therefore, loading antioxidants is an effective way to improve the structural stability of lipid membranes. However, the distribution and arrangement of antioxidants in lipid bilayers have not been reported to affect the structure and function of liposomes.
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Astaxanthin is a polar carotenoid with 10- and 20-fold higher antioxidant activity than β-carotene and lycopene, respectively.8 Pan et al.[9] found that astaxanthin contained in soybean lecithin lipids was able to inhibit lipid peroxidation by up to 70%. In addition, McNulty et al[10] investigated the effects of different molecular structures of carotenoids on the stability of phospholipid membranes rich in polyunsaturated fatty acids, and showed that β-carotenoids lacking terminal hydroxyl groups loaded into the lipid bilayers disturbed their orderly arrangement, whereas astaxanthin preserved the structural order of the membranes. The distance between the polar ends on both sides of the astaxanthin molecular backbone is 30 Å[11] , which is similar to the thickness of the lipid bilayer (25-32 Å)[12] , which allows astaxanthin to be anchored to both sides of the phospholipid membrane through the polar terminal loops on both sides of the structure across the lipid bilayer[10 , 13] . This bilateral anchoring to the lipid bilayer is different from the cholesterol mode of action of unilateral anchoring to the lipid bilayer[14] , and its regulatory effect on the structure of phospholipid membranes needs to be investigated in depth.
DHA-PC is a unique component of marine phospholipids, and the lipid bilayer formed by the self-assembly of its molecules needs to be loaded with stabilizers to improve the stability of the liposome structure. In this study, astaxanthin was used as a membrane stabilizer for DHA-PC liposomes, and DHA-PC liposomes loaded with astaxanthin (Astaxanthin-embedded DHA-PC lipo- some , DPC-AST) were prepared by ethanol injection. The composition ratio of polyunsaturated fatty acids in DHA-PC was analyzed by gas chromatography (GC), and then the preparation conditions of the liposomes were optimized, and the microstructural and physicochemical properties of the liposomes were analyzed in terms of particle size, polydispersity coefficient, zeta potential, and encapsulation rate, etc. The effects of astaxanthin on the mechanical properties of phospholipid membranes and on the resistance to lipid peroxidation were also analyzed by the fluorescence microprobe and the thiobarbituric acid reactant method, respectively. The effects of astaxanthin on the mechanical properties and lipid peroxidation resistance of phospholipid membranes were further analyzed by fluorescence probe and thiobarbituric acid reactant methods.
1 Materials and Methods
1 . 1 MATERIALS
DHA-PC (purity >80%) was purified from squid (Stheno-teuthis oualaniensis) eggs[5] ; astaxanthin (purity 95%) was purchased from Shanghai Aradin Biochemical Technology Company Limited; trichloroacetic acid and 2-thiobarbituric acid were purchased from Sinopharm Chemical Reagent Company Limited; ammonium 8-anilino-1-naphthalene sulfonate, 1,6-diphenyl-1,3,5,-hexatriene were purchased from Shanghai Yuan Ye Biotechnology Co. Ltd.; 8-Anilino-1-naphthalenesulfonic acid ammonium salt, 1,6-diphenyl-1,3,5,-hexatriene were purchased from Shanghai Yuanye Biotechnology Co.
1 . 2 Instruments
Gas Chromatograph :Model 78020, Agilent Technology Co., Ltd; Thermal Stirrer : Model DF-1001S, Shanghai Qiangqiang Instrument & Equipment Co. Nano ZS90, Malvern Instruments, UK; High Performance Liquid Chromatograph (HPLC): Model 1260, Agilent Technologies Ltd, USA; Multiple Light Scattering Instrument (MLSI): LAB, Formulaction, France; Multi-functional Enzyme Labeling Instrument (MELI): Spark 10 M, TECAN, Switzerland; Fluorescence Spectrophotometer (F-460), Hitachi, Japan; UV-Vis Spectrophotometer (UV-2355), Unocal Instruments Ltd, Shanghai, China; Fluorescence Spectrophotometer (F-460), Hitachi, Japan Fluorescence spectrophotometer: F-4000, Hitachi, Japan.
1 . 3 Test methods
1 . 3 . 1 Gas Chromatography Analysis of DHA-PC Fatty Acid Composition Reference Zhang
Gas chromatography (GC) analysis of DHA-PC was performed by the method of Sam et al[15] . The retention time of the methyl ester mixture was compared with that of the sample to characterize the sample. The column was a Sup elco-wax quartz capillary column (30 m×0.25 mm, 0.25 μm); the column temperature was 170 ℃ (0 min) → 5 ℃/min → 2 40 ℃ (222 min) with an equilibrium time of 1 min; the temperature of the injection port was 260 ℃; the temperature of the detector was 260 ℃; the injection volume was 2 μL; the flow rate was 200∶1; the hydrogen flow rate was 20∶1; and the sample was analyzed by the GC-HCA-PC. The injection volume was 2 μL; the split ratio was 2.0∶1; the hydrogen flow rate was 30 mL/min, the air flow rate was 40 mL/min, and the carrier gas was high-purity nitrogen.
1 . 3 . 2 Preparation and optimization of DHA-PC loaded with astaxanthin
1 . 3 . 2 . 1 Preparation of DPC-AST
The liposomes were prepared[16] . According to the recipe, 1 mL-5 mL of ethanol solution of astaxanthin was taken and mixed by shaking, and then the mixed solution was injected into the aqueous phase and stirred at a constant temperature of 37 ℃ for 40 min, and the ethanol was removed by rotary evaporation. The final concentration of DHA-PC in the system was 0.25 mg/mL.
1 . 3 . 2 . 2 Effect of Water to Ethanol Volume Ratio on DPC-AST Particle Size and Polydispersit y Index (PDI)
The mass ratio of DHA-PC to astaxanthin was fixed at 1:0.10, and the volume ratios of the aqueous and ethanol phases were set at 2:1, 3:1, 4:1, and 5:1 for the preparation of DPC-AST, respectively. The particle size and PDI of the liposomes were determined by laser dynamic light scattering (LDLS).
1 . 3 . 2 . 3 Effect of mass ratio and incubation time on the aggregation behavior of astaxanthin
The volume ratio of water to ethanol was fixed at 2∶1, and the mass ratios of DHA-PC to astaxanthin were set at 1∶0.0.0 2, 1∶0.0.0 5, and 1∶0.1 0, respectively, and the ethanol solutions of DHA-PC and astaxanthin were injected into the aqueous phase uniformly, and stirred at a constant temperature of 37 ℃ for 60 min, and the UV-Vis absorption spectra were taken in the range of 3,500~7,000 nm at different points of the incubation time.
1 . 3 . 2 . 4 Effect of mass ratio on the microstructure of DPC-AST
DPC-AST was prepared by fixing the volume ratio of water to ethanol at 2:1 and the quality ratios of DHA-PC to astaxanthin at 1:0.02, 1:0.05 and 1:0.10, respectively. The particle size, PDI and zeta potential of the liposomes were determined by laser dynamic light scattering.
1 . 3 . 3 Determination of astaxanthin content by HPLC method
The astaxanthin content in liposomes was determined by the method of reference [17]. The load was determined as the ratio of astaxanthin in liposomes to the total amount of each substance in liposomes by the ratio of the total input of astaxanthin standardized tretinoin. The HPLC conditions were as follows: the detector was a DAD detector; the chromatographic column was a YMC Carotenoid C30 (4.6 mm×250 mm, 5 μm); the mobile phases were methanol and methyl tert-butyl ether; the flow rate was 1 mL/min; the detection wavelength was 478 nm; the temperature of the column was 30 ℃; and the injection volume was 30 μL.
1 . 3 . 4 Evaluation of Lipid Structural Stability by Multiple Light Scattering
The structural stability of liposomes was determined by applying 10 mL of the prepared liposome suspension to a specific vial of the multiple light scattering instrument. The parameters were set to scan every 5 min for 4 hours.
1 . 3 . 5 Fluorescence polarization method for determination of phospholipid membrane fluidity
The fluidity of liposome membranes was determined by using a DPH fluorescent probe[18] . A suspension of liposomes was mixed with 500 μL of DPH tetrahydrofuran solution (0.086 mmol/L), and the solution was incubated at 37 ℃ for 1 h. The fluorescence polarization of the DPH probe was measured by a multifunctional enzyme marker. The excitation wavelength was 360 nm and the emission wavelength was 430 nm.
1 . 3 . 6 Antilipid peroxidation capacity of astaxanthin by thiobarbituric acid assay
The trichloroacetic acid-thiobarbituric acid-hydrochloric acid (TBA-TCA-HCl) solution was prepared by the method of Huang et al[19] . The astaxanthin-loaded and empty liposomes were incubated in a water bath at 25 ℃ with the lid open for 24 h. During this period, samples were taken every 4 h. 5 mL of TBA-TCA-HCl solution was added, and the liposomes were cooled in an ice bath after 30 min in a boiling water bath. The fluorescence intensity of TBARS was determined by fluorescence spectrophotometry according to the method of He et al[20] , and the parameters of the fluorescence spectrophotometer were set as follows: excitation wavelength of 515 nm, emission wavelength of 553 nm, excitation voltage of 600 V, and the width of the slit was 10 nm. The TBARS change rate was calculated as the ratio of the increase in TBARS fluorescence intensity at time t relative to the 0 h sample to the 0 h sample.
1 . 4 Data processing and analysis
Three parallel experiments were conducted in each group and the data were analyzed statistically (ANOVA) using SPSS software (IBM SPSS statistics 25) and were considered significant at P < 0.0 5. Graphing was performed using Origin 2009 software.
2 Results and analysis
2 . 1 Fatty acid composition of DHA-PC
Marine animal by-products are rich in phospholipids, with squid eggs containing 74.4 g of phospholipids per 100 g of total lipids[21] , making squid eggs a good source of marine phospholipids. The fatty acid composition of DHA-PC extracted from squid eggs is shown in Table 1.17 fatty acids were detected in DHA-PC, of which the three most prevalent fatty acids were palmitic acid (C1 6:0), DHA (C2 2:6), and EPA (C2 0:5), with 2.9.54%, 2.6.50%, and 13.59%, respectively, whereas DHA only accounted for 2%, 2.4.5%, 2.6.5%, and 2.6.5% of the widely commercially extracted yolk phosphatidylcholine (YPC). The percentage of DHA in the widely commercialized egg yolk phosphatidylcholine was only 0.37%[22] . In addition, Cheng Xinwei analyzed the fatty acid composition of dorado eggs and found that the fatty acid content of DHA and EPA was 41.90%, which was not much different from that of DHA and EPA in DHA-PC (40.09%). Phospholipids extracted from low-value marine animal processing by-products are rich in polyunsaturated fatty acids such as DHA and EPA, and marine phospholipids are excellent raw materials for the production of natural phospholipid liposomes.
2 . 2 Preparation and optimization of DHA-PC liposomes loaded with astaxanthin (DPC-AST)
2 . 2 . 2 . 1 Effect of water to ethanol volume ratio on particle size and PDI of DPC-AST
Like other phospholipids, DHA-PC can self-assemble in water to form liposomes. In the present study, liposomes were prepared by ethanol injection, and ethanol was chosen as an acceptable solvent for in vivo administration at a low concentration. The mass ratio of DHA-PC to astaxanthin was fixed at 1:0.10, and the volume ratio of water to ethanol was adjusted to investigate the effects of different volume ratios of water to ethanol on the average particle size and polydispersity index (PDI) of DPC-AST, and the results were shown in Table 2. When the volume ratio of water to ethanol was 2∶1, the average particle size of DPC-AST was (386 ± 8) nm, and the PDI was 0.29 ± 0.04. The results were shown in Table 2. As the volume of the aqueous phase increased, the average particle size of DPC-AST increased from 364 nm to 716 nm, and the PDI values were significantly higher than 0.3. The PDI values of DPC-AST and DPC-AST in the aqueous phase were significantly higher than 0.3. The PDI reflects the distribution of the particles in the system, and a PDI of less than 0.3 means that the liposome particles are concentrated and uniformly dispersed[19] . Therefore, DPC-AST was most stable when the volume ratio of water to ethanol was 2:1. Sogali et al[24] found that the volume ratio of water to ethanol had a significant effect on the particle size and PDI value of liposomes, and the optimal particle size of liposomes was in the range of 254-361 nm and the PDI was in the range of 0.35-0.43 at a solvent volume ratio of 2.5:1. Therefore, in the preparation of liposomes by ethanol injection, the volume ratio of water to ethanol is an important factor affecting the particle size distribution of liposomes.
2 . 2 . 2 Effect of mass ratio and incubation time on the aggregation behavior of astaxanthin
It is well known that the mass ratio is a key parameter in the preparation of liposomes, which affects the particle size and encapsulation rate of liposomes. The effect of the mass ratio of DHA-PC to astaxanthin on the self-assembly of DPC-AST was further investigated by controlling the ratio of water to ethanol at 2:1. It is worth noting that, Ding Lijun[25] found that after injecting astaxanthin and saturated DPPC phospholipids into the aqueous phase, the aggregation of astaxanthin could be generated by incubation in hydrous ethanol solution for different times. Therefore, in the present study, we analyzed the mass ratio and incubation time together, and investigated the effect of incubation time on the aggregation behavior of astaxanthin at different mass ratios of DHA-PC and astaxanthin by scanning ultraviolet visible absorption spectroscopy (UV-VIS), and the results are shown in Fig. 1. When the mass ratio of DHA-PC to astaxanthin was 1:0.02, the maximum absorption wavelength of the solution was near 480 nm at 2.5 min of incubation (Fig. 1(A)), indicating that astaxanthin existed mainly in the form of free monomers at that time. When the incubation time was increased to 40 min, the maximum absorption wavelength of the solution shifted to 490 nm, indicating that astaxanthin molecules started to undergo molecular aggregation. However, the main form of astaxanthin is still free single molecule.
Figures 1(B) and 1(C) show the time-dependent behavior of astaxanthin aggregation at mass ratios of 1:0.05 and 1:0.10 for DHA-PC and astaxanthin, respectively. At these two mass ratios, the maximum absorption peak of astaxanthin shifted to the vicinity of 5 20 nm with the incubation time, accompanied by a side-by-side peak at 5 67 nm, indicating that the astaxanthin completely formed J aggregates. The difference is that the time for astaxanthin to form J-aggregates was 30 min at a mass ratio of 1:0.05, while the time for astaxanthin to form J-aggregates was 20 min at a mass ratio of 1:0.10, which indicates that the greater the concentration of astaxanthin, the easier it is for the molecular aggregation to occur in the hydration solvent. The above results show that the mass ratio of DHA-PC to astaxanthin and the incubation time can have an important effect on the distribution of astaxanthin molecules loaded into the lipid bilayer, when the mixing time is more than 40 min, the astaxanthin loading tends to be stabilized, in which the astaxanthin loaded as a single molecule when the mass ratio of DHA-PC to astaxanthin was 1∶0.02, and mainly as J-aggregates when the mass ratios were 1∶0.05 and 1∶1.0. At the mass ratio of 1:0.05 and 1:10, astaxanthin was mainly loaded into the lipid bilayer in the form of J aggregates.
A series of astaxanthin-carrying liposomes were produced by vortexing aqueous ethanol solutions of DHA-PC with astaxanthin in the ratios of 1:0.02, 1:0.05 and 1:0.10 to remove ethanol. The liposomes were named DPC-AST 2%, DPC-AST 4.8% and DPC-AST 9% according to the ratio of the input mass of astaxanthin to the total mass of astaxanthin and DHA-PC, and the empty-carrier DHA-PC liposomes (DPCs) were prepared, and the suspensions of the above mentioned liposomes showed different colors due to the differences in the form of the loaded form and the content of astaxanthin (see Figure 1(D)). The 2% of DPC-AST loaded as a single molecule of astaxanthin showed a light orange color, whereas the 4.8% and 9% of DPC-AST loaded as a J-polymer of astaxanthin showed a light pink color and a dark pink color, respectively.
2 . 2 . 3 Effect of mass ratio on the microstructure, encapsulation rate and loading of liposomes
Liposomes with different astaxanthin loading forms and dosages were obtained according to the optimized preparation conditions, and the effects of the ratio of DHA-PC to astaxanthin on the microstructure and encapsulation rate of the liposomes were further compared, as shown in Table 3. Compared with the empty DPC (57.4 nm), the particle size of DPC-AST increased significantly (P < 0.05) to 200-40 nm after loading astaxanthin and showed a load-dependent relationship. The reasons for this were twofold: first, as the percentage of astaxanthin in the DHA-PC and astaxanthin system increased from 2% to 9%, the astaxanthin loading increased accordingly, resulting in an increase in the thickness of the lipid bilayer and an increase in the particle size. Jing YK et al[26] also found that the particle size of liposomes increased with the increase of astaxanthin. Secondly, the differences in particle size caused by the loading of astaxanthin in liposomes.
As mentioned above, when the mass ratio of DHA-PC to astaxanthin was 1:0.02, the astaxanthin contained in the lipid bilayer was mainly in the form of a single molecule, whereas when the mass ratios were 1:0.05 and 1:0.10, the astaxanthin contained in the lipid bilayer was mainly in the form of J-aggregates. Lu et al.[27] analyzed by absorption spectroscopy simulation and found that the calculated distances between each astaxanthin molecule in the astaxanthin J-aggregate were 0.66-8 nm, and the distances between each astaxanthin molecule in the astaxanthin J-aggregation were 0.6628 nm, and the distance between each astaxanthin molecule was 0.6628 nm. 8 nm. Therefore, the spatial occupation of stacked astaxanthin J aggregates is much larger than their single molecule length (30 Å), which leads to a significant increase in liposome particle size. The PDI value for DPC was 0.62, indicating that the liposome particles formed by DHA-PC alone were not uniformly distributed. However, loading astaxanthin could reduce the PDI value of liposomes to about 0.3, which indicated that loading astaxanthin could narrow the particle size distribution of liposomes without the help of ultrasound and high-pressure homogenization, and improved the dispersion stability and homogeneity of liposomes to a certain extent. In addition, the potentials of DPC and DPC-AST were in the range of -27~-33 mV, indicating that the loading of astaxanthin had no significant effect on the distribution of charge groups on the surface of liposome particles.
The encapsulation rate and load capacity are indicators of the ability of the carrier structure to encapsulate the loaded molecules. Comparing the encapsulation rates of lipids with different mass ratios, it can be seen that the encapsulation efficiency of astaxanthin in the phospholipid membrane structure was higher than 80%, which indicated that DHA-PC could encapsulate astaxanthin efficiently. As the percentage of astaxanthin increased from 2% to 9%, the encapsulation rate tended to increase and then decrease. The encapsulation rate of DPC-AST 4.8 % was the highest at 93.5 %, while the encapsulation rate of DPC-AST 9 % was the lowest at 84.6 %. This is because at 9% of astaxanthin, the distribution density of astaxanthin in the lipid bilayer was saturated and could not accommodate more astaxanthin loading. In addition, the loading capacity of liposomes increased with the increase of astaxanthin percentage, suggesting that astaxanthin could be effectively loaded into the lipid bilayer in the range of 2% to 9%.
2 . 3 Astaxanthin stabilizes liposome membrane structure
Liposomes are thermodynamically unstable systems, and the membrane structure is usually destabilized due to flocculation and migration of particles, so it is important to investigate the changes of liposome particles in order to study their stability. In contrast to general stability studies, multiple light scattering (MLS) can be used to monitor changes in particle size and position due to flocculation and agglomeration in real time without diluting the sample. The variation of transmission rate (RT) from the bottom of the solution to the top of the liquid surface over the measurement time can reflect the stability changes of colloidal particles such as settling, floating or flocculation[28-29] . Therefore, in the present study, we used a multiple light scattering (MLS) instrument to fully characterize all the physical destabilizing effects of astaxanthin loading on the membrane structure of liposomes by determining the changes in RT values of different heights of liposome suspensions over a period of 4 h in the absence of environmental stimuli, i.e., liposome suspensions were kept in a non-disturbed and non-contact condition. As shown in Fig. 2, the horizontal coordinates of the scans represent the height of the samples, and the bottom, middle and top of the samples are represented from left to right. The vertical coordinate represents the RT, and the curves with different colors represent the changes of RT at different moments. The upward and downward shifts of the left and right curves are related to the sinking and floating of liposomes, and the flat shift of the middle curve is related to the change of particle size.
The RT curves at the bottom and middle of the DPC and DPC-AST samples did not change significantly with time, indicating that there was no significant deposition or aggregation of lipids at the bottom and middle of the samples. At the top of the sample, the RT curves shifted upward with time (see the arrows in Fig. 2), suggesting that there may be some particles in the upper layer of the top of the sample sinking and clarifying, leading to an increase in RT. Compared with DPC and DPC-AST 2%, the top RT curves of DPC-AST 4.8% and DPC-AST 9% showed less variation with time, indicating that the top of the samples were less clarified over time and the structural stability of the astaxanthin-containing liposomes was better.
The stability of the liposomes was further quantified by using multiple light scattering analysis software to express the cumulative value of each change in RT at 25 ℃ for 4 h as the Turbis can instability index (TSI). The larger the TSI, the more unstable the sample is[29] . Figure 3 shows that the TSI of DPC increased rapidly to 0.91 in the first 300 s, and by the end of the test, the TSI of DPC was 1.20. The TSI of DPC-AST increased more rapidly than that of DPC-AST. The rate of increase of DPC-AST was slower than that of DPC, and the TSI values of DPC-AST 2%, DPC-AST 4.8%, and DPC-AST 9% were 1.15, 0.58, and 0.55, respectively, which were smaller than those of DPC at the end of the test, suggesting that the incorporation of astaxanthin could improve the structural stability of DPC. Further comparison of the TSI values of DPC-AST at the same time showed that the TSI values of the liposomes were the smallest when astaxanthin accounted for 4.8% and 9%, i.e., astaxanthin was embedded into the lipid bilayer in the form of J-aggregates, indicating that the loading of astaxanthin J-aggregates was most advantageous in stabilizing the structure of the lipid bilayer.
2 . 4 Regulation of liposome membrane fluidity by astaxanthin
Mobility is an important property of phospholipid bilayers that can affect the permeability of most substances and hence the structural stability of lipids[30] . The presence of unsaturated bonds in the phospholipid backbone reduces the orderliness of the intermolecular arrangement of membrane lipids, thereby increasing membrane fluidity. 1,6-Diphenyl-1,3,5,-hexatriene (DPH) is a lipophilic fluorescent probe that can be inserted into the central hydrophobic region formed by the arrangement of fatty acid chains in the tails of phospholipids, and the change of its fluorescence polarization can reflect the mobility of the lipid chains[31] . Figure 4 shows the effect of astaxanthin in DHA-PC and astaxanthin on the fluorescence polarization of DPH in the lipid bilayer of liposomes. Compared with the fluorescence polarization measured by unloaded DPC (0.11), the DPC-AST loaded with astaxanthin showed higher fluorescence polarization (P < 0.05), which indicated that the astaxanthin loaded with astaxanthin restricted the movement of DPH, and reduced the hydrophobic region of the central lipophobic region of the liposomes to a certain extent. This indicated that astaxanthin loading restricted the movement of DPH and to some extent reduced the mobility of fatty acid chains in the central hydrophobic region of the liposome.
Surprisingly, as the percentage of astaxanthin increased from 2% to 9%, the fluorescence polarization did not increase correspondingly, but rather decreased from 0.2 to about 0.1, indicating that astaxanthin loaded into the lipid bilayer could regulate the mobility of the central hydrophobic region of the phospholipid membrane, which was presumably related to the form of the loaded astaxanthin aggregates. In DPC-AST 2%, astaxanthin was mainly embedded in the lipid bilayer as a single molecule with a molecular length of about 30 Å. The interaction between α-hydroxyketones on both sides of the astaxanthin backbone and the hydroxyl groups of the neighboring phospholipid molecules was enhanced, and the conjugated polyenyl chain of the astaxanthin backbone limited the movement of the flexible polyunsaturated fatty acid chains in the hydrophobic region, resulting in the reduction of the fluidity of the lipid membrane. Other studies have shown that the anchoring of astaxanthin at the lipid-water interface on both sides of the membrane facilitates van der Waals forces between astaxanthin-conjugated polyene chains and lipid acyl chains in the internal hydrophobic region[32] , resulting in increased rigidity of liposome membranes after astaxanthin loading[13] . However, when the percentage of astaxanthin increases to 4.8 % and 9 %, the astaxanthin loaded in the liposome tends to be a J-aggregate, i.e., the astaxanthin molecules are loosely stacked in a head-to-tail "ladder" fashion, and only one side of the aggregated astaxanthin backbone, α-hydroxyketone, participates in non-covalent interactions with the hydroxyl groups of neighboring phospholipid molecules, resulting in increased movement of the phospholipid molecules and increased membrane fluidity. As a result, the movement of phospholipid molecular chains increased and the membrane fluidity increased. The above results suggest that the mode of distribution of astaxanthin in lipid bilayers, i.e., monomer or aggregate distribution, can regulate the fluidity of liposome membranes.
2 . 5 Antilipid peroxidation capacity
The presence of polyunsaturated fatty acid chains in the molecular skeleton of phospholipids can easily lead to lipid peroxidation during processing or storage, resulting in structural destabilization and leakage of oxidation products or drugs[3 3] . In order to evaluate the effect of astaxanthin loading on the oxidative stability of DHA-PC liposomes, the increase of TBARS produced by DPC and DPC-AST 9% was determined by the thiobarbituric acid reactants (TBARS) method over 24 h at room temperature. Figure 5 shows that TBARS in DPC increased by 90.71% in 24 h, indicating that liposomes are capable of spontaneous oxidation when exposed to atmospheric conditions. In comparison, DPC-AST 9% increased by only 35.86% under the same conditions, indicating that the loading of astaxanthin in liposomes significantly reduced the production of TBARS. Tan et al[34] reported that loading liposomes with carotenoids, such as lutein, inhibited the production of TBARS from spontaneous oxidation of liposomes at room temperature for 2 h, and the negative increase in TBARS was found when compared with that of the unloaded liposomes (37%). Compared with the increase of TBARS in unloaded liposomes (37%), the increase of TBARS in liposomes loaded with 2% lutein was only about 10%.
3 Conclusion
In this paper, DHA-PC liposomes loaded with astaxanthin were prepared by ethanol injection, and the key preparation parameters such as solvent ratio and solvent mixing time were determined. By comparing the effects of different percentages of astaxanthin on the encapsulation rate, self-aggregation and fluidity of liposomes, it was found that the formation of astaxanthin aggregates was an effective way to increase the astaxanthin loading in liposomes, and the aggregation state of astaxanthin embedded in the lipid bilayers could regulate the fluidity of the lipid membranes, which could improve the structural stability of the lipid membranes in terms of the membrane mechanical performance and antioxidant properties. Of course, the stability of astaxanthin-loaded liposomes in the process of storage and in vivo transportation needs further study. The results of this study can provide theoretical support for the application of natural phospholipids, especially marine phospholipids rich in polyunsaturated fatty acids, in foods and medicines.
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