siRNA 负载脂质体的制备与表征

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Abstract 摘要

The major challenge for RNAi-based therapy is the fabrication of the delivery system that meet the requirement of clinical applicability. Liposome-derived nanoparticles (NPs) are one of the best investigated systems for in vivo siRNA delivery. In the recent years, we have successfully redesigned the conventional cationic liposomes into Liposome/Protamine/hyaluronic acid (LPH) NPs and Lipid-Calcium-Phosphate (LCP) NPs in order to increase the in vivo gene silencing effect and reduce the toxicity. Here we describe the preparation of LPH and LCP NPs loaded with siRNA, and characterization analysis including size distribution, trapping efficiency, and in vivo activity. This protocol could be used for in vivo delivery of siRNA to target genes in cancer cells.

RNA 干扰（RNAi）疗法面临的主要挑战是如何构建符合临床应用要求的递送系统。脂质体衍生的纳米颗粒（NPs）是目前研究最为深入的体内 siRNA 递送系统之一。近年来，我们成功将传统的阳离子脂质体改造为脂质体/胍蛋白/透明质酸（LPH）纳米颗粒和脂质-钙-磷（LCP）纳米颗粒，以提高体内基因沉默效果并降低毒性。本文介绍了载有 siRNA 的 LPH 和 LCP 纳米颗粒的制备方法及其表征分析，包括粒径分布、封装效率和体内活性。这一方案可用于将 siRNA 有效递送到癌细胞中的靶基因。

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1 Introduction

RNA interference (RNAi) is a revolutionary tool to suppress the expression of the target gene with relevance to any disease. However, the siRNA will have to be delivered to the diseased cells in an efficient manner. The major challenges in achieving efficient siRNA delivery in vivo include low stability in blood serum, rapid excretion by renal clearance, nonspecific tissue distribution, poor cellular uptake, and inefficient intracellular release. Numerous materials have been explored to overcome these multiscale extracellular and intracellular barriers to the delivery of siRNA. Although viral vectors provide excellent in vivo activity of RNAi, safety concerns and difficulties with large-scale manufacture limit their use in clinic. Therefore, nonviral vectors such as liposome-based vehicles have been intensively explored as safe and effective alternatives. The only siRNA drug approved by FDA uses a nonviral vector. Despite the successful examples of using cationic liposomes for in vivo siRNA delivery, their short half-life in plasma and the toxicity of cationic lipids still limit the clinical application. To address these issues, we have redesigned the conventional cationic liposomes by incorporating a “core-shell” structure into the nanoparticles (NPs). This concept was originally developed earlier in our lab for plasmid DNA delivery, which involves pre-complexation of plasmid DNA with protamine sulfate followed by the addition of cationic liposomes. The DNA was condensed by protamine sulfate, a highly positively charged peptide that is approved by FDA for clinical use. When this method was later adapted for siRNA delivery, the cationic polymers formed a looser complex with siRNA than with the plasmid DNA, resulting in formulation of unstable particles and reduced delivery efficiency. Hyaluronic acid (HA), a polyanionic polysaccharide, was therefore employed in order to provide multivalent charges and improve the particle condensation. We mixed siRNA with HA before complexing with protamine. These components spontaneously self-assemble to form NPs. Steric stabilization was introduced by post-inserting PEGylated lipid onto the surface of LPH. LPH demonstrated efficient gene silencing via systemic delivery of siRNA in tumor-xenograft animal models. The sub-cellular distribution study in the cancer cells indicated that the siRNA might be sequestered in the endosomes, and incorporating an endosomal lytic mechanism might further improve the NP formulation. We then formulated the NP core with calcium phosphate (CaP) which could dissolve at the acidic endosome pH. This gave the birth of Lipid-Calcium-Phosphate (LCP) NPs. We proposed and later proved that dissolved CaP significantly increased the osmotic pressure of the endosome and induced swelling and rupture of the organelle, resulting in the release of the encapsulated siRNA. Here we describe methods for preparing siRNA-loaded LPH and LCP. Technical details of preparation and characterization of NPs are presented. This protocol could be used to deliver siRNA into cancer cells.

RNA 干扰（RNAi）技术是一种革命性的工具，能够有效抑制与疾病相关的靶基因表达。然而，成功递送 siRNA 到病变细胞是 RNAi 疗法的主要挑战。体内高效递送 siRNA 面临的主要困难包括：血清中的稳定性差、肾脏快速排泄、组织分布不精准、细胞摄取效率低以及细胞内释放不充分。为了克服这些在细胞外和细胞内的多重障碍，已经开发出多种材料来提高 siRNA 的递送效果。尽管病毒载体在体内具有较好的 RNAi 活性，但其安全隐患和难以大规模生产的问题限制了其在临床中的使用。因此，非病毒载体，尤其是基于脂质体的递送系统，已成为一种安全、有效的替代方案。事实上，FDA 批准的唯一 siRNA 药物就使用了非病毒载体。尽管阳离子脂质体在体内 siRNA 递送方面有许多成功案例，但其在血浆中的短暂半衰期和阳离子脂质的毒性依然限制了其临床应用。为解决这些问题，我们对传统的阳离子脂质体进行了改造，在纳米颗粒中引入了“核壳”结构。这一概念最早由我们的实验室提出，用于质粒 DNA 的递送，方法是先用鱼精蛋白与质粒 DNA 形成复合物，然后加入阳离子脂质体。鱼精蛋白硫酸盐是一种高度带正电的肽类，已获 FDA 批准用于临床，它能将质粒 DNA 有效压缩。当该方法用于 siRNA 递送时，发现阳离子聚合物与 siRNA 的结合相对松散，导致颗粒不稳定，递送效率降低。因此，我们引入了透明质酸（HA），一种多阴离子多糖，以增强颗粒的凝聚效果。我们将 siRNA 与透明质酸混合，再与鱼精蛋白复合，形成纳米颗粒。通过在 LPH 表面插入 PEG 化脂质，实现了纳米颗粒的空间稳定性。LPH 在肿瘤异种移植动物模型中通过系统性递送 siRNA，显示了良好的基因沉默效果。在癌细胞内的亚细胞分布研究表明，siRNA 可能被滞留在内涵体中，因此引入内涵体溶解机制可能会进一步提高纳米颗粒的效果。为此，我们将纳米颗粒核心设计为磷酸钙（CaP），其能在酸性内涵体 pH 环境下溶解，形成了脂质-钙-磷（LCP）纳米颗粒。我们提出并验证了溶解的磷酸钙显著提高了内涵体的渗透压，导致内涵体膨胀并破裂，从而释放出封装的 siRNA。本文详细介绍了 siRNA 载体 LPH 和 LCP 的制备方法及纳米颗粒的表征分析，所述方案可用于将 siRNA 递送至癌细胞。

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2 Materials 材料

2.1 Preparation of LPH LPH 的制备

1. 1,2-Dioleoyl-3-trimethylammonium -propane (DOTAP) (Avanti Polar Lipids, Alabaster, AL). 1,2-二油酰基-3-三甲基氨基丙烷（DOTAP），供应商：Avanti Polar Lipids（美国阿拉巴马州，Alabaster）。

2. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)2000] (DSPE-PEG2000) (Avanti Polar Lipids, Alabaster, AL).1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[聚乙二醇 2000]（DSPE-PEG2000），供应商：Avanti Polar Lipids（美国阿拉巴马州，Alabaster）。

3. Cholesterol. 胆固醇

4. Anti-luciferase siRNA with FAM labeling, 5′-CTT ACG CTG AGT ACT TCG A-3′. FAM 标记的抗荧光素酶 siRNA，序列为 5′-CTT ACG CTG AGT ACT TCG A-3′。

5. Protamine sulfate (fraction X from salmon). 鱼精蛋白硫酸盐（取自鲑鱼的 X 级部分）。

6. Hyaluronic acid sodium salt from Streptococcus equi (HA). 从马链球菌（Streptococcus equi）提取的透明质酸钠（HA）。

7. 25-mL Round-bottomed spherical flask. 25 毫升的圆底烧瓶。

8. Rotary evaporator. 旋转蒸发仪。

9. Mini-extruder set (Avanti Polar Lipids, Alabaster, AL). 迷你挤出器套装（Avanti Polar Lipids，美国阿拉巴马州，Alabaster）。

10. Polycarbonate Membranes with pore size of 1.0, 0.4 and 0.1μm (Avanti Polar Lipids, Alabaster, AL). 孔径为 1.0、0.4、0.1 微米的聚碳酸酯膜，供应商：Avanti Polar Lipids（美国阿拉巴马州，Alabaster）。

11. 1.5-mL Microcentrifuge tube. 1.5 毫升微量离心管。

2.2 Characterization of LPH LPH 的表征

1. Dynamic light scattering (DLS) particle sizer analyzer. 动态光散射（DLS）粒径分析仪。

2. Sepharose CL4B size exclusion column (PharmaciaBiotech, Uppsala, Sweden). Sepharose CL4B 尺寸排阻柱，供应商：Pharmacia Biotech（瑞典乌普萨拉）。

3. Lysis buffer, 2 mM EDTA with 0.05% Triton X-100 in pH 7.8 Tris buffer. 裂解缓冲液，成分为 2 mM EDTA 和 0.05% Triton X-100 的 pH 7.8 Tris 缓冲液。

4. Plate reader. 微孔板阅读器。

2.3 Preparation of LCP LCP 的制备

1. Dioleoylphosphatidic acid (DOPA) (Avanti Polar Lipids, Alabaster, AL).二油酰基磷脂酸（DOPA），供应商：Avanti Polar Lipids（美国阿拉巴马州，Alabaster）。

2. Anti-luciferase siRNA, 5′-CTT ACG CTG AGT ACT TCG A-3′. 抗荧光素酶 siRNA，序列为 5′-CTT ACG CTG AGT ACT TCG A-3′。

3. Control siRNA, 5′-AAT TCT CCG AAC GTG TCA CGT-3′. 对照 siRNA，序列为 5′-AAT TCT CCG AAC GTG TCA CGT-3′。

4. Anti-luciferase siRNA with FAM labeling, 5′-CTT ACG CTG AGT ACT TCG A-3′. FAM 标记的抗荧光素酶 siRNA，序列为 5′-CTT ACG CTG AGT ACT TCG A-3′。

5. Cyclohexane. 环己烷。

6. Igepal CO-520. 聚(氧乙烯)壬基苯基醚

7. Triton X-100.

8. Hexanol. 乙醇

9. 100-mL Round-bottomed spherical flask. 100 毫升的圆底烧瓶

10. 500 mM Calcium chloride (CaCl₂). 500 mM 氯化钙

11. 25 mM Disodium hydrogen phosphate (Na₂HPO₄), pH = 9.0. 25 mM 磷酸氢二钠

12. Ultracentrifuge. 超速离心机

13. Ethanol, 200 proof (100%), USP. 无水乙醇（200 proof，100%）

14. 7-mL Borosilicate glass scintillation vial. 7 毫升的硼硅酸盐西林瓶

15. Bath-type sonicator.浴式超声波清洗器

16. Vacuum desiccator. 真空干燥器

2.4 Characterization of LCP LCP 的表征

1. DLS particle sizer analyzer. 动态光散射（DLS）粒径分析仪

2. Lysis buffer, 2 mM EDTA with 0.05% Triton X-100 in pH 7.8 Tris buffer. 裂解缓冲液，成分为 2 mM EDTA 和 0.05% Triton X-100 的 pH 7.8 Tris 缓冲液

3. Plate reader. 微孔板阅读器

4. 200 Mesh carbon-coated copper grids (Ted Pella, Inc., Redding, CA).200 目碳膜涂层铜网，供应商：Ted Pella, Inc.（美国加利福尼亚州雷丁）

5. 2% Uranyl acetate. 2%醋酸铀染色剂

6. Transmission electron microscope (TEM). 透射电子显微镜（TEM）

2.5 In Vivo Luciferase Silencing Effect of LCP LCP 在体内的荧光素酶基因沉默效果

1. Female athymic nude mice (age 6–8 weeks, Charles River Laboratories, Wilmington, MA).雌性无胸腺裸鼠（年龄 6 到 8 周），供应商：Charles River Laboratories（美国马萨诸塞州 Wilmington）

2. Lysis buffer, 2 mM EDTA with 0.05% Triton X-100 in pH 7.8 Tris buffer. 裂解缓冲液，成分为 2 mM EDTA 和 0.05% Triton X-100 的 pH 7.8 Tris 缓冲液

3. Luciferase substrate (Luciferase Assay System, Promega Co., Madison, WI). 荧光素酶底物（供应商：Promega Co.，美国威斯康星州 Madison，荧光素酶检测试剂盒）

4. Plate reader. 微孔板阅读器

5. Protein assay kit (Micro BCA™ Protein Assay Kit, Pierce). 蛋白质检测试剂盒（Micro BCA™蛋白质检测试剂盒，供应商：Pierce）

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3 Methods

3.1 Preparation of LPH LPH 的制备步骤

1. Add 1 mL of DOTAP (10 mM) and 1 mL of cholesterol (10 mM) (see Note 1) in a 1:1 molar ratio to a 25-mL round-bottomed flask. 将 1 mL 的 DOTAP（10 mM）和 1 mL 的胆固醇（10 mM）按 1:1 摩尔比加入 25 mL 的圆底烧瓶中（参见 注释 1）。

2. Dry the lipid mixture under vacuum by using a rotary evaporator to form a thin film on the wall of the flask. 使用旋转蒸发仪，在真空下干燥脂质混合物，使其在烧瓶壁上形成一层薄膜。

3. Add 2 mL of water (see Note 2) to the flask and hydrate the lipid film to give a final concentration of total lipids at 40 mM. 向烧瓶中加入 2 mL 水（参见 注释 2），水化脂质薄膜，使最终脂质浓度达到 40 mM。

4. Sonicate the liposomes in a bath-type sonicator. 使用浴式超声波清洗器对脂质体进行超声处理。

5. Incubate the liposomes at 50 °C for 10 min and then sequentially extruded through polycarbonate membranes with the following pore sizes: 1.0, 0.4 and 0.1μm. This size extrusion method produces unilamellar DOTAP liposomes with particles around 100 nm. 将脂质体在 50°C 下孵育 10 分钟，然后依次通过孔径为 1.0、0.4 和 0.1μm 的聚碳酸酯膜进行挤出。此尺寸挤出法可生成粒径约 100 nm 的单层 DOTAP 脂质体。

6. Mixing 12.5μg siRNA (see Note 3) and 12.5μg HA (weight ratio 1:1) in 100μL of water in a 1.5-mL tube. 将 12.5μg siRNA（参见 注释 3）和 12.5μg 透明质酸（HA）（重量比 1:1）混合在 100μL 水中，置于 1.5 mL 离心管中。

7. Add 13μg protamine in 100μL of water to the mixture, and keep the mixture at room temperature for 10 min to form the core (see Note 5). 向该混合物中加入 13μg 鱼精蛋白溶于 100μL 水中，并在室温下放置 10 分钟，以形成核心结构（参见 注释 5）。

8. Add 60μL of DOTAP/cholesterol liposomes to the core, and keep the mixture at room temperature for 10 min to form LPH.向核心结构中加入 60μL DOTAP/胆固醇脂质体，并在室温下保持混合物 10 分钟，形成 LPH。

9. Add 15–30μL of micelle suspension of DSPE-PEG2000 (25 mg/mL in water) to the 260μL preformed LPH and then incubated at 50 °C for 10 min.向 260μL 预形成的 LPH 中加入 15-30μL DSPE-PEG2000（25 mg/mL 水溶液）的胶束悬浮液，然后在 50°C 下孵育 10 分钟。

10. Allow the PEGylated LPH to cool to room temperature before storage or further application (see Note 5, Fig. 1).让 PEG 化的 LPH 冷却至室温后再进行储存或进一步应用（参见 注释 5，见图 1）。

Fig. 1 Illustration for the preparation of LPH. (Reprint from 10 with permission)

3.2 Characterization of LPH LPH 的表征

3.2.1 Particle Size and Zeta Potential 粒径和 Zeta 电位

1. Dilute a dispersion of LPH into an appropriate concentration with water.用水将 LPH 分散液稀释至适当浓度。

2. Use the DLS method to determine the particle size and zeta potential of LPH (see Note 6).使用动态光散射（DLS）法测定 LPH 的粒径和 Zeta 电位（参见 注释 6）。

3.2.2 Trapping Efficiency of siRNA siRNA 的包封效率

1. Prepare the standard siRNA solutions by diluting the FAM-labeled siRNA in the lysis buffer.通过在裂解缓冲液中稀释 FAM 标记的 siRNA，制备标准 siRNA 溶液。

2. Prepare LPH with the FAM-labeled siRNA. 用 FAM 标记的 siRNA 制备 LPH。

3. Pass the 200μL of LPH through a Sepharose CL4B size exclusion column and collect the resulting eluents of both unentrapped siRNA and LPH.将 200μL 的 LPH 通过 Sepharose CL4B 尺寸排阻柱，收集未包封的 siRNA 和 LPH 的洗脱液。

4. Dissolve all the LPH samples in the lysis buffer. 将所有 LPH 样品溶解在裂解缓冲液中。

5. Add 100μL of standard solutions and LPH samples into a 96-well plate, and measure the fluorescence intensity at 525 nm by using a plate reader.将 100μL 的标准溶液和 LPH 样品加入 96 孔板中，使用酶标仪在 525 nm 处测量荧光强度。

6. Calculate the amount of entrapped siRNA according to the standard calibration curve. 根据标准曲线计算包封的 siRNA 量。

3.3 Preparation of LCP LCP 的制备

1. Mix cyclohexane/Igepal CO-520 (71:29, V:V) and cyclohexane/Triton X-100/hexanol (75:15:10, V:V:V) (3:1) to make the oil phase.混合环己烷/Igepal CO-520（71:29，体积比）和环己烷/Triton X-100/己醇（75:15:10，体积比）(3:1) 制备油相。

2. Mix 300μL of 500 mM CaCl₂ with 100μL of 2 mg/mL siRNA, and disperse the mixture in 15 mL oil phase to form a water-in-oil reverse micro-emulsion (siRNA emulsion).将 300μL 的 500 mM CaCl₂与 100μL 的 2 mg/mL siRNA 混合，并分散于 15 mL 油相中以形成水包油反相微乳（siRNA 乳液）。

3. Disperse 300μL of 25 mM Na₂HPO₄ (pH = 9.0) in a separate 15 mL oil phase, then add 200μL of DOPA (20 mg/mL) in chloroform to the mixture to form the phosphate microemulsion.将 300μL 的 25 mM Na₂HPO₄（pH = 9.0）分散在单独的 15 mL 油相中，然后加入 200μL 的 DOPA（20 mg/mL，溶于氯仿）以形成磷酸盐微乳。

4. Mix the two emulsions and keep stirring the mixture for 30 min.混合两种微乳液，并持续搅拌 30 分钟。

5. After that, add 30 mL of absolute ethanol to break the micro-emulsion.加入 30 mL 无水乙醇破坏微乳。

6. Centrifuge the mixture at 12,000 × g for at least 15 min to precipitate the CaP-siRNA cores.以 12,000 × g离心至少 15 分钟，以沉淀 CaP-siRNA 核心。

7. Wash the precipitated pellets with ethanol for 2–3 times to remove cyclohexane and surfactants.用乙醇清洗沉淀颗粒 2–3 次，去除环己烷和表面活性剂。

8. After being extensively washed, re-disperse the pellets in 1 mL of chloroform and store in a glass vial for further modification, discard excess salts or aggregates.在充分清洗后，将沉淀颗粒重新分散于 1 mL 氯仿中，储存在玻璃瓶中以进行进一步修饰，弃去多余的盐或聚集体。

9. To create the outer leaflet lipid coating, mix 1 mL of CaP-siRNA cores, 100μL of 20 mM DOTAP/Cholesterol (molar ratio 1:1), and 50μL of 10 mM DSPE-PEG2000 in a glass vial.为了形成外层脂质涂层，将 1 mL CaP-siRNA 核心、100μL 的 20 mM DOTAP/胆固醇（摩尔比 1:1）和 50μL 的 10 mM DSPE-PEG2000 混合在玻璃瓶中。

10. Dry the mixture with N2 gas to remove the chloroform solvent.用氮气干燥混合物，去除氯仿溶剂。

11. Vacuum desiccate the dried film for at least 10 min.用真空干燥干燥的薄膜至少 10 分钟。

12. Add a small volume of ethanol and 1 mL water to the vial, then hydrate the film and suspended the particles under vortexing.向玻璃瓶中加入少量乙醇和 1 mL 水，然后水化薄膜，在涡旋器上悬浮颗粒。

13. Sonicate the sample in a bath-type sonicator.使用浴式超声仪对样品进行超声处理。

3.4 Characterization of LCP (Fig. 2) LCP 的表征

3.4.1 Particle Size and Zeta Potential 粒径和 Zeta 电位

1. Dilute a dispersion of LCP into an appropriate concentration with water.用水将 LCP 分散液稀释至适当浓度。

2. Use the DLS method to determine the particle size and zeta potential of LCP (see Note 7).使用动态光散射（DLS）法测定 LCP 的粒径和 Zeta 电位（参见 注释 7）。

Fig. 2 Illustration for the preparation of LCP. (Figure is adapted from with permission)

3.4.2 Transmission Electron Microscopy 透射电子显微镜（TEM）

1. Place a drop of 5μL dispersion of LCP onto the grid and allow to deposit for 5 min.将 5μL 的 LCP 分散液滴到网格上，静置 5 分钟。

2. Blot the excess liquid by touching with a piece of filter paper.使用滤纸轻轻触碰样品表面，去除多余液体。

3. Stain the sample with 2% uranyl acetate (5μL), followed by overnight air drying at room temperature.使用 2%的醋酸铀（5μL）对样品进行染色，然后在室温下空气干燥过夜。

4. Acquire TEM images (see Note 8).获取 TEM 图像（参见 注释 8）。

3.4.3 Trapping Efficiency of siRNA siRNA 的包封效率

1. Prepare the standard siRNA solutions by diluting the FAM-labeled siRNA in the lysis buffer.通过在裂解缓冲液中稀释 FAM 标记的 siRNA，制备标准 siRNA 溶液。

2. Prepare LCP with the FAM-labeled siRNA, and dissolve the NPs in the lysis buffer.用 FAM 标记的 siRNA 制备 LCP，并将纳米颗粒溶解在裂解缓冲液中。

3. Add 100μL of standard solutions and LCP solution into a 96-well plate, and measure the fluorescence intensity at 525 nm by using a plate reader.将 100μL 的标准溶液和 LCP 溶液加入 96 孔板中，使用酶标仪在 525 nm 处测量荧光强度。

4. Calculate the amount of entrapped siRNA according to the standard calibration curve.根据标准曲线计算包封的 siRNA 量。

3.5 In Vivo Luciferase Silencing Effect of LCP LCP 在体内的荧光素酶沉默效果

1. Inject luciferase labeled H-460 cells (2 × 10⁵) subcutaneously into the lower back of female nude mice, and allow the tumor to grow to a size of about 400 mm³. Randomly assign the mice to three treatment groups: normal saline, anti-luciferase siRNA, or the control siRNA (n = 5 for each group).将标记荧光素酶的 H-460 细胞（2 × 10⁵个细胞）皮下注射到雌性裸鼠的下背部，允许肿瘤生长至约 400 mm³。然后随机将小鼠分为三组：生理盐水组、抗荧光素酶 siRNA 组或对照 siRNA 组（每组n = 5）。

2. Inject normal saline or LCP into the mice via tail-vein, LCP contains anti-luciferase siRNA or the control siRNA.通过尾静脉注射生理盐水或 LCP 到小鼠体内，LCP 含有抗荧光素酶 siRNA 或对照 siRNA。

3. After 24 h, sacrifice the mice and harvest the tumors. Weigh the tumor and homogenize it in the lysis buffer (the volume of lysis buffer (mL) to tumor weight (g) ratio is 2). Incubate the homogenized tumor at 4 °C for 1 h.在 24 小时后，处死小鼠并收获肿瘤。称重肿瘤并将其在裂解缓冲液中匀浆（裂解缓冲液的体积（mL）与肿瘤重量（g）的比例为 2）。将匀浆的肿瘤在 4°C 下孵育 1 小时。

4. Mixed 20μL of lysate with 80μL of substrate in a 96-well plate and measure the luminescence intensity by using a plate reader.在 96 孔板中，将 20μL 裂解液与 80μL 底物混合，使用酶标仪测量发光强度。

5. Determine the total protein concentration in the lysate by using a Micro BCA™ Protein Assay Kit. The activity of luciferase is shown as the luminescence intensity per μg protein (see Notes 9, 10 and 11).使用 Micro BCA™蛋白质定量试剂盒测定裂解液中的蛋白质总浓度。荧光素酶活性以每微克蛋白质的发光强度表示（参见 注释 9、10 和 11）。

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4 Notes

1. Unless stated otherwise, all lipids are dissolved in chloroform to prepare the stock solutions at specific concentrations.除非另有说明，所有脂质均溶解在氯仿中，以特定浓度制备储备溶液。

2. Unless stated otherwise, Milli Q water should be used in water and all solutions.除非另有说明，水和所有溶液应使用 Milli Q 水。

3. Unless stated otherwise, all siRNAs are purchased in the deprotected, desalted, annealed form.除非另有说明，所有 siRNA 均以去保护、脱盐、退火的形式购买。

4. The LPH formulation is optimized by fine-tuning the ratio of siRNA, HA, and protamine with different amount of cationic liposomes. The particle size, zeta potential, encapsulation efficiency, and in vitro intracellular siRNA delivery are analyzed for optimization. Slightly excess amount of the cationic lipid is required to fully coat the LPH. Further increase of the lipid/siRNA ratio elevates the zeta potential and decreases the delivery efficiency. This might be due to the competitive binding with the cells from the excess cationic liposomes.LPH 的配方通过微调 siRNA、HA 和鱼精蛋白的比例，并加入不同量的阳离子脂质体进行优化。优化分析包括颗粒大小、电位、封装效率和体外细胞内 siRNA 递送。需要稍多的阳离子脂质才能完全包覆 LPH，进一步增加脂质/siRNA 的比例会提高电位，但降低递送效率，这可能是由于过多的阳离子脂质体与细胞竞争结合所致。

5. Postinsertion of DSPE-PEG into the lipid bilayers of LPH is to shield the positive charge and prevent aggregation with serum proteins. The in vivo activity can be further improved if a targeting ligand such as aminoethylanisamide is attached to the surface of the NPs.将 DSPE-PEG 后插入 LPH 的脂质双层中，以屏蔽正电荷并防止与血清蛋白的聚集。如果在 NPs 表面附加靶向配体如氨基乙基茴香酰胺，体内活性可以进一步提高。

6. The particle size, zeta potential, and trapping efficiency of LPH are approximately 120 nm, 25 mV, and 90%, respectively.LPH 的颗粒大小、电位和包封效率大约为 120 nm、25 mV 和 90%。

7. The particle size, zeta potential, and trapping efficiency of LCP are approximately 40 nm, 0–15 mV, and 40%, respectively.LCP 的颗粒大小、电位和包封效率大约为 40 nm、0–15 mV 和 40%。

8. Figure 3 shows the TEM image of LCP with the “core-shell” structure. The coating lipid membrane of the NP could be seen by negative staining (arrows in Fig. 3b). The average size of LCP measure by TEM was about 25–30 nm in diameter, which was slightly smaller than the hydrodynamic diameter (40 nm) obtained by dynamic light scattering since TEM images were obtained under a dehydrated condition. It has been suggested that the optimal particle size for a cancer-targeting nanoparticle carrier is 20–100 nm. Therefore LCP are large enough to avoid renal filtration and small enough to extravasate through the leaky vasculatures in the tumor.图 3 展示了具有“核壳”结构的 LCP 的 TEM 图像。通过负染色可以观察到 NP 的包膜（图 3b 中的箭头）。TEM 测量的 LCP 平均大小约为 25–30 nm，比动态光散射法测得的水动力直径（40 nm）略小，因为 TEM 图像是在脱水条件下获取的。研究表明，癌症靶向纳米载体的最佳颗粒大小为 20–100 nm。LCP 足够大以避免肾脏过滤，同时又足够小可以通过肿瘤中漏出的血管。

9. The asymmetric structure of the lipid layer is verified by the measurement of zeta potential and the quenching efficiency of a fluorescence lipid marker.通过电位测量和荧光脂质标记的猝灭效率，验证了脂质层的不对称结构。

10. Beside the commonly accepted proton sponge effect and ion-pair formation mechanisms, we proposed a CaP dissolution-driven mechanism for cargo release from the endosome. Hypoxia-induced endosome acidification is well known in the tumor cells. Protons will penetrate the lipid membrane of LCP and dissolve the CaP core. The release of calcium ions increased the osmotic pressure and caused endosome swelling and bursting to release the entrapped siRNA. This de-assembly process of LCP has been verified by monitoring the particle size at an acidic pH and fluorescent intensity of Fura-2-acetoxymethyl ester (a cellular calcium concentration indicator) in cells.除了常见的质子海绵效应和离子对形成机制外，我们提出了钙磷酸盐（CaP）溶解驱动的内涵体释放机制。肿瘤细胞中的缺氧诱导内涵体酸化。质子穿透 LCP 的脂质膜，溶解 CaP 核心，释放的钙离子增加渗透压，引发内涵体膨胀和破裂，释放包封的 siRNA。通过在酸性 pH 下监测颗粒大小和细胞内钙浓度指示剂 Fura-2-acetoxymethyl ester 的荧光强度，已验证 LCP 的解组装过程。

11. Figure 4 shows the in vivo silencing effect of luciferase siRNA delivered with LCP. The estimated ED₅₀ for LCP-mediated delivery of siRNA is approximately 0.6 mg/kg. Similar studies have been conducted using LPH.图 4 显示了 LCP 递送 siRNA 体内沉默荧光素酶的效果。LCP 介导的 siRNA 递送的 ED₅₀约为 0.6 mg/kg。类似研究已用 LPH 进行。

12. Different composition of outer leaflet lipids could be used to coat LCP. When either DOTAP or DOPC was mentioned as the outer leaflet lipid, DOTAP or DOPC was mixed with the same molar amount of cholesterol. The outer leaflet lipid plays an important role in determining the biodistribution of LCP.LCP 的外层脂质可以使用不同的成分进行包裹。当 DOTAP 或 DOPC 作为外层脂质时，它们与相同摩尔量的胆固醇混合。外层脂质在决定 LCP 的生物分布中起着重要作用。

13. We have demonstrated that LCP with a neutral (DOPC) or a cationic lipid (DOTAP) exhibited similar level of in vitro silencing effect. However, in vivo studies have indicated that the LCP containing a cationic lipid (DOTAP) delivered siRNA more efficiently than the one containing neutral lipid (DOPC). For the in vivo application, the amount of NP that is delivered to the same endosome might not be sufficient; therefore cationic lipid might still be important for endosome escape.我们已证明，含有中性脂质（DOPC）或阳离子脂质（DOTAP）的 LCP 在体外表现出相似的沉默效果。然而，体内研究表明，含有阳离子脂质（DOTAP）的 LCP 比含有中性脂质的 LCP 更有效地递送 siRNA。在体内应用中，递送到同一个内涵体的 NP 数量可能不足，因此阳离子脂质在内涵体逃逸中可能仍然重要。

14. Like LPH, the in vivo activity of LCP could be further improved by using a targeting ligand such as aminoethylanisamide which was tethered to the distal end of PEG. LCP-PEG-AA had a significantly improved silencing effect compared to untargeted LCP-PEG.类似于 LPH，LCP 的体内活性可以通过在 PEG 末端附加靶向配体（如氨基乙基茴香酰胺）进一步改善。LCP-PEG-AA 的沉默效果显著优于未靶向的 LCP-PEG。

15. Compared to the conventional cationic liposomes, the LPH and LCP have a lipid layer supported by the complex core, which could accommodate a higher density of PEGylation, resulting in a better shielding of charge and reduced opsonization by serum proteins. This gives both LPH and LCP a relatively low degree of uptake by the liver and spleen, and a high level of accumulation in the tumor.与传统阳离子脂质体相比，LPH 和 LCP 具有由复杂核心支撑的脂质层，可以容纳更高密度的 PEG 化，从而更好地屏蔽电荷并减少血清蛋白的调理作用。这使得 LPH 和 LCP 具有相对较低的肝脏和脾脏摄取率，同时在肿瘤中积累较多。

16. In vitro study suggested that LCP might outperform LPH in the gene silencing effect. However, since LCP require multiple steps of preparation, while LPH are formulated in a self-assembly manner in a few steps, LPH are now more frequently used in our lab for in vivo siRNA delivery.体外研究表明，LCP 在基因沉默效果上可能优于 LPH。然而，由于 LCP 需要多步制备，而 LPH 是通过自组装方式在几步内制备完成的，因此我们实验室现在更常使用 LPH 进行体内 siRNA 递送。

Fig. 3 Characterization of LCP using TEM. TEM images of LCP coated with DOTAP and DSPE–PEG without (a) and with (b) negative staining. Arrows in (b) show lipid bilayer surrounding the CaP core. (Data are taken from [12 ( clbr://internal.invalid/OEBPS/html/485053_1_En_10_Chapter.xhtml#CR12 )] with permission)

Fig. 4 In vivo silencing effect of luciferase siRNA delivered with LCP. Luc luciferase siRNA, Con control siRNA. Numbers in X-axis indicate the injected dose of siRNA in mg/kg. *Indicates p* < 0.05. (Data are taken from with permission)