the nitrogen gas to evaporate the t

以蒸发溶剂的痕迹的氮气，然后以1:1的比例与油酸混合：2（重量/重量），并加入到用0.1-0.2％吐温80，并使用磁力搅拌器混合的CS溶液（REMI CM- 101个加，印度）30分钟。然后SA溶液逐滴加入到该CS-叶黄素-油酸-吐温80混合物，再混合30分钟，然后使用探针超声波仪（PCI，印度孟买）15分钟超声处理在50Hz下在冰上，随后高速均化在15000rpm离心5分钟。<br>2.6。混合胶束的制备<br>在磷酸盐缓冲盐水（10毫升）中制备含有单油酰基-甘油（2.5毫摩尔），牛磺胆酸钠（12毫摩尔），油酸（7.5毫摩尔）和叶黄素（600μM）的混合胶束。这些化学物质和油酸溶解在氯仿和甲醇以达到最终浓度。将溶剂蒸发，使用氮气至干。胶束是使用涡旋混合器（REMI CM-101加，印度），随后通过超声处理悬浮于磷酸盐缓冲盐水（pH 7.4）在剧烈混合（PCI，印度孟买）搅拌15分钟，得到混合胶束的澄清溶液[ 31,26]。<br>2.7。LNCS的表征<br>2.7.1。HPLC和LC-MS<br>纯化和提取叶黄素形式LNCS通过HPLC（联盟2690，沃特斯，UK）进行分析。从LNC标准叶黄素和叶黄素的萃取物注射（20μL）到配有C-18柱（250 *4.6平方毫米; SGE公司，印度孟买）HPLC系统和PDA检测器。以1mL / min的流速通过在444处监测含0.1％乙酸铵;（：2 2 V / V / V 6乙腈/甲醇/ DCM）叶黄素是使用流动相分离。为了认证纯化叶黄素，LC-MS的质谱（大气压化学电离模式; APCI +）的混合物在正离子模式按照Lakshminarayana等进行。[27]的方法稍加修改。简言之，正离子所使用的HPLC系统重新编码（联盟2695，沃特斯，UK）连接到Q-TOF的Ultima质谱仪（沃特斯2996模块化 UK）配备有大气压化学电离模块（APCI +）和源探针在130℃和500℃分别加热。在LC-MS条件，例如高电压透镜，电晕，锥孔电压，鞘，将氮气以0.5和5KV，30V和100和300升/小时分别优化。将分光光度计在正离子模式下校准，（M + H）离子信号记录并根据标准叶黄素证实。<br>2.7.2。粒度分析和LNCS的zeta电位测量<br>LNCS的粒径，多分散性指数（PDI）和动态光散射（DLS），通过粒度分析仪（马尔文纳米ZS 90）和DLS的Zetasizer NanoZS（Malvern公司，伍斯特郡，UK）进行分析。样品（一式三份），通过使用超声波水浴（PCI分析，WC-01），以获得良好分散的悬浮在蒸馏水（10mL）中，并进一步超声处理LNCS（0.1mL）中分散6分钟制备。该仪器配有垂直偏振光源，并通过测量粒度和PDI在室温下（27℃）90°散射的固定角之前氩离子激光器。数值以平均值±标准差。LNCS的Zeta电位值为（N = 3）在25℃，150V，通过电泳光散射分析，并且还测量的多分散指数反映的粒度分布的宽度。粒子的电荷传导原理计算来确认LNC [32]的稳定性。Zeta电位为

the nitrogen gas to evaporate the traces of solvents, then mixed with oleic acid at a ratio of 1:2 (w/w) and added to the CS solution with 0.1-0.2 % tween 80 and mixed using the magnetic stirrer (REMI CM-101 plus, India) for 30 min. Then SA solution was added dropwise to the CS-lutein-oleic acid-tween 80 mixture, mixed for another 30 min and then sonicated using the probe sonicator (PCI, Mumbai, India) for 15 min at 50Hz on ice followed by high-speed homogenization at 15000 rpm for 5 min.<br>2.6. Preparation of mixed micelle<br>Mixed micelles containing mono-oleoyl-glycerol (2.5 mM), sodium taurocholate (12 mM), oleic acid (7.5 mM) and lutein (600 μM) were prepared in phosphate buffered saline (10 mL). These chemicals and oleic acid were dissolved in chloroform and methanol to reach the final concentration. The solvents were evaporated to dryness using nitrogen. The micelle was resuspended in phosphate buffered saline (pH 7.4) with vigorous mixing using a vortex mixer (REMI CM-101 plus, India) followed by sonication (PCI, Mumbai, India) for 15 min to obtain a clear solution of mixed micelles [31,26].<br>2.7. Characterization of LNCs<br>2.7.1. HPLC and LC-MS<br>Purified and extracted lutein form LNCs were analyzed by HPLC (Alliance 2690, Waters, UK). Standard lutein and lutein extracts from LNC were injected (20 μL) onto the HPLC system equipped with C-18 column (250*4.6 mm2; SGE company, Mumbai, India) and PDA detector. Lutein was separated using the mobile phase (acetonitrile/methanol/DCM; 6:2:2 v/v/v) containing 0.1% ammonium acetate at a flow rate of 1 mL/min by monitoring at 444 nm. To authenticate the mass spectrum of the purified lutein, LC-MS (Atmospheric Pressure Chemical Ionization mode; APCI+) was performed in positive ion mode as per Lakshminarayana et al. [27] with slight modifications. In brief, the positive ions are recoded using the HPLC system (Alliance 2695, Waters, UK) connected to a Q-Tof Ultima mass spectrometer (Waters 2996 Modular, UK) equipped with atmospheric pressure chemical ionization module (APCI+) and the source probe were heated at 130 °C and 500 °C respectively. The LC-MS conditions such as high voltage lens, corona, cone voltage, sheath, and nitrogen gas were optimized at 0.5 and 5KV, 30V and 100 and 300 L/h respectively. The spectrophotometer was calibrated in a positive ion mode and the (M + H) ion signals were recorded and confirmed against the standard lutein.<br>2.7.2. Particle size analysis and zeta potential measurement of LNCs<br>Particle size, polydispersity index (PDI) and dynamic light scattering (DLS) of LNCs were analyzed by particle size analyzer (Malvern nano Zs 90) and DLS zetasizer NanoZS (Malvern Instruments Ltd., Worcestershire, UK). Samples (in triplicate) were prepared by dispersing the LNCs (0.1 mL) in distilled water (10 mL) and further sonicating for 6 min using an ultrasonic water bath (PCI analytics, WC-01) to obtain a well-dispersed suspension. The instrument is furnished with the vertically polarised light source, and by an argon-ion laser before measuring the particle size and PDI at a fixed angle of 90° scattering at room temperature (27°C). Values are presented as mean ± SD. Zeta potential value of LNCs was (n=3) analyzed by electrophoretic light scattering at 25 °C, 150 V, and polydispersity index was also measured reflecting the width of the particle size distribution. The charge conductivity principle of particles was calculated to confirm the stability of the LNC [32]. Zeta potential was

氮气蒸发微量溶剂，然后以1:2（w/w）的比例与油酸混合，并添加到含有0.1-0.2%吐温80的CS溶液中，并使用磁力搅拌器（REMI CM-101 plus，印度）混合30分钟。然后将SA溶液逐滴添加到CS叶黄素油酸吐温80混合物中，再混合30分钟，然后用探头超声仪（PCI，孟买，印度）在50赫兹的冰上超声15分钟，然后在15000转/分的速度下高速均质5分钟。<br>2.6条。混合胶束的制备<br>在磷酸盐缓冲液（10ml）中制备了含单油酰甘油（2.5m M）、牛磺胆酸钠（12mm）、油酸（7.5mm）和叶黄素（600μM）的混合胶束。这些化学物质和油酸溶解在氯仿和甲醇中达到最终浓度。溶剂用氮气蒸干。胶束在磷酸盐缓冲盐水（pH7.4）中重新悬浮，并使用涡流混合器（REMI CM-101 plus，印度）进行剧烈混合，然后进行15分钟的超声处理（PCI，孟买，印度），以获得混合胶束的澄清溶液[31,26]。<br>2.7条。LNCs的表征<br>2.7.1条。高效液相色谱和液相色谱-质谱<br>用高效液相色谱法（Alliance 2690，Waters，UK）分析从LNCs中纯化和提取的叶黄素。将LNC的标准叶黄素和叶黄素提取物（20μL）注入配备C-18柱（250*4.6平方毫米；SGE公司，印度孟买）和PDA检测器的高效液相色谱系统。用流动相（乙腈/甲醇/DCM；6:2:2v/v/v）分离叶黄素，流动相中含有0.1%醋酸铵，流速为1ml/min，在444nm处监测。为了验证纯化的叶黄素的质谱，按照Lakshminarayana等人的方法，在正离子模式下进行LC-MS（大气压化学电离模式；APCI+）。[27]稍加修改。简言之，正离子使用连接到配有大气压化学电离模块（APCI+）的Q-Tof Ultima质谱仪（Waters 2996模块，UK）的高效液相色谱系统（Alliance 2695，Waters，UK）重新编码，源探针分别在130°C和500°C下加热。在高压透镜、电晕、锥压、鞘层、氮气等条件下，分别在0.5和5KV、30V和100、300l/h下对LC-MS进行了优化。在正离子模式下校准分光光度计，记录（M+H）离子信号，并对照标准叶黄素进行确认。<br>2.7.2条。LNCs的粒度分析及zeta电位测量<br>采用粒度分析仪（Malvern nano Zs 90）和DLS zetasizer NanoZS（Malvern Instruments Ltd.，英国伍斯特郡）对LNCs的粒径、多分散指数（PDI）和动态光散射（DLS）进行了分析。通过将LNCs（0.1 mL）分散在蒸馏水中（10 mL）并使用超声波水浴（PCI analytics，WC-01）进一步超声6分钟来制备样品（一式三份），以获得均匀分散的悬浮液。仪器配备垂直偏振光光源，在室温（27°C）下以90°散射的固定角度测量粒度和PDI之前，使用氩离子激光器。数值表示为平均值±标准偏差。用电泳光散射法分析了25℃、150v下LNCs的Zeta电位值（n=3），并测定了反映粒径分布宽度的多分散指数。计算了粒子的电荷导电原理，以确定LNC的稳定性。Zeta电位