TLD：小囊泡尺寸优化型脂质体阿霉素制剂及其抗肿瘤疗效提升

TLD 是一种处于临床阶段的脂质体阿霉素制剂，具有较低的药脂比和小粒径尺寸，冷冻透射电镜（cryo-TEM）测定为仅 35.6 nm，动态光散射（DLS）测定为 65.2 nm。
在体外，针对人乳腺癌细胞系 MCF7，TLD 的 IC50 值低于 Caelyx（分别为 13.1 µg/mL 和 21.3 µg/mL）。
在体内，无论是在小鼠乳腺癌模型 4T1 还是纤维肉瘤模型 WEHI-164 中，TLD 的疗效均与 Caelyx 相当。

Abstract
Talidox (TLD) is an innovative liposomal doxorubicin formulation designed to enhance drug delivery efficiency and reduce systemic toxicity over existing liposomal doxorubicin formulations. This publication consolidates 10 years of preclinical research on TLD, integrating physicochemical characterization, and in vitro and in vivo efficacy. TLD has a reduced particle size and optimized drug-to-lipid ratio compared to Caelyx, aiming to improve tumor penetration and uptake and therefore therapeutic efficacy. The preclinical studies in breast cancer and soft tissue sarcoma models highlight the improved efficacy of TLD over free doxorubicin combined with a very good safety profile. This is complemented by the already published favorable risk-benefit ratio of TLD found in clinical trials.

摘要
Talidox (TLD) 是一种创新的脂质体阿霉素制剂，旨在相较于现有脂质体阿霉素制剂提高药物递送效率并降低全身毒性。本综述汇集了 TLD 十年的临床前研究成果，整合了其物理化学表征以及体外和体内疗效数据。与 Caelyx 相比，TLD 具有更小的粒径和优化的药脂比，旨在改善肿瘤渗透和摄取，从而提高治疗效果。在乳腺癌和软组织肉瘤模型中的临床前研究凸显了 TLD 相较于游离阿霉素疗效的提升，同时兼具良好的安全性。这与已发表的临床试验中发现的 TLD 良好的风险-获益比相得益彰。

Keywords：Liposomal doxorubicin，Nanomedicine，Tumor uptake，Pharmacokinetics，Biodistribution，Breast cancer，Soft tissue sarcoma，Talidox

关键词： 脂质体阿霉素，纳米医学，肿瘤摄取，药代动力学，生物分布，乳腺癌，软组织肉瘤，Talidox

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2.2. Preparation of TLD
For TLD production, DSPC and cholesterol were first dissolved together in a volume of absolute ethanol corresponding to 4 % v/v of the final bulk volume of liposomes.The resulting lipid mixture was then rapidly added to a 150 mM ammonium sulfate solution. The lipid mixture was sonicated with the ultrasonic liquid processor ISP-3000 (Industrial Sonomechanics, Miami, FL, USA) in continuous mode with an internal temperature of up to 70 °C until the liposomes achieved a target size (mean hydrodynamic diameter) in the range of 50 nm to 55 nm. This process was typically performed at a scale > 3 L. Notably, the sonication process facilitated the evaporation of a significant portion of ethanol. Subsequently, the liposomes underwent pegylation with DSPE-mPEG2000 at 65 °C for 30 minutes. Finally, to ensure the removal of the ammonium sulfate solution and any remaining ethanol, the extraliposomal buffer was exchanged with 10 mM HEPES buffer, pH 6.8 – 7.2, using tangential flow filtration (TFF). Subsequently, doxorubicin was remote loaded at 8.3 mM for 30 min at 65 °C and any residual free doxorubicin was eliminated through an additional TFF process against HEPES buffer. The doxorubicin content was determined using reverse-phase liquid chromatography. Briefly, a Titan C18 column (100×2.1 mm; 1.9 µm particle size) was used. Chromatography was isocratic using 50:50 acetonitrile: aqueous solution with 2.88 g/L SDS and 2.25 g/L orthophosphoric acid (85 %) at a flow rate of 0.4 mL/min. The column thermostat was set to 35 °C and injection volume was 2.5 µl and UV detection at λ = 254 nm. The sample preparation was a two-step dilution in methanol to a final dilution of 1:50. Internally, the method has been validated according to ICH Q2(R2).

2.2. TLD 的制备
TLD 的生产过程如下：首先，将 DSPC 和胆固醇共同溶解于一定体积的无水乙醇中，该乙醇体积相当于最终脂质体总体积的 4% (v/v)。随后，将所得脂质混合物快速加入到 150 mM 硫酸铵溶液中。使用 ISP-3000 型超声波液体处理器（Industrial Sonomechanics, Miami, FL, USA）对脂质混合物进行连续模式超声处理，内部温度最高达 70°C，直至脂质体达到目标尺寸（平均流体动力学直径），范围在 50 nm 至 55 nm 之间。此过程通常在规模大于 3 升的条件下进行。值得注意的是，超声处理促进了大部分乙醇的蒸发。接着，在 65°C 下使用 DSPE-mPEG2000 对脂质体进行聚乙二醇化修饰，持续 30 分钟。最后，为了确保去除硫酸铵溶液和任何残留的乙醇，使用切向流过滤 (TFF) 技术，将脂质体外缓冲液置换为 pH 值在 6.8 – 7.2 之间的 10 mM HEPES 缓冲液。随后，在 65°C 下以 8.3 mM 的浓度对阿霉素进行远程装载，持续 30 分钟，任何残留的游离阿霉素均通过额外的针对 HEPES 缓冲液的 TFF 过程去除。采用反相液相色谱法测定阿霉素含量。简要步骤如下：使用 Titan C18 色谱柱（100×2.1 mm；粒径 1.9 µm）。色谱分析采用等度洗脱，流动相为乙腈：水溶液（含 2.88 g/L SDS 和 2.25 g/L 85% 正磷酸）= 50:50，流速为 0.4 mL/min。柱温箱温度设定为 35°C，进样体积为 2.5 µl，紫外检测波长为 λ = 254 nm。样品制备采用两步稀释法，使用甲醇稀释至最终稀释比例为 1:50。该方法内部已根据 ICH Q2(R2) 指南完成验证。