一、Materials and Methods 材料與方法

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.

中文釋義：采用芬蘭Kibron MicroTrough XL型LB膜分析儀開展磷脂Langmuir單分子膜實驗，使用Wilhelmy法鉑銥材質DyneProbe探針測定界面膜的表面壓力。整套朗繆爾槽體加裝防風防護罩以消除氣流干擾，一體式溫控裝置將實驗溫度恒定控制在22.0 ± 0.5 ℃。完整的表面壓力對應單分子平均占有面積等溫線數據由配套DeltaGraph軟件記錄并導出。所有平行測試統一將滑障壓縮速率設定為每分鐘5平方厘米，最大程度降低壓縮過程中單分子膜松弛帶來的檢測誤差。

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.

中文釋義：DPPC、POPC、POPG、膽固醇等磷脂原料溶解于體積比9:1的氯仿-甲醇混合溶劑，配制成濃度1 mM的磷脂儲備液。待測藥物溶解于20 mM Tris、150 mM NaCl、pH 7.4緩沖液作為水相亞相；緩沖液使用前經0.22 μm濾膜過濾，并超聲脫氣30分鐘去除液相內溶解氣體。若難溶性藥物需要二甲基亞砜助溶，亞相中有機溶劑終體積分數控制在1%以內，同時設置溶劑空白對照組扣除溶劑本身界面活性干擾。

使用哈密頓微量進樣針將磷脂有機溶液逐點滴加在氣液界面鋪展成膜，鋪展后靜置15至20分鐘保證有機溶劑完全揮發，再啟動滑障壓縮程序。藥物干預實驗組通過槽體側孔緩慢將藥物注入亞相底層，僅對下層水相低速輕柔攪拌，避免破壞完整的界面磷脂單層膜。注藥后體系孵育30分鐘達到吸附平衡，再測定表面壓力-分子面積等溫線。

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 evaluate the ability of drugs to insert into phospholipid membranes.

中文釋義：每組表面壓力-分子面積等溫線測定至少開展三次獨立平行實驗，保障數據可重復性。從原始等溫曲線中提取多項定量特征參數：零表面壓力下極限分子平均占有面積、磷脂單層膜崩潰壓力、界面膜壓縮彈性模量，以及加藥后等溫曲線整體偏移幅度。額外補充恒面積模式下最大插入壓力、表面壓力升高值作為輔助定量指標，量化評價藥物嵌入磷脂膜的能力。

二、Results 結果部分 圖表描述標準句式

1. 圖題與圖注

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. 等溫曲線偏移核心描述句子

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.

中文釋義：與純磷脂空白組等溫曲線對比，孵育藥物后的磷脂單層膜等溫線在同等表面壓力條件下，沿分子面積坐標軸明顯向左偏移。該左移現象直接證明藥物分子插入磷脂單層膜的疏水脂肪酸鏈區域，增大了界面上單個磷脂分子的平均占有面積。

3. 膜崩潰壓力變化描述句式

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.

中文釋義：加入待測化合物后，磷脂界面單層膜的崩潰壓力出現明顯下降，該現象證明插入的藥物分子削弱了磷脂分子間的堆積作用力，降低了界面單分子膜的力學結構穩定性。反之，崩潰壓力無顯著變化僅代表藥物發生微弱的膜表面靜電吸附，并未深度嵌入磷脂雙分子層內部結構。

4. 壓縮模量結果表述句式

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.

中文釋義：根據等溫曲線斜率計算得出的壓縮彈性模量在加藥組數值更低，該結果說明藥物分子插入提升了磷脂單層膜的流動性、降低膜結構剛性，直觀體現受試化合物對仿生磷脂膜的擾動與增流作用。

三、Discussion 討論部分機理闡釋

1. 藥物與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. 磷脂電荷選擇性差異討論模板

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. 方法學價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、投稿統一規范

1. 儀器全稱首次出現：Kibron Langmuir-Blodgett (LB) film balance，后文簡寫為Kibron LB trough；

2. 曲線統一名稱：surface pressure-average molecular area Langmuir isotherms；

3. 物理量統一文字表述：surface pressure（表面壓力）、average molecular area（平均分子占有面積）、limiting molecular area（極限分子面積）、collapse pressure（崩潰壓力）、compression elastic modulus（壓縮彈性模量）、maximum insertion pressure（最大插入壓力）；

4. 統計學固定寫法：Data are expressed as mean ± standard deviation of three independent parallel measurements；

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