一、Materials and Methods 材料与方法

1. Instrumentation

The Langmuir phospholipid monolayer experiments were carried out on a Kibron MicroTrough XL Langmuir-Blodgett film balance (Kibron Company, Helsinki, Finland). A Wilhelmy-type DyneProbe platinum-iridium probe was adopted to measure the surface pressure of the interfacial film. The whole Langmuir trough was covered with a windproof shield to avoid airflow disturbance, and the built-in thermostat kept the experimental temperature stable at 22.0 ± 0.5 °C. The full set of surface pressure versus average molecular area isotherm data was recorded and exported by the original DeltaGraph supporting software. The barrier compression speed was fixed at 5 cm² per minute for all parallel tests, which could effectively reduce the measurement error caused by monolayer relaxation during compression.

中文释义：采用芬兰Kibron MicroTrough XL型91免费看污视频开展磷脂Langmuir单分子膜实验，使用Wilhelmy法铂铱材质DyneProbe探针测定界面膜的表面压力。整套朗缪尔槽体加装防风防护罩以消除气流干扰，一体式温控装置将实验温度恒定控制在22.0 ± 0.5 ℃。完整的表面压力对应单分子平均占有面积等温线数据由配套DeltaGraph软件记录并导出。所有平行测试统一将滑障压缩速率设定为每分钟5平方厘米，最大程度降低压缩过程中单分子膜松弛带来的检测误差。

2. Lipids, test compounds and aqueous subphase preparation

Phospholipid raw materials including DPPC, POPC, POPG and cholesterol were dissolved in the mixed solvent of chloroform and methanol with a volume ratio of 9:1, and the final concentration of lipid stock solution was prepared to 1 mM. The tested drug substance was dissolved in Tris-HCl buffer solution (20 mM Tris, 150 mM NaCl, pH 7.4) as the aqueous subphase. Before use, the buffer was filtered with a 0.22 μm filter membrane and treated with ultrasonic degassing for 30 minutes to remove dissolved gas inside the liquid. If dimethyl sulfoxide was needed to dissolve poorly soluble drugs, the final volume proportion of dimethyl sulfoxide in the subphase was controlled below 1%, and a solvent blank control group was set up to deduct the interference of solvent surface activity.

The lipid monolayer was formed by dropwise spreading the lipid organic solution on the gas-liquid interface with a Hamilton micro-syringe. After spreading, the system stood for 15 to 20 minutes to completely volatilize the organic solvent before starting barrier compression. For the experimental groups with drug intervention, the target compound was slowly injected into the bottom of the subphase through the side injection hole of the trough, with low-speed gentle stirring only acting on the lower water phase to prevent damage to the intact interfacial phospholipid monolayer. After drug injection, the system was incubated for 30 minutes to reach adsorption equilibrium, and then the surface pressure-molecular area isotherm was measured.

中文释义：DPPC、POPC、POPG、胆固醇等磷脂原料溶解于体积比9:1的氯仿-甲醇混合溶剂，配制成浓度1 mM的磷脂储备液。待测药物溶解于20 mM Tris、150 mM NaCl、pH 7.4缓冲液作为水相亚相；缓冲液使用前经0.22 μm滤膜过滤，并超声脱气30分钟去除液相内溶解气体。若难溶性药物需要二甲基亚砜助溶，亚相中有机溶剂终体积分数控制在1%以内，同时设置溶剂空白对照组扣除溶剂本身界面活性干扰。

使用哈密顿微量进样针将磷脂有机溶液逐点滴加在气液界面铺展成膜，铺展后静置15至20分钟保证有机溶剂完全挥发，再启动滑障压缩程序。药物干预实验组通过槽体侧孔缓慢将药物注入亚相底层，仅对下层水相低速轻柔搅拌，避免破坏完整的界面磷脂单层膜。注药后体系孵育30分钟达到吸附平衡，再测定表面压力-分子面积等温线。

3. Parallel setting and key parameter calculation

Each group of surface pressure-molecular area isotherm measurement was repeated at least three independent parallel experiments to ensure data repeatability. Multiple quantitative characteristic parameters were extracted from the original isotherm curves: the limiting average molecular occupied area under zero surface pressure, collapse pressure of phospholipid monolayer film, compression elastic modulus of the interfacial film, and the overall offset degree of the isotherm curve after drug treatment. Auxiliary quantitative indicators such as maximum insertion pressure and surface pressure increment under constant area mode were further supplemented to quantitatively evalsuate the ability of drugs to insert into phospholipid membranes.

中文释义：每组表面压力-分子面积等温线测定至少开展三次独立平行实验，保障数据可重复性。从原始等温曲线中提取多项定量特征参数：零表面压力下极限分子平均占有面积、磷脂单层膜崩溃压力、界面膜压缩弹性模量，以及加药后等温曲线整体偏移幅度。额外补充恒面积模式下最大插入压力、表面压力升高值作为辅助定量指标，量化评价药物嵌入磷脂膜的能力。

二、Results 结果部分 图表描述标准句式

1. 图题与图注

Figure X. Surface pressure versus average molecular area Langmuir isotherms of pure phospholipid monolayers and phospholipid monolayers incubated with different concentrations of tested drug on the buffer-air interface, measured by Kibron LB film balance. The black curve represents blank phospholipid monolayer control, and colored curves correspond to lipid monolayers after injecting target drug into the subphase. All measurements were carried out at 22 °C with a barrier compression rate of 5 cm² per minute. Each curve is the average result of three parallel replicates.

2. 等温曲线偏移核心描述句子

Compared with the isotherm curve of pure phospholipid blank group, the isotherm of the phospholipid monolayer incubated with drug shifted significantly to the left along the molecular area axis under the same surface pressure condition. This leftward offset directly indicated that drug molecules inserted into the hydrophobic fatty acid chain region of the phospholipid monolayer, which increased the average occupied area of each single phospholipid molecule on the interface.

中文释义：与纯磷脂空白组等温曲线对比，孵育药物后的磷脂单层膜等温线在同等表面压力条件下，沿分子面积坐标轴明显向左偏移。该左移现象直接证明药物分子插入磷脂单层膜的疏水脂肪酸链区域，增大了界面上单个磷脂分子的平均占有面积。

3. 膜崩溃压力变化描述句式

After adding the tested compound, the collapse pressure of the phospholipid interfacial monolayer decreased obviously. This phenomenon proved that inserted drug molecules weakened the intermolecular stacking force between phospholipid molecules and reduced the mechanical structural stability of the interfacial single-layer film. On the contrary, nearly unchanged collapse pressure only reflected weak electrostatic adsorption on the membrane surface, rather than deep embedding into the interior of phospholipid bilayer structure.

中文释义：加入待测化合物后，磷脂界面单层膜的崩溃压力出现明显下降，该现象证明插入的药物分子削弱了磷脂分子间的堆积作用力，降低了界面单分子膜的力学结构稳定性。反之，崩溃压力无显著变化仅代表药物发生微弱的膜表面静电吸附，并未深度嵌入磷脂双分子层内部结构。

4. 压缩模量结果表述句式

The compression elastic modulus calculated according to the slope of the isotherm curve was lower in the drug-treated group. This result illustrated that the insertion of drug molecules increased the fluidity of the phospholipid monolayer and reduced the rigidity of the membrane structure, which intuitively reflected the disturbing and fluidizing effect of the test compound on biomimetic phospholipid membrane.

中文释义：根据等温曲线斜率计算得出的压缩弹性模量在加药组数值更低，该结果说明药物分子插入提升了磷脂单层膜的流动性、降低膜结构刚性，直观体现受试化合物对仿生磷脂膜的扰动与增流作用。

三、Discussion 讨论部分机理阐释

1. 药物与磷脂作用模式判定完整段落

The left offset of surface pressure-molecular area isotherms, the reduction of monolayer collapse pressure and the decline of compression elastic modulus comprehensively confirmed that the tested drug had strong insertion capacity into phospholipid monolayers, instead of only superficial weak adsorption on the membrane surface. Combined with the maximum insertion pressure data obtained from constant pressure control experiments, we could further confirm that the compound could penetrate into densely arranged lipid domains under the physiological lateral pressure of cell membrane (about 25 mN/m). This research provided direct biophysical evidence at the molecular level for the membrane-targeted action mechanism of the drug.

2. 磷脂电荷选择性差异讨论模板

Obvious differences in the offset amplitude of isotherm curves were observed between negatively charged POPG anionic phospholipid monolayer and neutral POPC phospholipid monolayer. The result revealed that the interaction between the drug and phospholipid was initially driven by electrostatic attraction, followed by hydrophobic insertion into the fatty acid tail region of lipid molecules. Such selective binding characteristic partly explained the differential cytotoxic effect of the drug on negatively charged bacterial cell membrane and neutral mammalian cell membrane.

3. 方法学价值拔高投稿常用句式

The biomimetic phospholipid monolayer characterization method based on Kibron LB film balance is a reliable in vitro two-dimensional cell membrane simulation model. Compared with liposome particle size detection and content leakage experiment, surface pressure-molecular area isotherm can quantitatively visualize the molecular-scale embedding behavior and membrane disturbance degree of drugs, which supplements and perfects the research system of drug-phospholipid membrane interaction mechanism.

四、投稿统一规范

1. 仪器全称首次出现：Kibron Langmuir-Blodgett (LB) film balance，后文简写为Kibron LB trough；

2. 曲线统一名称：surface pressure-average molecular area Langmuir isotherms；

3. 物理量统一文字表述：surface pressure（表面压力）、average molecular area（平均分子占有面积）、limiting molecular area（极限分子面积）、collapse pressure（崩溃压力）、compression elastic modulus（压缩弹性模量）、maximum insertion pressure（最大插入压力）；

4. 统计学固定写法：Data are expressed as mean ± standard deviation of three independent parallel measurements；

5. 重复性描述：Three repeated tests presented high overlapping degree of isotherm curves, which verified good experimental repeatability.