Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO₄ as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSL released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2⁺, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8⁺ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (no-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 μg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Resiquimod (R848) 是一种 Toll 样受体 7 和 8 (TLR7/8) 激动剂，具有有效的抗肿瘤和免疫刺激活性。然而，由于 R848 在水中的溶解度差和全身免疫激活，因此 R848 的全身递送耐受性较差。为了解决这些限制，我们使用热敏脂质体 (TSL) 作为递送载体，开发了一种含有 R848 的静脉注射制剂。将R848远程加载到由DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%)组成的TSL中，内部含有100 mM FeSO ₄作为捕获剂。优化后的 R848 负载 TSL（R848-TSL）的最终 R848 与脂质比率为 0.09（ w /w），比之前报道的值高 10 倍。 R848-TSL 在 42 °C 下 5 分钟内释放了 80% 的 R848。然后将这些 TSL 与 αPD-1（一种免疫检查点抑制剂）和超声介导的热疗联合用于neu缺失 (NDL) 小鼠乳腺癌模型（Her2 ⁺ ，ER/PR 阴性）。与 αPD-1 结合，局部注射 R848-TSL 显示出卓越的疗效，在 11 只荷瘤小鼠中，有 8 只在 100 天内在治疗部位和远隔部位实现了 NDL 肿瘤完全消退。免疫组织化学证实 R848-TSL 增强了 CD8 ⁺ T 细胞浸润和积累。 R848-TSL 的全身递送，结合局部热疗和 αPD-1，抑制了肿瘤生长，并将中位生存期从 28 天（无治疗对照）延长至 94 天。在重新注射肿瘤细胞后，先前治愈的小鼠没有一只出现肿瘤，而年龄匹配的对照小鼠则为 100%。 R848的剂量（瘤内注射10μg或静脉注射6mg/kg，最多4次）耐受良好，没有体重减轻或器官肥大。总之，我们开发了可局部或系统给药的 R848-TSL，与 αPD-1 联合使用可在乳腺癌小鼠模型中实现肿瘤消退并提高生存率。