Erythrocyte leveraged chemotherapy (ELeCt): Nanoparticle assembly on erythrocyte surface to combat lung metastasis
红细胞助力化疗（ELeCt）：红细胞表面纳米颗粒组装用于对抗肺转移
Authors Info & Affiliations
赵宗敏 HTTPS://ORCID.ORG/0000-0001-8979-844X, 安维艾·乌基德夫 HTTPS://ORCID.ORG/0000-0002-6757-7942, 高永生 HTTPS://ORCID.ORG/0000-0002-2347-1855, 金在英 HTTPS://ORCID.ORG/0000-0002-4883-2768, 及 萨米尔·米特拉戈特里 HTTPS://ORCID.ORG/0000-0002-2459-8305 作者信息与所属机构
赵宗敏 HTTPS://ORCID.ORG/0000-0001-8979-844X, 安维艾·乌基德夫 HTTPS://ORCID.ORG/0000-0002-6757-7942, 高永生 HTTPS://ORCID.ORG/0000-0002-2347-1855, 金在英 HTTPS://ORCID.ORG/0000-0002-4883-2768, 及 萨米尔·米特拉戈特里 HTTPS://ORCID.ORG/0000-0002-2459-8305 作者信息与所属机构
Science Advances 科学进展
13 Nov 2019 2019 年 11 月 13 日
Vol 5, Issue 11 第 5 卷，第 11 期
Abstract 摘要
Despite being the mainstay of cancer treatment, chemotherapy has shown limited efficacy for the treatment of lung metastasis due to ineffective targeting and poor tumor accumulation. Here, we report a highly effective erythrocyte leveraged chemotherapy (ELeCt) platform, consisting of biodegradable drug nanoparticles assembled onto the surface of erythrocytes, to enable chemotherapy for lung metastasis treatment. The ELeCt platform significantly extended the circulation time of the drug nanoparticles and delivered 10-fold higher drug content to the lung compared with the free nanoparticles. In both the early- and late-stage melanoma lung metastasis models, the ELeCt platform enabled substantial inhibition of tumor growth that resulted in significant improvement of survival. Further, the ELeCt platform can be used to deliver numerous approved chemotherapeutic drugs. Together, the findings suggest that the ELeCt platform offers a versatile strategy to enable chemotherapy for effective lung metastasis treatment.
尽管化疗是癌症治疗的主流方法，但由于靶向性差和肿瘤内积累不足，其在治疗肺转移方面效果有限。本文报道了一种高效的红细胞辅助化疗（Erythrocyte Leveraged Chemotherapy, ELeCt）平台，该平台由可生物降解的药物纳米颗粒组装在红细胞表面构成，旨在实现针对肺转移的化疗。ELeCt 平台显著延长了药物纳米颗粒的循环时间，并使药物在肺部的含量比自由纳米颗粒提高了十倍。在早、晚期黑色素瘤肺转移模型中，ELeCt 平台均能大幅抑制肿瘤生长，显著改善生存率。此外，ELeCt 平台还可用于递送多种已获批准的化疗药物。综上所述，研究结果表明，ELeCt 平台为实现有效治疗肺转移提供了多功能的化疗策略。
尽管化疗是癌症治疗的主流方法，但由于靶向性差和肿瘤内积累不足，其在治疗肺转移方面效果有限。本文报道了一种高效的红细胞辅助化疗（Erythrocyte Leveraged Chemotherapy, ELeCt）平台，该平台由可生物降解的药物纳米颗粒组装在红细胞表面构成，旨在实现针对肺转移的化疗。ELeCt 平台显著延长了药物纳米颗粒的循环时间，并使药物在肺部的含量比自由纳米颗粒提高了十倍。在早、晚期黑色素瘤肺转移模型中，ELeCt 平台均能大幅抑制肿瘤生长，显著改善生存率。此外，ELeCt 平台还可用于递送多种已获批准的化疗药物。综上所述，研究结果表明，ELeCt 平台为实现有效治疗肺转移提供了多功能的化疗策略。
INTRODUCTION 引言
Cancer has been one of the leading causes of mortality over the last few decades (1). While early detection of tumor cells in specific tissues or the blood has improved the survival of patients with cancer, current standard-of-care interventions, including surgery, radiation therapy, or chemotherapy, have limited efficacy if cancer is not detected early (1–4). Early detection, however, is not often feasible, and in most patients, tumors have metastasized to secondary locations by the time of diagnosis (2, 4).
癌症在过去几十年中一直是导致死亡的主要原因之一（1）。尽管在特定组织或血液中早期检测肿瘤细胞已提高了癌症患者的生存率，但目前包括手术、放疗或化疗在内的标准治疗手段，若癌症未能早期发现，其疗效有限（1-4）。然而，早期检测并不总是可行，多数患者在确诊时肿瘤已发生转移（2, 4）。
癌症在过去几十年中一直是导致死亡的主要原因之一（1）。尽管在特定组织或血液中早期检测肿瘤细胞已提高了癌症患者的生存率，但目前包括手术、放疗或化疗在内的标准治疗手段，若癌症未能早期发现，其疗效有限（1-4）。然而，早期检测并不总是可行，多数患者在确诊时肿瘤已发生转移（2, 4）。
According to the National Cancer Institute, the most common site of metastasis for a variety of primary cancers is the lung, owing to its high vascular density. Lung metastasis is highly fatal if not treated, and currently, there is no specific treatment for it (5, 6). Systemic chemotherapy is one of the standard treatment options for lung metastasis (7, 8). However, its efficacy has been far from desirable due to ineffective targeting and poor accumulation in the lungs. Nanotechnology has played a pivotal role in enhancing the treatment of advanced metastatic cancers (9–11) and therefore can be applied in the case of lung metastasis as well. However, traditional nanoparticle (NP) delivery often fails to accumulate at the desired site of action due to the existence of biological barriers that impede the intravascularly injected NPs (12–17). Active targeting using tissue-specific ligands has often been explored as a strategy to improve tissue accumulation but has only resulted in modest improvement of therapeutic efficacy and decreased translational capability due to increased cost of production (18–26).
根据美国国家癌症研究所的数据，多种原发性癌症最常见的转移部位是肺部，这归因于其高血管密度。若不加以治疗，肺转移具有高度致命性，而目前尚无针对肺转移的特定治疗方法（5, 6）。全身化疗是肺转移的标准治疗方案之一（7, 8），但由于靶向效果不佳及在肺部积聚不良，其疗效远未达到理想状态。纳米技术在提升晚期转移性癌症治疗方面发挥了关键作用（9–11），因此同样可应用于肺转移的治疗。然而，传统的纳米颗粒（NP）递送往往因生物屏障的存在而无法在目标作用部位有效积聚，这些屏障阻碍了静脉注射的 NPs（12–17）。主动靶向利用组织特异性配体作为策略，已被广泛探索以提高组织积聚，但仅带来了治疗效果的适度提升，且由于生产成本增加，导致其转化能力下降（18–26）。
根据美国国家癌症研究所的数据，多种原发性癌症最常见的转移部位是肺部，这归因于其高血管密度。若不加以治疗，肺转移具有高度致命性，而目前尚无针对肺转移的特定治疗方法（5, 6）。全身化疗是肺转移的标准治疗方案之一（7, 8），但由于靶向效果不佳及在肺部积聚不良，其疗效远未达到理想状态。纳米技术在提升晚期转移性癌症治疗方面发挥了关键作用（9–11），因此同样可应用于肺转移的治疗。然而，传统的纳米颗粒（NP）递送往往因生物屏障的存在而无法在目标作用部位有效积聚，这些屏障阻碍了静脉注射的 NPs（12–17）。主动靶向利用组织特异性配体作为策略，已被广泛探索以提高组织积聚，但仅带来了治疗效果的适度提升，且由于生产成本增加，导致其转化能力下降（18–26）。
To achieve efficient drug delivery to enable chemotherapy for effective lung metastasis treatment, we used the unique physiology of the target site and developed a two-pronged strategy [erythrocyte leveraged chemotherapy (ELeCt)]—biodegradable drug NPs assembled on the surface of erythrocyte (Fig. 1AOpens in image viewer
为了实现高效的药物递送，从而为有效治疗肺转移提供化疗支持，我们利用了目标部位的独特生理特性，并开发了一种双管齐下的策略——红细胞增强化疗（ELeCt），即在红细胞表面组装可生物降解的药物纳米颗粒（图 1A）。). Erythrocytes act as a primary drug delivery system, capable of responsively dislodging the particles in the lung endothelium and tumor nodules in response to the high shear stress experienced by erythrocytes in narrow lung capillaries (27, 28). The biodegradable NPs themselves are capable of encapsulating large amounts of chemotherapeutics and having a characteristic controlled-release mechanism (29, 30). They act as a secondary drug delivery system enabling sustained delivery of the cargo. In this study, superior accumulation and therapeutic efficacy of this lung physiology-assisted NP strategy were demonstrated using a model chemotherapeutic doxorubicin (DOX). This concept was successfully used to combat lung metastasis and improve survival in early- and late-stage melanoma lung metastasis models. The ability to incorporate a plethora of current clinical chemotherapy drugs and drug combinations in the biodegradable NPs and subsequently assemble onto the erythrocytes was demonstrated. The particles also readily assembled to human erythrocytes and dislodged in a shear-dependent manner. Together, ELeCt offers a versatile, potent, and translatable platform to combat lung metastasis.
红细胞作为主要的药物递送系统，能够在狭窄的肺毛细血管中因高剪切应力作用下，响应性地从肺内皮和肿瘤结节中释放纳米颗粒（27, 28）。可生物降解的纳米颗粒本身能够封装大量化疗药物，并具有特征性的控释机制（29, 30）。它们作为次级药物递送系统，实现药物的持续递送。本研究通过模型化疗药物阿霉素（DOX）展示了这一肺生理辅助纳米颗粒策略在药物积累和治疗效果上的优越性。该概念成功应用于对抗肺转移，并提高了早期和晚期黑色素瘤肺转移模型中的生存率。研究表明，可生物降解的纳米颗粒能够整合多种当前临床化疗药物及药物组合，并随后装配到红细胞上。这些颗粒也能轻易地装配到人红细胞上，并在剪切力依赖的方式下释放。 ELeCt 联合应用，提供了一个多用途、高效且可转化的平台，用于对抗肺转移。
为了实现高效的药物递送，从而为有效治疗肺转移提供化疗支持，我们利用了目标部位的独特生理特性，并开发了一种双管齐下的策略——红细胞增强化疗（ELeCt），即在红细胞表面组装可生物降解的药物纳米颗粒（图 1A）。). Erythrocytes act as a primary drug delivery system, capable of responsively dislodging the particles in the lung endothelium and tumor nodules in response to the high shear stress experienced by erythrocytes in narrow lung capillaries (27, 28). The biodegradable NPs themselves are capable of encapsulating large amounts of chemotherapeutics and having a characteristic controlled-release mechanism (29, 30). They act as a secondary drug delivery system enabling sustained delivery of the cargo. In this study, superior accumulation and therapeutic efficacy of this lung physiology-assisted NP strategy were demonstrated using a model chemotherapeutic doxorubicin (DOX). This concept was successfully used to combat lung metastasis and improve survival in early- and late-stage melanoma lung metastasis models. The ability to incorporate a plethora of current clinical chemotherapy drugs and drug combinations in the biodegradable NPs and subsequently assemble onto the erythrocytes was demonstrated. The particles also readily assembled to human erythrocytes and dislodged in a shear-dependent manner. Together, ELeCt offers a versatile, potent, and translatable platform to combat lung metastasis.
红细胞作为主要的药物递送系统，能够在狭窄的肺毛细血管中因高剪切应力作用下，响应性地从肺内皮和肿瘤结节中释放纳米颗粒（27, 28）。可生物降解的纳米颗粒本身能够封装大量化疗药物，并具有特征性的控释机制（29, 30）。它们作为次级药物递送系统，实现药物的持续递送。本研究通过模型化疗药物阿霉素（DOX）展示了这一肺生理辅助纳米颗粒策略在药物积累和治疗效果上的优越性。该概念成功应用于对抗肺转移，并提高了早期和晚期黑色素瘤肺转移模型中的生存率。研究表明，可生物降解的纳米颗粒能够整合多种当前临床化疗药物及药物组合，并随后装配到红细胞上。这些颗粒也能轻易地装配到人红细胞上，并在剪切力依赖的方式下释放。 ELeCt 联合应用，提供了一个多用途、高效且可转化的平台，用于对抗肺转移。
Fig. 1 Schematic illustration of the ELeCt platform and characterization of drug (DOX)–loaded biodegradable PLGA NPs.
图 1. ELeCt 平台示意图及载药（DOX）可生物降解 PLGA 纳米粒的表征。
图 1. ELeCt 平台示意图及载药（DOX）可生物降解 PLGA 纳米粒的表征。
(A) Schematic illustration of the composition and mechanism of the biodegradable drug NP assembling on the erythrocyte platform (ELeCt) for lung metastasis treatment. (B to D) Average size (B), zeta potential (C), and drug loading contents (D) of plain and drug-loaded NPs. (E) SEM images showing the morphological features of the NPs. Scale bars, 200 nm. (F) Size distribution of plain and drug-loaded NPs. (G) Drug release kinetics from the biodegradable NPs in a complete medium (n = 4). (H and I) Flow cytometry histogram plots (H) and CLSM images (I) showing the interaction of drug-loaded NPs with B16F10-Luc melanoma cells. In (I), cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). (J and K) Dose-response curve (J) and median inhibitory concentration (IC50) values (K) of B16F10-Luc cells after being treated with different formulations for 24 hours (n = 6). n.s., not significantly different (Student’s t test).
(A) 用于治疗肺转移的可生物降解药物纳米颗粒在红细胞平台（ELeCt）上组装的组成及机制示意图。(B 至 D) 纯纳米颗粒与载药纳米颗粒的平均尺寸（B）、zeta 电位（C）及药物负载量（D）。(E) 扫描电镜图像显示纳米颗粒的形态特征。比例尺，200 纳米。(F) 纯纳米颗粒与载药纳米颗粒的尺寸分布。(G) 在完全培养基中可生物降解纳米颗粒的药物释放动力学（n = 4）。(H 和 I) 流式细胞术直方图（H）及共聚焦激光扫描显微镜（CLSM）图像（I）展示载药纳米颗粒与 B16F10-Luc 黑色素瘤细胞的相互作用。在 (I) 中，细胞核用 4′,6-二脒基-2-苯基吲哚（DAPI）染色。(J 和 K) B16F10-Luc 细胞经不同配方处理 24 小时后的剂量-反应曲线（J）及半数抑制浓度（IC50）值（K）（n = 6）。n.s.，无显著差异（学生 t 检验）。
(A) 用于治疗肺转移的可生物降解药物纳米颗粒在红细胞平台（ELeCt）上组装的组成及机制示意图。(B 至 D) 纯纳米颗粒与载药纳米颗粒的平均尺寸（B）、zeta 电位（C）及药物负载量（D）。(E) 扫描电镜图像显示纳米颗粒的形态特征。比例尺，200 纳米。(F) 纯纳米颗粒与载药纳米颗粒的尺寸分布。(G) 在完全培养基中可生物降解纳米颗粒的药物释放动力学（n = 4）。(H 和 I) 流式细胞术直方图（H）及共聚焦激光扫描显微镜（CLSM）图像（I）展示载药纳米颗粒与 B16F10-Luc 黑色素瘤细胞的相互作用。在 (I) 中，细胞核用 4′,6-二脒基-2-苯基吲哚（DAPI）染色。(J 和 K) B16F10-Luc 细胞经不同配方处理 24 小时后的剂量-反应曲线（J）及半数抑制浓度（IC50）值（K）（n = 6）。n.s.，无显著差异（学生 t 检验）。
RESULTS 结果
Drug-loaded biodegradable NPs could efficiently interact with target cancer cells
载药可生物降解的纳米颗粒能够高效地与目标癌细胞相互作用
We used DOX as a model drug and prepared drug-loaded biodegradable polymeric [poly(lactic-co-glycolic acid) (PLGA)] NPs using the nanoprecipitation method. The drug-loaded PLGA NPs had a diameter of 136.0 ± 2.7 nm, which was slightly larger than the plain NPs (Fig. 1BOpens in image viewer
我们选用阿霉素（DOX）作为模型药物，并通过纳米沉淀法制备了载有药物的可生物降解聚合物[聚（乳酸-共-乙醇酸）（PLGA）]纳米颗粒（NPs）。载药的 PLGA NPs 直径为 136.0 ± 2.7 nm，略大于未载药的 NPs（图 1B）。). The encapsulation of DOX made the surface of the drug-loaded NPs slightly positive (10.45 ± 0.84 mV) (Fig. 1COpens in image viewer
DOX 的封装使得载药纳米颗粒的表面略呈正电性（10.45 ± 0.84 mV）（图 1C）。), and this can be attributed to the presence of DOX on the NP surface. The drug-loaded PLGA NPs exhibited a high drug loading capacity (196.7 ± 5.8 mg/g) (Fig. 1DOpens in image viewer
），这可归因于 NP 表面存在 DOX。载药 PLGA 纳米颗粒显示出较高的药物负载能力（196.7 ± 5.8 mg/g）（图 1D）。). We characterized the morphology of the NPs using scanning electron microscopy (SEM). SEM images shown in Fig. 1EOpens in image viewer
我们利用扫描电子显微镜（SEM）对纳米颗粒（NPs）的形态进行了表征。图 1E 展示了 SEM 图像。 revealed that both the plain and the drug-loaded PLGA NPs were spherical and relatively monodispersed. The dynamic light scattering data (Fig. 1FOpens in image viewer
显示，无论是空白还是载药的 PLGA 纳米颗粒均为球形且相对单分散。动态光散射数据（图 1F）) confirmed the uniform size distribution of the prepared NPs. To test whether the drug could be released from the PLGA NPs, we assayed their release profile in a complete medium. A burst followed by a sustained-release profile was observed, and most of the drug was released within the first 6 hours (Fig. 1GOpens in image viewer
) 证实了所制备纳米颗粒的均匀尺寸分布。为了测试药物是否可以从 PLGA 纳米颗粒中释放，我们在完全培养基中检测了它们的释放曲线。结果显示，药物首先经历了爆发式释放，随后是持续释放过程，大部分药物在最初的 6 小时内释放（图 1G）。). Efficient interaction of drug NPs with the target cancer cells is critical for successful drug delivery and efficacy. In this study, we used B16F10-Luc melanoma cells as a model to evaluate the interaction between the drug-loaded biodegradable PLGA NPs and the target cancer cells. As shown in Fig. 1HOpens in image viewer
）。药物纳米颗粒与靶向癌细胞的高效相互作用对于成功的药物递送和疗效至关重要。在本研究中，我们采用 B16F10-Luc 黑色素瘤细胞作为模型，评估载药可生物降解的 PLGA 纳米颗粒与靶向癌细胞之间的相互作用。如图 1H 所示。, the drug-loaded PLGA NPs appeared to be internalized by B16F10-Luc cells quickly and efficiently. Within 20 min of the incubation, a substantial portion of the cells had drug-loaded NPs in them. The confocal laser scanning microscopy (CLSM) images shown in Fig. 1IOpens in image viewer
药物负载的 PLGA 纳米颗粒似乎能迅速且有效地被 B16F10-Luc 细胞内化。在孵育 20 分钟后，相当一部分细胞内已含有药物负载的纳米颗粒。图 1I 展示的共聚焦激光扫描显微镜（CLSM）图像证实了这一点。 confirmed the efficient interactions between the NPs and the B16F10-Luc cells. Noticeably, the increase in DOX fluorescence within the cell nucleus suggested an effective intracellular delivery and sufficient release of the loaded drug. We further evaluated the in vitro antitumor efficacy of the drug-loaded PLGA NPs in a two-dimensional culture of the same cell line. As indicated by the dose-response curve (Fig. 1JOpens in image viewer
证实了纳米颗粒（NPs）与 B16F10-Luc 细胞之间的高效相互作用。值得注意的是，细胞核内 DOX 荧光增强表明药物有效内化及负载药物的充分释放。我们进一步评估了负载药物的 PLGA 纳米颗粒在同一细胞系二维培养中的体外抗肿瘤效果。如图 1J 所示的剂量-反应曲线所示。) and IC50 (median inhibitory concentration) values (Fig. 1KOpens in image viewer
)和 IC 50 （半抑制浓度）值（图 1K）), the drug-loaded PLGA NPs exhibited a slightly weaker cell killing efficacy compared with the free drug. However, the difference between them was not significant.
载药的 PLGA 纳米颗粒在细胞杀伤效果上略逊于游离药物，但两者间的差异并不显著。
我们选用阿霉素（DOX）作为模型药物，并通过纳米沉淀法制备了载有药物的可生物降解聚合物[聚（乳酸-共-乙醇酸）（PLGA）]纳米颗粒（NPs）。载药的 PLGA NPs 直径为 136.0 ± 2.7 nm，略大于未载药的 NPs（图 1B）。). The encapsulation of DOX made the surface of the drug-loaded NPs slightly positive (10.45 ± 0.84 mV) (Fig. 1COpens in image viewer
DOX 的封装使得载药纳米颗粒的表面略呈正电性（10.45 ± 0.84 mV）（图 1C）。), and this can be attributed to the presence of DOX on the NP surface. The drug-loaded PLGA NPs exhibited a high drug loading capacity (196.7 ± 5.8 mg/g) (Fig. 1DOpens in image viewer
），这可归因于 NP 表面存在 DOX。载药 PLGA 纳米颗粒显示出较高的药物负载能力（196.7 ± 5.8 mg/g）（图 1D）。). We characterized the morphology of the NPs using scanning electron microscopy (SEM). SEM images shown in Fig. 1EOpens in image viewer
我们利用扫描电子显微镜（SEM）对纳米颗粒（NPs）的形态进行了表征。图 1E 展示了 SEM 图像。 revealed that both the plain and the drug-loaded PLGA NPs were spherical and relatively monodispersed. The dynamic light scattering data (Fig. 1FOpens in image viewer
显示，无论是空白还是载药的 PLGA 纳米颗粒均为球形且相对单分散。动态光散射数据（图 1F）) confirmed the uniform size distribution of the prepared NPs. To test whether the drug could be released from the PLGA NPs, we assayed their release profile in a complete medium. A burst followed by a sustained-release profile was observed, and most of the drug was released within the first 6 hours (Fig. 1GOpens in image viewer
) 证实了所制备纳米颗粒的均匀尺寸分布。为了测试药物是否可以从 PLGA 纳米颗粒中释放，我们在完全培养基中检测了它们的释放曲线。结果显示，药物首先经历了爆发式释放，随后是持续释放过程，大部分药物在最初的 6 小时内释放（图 1G）。). Efficient interaction of drug NPs with the target cancer cells is critical for successful drug delivery and efficacy. In this study, we used B16F10-Luc melanoma cells as a model to evaluate the interaction between the drug-loaded biodegradable PLGA NPs and the target cancer cells. As shown in Fig. 1HOpens in image viewer
）。药物纳米颗粒与靶向癌细胞的高效相互作用对于成功的药物递送和疗效至关重要。在本研究中，我们采用 B16F10-Luc 黑色素瘤细胞作为模型，评估载药可生物降解的 PLGA 纳米颗粒与靶向癌细胞之间的相互作用。如图 1H 所示。, the drug-loaded PLGA NPs appeared to be internalized by B16F10-Luc cells quickly and efficiently. Within 20 min of the incubation, a substantial portion of the cells had drug-loaded NPs in them. The confocal laser scanning microscopy (CLSM) images shown in Fig. 1IOpens in image viewer
药物负载的 PLGA 纳米颗粒似乎能迅速且有效地被 B16F10-Luc 细胞内化。在孵育 20 分钟后，相当一部分细胞内已含有药物负载的纳米颗粒。图 1I 展示的共聚焦激光扫描显微镜（CLSM）图像证实了这一点。 confirmed the efficient interactions between the NPs and the B16F10-Luc cells. Noticeably, the increase in DOX fluorescence within the cell nucleus suggested an effective intracellular delivery and sufficient release of the loaded drug. We further evaluated the in vitro antitumor efficacy of the drug-loaded PLGA NPs in a two-dimensional culture of the same cell line. As indicated by the dose-response curve (Fig. 1JOpens in image viewer
证实了纳米颗粒（NPs）与 B16F10-Luc 细胞之间的高效相互作用。值得注意的是，细胞核内 DOX 荧光增强表明药物有效内化及负载药物的充分释放。我们进一步评估了负载药物的 PLGA 纳米颗粒在同一细胞系二维培养中的体外抗肿瘤效果。如图 1J 所示的剂量-反应曲线所示。) and IC50 (median inhibitory concentration) values (Fig. 1KOpens in image viewer
)和 IC 50 （半抑制浓度）值（图 1K）), the drug-loaded PLGA NPs exhibited a slightly weaker cell killing efficacy compared with the free drug. However, the difference between them was not significant.
载药的 PLGA 纳米颗粒在细胞杀伤效果上略逊于游离药物，但两者间的差异并不显著。
Drug-loaded biodegradable NPs efficiently assembled onto erythrocytes
载药可生物降解纳米颗粒高效组装到红细胞表面
We first evaluated whether the drug-loaded PLGA NPs could efficiently assemble onto the mouse erythrocytes. To do this, we incubated mouse erythrocytes with the NPs at a range of NP-to-erythrocyte ratios (50:1 to 800:1) and detected the binding of NPs using flow cytometry. As shown in Fig. 2 (A and B)Opens in image viewer
我们首先评估了载药 PLGA 纳米颗粒是否能高效地组装到小鼠红细胞上。为此，我们将小鼠红细胞与不同纳米颗粒与红细胞比例（50:1 到 800:1）的纳米颗粒一起孵育，并利用流式细胞术检测纳米颗粒的结合情况。如图 2（A 和 B）所示。, the drug-loaded PLGA NPs indeed assembled onto the mouse erythrocytes efficiently. Particularly, 81.6% of erythrocytes were found to carry NPs when being incubated with NPs at a ratio of 200:1, and this number increased to >96% on further increasing the incubation ratio. The binding efficiency of the NPs to the erythrocytes was also quantified. Unexpectedly, a substantial portion (39.3 to 54.5%) of the incubated NPs assembled onto the mouse erythrocytes, depending on the feed ratio of the NPs to the erythrocytes (Fig. 2COpens in image viewer
载药的 PLGA 纳米颗粒确实能高效地组装到小鼠红细胞表面。特别是，当以 200:1 的比例与纳米颗粒共孵育时，发现 81.6%的红细胞携带有纳米颗粒，而进一步提高孵育比例后，这一比例增至超过 96%。纳米颗粒与红细胞的结合效率也得到了量化。出乎意料的是，根据纳米颗粒与红细胞的投料比，有相当一部分（39.3%至 54.5%）的纳米颗粒会组装到小鼠红细胞上（图 2C）。). Because of this high binding efficiency and the high drug loading capacity of the NPs, the mouse erythrocytes were able to carry a high drug dose (as high as 294.1 μg per 3 × 108 erythrocytes) (Fig. 2DOpens in image viewer
).由于这种高结合效率以及纳米颗粒的高药物负载能力，小鼠红细胞能够携带高剂量的药物（每 3×10 8 个红细胞高达 294.1 微克）（图 2D）。). In addition, the drug dose on the mouse erythrocytes could be easily tuned by manipulating the feed ratio of the NPs to the erythrocytes. Next, we visualized the assembly of drug-loaded PLGA NPs onto the mouse erythrocytes using CLSM and SEM. As shown in Fig. 2 (E and F)Opens in image viewer
此外，通过调节纳米颗粒与小鼠红细胞的投料比例，可以轻松调整小鼠红细胞上的药物剂量。接下来，我们利用共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）观察了载药 PLGA 纳米颗粒在红细胞表面的组装情况。如图 2（E 和 F）所示。, both the CLSM and SEM data confirmed the efficient assembly of the NPs onto the mouse erythrocytes. Meanwhile, the mouse erythrocytes maintained their biconcave shapes after being hitchhiked by the drug-loaded PLGA NPs (Fig. 2 (E and F)Opens in image viewer
, 共聚焦显微镜（CLSM）和扫描电子显微镜（SEM）数据均证实了纳米颗粒（NPs）在鼠红细胞表面上的高效组装。同时，搭载药物的聚乳酸-羟基乙酸共聚物（PLGA）纳米颗粒附着后，鼠红细胞仍保持其双凹形态（图 2（E 和 F））。), indicating the assembly of the NPs had caused minimal damage to the carrier erythrocytes. To test the translational potential of the erythrocyte hitchhiking platform, we evaluated the assembly of the drug-loaded PLGA NPs onto the human erythrocytes. Both the CLSM and SEM images shown in Fig. 2 (G and H)Opens in image viewer
), 表明纳米颗粒的组装对载体红细胞造成的损伤最小。为了测试红细胞搭便车平台的转化潜力，我们评估了载药 PLGA 纳米颗粒在人红细胞上的组装情况。图 2（G 和 H）中的 CLSM 和 SEM 图像均展示了这一过程。 suggested that the drug NPs could efficiently assemble onto the human erythrocytes as well. In addition, we also evaluated the assembly of drug-loaded PLGA NPs to human erythrocytes at different NP-to-erythrocyte feed ratios (200:1 to 1600:1). Similar to the murine counterparts, the drug-loaded PLGA NPs assembled onto the human erythrocytes with high efficiency (38.7 to 45.7%) at various NP-to-erythrocyte feed ratios (Fig. 2 (I and J)Opens in image viewer
研究表明，药物纳米颗粒（NPs）能够高效地组装到人红细胞上。此外，我们还评估了不同纳米颗粒与红细胞投料比（200:1 至 1600:1）下，载药的聚乳酸-羟基乙酸共聚物（PLGA NPs）与人红细胞的组装情况。与小鼠红细胞类似，载药的 PLGA NPs 在不同投料比下均能高效地组装到人红细胞上，效率高达 38.7%至 45.7%（图 2（I 和 J））。). Moreover, the drug dose on human erythrocytes could be tuned by changing the incubation ratio, and a very high drug dose (209.1 μg per 1.5 × 108 erythrocytes) could be hitchhiked to human erythrocytes when being incubated at a 1600:1 NP-to-erythrocyte ratio (Fig. 2KOpens in image viewer
此外，通过调整培养比例，可以调节人红细胞上的药物剂量，当以 1600:1 的纳米颗粒与红细胞比例进行培养时，可将极高剂量的药物（每 1.5 × 10^9 个红细胞 209.1 微克）搭载到人红细胞上（图 2K）。).
我们首先评估了载药 PLGA 纳米颗粒是否能高效地组装到小鼠红细胞上。为此，我们将小鼠红细胞与不同纳米颗粒与红细胞比例（50:1 到 800:1）的纳米颗粒一起孵育，并利用流式细胞术检测纳米颗粒的结合情况。如图 2（A 和 B）所示。, the drug-loaded PLGA NPs indeed assembled onto the mouse erythrocytes efficiently. Particularly, 81.6% of erythrocytes were found to carry NPs when being incubated with NPs at a ratio of 200:1, and this number increased to >96% on further increasing the incubation ratio. The binding efficiency of the NPs to the erythrocytes was also quantified. Unexpectedly, a substantial portion (39.3 to 54.5%) of the incubated NPs assembled onto the mouse erythrocytes, depending on the feed ratio of the NPs to the erythrocytes (Fig. 2COpens in image viewer
载药的 PLGA 纳米颗粒确实能高效地组装到小鼠红细胞表面。特别是，当以 200:1 的比例与纳米颗粒共孵育时，发现 81.6%的红细胞携带有纳米颗粒，而进一步提高孵育比例后，这一比例增至超过 96%。纳米颗粒与红细胞的结合效率也得到了量化。出乎意料的是，根据纳米颗粒与红细胞的投料比，有相当一部分（39.3%至 54.5%）的纳米颗粒会组装到小鼠红细胞上（图 2C）。). Because of this high binding efficiency and the high drug loading capacity of the NPs, the mouse erythrocytes were able to carry a high drug dose (as high as 294.1 μg per 3 × 108 erythrocytes) (Fig. 2DOpens in image viewer
).由于这种高结合效率以及纳米颗粒的高药物负载能力，小鼠红细胞能够携带高剂量的药物（每 3×10 8 个红细胞高达 294.1 微克）（图 2D）。). In addition, the drug dose on the mouse erythrocytes could be easily tuned by manipulating the feed ratio of the NPs to the erythrocytes. Next, we visualized the assembly of drug-loaded PLGA NPs onto the mouse erythrocytes using CLSM and SEM. As shown in Fig. 2 (E and F)Opens in image viewer
此外，通过调节纳米颗粒与小鼠红细胞的投料比例，可以轻松调整小鼠红细胞上的药物剂量。接下来，我们利用共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）观察了载药 PLGA 纳米颗粒在红细胞表面的组装情况。如图 2（E 和 F）所示。, both the CLSM and SEM data confirmed the efficient assembly of the NPs onto the mouse erythrocytes. Meanwhile, the mouse erythrocytes maintained their biconcave shapes after being hitchhiked by the drug-loaded PLGA NPs (Fig. 2 (E and F)Opens in image viewer
, 共聚焦显微镜（CLSM）和扫描电子显微镜（SEM）数据均证实了纳米颗粒（NPs）在鼠红细胞表面上的高效组装。同时，搭载药物的聚乳酸-羟基乙酸共聚物（PLGA）纳米颗粒附着后，鼠红细胞仍保持其双凹形态（图 2（E 和 F））。), indicating the assembly of the NPs had caused minimal damage to the carrier erythrocytes. To test the translational potential of the erythrocyte hitchhiking platform, we evaluated the assembly of the drug-loaded PLGA NPs onto the human erythrocytes. Both the CLSM and SEM images shown in Fig. 2 (G and H)Opens in image viewer
), 表明纳米颗粒的组装对载体红细胞造成的损伤最小。为了测试红细胞搭便车平台的转化潜力，我们评估了载药 PLGA 纳米颗粒在人红细胞上的组装情况。图 2（G 和 H）中的 CLSM 和 SEM 图像均展示了这一过程。 suggested that the drug NPs could efficiently assemble onto the human erythrocytes as well. In addition, we also evaluated the assembly of drug-loaded PLGA NPs to human erythrocytes at different NP-to-erythrocyte feed ratios (200:1 to 1600:1). Similar to the murine counterparts, the drug-loaded PLGA NPs assembled onto the human erythrocytes with high efficiency (38.7 to 45.7%) at various NP-to-erythrocyte feed ratios (Fig. 2 (I and J)Opens in image viewer
研究表明，药物纳米颗粒（NPs）能够高效地组装到人红细胞上。此外，我们还评估了不同纳米颗粒与红细胞投料比（200:1 至 1600:1）下，载药的聚乳酸-羟基乙酸共聚物（PLGA NPs）与人红细胞的组装情况。与小鼠红细胞类似，载药的 PLGA NPs 在不同投料比下均能高效地组装到人红细胞上，效率高达 38.7%至 45.7%（图 2（I 和 J））。). Moreover, the drug dose on human erythrocytes could be tuned by changing the incubation ratio, and a very high drug dose (209.1 μg per 1.5 × 108 erythrocytes) could be hitchhiked to human erythrocytes when being incubated at a 1600:1 NP-to-erythrocyte ratio (Fig. 2KOpens in image viewer
此外，通过调整培养比例，可以调节人红细胞上的药物剂量，当以 1600:1 的纳米颗粒与红细胞比例进行培养时，可将极高剂量的药物（每 1.5 × 10^9 个红细胞 209.1 微克）搭载到人红细胞上（图 2K）。).
Fig. 2 Doxorubicin-loaded biodegradable PLGA NPs efficiently assemble onto mouse and human erythrocytes.
图 2. 载有阿霉素的可生物降解 PLGA 纳米颗粒高效地组装在小鼠和人类红细胞上。
图 2. 载有阿霉素的可生物降解 PLGA 纳米颗粒高效地组装在小鼠和人类红细胞上。
(A) Flow cytometry analysis of assembly of DOX-loaded PLGA NPs to mouse erythrocytes at different NP-to-erythrocyte ratios (left to right: 0:1, 50:1, 200:1, 400:1, and 800:1). (B) Percentage of mouse erythrocytes carrying at least one NP. (C) Nanoparticle binding efficiency and (D) drug dose on mouse erythrocytes at different NP–to–mouse erythrocyte ratios. (E) CLSM and (F) SEM images of mouse erythrocytes with drug-loaded NPs assembled on them. Scale bars in (F), 2 μm. (G) CLSM and (H) SEM images of human erythrocytes with drug-loaded NPs assembled on them. Scale bars in (H), 2 μm. (I) Flow cytometry assay of the assembly of drug-loaded NPs to human erythrocytes at different NP-to-erythrocyte ratios (left to right: 0:1, 200:1, 800:1, and 1600:1). (J) Nanoparticle binding efficiency and (K) drug dose on human erythrocytes at different NP-to-erythrocyte ratios.
(A) 流式细胞术分析不同纳米颗粒与小鼠红细胞比例下（从左至右：0:1, 50:1, 200:1, 400:1 和 800:1）DOX 负载的 PLGA 纳米颗粒与小鼠红细胞的组装情况。(B) 携带至少一个纳米颗粒的小鼠红细胞百分比。(C) 纳米颗粒结合效率及 (D) 不同纳米颗粒与小鼠红细胞比例下的药物剂量。(E) 共聚焦激光扫描显微镜及 (F) 扫描电子显微镜下显示小鼠红细胞表面组装有药物负载纳米颗粒的图像。(F) 中的比例尺为 2 μm。(G) 共聚焦激光扫描显微镜及 (H) 扫描电子显微镜下显示人红细胞表面组装有药物负载纳米颗粒的图像。(H) 中的比例尺为 2 μm。(I) 流式细胞术检测不同纳米颗粒与人红细胞比例下（从左至右：0:1, 200:1, 800:1 和 1600:1）药物负载纳米颗粒与人红细胞的组装情况。(J) 纳米颗粒结合效率及 (K) 不同纳米颗粒与人红细胞比例下的药物剂量。
(A) 流式细胞术分析不同纳米颗粒与小鼠红细胞比例下（从左至右：0:1, 50:1, 200:1, 400:1 和 800:1）DOX 负载的 PLGA 纳米颗粒与小鼠红细胞的组装情况。(B) 携带至少一个纳米颗粒的小鼠红细胞百分比。(C) 纳米颗粒结合效率及 (D) 不同纳米颗粒与小鼠红细胞比例下的药物剂量。(E) 共聚焦激光扫描显微镜及 (F) 扫描电子显微镜下显示小鼠红细胞表面组装有药物负载纳米颗粒的图像。(F) 中的比例尺为 2 μm。(G) 共聚焦激光扫描显微镜及 (H) 扫描电子显微镜下显示人红细胞表面组装有药物负载纳米颗粒的图像。(H) 中的比例尺为 2 μm。(I) 流式细胞术检测不同纳米颗粒与人红细胞比例下（从左至右：0:1, 200:1, 800:1 和 1600:1）药物负载纳米颗粒与人红细胞的组装情况。(J) 纳米颗粒结合效率及 (K) 不同纳米颗粒与人红细胞比例下的药物剂量。
ELeCt enabled enhanced and targeted delivery of the NP drugs to the lungs bearing metastasis
ELeCt 实现了纳米颗粒药物对带有转移灶的肺部的增强与靶向递送
We first conducted a pharmacokinetic study to examine the blood circulation time of different drug formulations. As shown in Fig. 3AOpens in image viewer
我们首先进行了药代动力学研究，以检测不同药物制剂的血液循环时间。如图 3A 所示。, by assembling drug NPs to erythrocytes, a higher drug concentration in the blood was achieved at all the tested time points, indicating an extended circulation time of the hitchhiked formulation. Mouse lung capillaries have an average diameter of 5 μm, narrowing down up to sizes as small as 1 μm, three to four times smaller than the mouse erythrocyte diameter (27). Upon intravenous administration, the drug-loaded NPs assembled onto erythrocytes are expected to detach from the carrier erythrocytes because of the high shear stress and be deposited in the narrow lung capillaries. To test this hypothesis, we first performed an in vitro shear study in which the erythrocytes carrying the drug-loaded NPs were sheared for 20 min at a low (~1 Pa) or high (6 Pa) shear stress. As shown in Fig. 3BOpens in image viewer
通过将药物纳米颗粒（NPs）组装到红细胞上，在所有测试时间点均实现了更高的血液药物浓度，这表明搭载的制剂具有延长的循环时间。小鼠肺毛细血管的平均直径为 5 微米，最窄处可达 1 微米，比小鼠红细胞直径（27）小三到四倍。静脉注射后，载有药物的纳米颗粒组装到红细胞上，预计会因高剪切应力而脱离载体红细胞，并沉积在狭窄的肺毛细血管中。为验证这一假设，我们首先在体外进行了剪切实验，将携带药物纳米颗粒的红细胞在低剪切应力（~1 帕）或高剪切应力（6 帕）下剪切 20 分钟。如图 3B 所示。, detachment of the drug NPs from the mouse erythrocytes was evidently shear dependent, providing a basis for specific delivery of drug NPs to the diseased lungs. Particularly, 76% of the hitchhiked drug NPs were sheared off at the lung-corresponding shear stress (6 Pa), using a rheometer. Moreover, this shear-dependent detachment of drug NPs was also observed with the human erythrocytes, bolstering the translational potential of this ELeCt platform. To test whether the drug NPs could be sheared off and deposited in the lungs that bear metastasis in vivo, we conducted a biodistribution study in mice bearing B16F10-Luc melanoma lung metastasis and quantified the amount of drug, in this case DOX. As shown in Fig. 3 (C and D)Opens in image viewer
药物纳米颗粒从小鼠红细胞上的脱离显然依赖于剪切力，这为药物纳米颗粒定向输送至病变肺部提供了基础。特别是，使用流变仪在对应于肺部的剪切应力（6 帕）下，76%的搭便车药物纳米颗粒被剪切下来。此外，这种剪切依赖性的药物纳米颗粒脱离现象也在人红细胞上观察到，增强了这一 ELeCt 平台在转化应用中的潜力。为了测试药物纳米颗粒是否能在体内被剪切并沉积在带有转移的肺部，我们对携带 B16F10-Luc 黑色素瘤肺转移的小鼠进行了生物分布研究，并量化了药物（此处为 DOX）的含量。如图 3（C 和 D）所示。, by assembly onto erythrocytes, the drug-loaded NPs delivered 16.6-fold higher drug content to the diseased lungs as compared with their free NP counterparts, 20 min after administration. Even at a longer time point (6 hours), erythrocyte hitchhiking deposited 8.7-fold higher drug content in the lungs as compared with their unhitchhiked counterparts. In addition, erythrocyte hitchhiking delivered a 6.9-fold higher drug content to the lungs with melanoma metastasis as compared with the free drug injection, 20 min after administration. Next, we investigated the distribution of the drug NPs sheared off from the carrier erythrocytes within the lungs bearing metastasis. As shown in Fig. 3EOpens in image viewer
通过装配到红细胞上，载药纳米颗粒在给药后 20 分钟内将药物含量输送到患病肺部的效率提高了 16.6 倍，相较于自由纳米颗粒。即使在更长的时间点（6 小时），红细胞搭便车方式在肺部沉积的药物含量也比未搭便车的纳米颗粒高 8.7 倍。此外，红细胞搭便车方式在给药后 20 分钟内将药物输送到带有黑色素瘤转移的肺部的含量，比自由药物注射高 6.9 倍。接着，我们研究了从载体红细胞上剥离的药物纳米颗粒在含有转移的肺部内的分布情况。如图 3E 所示。, consistent with the biodistribution data, more drug NPs were found in the lung section being treated with erythrocytes with NPs assembled on them compared with that being treated with the NPs alone. Evidently, a substantial portion, although not all, of the deposited NPs went deep into the tumor metastasis nodules, suggesting the biodegradable drug NP assembling on erythrocyte was able to precisely deliver the payload chemotherapeutic agents to their desired site of action.
与生物分布数据一致，与仅使用纳米颗粒处理相比，在红细胞表面组装有纳米颗粒的红细胞处理过的肺部切片中发现了更多的药物纳米颗粒。显然，尽管并非全部，但相当一部分沉积的纳米颗粒深入到了肿瘤转移结节内部，这表明在红细胞上组装的生物可降解药物纳米颗粒能够精确地将化疗药物输送到其作用靶点。
我们首先进行了药代动力学研究，以检测不同药物制剂的血液循环时间。如图 3A 所示。, by assembling drug NPs to erythrocytes, a higher drug concentration in the blood was achieved at all the tested time points, indicating an extended circulation time of the hitchhiked formulation. Mouse lung capillaries have an average diameter of 5 μm, narrowing down up to sizes as small as 1 μm, three to four times smaller than the mouse erythrocyte diameter (27). Upon intravenous administration, the drug-loaded NPs assembled onto erythrocytes are expected to detach from the carrier erythrocytes because of the high shear stress and be deposited in the narrow lung capillaries. To test this hypothesis, we first performed an in vitro shear study in which the erythrocytes carrying the drug-loaded NPs were sheared for 20 min at a low (~1 Pa) or high (6 Pa) shear stress. As shown in Fig. 3BOpens in image viewer
通过将药物纳米颗粒（NPs）组装到红细胞上，在所有测试时间点均实现了更高的血液药物浓度，这表明搭载的制剂具有延长的循环时间。小鼠肺毛细血管的平均直径为 5 微米，最窄处可达 1 微米，比小鼠红细胞直径（27）小三到四倍。静脉注射后，载有药物的纳米颗粒组装到红细胞上，预计会因高剪切应力而脱离载体红细胞，并沉积在狭窄的肺毛细血管中。为验证这一假设，我们首先在体外进行了剪切实验，将携带药物纳米颗粒的红细胞在低剪切应力（~1 帕）或高剪切应力（6 帕）下剪切 20 分钟。如图 3B 所示。, detachment of the drug NPs from the mouse erythrocytes was evidently shear dependent, providing a basis for specific delivery of drug NPs to the diseased lungs. Particularly, 76% of the hitchhiked drug NPs were sheared off at the lung-corresponding shear stress (6 Pa), using a rheometer. Moreover, this shear-dependent detachment of drug NPs was also observed with the human erythrocytes, bolstering the translational potential of this ELeCt platform. To test whether the drug NPs could be sheared off and deposited in the lungs that bear metastasis in vivo, we conducted a biodistribution study in mice bearing B16F10-Luc melanoma lung metastasis and quantified the amount of drug, in this case DOX. As shown in Fig. 3 (C and D)Opens in image viewer
药物纳米颗粒从小鼠红细胞上的脱离显然依赖于剪切力，这为药物纳米颗粒定向输送至病变肺部提供了基础。特别是，使用流变仪在对应于肺部的剪切应力（6 帕）下，76%的搭便车药物纳米颗粒被剪切下来。此外，这种剪切依赖性的药物纳米颗粒脱离现象也在人红细胞上观察到，增强了这一 ELeCt 平台在转化应用中的潜力。为了测试药物纳米颗粒是否能在体内被剪切并沉积在带有转移的肺部，我们对携带 B16F10-Luc 黑色素瘤肺转移的小鼠进行了生物分布研究，并量化了药物（此处为 DOX）的含量。如图 3（C 和 D）所示。, by assembly onto erythrocytes, the drug-loaded NPs delivered 16.6-fold higher drug content to the diseased lungs as compared with their free NP counterparts, 20 min after administration. Even at a longer time point (6 hours), erythrocyte hitchhiking deposited 8.7-fold higher drug content in the lungs as compared with their unhitchhiked counterparts. In addition, erythrocyte hitchhiking delivered a 6.9-fold higher drug content to the lungs with melanoma metastasis as compared with the free drug injection, 20 min after administration. Next, we investigated the distribution of the drug NPs sheared off from the carrier erythrocytes within the lungs bearing metastasis. As shown in Fig. 3EOpens in image viewer
通过装配到红细胞上，载药纳米颗粒在给药后 20 分钟内将药物含量输送到患病肺部的效率提高了 16.6 倍，相较于自由纳米颗粒。即使在更长的时间点（6 小时），红细胞搭便车方式在肺部沉积的药物含量也比未搭便车的纳米颗粒高 8.7 倍。此外，红细胞搭便车方式在给药后 20 分钟内将药物输送到带有黑色素瘤转移的肺部的含量，比自由药物注射高 6.9 倍。接着，我们研究了从载体红细胞上剥离的药物纳米颗粒在含有转移的肺部内的分布情况。如图 3E 所示。, consistent with the biodistribution data, more drug NPs were found in the lung section being treated with erythrocytes with NPs assembled on them compared with that being treated with the NPs alone. Evidently, a substantial portion, although not all, of the deposited NPs went deep into the tumor metastasis nodules, suggesting the biodegradable drug NP assembling on erythrocyte was able to precisely deliver the payload chemotherapeutic agents to their desired site of action.
与生物分布数据一致，与仅使用纳米颗粒处理相比，在红细胞表面组装有纳米颗粒的红细胞处理过的肺部切片中发现了更多的药物纳米颗粒。显然，尽管并非全部，但相当一部分沉积的纳米颗粒深入到了肿瘤转移结节内部，这表明在红细胞上组装的生物可降解药物纳米颗粒能够精确地将化疗药物输送到其作用靶点。
Fig. 3 The ELeCt platform enables enhanced and targeted delivery of NP drugs to the lungs bearing metastasis.
图 3 ELeCt 平台实现了对带有转移灶的肺部进行 NP 药物的增强和靶向递送。
图 3 ELeCt 平台实现了对带有转移灶的肺部进行 NP 药物的增强和靶向递送。
(A) Pharmacokinetics of intravenously administered DOX formulations. Extended blood circulation time of DOX was achieved by erythrocyte hitchhiking compared with using free drug or NPs alone (n = 3). Significantly different [one-way analysis of variance (ANOVA)]: *P < 0.05 and **P < 0.01. (B) Hitchhiked drug-loaded NPs could specifically detach from mouse and human erythrocytes under the lung-corresponding shear stress. Samples were sheared for 20 min (n = 3). Low shear indicates rotary shear (~1 Pa), while high shear was at 6 Pa. Significantly different (Student’s t test): ***P < 0.001. (C) Drug accumulation in the lungs of mice bearing B16F10-Luc lung metastasis at 20 min and 6 hours after intravenous administration of different DOX formulations (n = 3). Significantly different (one-way ANOVA): *P < 0.05 and ***P < 0.001. (D) Comparison of the drug concentration in the lungs of erythrocyte hitchhiking group to that of the free drug and NP-alone groups (n = 3). (E) Drug distribution in the diseased lungs 20 min after intravenous administration of DOX formulations. Dashed lines indicate the edge of metastasis nodules.
(A) 静脉注射多柔比星（DOX）制剂的药代动力学。与单独使用游离药物或纳米颗粒（NPs）相比，通过红细胞搭便车实现了 DOX 的延长血液循环时间（n = 3）。显著差异[单因素方差分析（ANOVA）]：*P < 0.05 和 **P < 0.01。(B) 搭便车的载药纳米颗粒能够在肺部对应的剪切应力下从小鼠和人类红细胞上特异性脱离。样本在剪切应力下处理 20 分钟（n = 3）。低剪切表示旋转剪切（~1 Pa），而高剪切为 6 Pa。显著差异（学生 t 检验）：***P < 0.001。(C) 不同 DOX 制剂静脉注射后 20 分钟和 6 小时，携带 B16F10-Luc 肺转移的小鼠肺部药物积累情况（n = 3）。显著差异（单因素 ANOVA）：*P < 0.05 和 ***P < 0.001。(D) 红细胞搭便车组与游离药物和单独 NP 组在肺部药物浓度上的比较（n = 3）。(E) DOX 制剂静脉注射后 20 分钟，病肺中的药物分布。虚线表示转移结节的边缘。
(A) 静脉注射多柔比星（DOX）制剂的药代动力学。与单独使用游离药物或纳米颗粒（NPs）相比，通过红细胞搭便车实现了 DOX 的延长血液循环时间（n = 3）。显著差异[单因素方差分析（ANOVA）]：*P < 0.05 和 **P < 0.01。(B) 搭便车的载药纳米颗粒能够在肺部对应的剪切应力下从小鼠和人类红细胞上特异性脱离。样本在剪切应力下处理 20 分钟（n = 3）。低剪切表示旋转剪切（~1 Pa），而高剪切为 6 Pa。显著差异（学生 t 检验）：***P < 0.001。(C) 不同 DOX 制剂静脉注射后 20 分钟和 6 小时，携带 B16F10-Luc 肺转移的小鼠肺部药物积累情况（n = 3）。显著差异（单因素 ANOVA）：*P < 0.05 和 ***P < 0.001。(D) 红细胞搭便车组与游离药物和单独 NP 组在肺部药物浓度上的比较（n = 3）。(E) DOX 制剂静脉注射后 20 分钟，病肺中的药物分布。虚线表示转移结节的边缘。
The ELeCt platform inhibited lung metastasis progression and improved survival
ELeCt 平台抑制了肺转移的进展并提高了生存率
To evaluate the efficacy of the biodegradable drug NP assembly on the erythrocyte platform, we established a B16F10-Luc melanoma lung metastasis model and tested the antimetastatic efficacies in both the early and the late stages of the same model. We first tested the efficacy of the developed platform in controlling early-stage lung metastasis. As shown in Fig. 4AOpens in image viewer
为了评估生物可降解药物纳米颗粒在红细胞平台上的组装效果，我们建立了一个 B16F10-Luc 黑色素瘤肺转移模型，并在同一模型的早期和晚期阶段测试了其抗转移功效。我们首先测试了所开发平台在控制早期肺转移方面的效果。如图 4A 所示。, the lung metastasis model was established by intravenously injecting B16F10-Luc cells via the tail vein. Four doses of treatments were given every other day with the first dose being administered 1 day after the tumor cell injection. The lung metastasis burden was measured by the bioluminescence intensity in the lung. As indicated by the bioluminescence images (Fig. 4BOpens in image viewer
, 通过尾静脉静脉注射 B16F10-Luc 细胞建立了肺转移模型。每两天给予四次治疗，首次治疗在肿瘤细胞注射后 1 天进行。肺转移负担通过肺部的生物发光强度来测量。如图 4B 所示的生物发光图像所示。) and lung metastasis burden growth curve of individual mouse (Fig. 4COpens in image viewer
)以及单个老鼠的肺转移负担增长曲线（图 4C）), a significantly better inhibition of the lung metastasis progression was achieved by the ELeCt as compared with using the free drug or NPs alone. Two mice remained completely free of lung metastasis after being treated with the drug NPs assembled on erythrocytes for up to day 31 after tumor inoculation. We also calculated the overall lung metastasis burden based on the bioluminescence intensity in the lungs. As shown in Fig. 4DOpens in image viewer
与单独使用游离药物或纳米颗粒相比，通过 ELeCt 显著更好地抑制了肺转移的进展。在肿瘤接种后长达 31 天内，接受红细胞表面组装的药物纳米颗粒处理的两只小鼠完全未出现肺转移。我们还基于肺部的生物发光强度计算了总体肺转移负担。如图 4D 所示。, in the first 23 days after tumor inoculation, lung metastasis was almost completely inhibited in all mice being treated with the drug NPs assembled on erythrocytes. Particularly, as shown in Fig. 4EOpens in image viewer
在肿瘤接种后的前 23 天内，所有接受红细胞表面组装的药物纳米颗粒（NPs）处理的小鼠，其肺转移几乎被完全抑制。特别地，如图 4E 所示。, on day 16, free drug and drug NPs alone resulted in a 17.2- and 1.8-fold lower average bioluminescence intensity compared with the control, respectively. In a sharp contrast, ELeCt achieved a 204.8-fold lower average bioluminescence intensity compared with the control. Similar finding was also observed on day 23. As shown in Fig. 4FOpens in image viewer
在第 16 天，单独的游离药物和药物纳米颗粒相比对照组，分别导致生物发光强度平均降低了 17.2 倍和 1.8 倍。与此形成鲜明对比的是，ELeCt 相较于对照组，平均生物发光强度降低了 204.8 倍。类似的结果在第 23 天也被观察到。如图 4F 所示。, compared with using the drug NPs alone, the treatment using drug NPs assembled on erythrocytes led to a 302-fold lower average bioluminescence intensity. The Kaplan-Meier survival analysis (Fig. 4HOpens in image viewer
与单独使用药物纳米颗粒相比，使用装配在红细胞上的药物纳米颗粒进行治疗，平均生物发光强度降低了 302 倍。Kaplan-Meier 生存分析（图 4H）显示) further confirmed the significantly improved survival benefit of the ELeCt approach over using the NPs alone. The use of the free drug or NPs alone only improved survival slightly, increasing the median survival time from 29 to 32 days. In a sharp comparison, by the treatment with drug NPs assembled on erythrocytes, the animal median survival time was extended from 29 to 61 days. Moreover, one of seven mice continued to survive for at least 70 days. We also monitored the body weight change of mice during the entire treatment period. No significant body weight loss was detected for any of the treatments, compared with a sharp decline in the body weight during the free drug treatment (Fig. 4GOpens in image viewer
进一步证实了 ELeCt 方法相较于单独使用纳米颗粒（NPs）显著提升的生存优势。单独使用游离药物或纳米颗粒仅略微改善了生存期，使中位生存时间从 29 天增至 32 天。相比之下，通过使用红细胞表面组装的药物纳米颗粒进行治疗，动物的中位生存时间从 29 天延长至 61 天。此外，七只小鼠中有一只至少存活了 70 天。我们还监测了整个治疗期间小鼠的体重变化。与游离药物治疗期间体重急剧下降相比，所有治疗组均未检测到显著的体重减轻（图 4G）。), indicating that only the free drug administration caused obvious toxicity at the current drug dose.
表明仅在当前药物剂量下进行游离药物给药会引起明显的毒性。
为了评估生物可降解药物纳米颗粒在红细胞平台上的组装效果，我们建立了一个 B16F10-Luc 黑色素瘤肺转移模型，并在同一模型的早期和晚期阶段测试了其抗转移功效。我们首先测试了所开发平台在控制早期肺转移方面的效果。如图 4A 所示。, the lung metastasis model was established by intravenously injecting B16F10-Luc cells via the tail vein. Four doses of treatments were given every other day with the first dose being administered 1 day after the tumor cell injection. The lung metastasis burden was measured by the bioluminescence intensity in the lung. As indicated by the bioluminescence images (Fig. 4BOpens in image viewer
, 通过尾静脉静脉注射 B16F10-Luc 细胞建立了肺转移模型。每两天给予四次治疗，首次治疗在肿瘤细胞注射后 1 天进行。肺转移负担通过肺部的生物发光强度来测量。如图 4B 所示的生物发光图像所示。) and lung metastasis burden growth curve of individual mouse (Fig. 4COpens in image viewer
)以及单个老鼠的肺转移负担增长曲线（图 4C）), a significantly better inhibition of the lung metastasis progression was achieved by the ELeCt as compared with using the free drug or NPs alone. Two mice remained completely free of lung metastasis after being treated with the drug NPs assembled on erythrocytes for up to day 31 after tumor inoculation. We also calculated the overall lung metastasis burden based on the bioluminescence intensity in the lungs. As shown in Fig. 4DOpens in image viewer
与单独使用游离药物或纳米颗粒相比，通过 ELeCt 显著更好地抑制了肺转移的进展。在肿瘤接种后长达 31 天内，接受红细胞表面组装的药物纳米颗粒处理的两只小鼠完全未出现肺转移。我们还基于肺部的生物发光强度计算了总体肺转移负担。如图 4D 所示。, in the first 23 days after tumor inoculation, lung metastasis was almost completely inhibited in all mice being treated with the drug NPs assembled on erythrocytes. Particularly, as shown in Fig. 4EOpens in image viewer
在肿瘤接种后的前 23 天内，所有接受红细胞表面组装的药物纳米颗粒（NPs）处理的小鼠，其肺转移几乎被完全抑制。特别地，如图 4E 所示。, on day 16, free drug and drug NPs alone resulted in a 17.2- and 1.8-fold lower average bioluminescence intensity compared with the control, respectively. In a sharp contrast, ELeCt achieved a 204.8-fold lower average bioluminescence intensity compared with the control. Similar finding was also observed on day 23. As shown in Fig. 4FOpens in image viewer
在第 16 天，单独的游离药物和药物纳米颗粒相比对照组，分别导致生物发光强度平均降低了 17.2 倍和 1.8 倍。与此形成鲜明对比的是，ELeCt 相较于对照组，平均生物发光强度降低了 204.8 倍。类似的结果在第 23 天也被观察到。如图 4F 所示。, compared with using the drug NPs alone, the treatment using drug NPs assembled on erythrocytes led to a 302-fold lower average bioluminescence intensity. The Kaplan-Meier survival analysis (Fig. 4HOpens in image viewer
与单独使用药物纳米颗粒相比，使用装配在红细胞上的药物纳米颗粒进行治疗，平均生物发光强度降低了 302 倍。Kaplan-Meier 生存分析（图 4H）显示) further confirmed the significantly improved survival benefit of the ELeCt approach over using the NPs alone. The use of the free drug or NPs alone only improved survival slightly, increasing the median survival time from 29 to 32 days. In a sharp comparison, by the treatment with drug NPs assembled on erythrocytes, the animal median survival time was extended from 29 to 61 days. Moreover, one of seven mice continued to survive for at least 70 days. We also monitored the body weight change of mice during the entire treatment period. No significant body weight loss was detected for any of the treatments, compared with a sharp decline in the body weight during the free drug treatment (Fig. 4GOpens in image viewer
进一步证实了 ELeCt 方法相较于单独使用纳米颗粒（NPs）显著提升的生存优势。单独使用游离药物或纳米颗粒仅略微改善了生存期，使中位生存时间从 29 天增至 32 天。相比之下，通过使用红细胞表面组装的药物纳米颗粒进行治疗，动物的中位生存时间从 29 天延长至 61 天。此外，七只小鼠中有一只至少存活了 70 天。我们还监测了整个治疗期间小鼠的体重变化。与游离药物治疗期间体重急剧下降相比，所有治疗组均未检测到显著的体重减轻（图 4G）。), indicating that only the free drug administration caused obvious toxicity at the current drug dose.
表明仅在当前药物剂量下进行游离药物给药会引起明显的毒性。
Fig. 4 The ELeCt platform inhibits lung metastasis progression and improves survival in the early-stage B16F10-Luc metastasis model.
图 4 ELeCt 平台抑制早期 B16F10-Luc 转移模型中肺转移进展并提高生存率。
图 4 ELeCt 平台抑制早期 B16F10-Luc 转移模型中肺转移进展并提高生存率。
(A) Schematic chart of the treatment schedule. (B) Bioluminescence images of lung metastasis at different time points. EXP indicates “Expired.” (C) Lung metastasis progression curve as depicted from in vivo bioluminescence signal intensity. (D) Quantification of lung metastasis burden at different time points (n = 7). (E) Scatter plot comparing the lung metastasis burden in different treatment groups as depicted from bioluminescence signal intensity on day 16 (n = 7). Significantly different (Kruskal-Wallis test): *P < 0.05, **P < 0.01, and ****P < 0.0001. (F) Scatter plot comparison of the lung metastasis burden on day 23 (n = 7). Significantly different (Kruskal-Wallis test): *P < 0.05, **P < 0.01, and ****P < 0.0001. (G) Body weight change of mice during the treatment period (n = 7). (H) Survival of mice under different treatments as displayed by Kaplan-Meier curves (n = 7). Significantly different (log-rank test): *P < 0.05 and ***P < 0.001.
(A) 治疗方案示意图。(B) 不同时间点的肺转移生物发光图像。EXP 表示“已过期”。(C) 体内生物发光信号强度显示的肺转移进展曲线。(D) 不同时间点的肺转移负荷定量分析（n = 7）。(E) 第 16 天不同治疗组肺转移负荷的散点图比较，基于生物发光信号强度（n = 7）。显著差异（Kruskal-Wallis 检验）：*P < 0.05, **P < 0.01, 和 ****P < 0.0001。(F) 第 23 天肺转移负荷的散点图比较（n = 7）。显著差异（Kruskal-Wallis 检验）：*P < 0.05, **P < 0.01, 和 ****P < 0.0001。(G) 治疗期间小鼠体重变化（n = 7）。(H) 不同治疗下小鼠生存率的 Kaplan-Meier 曲线展示（n = 7）。显著差异（log-rank 检验）：*P < 0.05 和 ***P < 0.001。
(A) 治疗方案示意图。(B) 不同时间点的肺转移生物发光图像。EXP 表示“已过期”。(C) 体内生物发光信号强度显示的肺转移进展曲线。(D) 不同时间点的肺转移负荷定量分析（n = 7）。(E) 第 16 天不同治疗组肺转移负荷的散点图比较，基于生物发光信号强度（n = 7）。显著差异（Kruskal-Wallis 检验）：*P < 0.05, **P < 0.01, 和 ****P < 0.0001。(F) 第 23 天肺转移负荷的散点图比较（n = 7）。显著差异（Kruskal-Wallis 检验）：*P < 0.05, **P < 0.01, 和 ****P < 0.0001。(G) 治疗期间小鼠体重变化（n = 7）。(H) 不同治疗下小鼠生存率的 Kaplan-Meier 曲线展示（n = 7）。显著差异（log-rank 检验）：*P < 0.05 和 ***P < 0.001。
Next, we investigated the antimetastatic activity of the developed therapies in late-stage lung metastasis. As shown in Fig. 5AOpens in image viewer
接下来，我们研究了所开发疗法在晚期肺转移中的抗转移活性。如图 5A 所示。, after intravenous tumor cell injection, mice received four doses of therapies every other day with the first dose being administered a week after inoculation (day 7). According to the bioluminescence images (Fig. 5BOpens in image viewer
在静脉注射肿瘤细胞后，小鼠每隔一天接受四次治疗，首次治疗在接种一周后进行（第 7 天）。根据生物发光成像（图 5B）。) and lung metastasis growth curve (Fig. 5COpens in image viewer
)以及肺转移生长曲线（图 5C）) of individual mice, using the drug NPs alone did not lead to significant inhibition of lung metastasis progression. However, the drug NPs assembled on erythrocytes (ELeCt) were able to slow down the lung metastasis progression, although not as notably as in the early-stage metastasis model. The overall lung metastasis burden data shown in Fig. 5DOpens in image viewer
单独使用药物纳米颗粒（NPs）并未显著抑制肺转移的进展。然而，药物纳米颗粒组装在红细胞表面（ELeCt）能够减缓肺转移的进展，尽管这一效果在早期转移模型中不如早期阶段显著。图 5D 展示了整体肺转移负担的数据。 confirmed the better efficacy of the hitchhiked drug NPs over using the free NPs alone. In particular, on day 16 after tumor inoculation, the hitchhiked drug NPs exhibited a 2.4-fold better efficacy in terms of inhibiting metastasis growth. On day 16, the lungs were excised, and the surface metastatic nodules on the lungs were counted. The surface nodules data shown in Fig. 5EOpens in image viewer
证实了搭便车药物纳米颗粒在抑制转移生长方面的更佳疗效，优于单独使用自由纳米颗粒。特别是在肿瘤接种后的第 16 天，搭便车药物纳米颗粒显示出在抑制转移生长方面有 2.4 倍的更好效果。在第 16 天，肺部被切除，并对肺表面上的转移性结节进行计数。图 5E 中显示了肺表面结节的数据。 were consistent with the bioluminescence metastasis burden data evaluated with bioluminescence. A 2.3-fold better efficacy in reducing surface nodules was achieved by assembling the drug NPs to the erythrocytes. The hematoxylin and eosin (H&E) analysis of the lungs of mice confirmed this result (fig. S1). In addition, the body weight change data shown in Fig. 2FOpens in image viewer
与生物发光评估的转移负担数据一致。将药物纳米颗粒组装到红细胞上，实现了降低表面结节的疗效提高了 2.3 倍。苏木精和伊红（H&E）染色的肺部组织分析证实了这一结果（图 S1）。此外，图 2F 所示的体重变化数据也支持了这一结论。 and the H &E analysis data shown in fig. S2 suggested that no significant toxicity was associated with any of the treatments. We then conducted a separate study to evaluate the efficacy of the therapies in terms of extending the animal survival time. As shown in Fig. 5GOpens in image viewer
H&E 分析数据如图 S2 所示，表明所有治疗方案均未引起显著毒性。随后，我们进行了另一项研究，以评估这些疗法在延长动物生存时间方面的疗效。如图 5G 所示。, unlike in the early-stage metastasis model, the use of drug NPs alone did not provide any survival benefit. However, the treatment using drug NPs assembled on erythrocytes (ELeCt) significantly improved the animal survival, extending the median survival time from 28.5 to 37 days. In particular, one of eight mice that received the hitchhiked drug NPs continued to survive for at least 48 days.
与早期转移模型不同，单独使用药物纳米颗粒并未带来任何生存优势。然而，使用装配在红细胞上的药物纳米颗粒（ELeCt）进行治疗显著提高了动物的生存率，将中位生存时间从 28.5 天延长至 37 天。尤为值得注意的是，接受搭便车药物纳米颗粒治疗的八只小鼠中，有一只至少存活了 48 天。
接下来，我们研究了所开发疗法在晚期肺转移中的抗转移活性。如图 5A 所示。, after intravenous tumor cell injection, mice received four doses of therapies every other day with the first dose being administered a week after inoculation (day 7). According to the bioluminescence images (Fig. 5BOpens in image viewer
在静脉注射肿瘤细胞后，小鼠每隔一天接受四次治疗，首次治疗在接种一周后进行（第 7 天）。根据生物发光成像（图 5B）。) and lung metastasis growth curve (Fig. 5COpens in image viewer
)以及肺转移生长曲线（图 5C）) of individual mice, using the drug NPs alone did not lead to significant inhibition of lung metastasis progression. However, the drug NPs assembled on erythrocytes (ELeCt) were able to slow down the lung metastasis progression, although not as notably as in the early-stage metastasis model. The overall lung metastasis burden data shown in Fig. 5DOpens in image viewer
单独使用药物纳米颗粒（NPs）并未显著抑制肺转移的进展。然而，药物纳米颗粒组装在红细胞表面（ELeCt）能够减缓肺转移的进展，尽管这一效果在早期转移模型中不如早期阶段显著。图 5D 展示了整体肺转移负担的数据。 confirmed the better efficacy of the hitchhiked drug NPs over using the free NPs alone. In particular, on day 16 after tumor inoculation, the hitchhiked drug NPs exhibited a 2.4-fold better efficacy in terms of inhibiting metastasis growth. On day 16, the lungs were excised, and the surface metastatic nodules on the lungs were counted. The surface nodules data shown in Fig. 5EOpens in image viewer
证实了搭便车药物纳米颗粒在抑制转移生长方面的更佳疗效，优于单独使用自由纳米颗粒。特别是在肿瘤接种后的第 16 天，搭便车药物纳米颗粒显示出在抑制转移生长方面有 2.4 倍的更好效果。在第 16 天，肺部被切除，并对肺表面上的转移性结节进行计数。图 5E 中显示了肺表面结节的数据。 were consistent with the bioluminescence metastasis burden data evaluated with bioluminescence. A 2.3-fold better efficacy in reducing surface nodules was achieved by assembling the drug NPs to the erythrocytes. The hematoxylin and eosin (H&E) analysis of the lungs of mice confirmed this result (fig. S1). In addition, the body weight change data shown in Fig. 2FOpens in image viewer
与生物发光评估的转移负担数据一致。将药物纳米颗粒组装到红细胞上，实现了降低表面结节的疗效提高了 2.3 倍。苏木精和伊红（H&E）染色的肺部组织分析证实了这一结果（图 S1）。此外，图 2F 所示的体重变化数据也支持了这一结论。 and the H &E analysis data shown in fig. S2 suggested that no significant toxicity was associated with any of the treatments. We then conducted a separate study to evaluate the efficacy of the therapies in terms of extending the animal survival time. As shown in Fig. 5GOpens in image viewer
H&E 分析数据如图 S2 所示，表明所有治疗方案均未引起显著毒性。随后，我们进行了另一项研究，以评估这些疗法在延长动物生存时间方面的疗效。如图 5G 所示。, unlike in the early-stage metastasis model, the use of drug NPs alone did not provide any survival benefit. However, the treatment using drug NPs assembled on erythrocytes (ELeCt) significantly improved the animal survival, extending the median survival time from 28.5 to 37 days. In particular, one of eight mice that received the hitchhiked drug NPs continued to survive for at least 48 days.
与早期转移模型不同，单独使用药物纳米颗粒并未带来任何生存优势。然而，使用装配在红细胞上的药物纳米颗粒（ELeCt）进行治疗显著提高了动物的生存率，将中位生存时间从 28.5 天延长至 37 天。尤为值得注意的是，接受搭便车药物纳米颗粒治疗的八只小鼠中，有一只至少存活了 48 天。
Fig. 5 The ELeCt platform inhibits lung metastasis progression and extends survival in the late-stage B16F10-Luc metastasis model.
图 5 ELeCt 平台抑制晚期 B16F10-Luc 转移模型中肺转移进展并延长生存期。
图 5 ELeCt 平台抑制晚期 B16F10-Luc 转移模型中肺转移进展并延长生存期。
(A) Schematic illustration of the treatment schedule. (B) Bioluminescence images of lung metastasis progression at different time points. (C) Lung metastasis growth curve in mice treated with different DOX formulations. (D) Quantitative analysis of lung metastasis burden as depicted from bioluminescence signal intensity (n = 7). Significantly different (one-way ANOVA): *P < 0.05 and **P < 0.01. (E) Quantification of metastasis nodule numbers on excised lungs from mice in different treatment groups on day 16 (n = 7). Significantly different (one-way ANOVA): **P < 0.01 and ***P < 0.001. (F) Body weight change of mice during the treatment period (n = 7). (G) Kaplan-Meier survival curves of mice in different treatment groups. Significantly different (log-rank test): **P < 0.01 and ***P < 0.001.
(A) 治疗方案示意图。(B) 不同时间点的肺转移进展生物发光图像。(C) 不同 DOX 制剂处理小鼠的肺转移生长曲线。(D) 根据生物发光信号强度定量分析肺转移负担（n = 7）。显著差异（单因素方差分析）：*P < 0.05 和 **P < 0.01。(E) 第 16 天不同治疗组小鼠切除肺部转移结节数量的量化（n = 7）。显著差异（单因素方差分析）：**P < 0.01 和 ***P < 0.001。(F) 治疗期间小鼠体重变化（n = 7）。(G) 不同治疗组小鼠的 Kaplan-Meier 生存曲线。显著差异（对数秩检验）：**P < 0.01 和 ***P < 0.001。
(A) 治疗方案示意图。(B) 不同时间点的肺转移进展生物发光图像。(C) 不同 DOX 制剂处理小鼠的肺转移生长曲线。(D) 根据生物发光信号强度定量分析肺转移负担（n = 7）。显著差异（单因素方差分析）：*P < 0.05 和 **P < 0.01。(E) 第 16 天不同治疗组小鼠切除肺部转移结节数量的量化（n = 7）。显著差异（单因素方差分析）：**P < 0.01 和 ***P < 0.001。(F) 治疗期间小鼠体重变化（n = 7）。(G) 不同治疗组小鼠的 Kaplan-Meier 生存曲线。显著差异（对数秩检验）：**P < 0.01 和 ***P < 0.001。
Several chemotherapeutic agents could be loaded into biodegradable NPs and efficiently assembled onto erythrocytes
多种化疗药物可被装载到可生物降解的纳米颗粒中，并高效地组装到红细胞表面
To test the feasibility of using the ELeCt platform for the delivery of other chemotherapeutic agents, we selected six other common chemotherapeutic agents or their combinations, including camptothecin, paclitaxel, docetaxel, 5-fluorouracil, gemcitabine, methotrexate, and the combination of 5-fluorouracil and methotrexate, and loaded them into the biodegradable PLGA NPs. Despite having diverse physicochemical properties (shown in fig. S3 and table S1), the different chemotherapeutic agent–loaded NPs were able to assemble onto erythrocytes (Fig. 6Opens in image viewer
为了验证 ELeCt 平台用于递送其他化疗药物的可行性，我们选择了六种常见的化疗药物或其组合，包括喜树碱、紫杉醇、多西他赛、5-氟尿嘧啶、吉西他滨、甲氨蝶呤以及 5-氟尿嘧啶与甲氨蝶呤的组合，并将它们负载到可生物降解的 PLGA 纳米颗粒中。尽管这些化疗药物具有不同的理化性质（如图 S3 和表 S1 所示），但不同化疗药物负载的纳米颗粒均能够组装到红细胞表面（图 6）。). These data supported that the biodegradable drug NP assembling onto erythrocytes approach (ELeCt) can potentially be a versatile platform to deliver selected chemotherapies to lung metastasis that originated from different primary tumors.
这些数据支持了将生物可降解药物纳米颗粒组装到红细胞上的方法（ELeCt）可能成为一种多功能的平台，用于向源自不同原发肿瘤的肺转移输送选定的化疗药物。
为了验证 ELeCt 平台用于递送其他化疗药物的可行性，我们选择了六种常见的化疗药物或其组合，包括喜树碱、紫杉醇、多西他赛、5-氟尿嘧啶、吉西他滨、甲氨蝶呤以及 5-氟尿嘧啶与甲氨蝶呤的组合，并将它们负载到可生物降解的 PLGA 纳米颗粒中。尽管这些化疗药物具有不同的理化性质（如图 S3 和表 S1 所示），但不同化疗药物负载的纳米颗粒均能够组装到红细胞表面（图 6）。). These data supported that the biodegradable drug NP assembling onto erythrocytes approach (ELeCt) can potentially be a versatile platform to deliver selected chemotherapies to lung metastasis that originated from different primary tumors.
这些数据支持了将生物可降解药物纳米颗粒组装到红细胞上的方法（ELeCt）可能成为一种多功能的平台，用于向源自不同原发肿瘤的肺转移输送选定的化疗药物。
Fig. 6 Other chemotherapeutic agent–loaded biodegradable NPs can efficiently bind to erythrocytes.
图 6. 其他载有化疗药物的可生物降解纳米颗粒也能高效结合红细胞。
图 6. 其他载有化疗药物的可生物降解纳米颗粒也能高效结合红细胞。
The tested chemotherapeutic agents include camptothecin, paclitaxel, docetaxel, 5-fluorouracil, gemcitabine, methotrexate, and the combination of 5-fluorouracil and methotrexate. Scale bars, 1 μm.
测试的化疗药物包括喜树碱、紫杉醇、多西他赛、5-氟尿嘧啶、吉西他滨、甲氨蝶呤以及 5-氟尿嘧啶与甲氨蝶呤的组合。比例尺，1 微米。
测试的化疗药物包括喜树碱、紫杉醇、多西他赛、5-氟尿嘧啶、吉西他滨、甲氨蝶呤以及 5-氟尿嘧啶与甲氨蝶呤的组合。比例尺，1 微米。
DISCUSSION 讨论
Because of its unique physiological features like high blood throughput and high density of narrow capillaries, lung is one of the major organs into which the evaded tumor cells from primary tumor sites can spread (31). Patients with advanced cancer (30 to 55%) have lung metastasis (32). Treating lung metastasis is more challenging than treating the primary tumors because it typically progresses more aggressively (33). Systemic chemotherapy is one standard treatment option for lung metastasis. However, its efficacy is usually far from desirable, attributed to its ineffective targeting and poor accumulation in the lungs. Conventional NP-mediated drug delivery also fails to achieve good localization with the desired site of action (34). Here, we report an erythrocyte hitchhiking platform, ELeCt, consisting of drug-loaded biodegradable NPs assembled on erythrocytes for promoting chemotherapy for effective lung metastasis treatment. Excellent studies have shown NPs hitchhiking on erythrocytes to accumulate in lungs, including recently in metastatic lungs (35); however, the ability of such a mechanism to yield survival benefits has not been known. To that end, we successfully demonstrate the ability of ELeCt to slow down the progression and improve the survival in early- and late-stage experimental melanoma metastasis models, resembling early detection and mid-to-late detection clinical scenarios, respectively.
由于其独特的生理特性，如高血流量和密集的狭窄毛细血管网络，肺部是肿瘤细胞从原发肿瘤部位逃逸后扩散的主要器官之一（31）。晚期癌症患者（30%至 55%）常伴有肺转移（32）。治疗肺转移比治疗原发肿瘤更具挑战性，因为其通常进展更为迅速（33）。全身化疗是治疗肺转移的标准方案之一，但其效果往往不尽如人意，主要归因于靶向性差及在肺部积累不足。传统纳米颗粒（NP）介导的药物递送同样难以实现理想的靶向定位（34）。在此，我们介绍了一种红细胞搭便车平台——ELeCt，该平台通过在红细胞表面组装载药的可生物降解纳米颗粒，以促进化疗，从而实现对肺转移的有效治疗。已有出色研究表明，纳米颗粒搭乘红细胞可显著在肺部，包括转移性肺部积累（35）；然而，这种机制是否能带来生存获益尚未明确。 为此，我们成功展示了 ELeCt 在早期和晚期实验性黑色素瘤转移模型中减缓进展和提高生存率的能力，分别模拟了临床中早期发现和中晚期发现的情景。
由于其独特的生理特性，如高血流量和密集的狭窄毛细血管网络，肺部是肿瘤细胞从原发肿瘤部位逃逸后扩散的主要器官之一（31）。晚期癌症患者（30%至 55%）常伴有肺转移（32）。治疗肺转移比治疗原发肿瘤更具挑战性，因为其通常进展更为迅速（33）。全身化疗是治疗肺转移的标准方案之一，但其效果往往不尽如人意，主要归因于靶向性差及在肺部积累不足。传统纳米颗粒（NP）介导的药物递送同样难以实现理想的靶向定位（34）。在此，我们介绍了一种红细胞搭便车平台——ELeCt，该平台通过在红细胞表面组装载药的可生物降解纳米颗粒，以促进化疗，从而实现对肺转移的有效治疗。已有出色研究表明，纳米颗粒搭乘红细胞可显著在肺部，包括转移性肺部积累（35）；然而，这种机制是否能带来生存获益尚未明确。 为此，我们成功展示了 ELeCt 在早期和晚期实验性黑色素瘤转移模型中减缓进展和提高生存率的能力，分别模拟了临床中早期发现和中晚期发现的情景。
Conventional nanomedicines use the attachment of active targeting ligands to enhance the targeted delivery of chemotherapeutic payloads (10, 11, 36–40). The ELeCt platform developed in this work exploits a completely new paradigm, taking advantage of the unique physiology of the target sites (high shear stress) and responsive dislodging of the chemotherapeutic payloads. Our in vitro drug-release data showed that the biodegradable NPs were able to have burst followed by relatively sustained drug release. Our pharmacokinetic and biodistribution data suggested that the ELeCt platform has two important features compared with the free drug and NPs alone—extended blood circulation time and improved accumulation to lung metastasis. Actually, both features are favorable for lung metastasis treatment. The extended circulation time is consistent with previous reports (27, 41). By hitchhiking to erythrocytes, NPs experience less immune recognition by the reticuloendothelial system organs, enabling them to stay in circulation for a longer time (27, 28, 41). The higher concentration of payload drug in the blood endowed by the ELeCt would allow more drug to interact with and kill the circulating tumor cells. Our in vitro shear study data evidently proved that the detachment of drug NPs from erythrocytes is shear dependent, and this is the basis for using the platform to precisely deliver payload chemotherapeutics to the target lung metastasis sites. It should be noticed that a substantial portion of the drug NPs were also detached at the low shear stress. This factor emphasized the need for investigating the surface modification of the drug NPs to modulate the binding strength of drug NPs to erythrocytes for future explorations with this technology. Our biodistribution data suggested that the biodegradable NP assembly on erythrocyte (ELeCt) platform was able to deliver a high concentration of payload chemotherapeutics to the lung metastatic sites in a short period of time. Impressively, the ELeCt platform delivered 16.6-fold more drug to the lungs bearing metastasis in 20 min compared with using the drug NPs alone. In comparison, the conventional targeted nanomedicine approach using targeting ligands can rarely achieve such high delivery enhancement (17, 42). Moreover, it usually shows a maximum tumor accumulation at a significantly longer time point (12 to 24 hours), depending on the properties of the nanomedicine (43). The quick and targeted delivery of drug NPs by the ELeCt platform would bring benefits for inhibiting tumor growth. For instance, typical nanomedicines, independent of their material origins, usually have an initial burst drug release and thus cause premature drug leakage (44), potentially attenuating the therapeutic efficacy and often leading to toxicity. The quick and targeted delivery achieved by the ELeCt platform has the potential to circumvent this issue. In addition, not unexpectedly, the lung section imaging suggested that the deposited NPs were distributed throughout the lung sections, both the inside and the outside of the lung metastatic nodules. The NPs deposited outside of the metastasis nodules have the potential to serve as a drug reservoir to release drug that can relocate to the metastatic nodules within close proximity.
传统纳米药物通过附着活性靶向配体来增强化疗有效载荷的靶向递送（10, 11, 36–40）。本工作中开发的 ELeCt 平台采用了一种全新的范式，利用目标部位（高剪切应力）的独特生理特性以及对化疗有效载荷的响应性脱落。我们的体外药物释放数据显示，可生物降解的纳米颗粒能够先爆发式释放，随后是相对持续的药物释放。药代动力学和生物分布数据表明，与游离药物和单独的纳米颗粒相比，ELeCt 平台具有两个重要特征——延长血液循环时间和增强对肺转移的积累。实际上，这两个特征均有利于肺转移的治疗。延长的循环时间与先前报道一致（27, 41）。通过搭便车到红细胞上，纳米颗粒经历的网状内皮系统器官的免疫识别较少，从而使它们能够在循环中停留更长时间（27, 28, 41）。 ELeCt 赋予血液中更高浓度的有效载荷药物，使得更多药物能够与循环中的肿瘤细胞相互作用并将其杀灭。我们的体外剪切实验数据明确证实，药物纳米颗粒从红细胞上的脱落是剪切依赖性的，这为利用该平台精确递送有效载荷化疗药物至目标肺转移部位奠定了基础。值得注意的是，在低剪切应力下也有相当一部分药物纳米颗粒发生脱落。这一因素强调了未来探索中需研究药物纳米颗粒的表面修饰，以调节其与红细胞的结合强度。我们的生物分布数据显示，红细胞上的可生物降解纳米颗粒组装（ELeCt）平台能够在短时间内将高浓度的有效载荷化疗药物递送至肺转移部位。令人印象深刻的是，与单独使用药物纳米颗粒相比，ELeCt 平台在 20 分钟内向含有转移的肺部递送的药物量增加了 16.6 倍。 相比之下，传统使用靶向配体的靶向纳米药物方法很少能达到如此高的递送增强效果（17, 42）。此外，它通常在显著更长的时间点（12 至 24 小时）表现出最大肿瘤积累，这取决于纳米药物的性质（43）。ELeCt 平台实现的快速且靶向的药物 NPs 递送将为抑制肿瘤生长带来益处。例如，典型的纳米药物，无论其材料来源如何，通常都会经历初始的药物爆发释放，从而导致过早的药物泄漏（44），这可能会削弱治疗效果并常常引发毒性。ELeCt 平台实现的快速且靶向的递送有望规避这一问题。此外，不出所料，肺切片成像显示沉积的 NPs 分布于整个肺切片，包括肺转移结节的内部和外部。沉积在转移结节外部的 NPs 有可能作为药物储库，释放的药物能够重新定位到邻近的转移结节。
传统纳米药物通过附着活性靶向配体来增强化疗有效载荷的靶向递送（10, 11, 36–40）。本工作中开发的 ELeCt 平台采用了一种全新的范式，利用目标部位（高剪切应力）的独特生理特性以及对化疗有效载荷的响应性脱落。我们的体外药物释放数据显示，可生物降解的纳米颗粒能够先爆发式释放，随后是相对持续的药物释放。药代动力学和生物分布数据表明，与游离药物和单独的纳米颗粒相比，ELeCt 平台具有两个重要特征——延长血液循环时间和增强对肺转移的积累。实际上，这两个特征均有利于肺转移的治疗。延长的循环时间与先前报道一致（27, 41）。通过搭便车到红细胞上，纳米颗粒经历的网状内皮系统器官的免疫识别较少，从而使它们能够在循环中停留更长时间（27, 28, 41）。 ELeCt 赋予血液中更高浓度的有效载荷药物，使得更多药物能够与循环中的肿瘤细胞相互作用并将其杀灭。我们的体外剪切实验数据明确证实，药物纳米颗粒从红细胞上的脱落是剪切依赖性的，这为利用该平台精确递送有效载荷化疗药物至目标肺转移部位奠定了基础。值得注意的是，在低剪切应力下也有相当一部分药物纳米颗粒发生脱落。这一因素强调了未来探索中需研究药物纳米颗粒的表面修饰，以调节其与红细胞的结合强度。我们的生物分布数据显示，红细胞上的可生物降解纳米颗粒组装（ELeCt）平台能够在短时间内将高浓度的有效载荷化疗药物递送至肺转移部位。令人印象深刻的是，与单独使用药物纳米颗粒相比，ELeCt 平台在 20 分钟内向含有转移的肺部递送的药物量增加了 16.6 倍。 相比之下，传统使用靶向配体的靶向纳米药物方法很少能达到如此高的递送增强效果（17, 42）。此外，它通常在显著更长的时间点（12 至 24 小时）表现出最大肿瘤积累，这取决于纳米药物的性质（43）。ELeCt 平台实现的快速且靶向的药物 NPs 递送将为抑制肿瘤生长带来益处。例如，典型的纳米药物，无论其材料来源如何，通常都会经历初始的药物爆发释放，从而导致过早的药物泄漏（44），这可能会削弱治疗效果并常常引发毒性。ELeCt 平台实现的快速且靶向的递送有望规避这一问题。此外，不出所料，肺切片成像显示沉积的 NPs 分布于整个肺切片，包括肺转移结节的内部和外部。沉积在转移结节外部的 NPs 有可能作为药物储库，释放的药物能够重新定位到邻近的转移结节。
Our in vivo efficacy data suggested that the enhanced and targeted delivery of chemotherapeutics by the ELeCt platform could bring benefits for inhibiting both the early-stage and the late-stage lung metastasis growth. In the early-stage lung metastasis model, the treatments using free drug or drug NPs alone exhibited some slowdown of the progression of lung metastasis. However, their antimetastatic efficacy was not potent enough to significantly extend the animal survival. In comparison, the ELeCt platform was able to provide a 100- to 300-fold better antimetastatic efficacy compared with using the free drug or drug NPs alone. Its improved antimetastatic efficacy led to a significantly extended animal survival, extending the median survival time of mice bearing lung metastasis by 32 days, compared with the control group. The data suggested that the ELeCt platform has the potential to enable chemotherapy for effective treatment of early-stage lung metastasis. In the late-stage metastasis model, the administration of drug NPs alone failed to significantly inhibit the lung metastasis growth and to improve the survival time. The ELeCt platform was able to significantly slow down the lung metastasis progression and modestly improved animal survival. Evidently, the antimetastatic efficacy of the therapies is closely related to the start time of the therapies. The efficacy of the developed therapies to treat in an even later-stage lung metastasis has not been shown yet. In addition, future studies may also need to be done to unveil the effect of drug dose and schedule of the therapies on their antimetastatic efficacy.
我们的体内疗效数据显示，ELeCt 平台增强并靶向输送化疗药物，可为抑制早期和晚期肺转移生长带来益处。在早期肺转移模型中，单独使用游离药物或药物纳米颗粒的治疗显示出对肺转移进展的某种减缓，但其抗转移效果不足以显著延长动物生存期。相比之下，ELeCt 平台提供的抗转移效果比单独使用游离药物或药物纳米颗粒高出 100 至 300 倍。其增强的抗转移效果显著延长了动物的生存期，与对照组相比，携带肺转移的小鼠中位生存时间延长了 32 天。数据显示，ELeCt 平台有潜力实现对早期肺转移的有效化疗治疗。在晚期转移模型中，单独使用药物纳米颗粒未能显著抑制肺转移生长或改善生存时间。 ELeCt 平台能够显著减缓肺转移的进展，并适度提高动物的生存率。显然，这些疗法的抗转移效果与其开始时间密切相关。目前尚未展示所开发疗法在更晚期肺转移治疗中的效果。此外，未来的研究可能还需要揭示药物剂量和治疗方案对这些疗法抗转移效果的影响。
我们的体内疗效数据显示，ELeCt 平台增强并靶向输送化疗药物，可为抑制早期和晚期肺转移生长带来益处。在早期肺转移模型中，单独使用游离药物或药物纳米颗粒的治疗显示出对肺转移进展的某种减缓，但其抗转移效果不足以显著延长动物生存期。相比之下，ELeCt 平台提供的抗转移效果比单独使用游离药物或药物纳米颗粒高出 100 至 300 倍。其增强的抗转移效果显著延长了动物的生存期，与对照组相比，携带肺转移的小鼠中位生存时间延长了 32 天。数据显示，ELeCt 平台有潜力实现对早期肺转移的有效化疗治疗。在晚期转移模型中，单独使用药物纳米颗粒未能显著抑制肺转移生长或改善生存时间。 ELeCt 平台能够显著减缓肺转移的进展，并适度提高动物的生存率。显然，这些疗法的抗转移效果与其开始时间密切相关。目前尚未展示所开发疗法在更晚期肺转移治疗中的效果。此外，未来的研究可能还需要揭示药物剂量和治疗方案对这些疗法抗转移效果的影响。
The exact mechanism of the drug-loaded biodegradable NP assembling on erythrocytes is not clear. Previous studies from our laboratory and others have attributed the assembly of NPs to erythrocytes to the noncovalent interactions such as electrostatic interactions, hydrophobic interactions, and H-bonds between the polymeric NPs and domains on the red blood cell (RBC) membrane (27, 28, 35). The assembly is most likely a result of balance between surface tension forces caused by the NP-induced membrane stretching and the noncovalent interactions between the cell membrane and NPs. The balance of the two factors drives stable assembly of the particles onto erythrocytes (27). However, details of this mechanism need future investigation. Our drug NP binding data suggested that the model drug–loaded NPs, in this case, DOX, could assemble onto the mouse erythrocytes at a very high binding efficiency. This feature is critical for making the ELeCt platform work. The number of erythrocytes that can be administered has an upper limit, and only having a high drug dose on individual erythrocytes can achieve the therapeutic concentration of chemotherapeutics. In addition, our data also suggested that the drug dose on erythrocytes could be tuned by changing the feed incubation ratios of drug NPs to erythrocytes, thus providing the possibility of changing drug dosage according to specific lung metastasis conditions. Other than DOX, we were able to load different commonly used chemotherapeutic agents or their combinations to the biodegradable NPs. Moreover, these drug-loaded NPs could assemble onto the mouse erythrocytes as well. This opens a new window to use the ELeCt platform to treat lung metastasis originating from different primary sites. Lung metastasis can have different primary tumor origins like breast cancer, bladder cancer, melanoma, and many others. The metastasis derived from different origins is preferably treated by specific chemotherapeutic agents (45, 46). The ELeCt platform has the potential to be a versatile platform to treat different lung metastasis by loading optimal chemotherapeutic agents according to their primary tumor origins. The impact of the chemotherapeutics’ properties on the performance of the ELeCt platform should be further investigated in future studies. Our data also suggested that the drug-loaded biodegradable NPs efficiently assembled onto human erythrocytes and were detached from them under lung-corresponding shear stress. In addition, the material used to prepare the biodegradable NPs (PLGA) is part of several FDA-approved products (47). Therefore, this platform technology has a translational potential. However, this needs to be explored further in the future.
药物负载的可生物降解纳米颗粒在红细胞上组装的精确机制尚不明确。我们实验室及其他研究先前的研究表明，纳米颗粒与红细胞的组装归因于聚合物纳米颗粒与红细胞膜域之间的非共价相互作用，如静电相互作用、疏水相互作用及氢键（27, 28, 35）。这种组装很可能是纳米颗粒引起的膜拉伸所产生的表面张力与细胞膜与纳米颗粒之间非共价相互作用之间平衡的结果。这两种因素的平衡促使颗粒在红细胞上形成稳定的组装（27）。然而，该机制的细节仍需未来研究揭示。我们的药物纳米颗粒结合数据显示，在此情况下，模型药物多柔比星（DOX）负载的纳米颗粒能够以极高的结合效率组装到小鼠红细胞上。这一特性对于 ELeCt 平台的运作至关重要。可施用的红细胞数量存在上限，唯有在单个红细胞上实现高药物剂量，才能达到化疗药物的治疗浓度。 此外，我们的数据还表明，通过调整药物纳米颗粒与红细胞的进料孵育比例，可以调节红细胞上的药物剂量，从而根据特定的肺转移情况调整药物剂量。除了阿霉素（DOX）外，我们还能够将不同的常用化疗药物或其组合加载到可生物降解的纳米颗粒上。此外，这些载药纳米颗粒同样能够组装到小鼠红细胞表面。这为利用 ELeCt 平台治疗源自不同原发部位的肺转移开辟了新的途径。肺转移可能源自乳腺癌、膀胱癌、黑色素瘤等多种原发肿瘤。不同来源的转移通常需要特定的化疗药物进行最佳治疗（45, 46）。ELeCt 平台具有成为多功能平台以治疗不同肺转移的潜力，能够根据其原发肿瘤类型加载最优的化疗药物。未来研究应进一步探讨化疗药物性质对 ELeCt 平台性能的影响。 我们的数据还表明，载药的可生物降解纳米颗粒能够有效地组装在人红细胞上，并在肺部相应的剪切应力下从其表面脱落。此外，用于制备这些可生物降解纳米颗粒的材料（PLGA）是多个 FDA 批准产品的一部分（47）。因此，这一平台技术具有转化潜力。然而，这需要在未来的研究中进一步探索。
药物负载的可生物降解纳米颗粒在红细胞上组装的精确机制尚不明确。我们实验室及其他研究先前的研究表明，纳米颗粒与红细胞的组装归因于聚合物纳米颗粒与红细胞膜域之间的非共价相互作用，如静电相互作用、疏水相互作用及氢键（27, 28, 35）。这种组装很可能是纳米颗粒引起的膜拉伸所产生的表面张力与细胞膜与纳米颗粒之间非共价相互作用之间平衡的结果。这两种因素的平衡促使颗粒在红细胞上形成稳定的组装（27）。然而，该机制的细节仍需未来研究揭示。我们的药物纳米颗粒结合数据显示，在此情况下，模型药物多柔比星（DOX）负载的纳米颗粒能够以极高的结合效率组装到小鼠红细胞上。这一特性对于 ELeCt 平台的运作至关重要。可施用的红细胞数量存在上限，唯有在单个红细胞上实现高药物剂量，才能达到化疗药物的治疗浓度。 此外，我们的数据还表明，通过调整药物纳米颗粒与红细胞的进料孵育比例，可以调节红细胞上的药物剂量，从而根据特定的肺转移情况调整药物剂量。除了阿霉素（DOX）外，我们还能够将不同的常用化疗药物或其组合加载到可生物降解的纳米颗粒上。此外，这些载药纳米颗粒同样能够组装到小鼠红细胞表面。这为利用 ELeCt 平台治疗源自不同原发部位的肺转移开辟了新的途径。肺转移可能源自乳腺癌、膀胱癌、黑色素瘤等多种原发肿瘤。不同来源的转移通常需要特定的化疗药物进行最佳治疗（45, 46）。ELeCt 平台具有成为多功能平台以治疗不同肺转移的潜力，能够根据其原发肿瘤类型加载最优的化疗药物。未来研究应进一步探讨化疗药物性质对 ELeCt 平台性能的影响。 我们的数据还表明，载药的可生物降解纳米颗粒能够有效地组装在人红细胞上，并在肺部相应的剪切应力下从其表面脱落。此外，用于制备这些可生物降解纳米颗粒的材料（PLGA）是多个 FDA 批准产品的一部分（47）。因此，这一平台技术具有转化潜力。然而，这需要在未来的研究中进一步探索。
In summary, the ELeCt platform, drug-loaded biodegradable NP assembling on erythrocyte, was developed, which enables lung physiology–assisted shear-responsive targeted delivery of chemotherapeutic agents to treat lung metastasis. The drug NPs assembled on erythrocytes could be precisely dislodged in the lungs bearing metastasis in response to the intrinsic mechanical high shear stress. Various commonly used chemotherapeutic agents could be loaded into the biodegradable NPs and further made to successfully assemble onto the erythrocytes. This platform successfully delivered one-order-of-magnitude-higher content of the model drug (DOX) to the diseased lungs as compared with using the NPs alone. This platform enabled chemotherapy to effectively inhibit lung metastasis growth and significantly improve the survival. All in all, the ELeCt platform can be a versatile strategy to treat lung metastasis originating from different primary tumors, with a strong translational potential.
总之，ELeCt 平台——即药物负载的可生物降解纳米颗粒在红细胞表面组装——已成功研发，该平台实现了肺部生理辅助下的剪切应力响应型靶向递送化疗药物，以治疗肺转移。组装在红细胞上的药物纳米颗粒能够在承受高机械剪切应力的转移性肺部精确释放。多种常用化疗药物均可负载于这些可生物降解的纳米颗粒中，并成功组装到红细胞表面。与单独使用纳米颗粒相比，该平台将模型药物（DOX）递送至病变肺部的含量提高了整整一个数量级。这一平台使得化疗能够有效抑制肺转移的生长，并显著提升生存率。综上所述，ELeCt 平台可作为一种多用途策略，用于治疗源自不同原发肿瘤的肺转移，具有强大的转化应用潜力。
总之，ELeCt 平台——即药物负载的可生物降解纳米颗粒在红细胞表面组装——已成功研发，该平台实现了肺部生理辅助下的剪切应力响应型靶向递送化疗药物，以治疗肺转移。组装在红细胞上的药物纳米颗粒能够在承受高机械剪切应力的转移性肺部精确释放。多种常用化疗药物均可负载于这些可生物降解的纳米颗粒中，并成功组装到红细胞表面。与单独使用纳米颗粒相比，该平台将模型药物（DOX）递送至病变肺部的含量提高了整整一个数量级。这一平台使得化疗能够有效抑制肺转移的生长，并显著提升生存率。综上所述，ELeCt 平台可作为一种多用途策略，用于治疗源自不同原发肿瘤的肺转移，具有强大的转化应用潜力。
MATERIALS AND METHODS 材料与方法
Nanoparticle preparation and characterization
纳米颗粒的制备与表征
PLGA NPs encapsulating DOX were prepared using a nanoprecipitation method. Briefly, 5 mg of DOX was dissolved in 500 μl of methanol and 5 μl of triethylamine. This was added to 1 ml of acetone containing 20 mg of PLGA. The mixture was then injected into 10 ml of 1% polyvinyl alcohol solution under constant stirring using a syringe pump at 1 ml/min. The particles were kept under constant stirring overnight before removing the organic solvents using rotary evaporation. The formed particles were centrifuged at 12,000g for 15 min, and the supernatant was analyzed to quantify drug loading. The particles were then resuspended in deionized water and assessed for their size, zeta potential, and polydispersity index using dynamic light scattering (Malvern Zen3600) and SEM (Zeiss FESEM Supra 55VP, Zeiss FESEM Ultra 55). The NPs were washed for a total of two washes with deionized water before their final resuspension in phosphate-buffered saline (PBS). Nanoparticles containing other chemotherapeutic drugs were prepared using the similar nanoprecipitation technique described above with minor modifications (details are shown in the Supplementary Materials).
采用纳米沉淀法制备了包封 DOX 的 PLGA 纳米颗粒。简言之，将 5 mg DOX 溶于 500 μl 甲醇和 5 μl 三乙胺中，随后加入含有 20 mg PLGA 的 1 ml 丙酮溶液。混合物通过注射泵以 1 ml/min 的速度持续搅拌下注入 10 ml 1%聚乙烯醇溶液中。颗粒在持续搅拌下静置过夜，之后通过旋转蒸发去除有机溶剂。形成的颗粒在 12,000g 下离心 15 分钟，上清液用于定量药物负载。随后，颗粒在去离子水中重新悬浮，并通过动态光散射（Malvern Zen3600）和扫描电子显微镜（Zeiss FESEM Supra 55VP，Zeiss FESEM Ultra 55）评估其粒径、zeta 电位和多分散性指数。在进行最终的磷酸盐缓冲液（PBS）重悬之前，纳米颗粒用去离子水洗涤两次。其他化疗药物的纳米颗粒则采用上述类似的纳米沉淀技术制备，仅作细微调整（详见补充材料）。
采用纳米沉淀法制备了包封 DOX 的 PLGA 纳米颗粒。简言之，将 5 mg DOX 溶于 500 μl 甲醇和 5 μl 三乙胺中，随后加入含有 20 mg PLGA 的 1 ml 丙酮溶液。混合物通过注射泵以 1 ml/min 的速度持续搅拌下注入 10 ml 1%聚乙烯醇溶液中。颗粒在持续搅拌下静置过夜，之后通过旋转蒸发去除有机溶剂。形成的颗粒在 12,000g 下离心 15 分钟，上清液用于定量药物负载。随后，颗粒在去离子水中重新悬浮，并通过动态光散射（Malvern Zen3600）和扫描电子显微镜（Zeiss FESEM Supra 55VP，Zeiss FESEM Ultra 55）评估其粒径、zeta 电位和多分散性指数。在进行最终的磷酸盐缓冲液（PBS）重悬之前，纳米颗粒用去离子水洗涤两次。其他化疗药物的纳米颗粒则采用上述类似的纳米沉淀技术制备，仅作细微调整（详见补充材料）。
Blood collection and processing
血液采集与处理
Murine whole blood was collected via cardiac puncture using a heparin precoated syringe and stored in BD Microtainer blood collection tubes prior to use. Whole blood was centrifuged at 1000g for 10 min at 4°C to remove the serum and the buffy coat layers from the erythrocyte compartment. The isolated erythrocytes were further washed three times with cold PBS and centrifuged at 650g for 15 min at 4°C before their final resuspension at a concentration of 10% hematocrit in PBS (erythrocyte stock solution). Human whole blood obtained from BioIVT (NY, USA) was processed and stored using the same procedure as murine blood. Freshly processed erythrocytes were used for every experiment in this study.
小鼠全血通过使用肝素预涂层的注射器进行心脏穿刺采集，并储存在 BD Microtainer 血收集管中备用。全血在 4°C 下以 1000g 的离心力离心 10 分钟，以去除血清和白细胞层，从而分离出红细胞部分。分离的红细胞再用冷磷酸盐缓冲液（PBS）洗涤三次，并在 4°C 下以 650g 的离心力离心 15 分钟，最终以 10%血细胞比容的浓度重新悬浮于 PBS 中（红细胞储备液）。从 BioIVT（纽约，美国）获得的人全血采用与小鼠血液相同的程序进行处理和储存。本研究中每次实验均使用新鲜处理的红细胞。
小鼠全血通过使用肝素预涂层的注射器进行心脏穿刺采集，并储存在 BD Microtainer 血收集管中备用。全血在 4°C 下以 1000g 的离心力离心 10 分钟，以去除血清和白细胞层，从而分离出红细胞部分。分离的红细胞再用冷磷酸盐缓冲液（PBS）洗涤三次，并在 4°C 下以 650g 的离心力离心 15 分钟，最终以 10%血细胞比容的浓度重新悬浮于 PBS 中（红细胞储备液）。从 BioIVT（纽约，美国）获得的人全血采用与小鼠血液相同的程序进行处理和储存。本研究中每次实验均使用新鲜处理的红细胞。
Assembly of drug NPs to erythrocytes and characterization
药物纳米颗粒在红细胞上的组装及其特性研究
Equal volumes of erythrocyte stock solution and drug NP suspension were mixed in Axygen 1.5-ml Self-Standing Screw Cap Tubes and further thoroughly mixed by inversion and pipetting. The tubes were then allowed to rotate on a tube revolver (Thermo Fisher Scientific) for 40 min. The hitchhiked erythrocytes were then pelleted by centrifugation at 100g for 5 min at 4°C, unabsorbed particles were carefully removed, and the pellet was washed again with 1 ml of 1× PBS to remove loosely bound particles. The hitchhiked erythrocytes were finally resuspended at 10% (v/v) in 1× PBS and used for further characterization or in vivo studies.
等体积的红细胞储备液与药物纳米颗粒悬液在 Axygen 1.5 毫升自立式螺旋盖管中混合，并通过倒置和移液进一步充分混合。随后，将试管置于转管器（赛默飞世尔科技）上旋转 40 分钟。搭便车的红细胞通过在 4°C 下以 100g 离心 5 分钟进行沉淀，小心去除未吸收的颗粒，然后用 1 毫升 1×PBS 洗涤沉淀物，以去除松散结合的颗粒。最终，搭便车的红细胞以 10%（体积/体积）的浓度重新悬浮于 1×PBS 中，用于进一步的特性分析或体内研究。
等体积的红细胞储备液与药物纳米颗粒悬液在 Axygen 1.5 毫升自立式螺旋盖管中混合，并通过倒置和移液进一步充分混合。随后，将试管置于转管器（赛默飞世尔科技）上旋转 40 分钟。搭便车的红细胞通过在 4°C 下以 100g 离心 5 分钟进行沉淀，小心去除未吸收的颗粒，然后用 1 毫升 1×PBS 洗涤沉淀物，以去除松散结合的颗粒。最终，搭便车的红细胞以 10%（体积/体积）的浓度重新悬浮于 1×PBS 中，用于进一步的特性分析或体内研究。
Hitchhiking efficiency and the drug loading on erythrocytes were determined using fluorescence measurements. For quantification using fluorescence, 25 μl of erythrocytes was lysed using deionized water, and the drug content was quantified using DOX fluorescence [excitation (Ex)/emission (Em), 470/590 nm] on a plate reader (Tecan Safire 2, NC, USA). The percentage of erythrocytes carrying NPs for different NP-to-erythrocyte ratios was determined using flow cytometry (BD LSR Analyzer II, CA, USA) using DOX fluorescence (Em/Ex, 470/590 nm) and confirmed by confocal microscopy (Upright Zeiss LSM 710 NLO ready, Germany). Nanoparticle assembly to erythrocytes was confirmed using SEM (Zeiss FESEM Supra 55VP, Zeiss FESEM Ultra 55). Briefly, the hitchhiked erythrocytes were fixed using 2.5% glutaraldehyde solution and washed in an increasing ethanol gradient before being chemically dried using hexamethyldisilazane. Last, the samples were sputter coated (EMT 150T ES metal sputter coater, PA, USA) prior to imaging.
搭便车效率及药物在红细胞上的负载量通过荧光测量法确定。为进行荧光定量分析，取 25 μl 红细胞用去离子水裂解，并利用 DOX 荧光[激发(Ex)/发射(Em)，470/590 nm]在板读仪（Tecan Safire 2，NC，USA）上定量药物含量。不同纳米颗粒与红细胞比例下携带纳米颗粒的红细胞百分比通过流式细胞术（BD LSR Analyzer II，CA，USA）利用 DOX 荧光（Em/Ex，470/590 nm）测定，并由共聚焦显微镜（Upright Zeiss LSM 710 NLO ready，德国）确认。纳米颗粒在红细胞上的组装通过 SEM（Zeiss FESEM Supra 55VP，Zeiss FESEM Ultra 55）确认。简而言之，搭便车的红细胞先用 2.5%戊二醛溶液固定，在递增乙醇梯度中洗涤后，用六甲基二硅氮烷化学干燥。最后，样品在成像前进行喷金处理（EMT 150T ES 金属喷镀仪，PA，USA）。
搭便车效率及药物在红细胞上的负载量通过荧光测量法确定。为进行荧光定量分析，取 25 μl 红细胞用去离子水裂解，并利用 DOX 荧光[激发(Ex)/发射(Em)，470/590 nm]在板读仪（Tecan Safire 2，NC，USA）上定量药物含量。不同纳米颗粒与红细胞比例下携带纳米颗粒的红细胞百分比通过流式细胞术（BD LSR Analyzer II，CA，USA）利用 DOX 荧光（Em/Ex，470/590 nm）测定，并由共聚焦显微镜（Upright Zeiss LSM 710 NLO ready，德国）确认。纳米颗粒在红细胞上的组装通过 SEM（Zeiss FESEM Supra 55VP，Zeiss FESEM Ultra 55）确认。简而言之，搭便车的红细胞先用 2.5%戊二醛溶液固定，在递增乙醇梯度中洗涤后，用六甲基二硅氮烷化学干燥。最后，样品在成像前进行喷金处理（EMT 150T ES 金属喷镀仪，PA，USA）。
In vitro serum stability and shear studies
体外血清稳定性和剪切力研究
For serum stability studies, hitchhiked murine and human erythrocytes were incubated in 1 ml of fetal bovine serum (FBS) or human serum (from BioIVT) on a tube revolver at 12 rpm at 37°C. These conditions simulate low shear physiological environment. After incubation for 20 min, the cells were pelleted by centrifugation at 250g for 5 min and resuspended to 10% (v/v) in 1× PBS. Twenty-five microliters of erythrocytes was then lysed using deionized water, and the remaining drug content was quantified using DOX fluorescence (Ex/Em, 470/590 nm) on a plate reader (Tecan Safire 2).
为了进行血清稳定性研究，搭载的鼠源和人源红细胞在 1 毫升胎牛血清（FBS）或人血清（来自 BioIVT）中，于 37°C 下以 12 转/分的转速在试管旋转器上孵育。这些条件模拟了低剪切生理环境。孵育 20 分钟后，细胞通过 250g 离心 5 分钟沉淀，并重新悬浮至 10%（体积/体积）的 1×PBS 中。随后，取 25 微升红细胞用去离子水裂解，剩余药物含量通过 DOX 荧光（激发/发射波长，470/590 nm）在板读数仪（Tecan Safire 2）上定量。
为了进行血清稳定性研究，搭载的鼠源和人源红细胞在 1 毫升胎牛血清（FBS）或人血清（来自 BioIVT）中，于 37°C 下以 12 转/分的转速在试管旋转器上孵育。这些条件模拟了低剪切生理环境。孵育 20 分钟后，细胞通过 250g 离心 5 分钟沉淀，并重新悬浮至 10%（体积/体积）的 1×PBS 中。随后，取 25 微升红细胞用去离子水裂解，剩余药物含量通过 DOX 荧光（激发/发射波长，470/590 nm）在板读数仪（Tecan Safire 2）上定量。
For shear studies, hitchhiked murine and human erythrocytes were incubated in 10 ml of FBS or human serum. A rotatory shear (6 Pa) was applied to erythrocytes in serum using a cylindrical coquette viscometer (1 mm gap, AR-G2 rheometer, TA instruments, DE, USA) for 20 min. The samples were maintained at 37°C during the application of shear using a water jacket. These conditions simulate lung-corresponding high shear physiological environment. After 20 min, the cells were pelleted by centrifugation at 250g for 10 min and resuspended to 10% (v/v) in 1× PBS. Twenty-five microliters of erythrocytes was then lysed using deionized water, and the remaining drug content was quantified using DOX fluorescence (Ex/Em, 470/590 nm) on a plate reader (Tecan Safire 2).
在剪切研究中，搭载的鼠源和人源红细胞在 10 毫升胎牛血清或人血清中进行孵育。通过圆筒形库埃特粘度计（间隙 1 毫米，AR-G2 流变仪，TA 仪器，DE，美国）在血清中对红细胞施加旋转剪切力（6 帕），持续 20 分钟。利用水套将样品在剪切力施加过程中维持在 37°C。这些条件模拟了与肺部相应的高剪切生理环境。20 分钟后，细胞通过 250g 离心 10 分钟进行沉淀，并重新悬浮至 10%（体积/体积）的 1×PBS 中。随后，取 25 微升红细胞用去离子水裂解，剩余药物含量通过 DOX 荧光（激发/发射波长，470/590 纳米）在板读数仪（Tecan Safire 2）上定量。
在剪切研究中，搭载的鼠源和人源红细胞在 10 毫升胎牛血清或人血清中进行孵育。通过圆筒形库埃特粘度计（间隙 1 毫米，AR-G2 流变仪，TA 仪器，DE，美国）在血清中对红细胞施加旋转剪切力（6 帕），持续 20 分钟。利用水套将样品在剪切力施加过程中维持在 37°C。这些条件模拟了与肺部相应的高剪切生理环境。20 分钟后，细胞通过 250g 离心 10 分钟进行沉淀，并重新悬浮至 10%（体积/体积）的 1×PBS 中。随后，取 25 微升红细胞用去离子水裂解，剩余药物含量通过 DOX 荧光（激发/发射波长，470/590 纳米）在板读数仪（Tecan Safire 2）上定量。
Animals 动物
Female C57BL/6 mice (7 to 9 weeks of age) were purchased from Charles River Laboratories (MA, USA). All experiments were performed according to the approved protocols by the Institutional Animal Care and Use Committee of the Faculty of Arts and Sciences, Harvard University, Cambridge.
雌性 C57BL/6 小鼠（7 至 9 周龄）购自查尔斯河实验室（美国马萨诸塞州）。所有实验均按照哈佛大学文理学院动物管理和使用委员会批准的协议进行。
雌性 C57BL/6 小鼠（7 至 9 周龄）购自查尔斯河实验室（美国马萨诸塞州）。所有实验均按照哈佛大学文理学院动物管理和使用委员会批准的协议进行。
In vivo pharmacokinetics and biodistribution studies
体内药代动力学和生物分布研究
For the pharmacokinetics study, healthy female C57BL/6 mice were used. Free DOX, DOX-loaded NPs, and drug NPs assembled on erythrocytes (RBC-NPs) (n = 3 for all groups) were injected intravenously into the tail vein at a dose of 5.2 mg/kg. Blood samples were collected from the mice by submandibular bleed at 2 min, 15 min, 30 min, 2 hours, and 5 hours after the injection. The plasma was separated from the cellular component by centrifuging at 5000 rpm for 10 min. DOX was extracted from both the compartments (30 μl) using 150 μl of acetonitrile. The drug content was quantified using reversed-phase liquid chromatography–mass spectroscopy (LC-MS; Agilent 1290/6140 UHPLC, CA, USA) ran through an Agilent C-18 column (Poroshell 120, EC-C18, 3.0 mm by 100 mm, 2.7 μm) using a gradient mobile solvent.
在药代动力学研究中，使用了健康的雌性 C57BL/6 小鼠。自由 DOX、载 DOX 的纳米颗粒以及组装在红细胞表面的药物纳米颗粒（RBC-NPs）（各组 n=3）通过尾静脉以 5.2 mg/kg 的剂量静脉注射。在注射后 2 分钟、15 分钟、30 分钟、2 小时和 5 小时，通过下颌下出血从老鼠采集血液样本。通过以 5000 rpm 离心 10 分钟，将血浆与细胞成分分离。使用 150 微升的乙腈从两个部分（30 微升）中提取 DOX。使用反相液相色谱-质谱法（LC-MS；Agilent 1290/6140 UHPLC，CA，USA）对药物含量进行定量分析，该方法通过 Agilent C-18 柱（Poroshell 120，EC-C18，3.0 mm×100 mm，2.7 μm）进行梯度移动溶剂。
在药代动力学研究中，使用了健康的雌性 C57BL/6 小鼠。自由 DOX、载 DOX 的纳米颗粒以及组装在红细胞表面的药物纳米颗粒（RBC-NPs）（各组 n=3）通过尾静脉以 5.2 mg/kg 的剂量静脉注射。在注射后 2 分钟、15 分钟、30 分钟、2 小时和 5 小时，通过下颌下出血从老鼠采集血液样本。通过以 5000 rpm 离心 10 分钟，将血浆与细胞成分分离。使用 150 微升的乙腈从两个部分（30 微升）中提取 DOX。使用反相液相色谱-质谱法（LC-MS；Agilent 1290/6140 UHPLC，CA，USA）对药物含量进行定量分析，该方法通过 Agilent C-18 柱（Poroshell 120，EC-C18，3.0 mm×100 mm，2.7 μm）进行梯度移动溶剂。
For the biodistribution studies, 1 × 105 B16F10-Luc cells were injected intravenously into the tail vein of female C57BL/6 mice. Fourteen days after inoculation, mice were intravenously injected with free DOX, DOX-loaded NPs, and drug NPs assembled on erythrocytes (RBC-NPs) (n = 3 for all groups) into the tail vein at a dose of 5.2 mg/kg. Mice were euthanized at 20 min and 6 hours after the injection, and organs were harvested for further processing. Organs were rinsed using cold PBS three times to remove the residual blood. One milliliter of cold deionized water was added to each organ, and the organs were homogenized using a high shear homogenizer (IKA T 10 Basic ULTRA-TURRAX, NC, USA). DOX was extracted from the homogenates using acetonitrile (1:4 homogenate:acetonitrile), and the drug content was quantified using DOX fluorescence (Em/Ex, 470/590 nm) on a plate reader (Tecan Safire 2). The data are expressed as drug content (micrograms) normalized to the organ weight.
为了进行生物分布研究，将 1 × 10^6 个 B16F10-Luc 细胞通过尾静脉注射到雌性 C57BL/6 小鼠体内。接种后 14 天，小鼠通过尾静脉分别注射游离 DOX、载 DOX 纳米颗粒（NPs）以及装配在红细胞表面的药物纳米颗粒（RBC-NPs），剂量为 5.2 mg/kg（每组 n=3）。注射后 20 分钟和 6 小时，小鼠被安乐死，器官被采集以进行进一步处理。器官用冷 PBS 冲洗三次以去除残留血液。向每个器官中加入 1 毫升冷去离子水，并使用高剪切均质器（IKA T 10 Basic ULTRA-TURRAX，NC，USA）进行均质化处理。通过乙腈（均质物：乙腈为 1:4）从均质物中提取 DOX，并使用 DOX 荧光（Em/Ex，470/590 nm）在板读数器（Tecan Safire 2）上定量药物含量。数据以药物含量（微克）与器官重量的比值表示。
为了进行生物分布研究，将 1 × 10^6 个 B16F10-Luc 细胞通过尾静脉注射到雌性 C57BL/6 小鼠体内。接种后 14 天，小鼠通过尾静脉分别注射游离 DOX、载 DOX 纳米颗粒（NPs）以及装配在红细胞表面的药物纳米颗粒（RBC-NPs），剂量为 5.2 mg/kg（每组 n=3）。注射后 20 分钟和 6 小时，小鼠被安乐死，器官被采集以进行进一步处理。器官用冷 PBS 冲洗三次以去除残留血液。向每个器官中加入 1 毫升冷去离子水，并使用高剪切均质器（IKA T 10 Basic ULTRA-TURRAX，NC，USA）进行均质化处理。通过乙腈（均质物：乙腈为 1:4）从均质物中提取 DOX，并使用 DOX 荧光（Em/Ex，470/590 nm）在板读数器（Tecan Safire 2）上定量药物含量。数据以药物含量（微克）与器官重量的比值表示。
For NP distribution within the diseased lungs, 1 × 105 B16F10-Luc cells were injected intravenously into the tail vein of female C57BL/6 mice. Twenty-eight days after inoculation, mice were injected with DOX-loaded NPs and drug NPs assembled on erythrocytes (RBC-NPs). Twenty minutes after the injection, the mice were euthanized, and the intact lungs were collected. Lungs were washed twice with cold 1× PBS before being fixed in a 4% paraformaldehyde solution overnight. The fixed lungs were then frozen in Tissue-Tek OCT compound (Sakura Finetek) and sectioned using a cryostat (Leica CM1950, IL, USA). The sectioned tissue was mounted using Fluroshield to stain for DAPI (4′,6-diamidino-2-phenylindole) (Ex/Em, 340/488 nm) and was analyzed using a confocal microscope (Upright Zeiss LSM 710 NLO ready).
为了研究纳米颗粒（NP）在患病肺部的分布情况，将 1 × 10^6 个 B16F10-Luc 细胞通过尾静脉注射到雌性 C57BL/6 小鼠体内。接种后 28 天，向小鼠注射载有 DOX 的纳米颗粒以及组装在红细胞表面的药物纳米颗粒（RBC-NPs）。注射后 20 分钟，小鼠被安乐死，并收集完整的肺部。肺部先用冷 PBS 洗涤两次，然后置于 4%多聚甲醛溶液中过夜固定。固定后的肺部样本在 Tissue-Tek OCT 复合物（Sakura Finetek）中冷冻，并使用冷冻切片机（Leica CM1950，IL，美国）进行切片。切片组织用 Fluroshield 封片，进行 DAPI（4′,6-二脒基-2-苯基吲哚）染色（Ex/Em，340/488 nm），并通过共聚焦显微镜（Upright Zeiss LSM 710 NLO ready）进行分析。
为了研究纳米颗粒（NP）在患病肺部的分布情况，将 1 × 10^6 个 B16F10-Luc 细胞通过尾静脉注射到雌性 C57BL/6 小鼠体内。接种后 28 天，向小鼠注射载有 DOX 的纳米颗粒以及组装在红细胞表面的药物纳米颗粒（RBC-NPs）。注射后 20 分钟，小鼠被安乐死，并收集完整的肺部。肺部先用冷 PBS 洗涤两次，然后置于 4%多聚甲醛溶液中过夜固定。固定后的肺部样本在 Tissue-Tek OCT 复合物（Sakura Finetek）中冷冻，并使用冷冻切片机（Leica CM1950，IL，美国）进行切片。切片组织用 Fluroshield 封片，进行 DAPI（4′,6-二脒基-2-苯基吲哚）染色（Ex/Em，340/488 nm），并通过共聚焦显微镜（Upright Zeiss LSM 710 NLO ready）进行分析。
Efficacy studies on in vivo experimental lung metastasis model
体内实验性肺转移模型疗效研究
An experimental lung metastasis model was established by intravenous injection of 1 × 105 B16F10-Luc cells into the tail vein of female C57BL/6 mice. Efficacy for the treatment groups was evaluated in early-stage and late-stage metastatic models. Mice were randomized on the basis of the bioluminescence intensity in the lungs 1 day before the first injection of therapies. A control (saline) group and three treatment groups (DOX-NPs, RBC-NPs, and free DOX) at a dose of 5.2 mg/kg were evaluated for their efficacy (n = 7 for all groups, unless otherwise specified).
通过静脉注射 1×10^6 B16F10-Luc 细胞至雌性 C57BL/6 小鼠尾静脉，建立实验性肺转移模型。评估治疗组在早期和晚期转移模型中的疗效。根据治疗前一天肺部生物发光强度对小鼠进行随机分组。对照组（生理盐水）和三个治疗组（DOX-NPs、RBC-NPs 和游离 DOX）以 5.2 mg/kg 剂量评估其疗效（每组 n=7，除非另有说明）。
通过静脉注射 1×10^6 B16F10-Luc 细胞至雌性 C57BL/6 小鼠尾静脉，建立实验性肺转移模型。评估治疗组在早期和晚期转移模型中的疗效。根据治疗前一天肺部生物发光强度对小鼠进行随机分组。对照组（生理盐水）和三个治疗组（DOX-NPs、RBC-NPs 和游离 DOX）以 5.2 mg/kg 剂量评估其疗效（每组 n=7，除非另有说明）。
For the early-stage metastatic model, treatments were given starting the day after the inoculation. Four injections were given over 6 days, i.e., days 1, 3, 5, and 7 after inoculation. On days 6, 8, 10, 12,18, 23, and 31 after inoculation, the mice were imaged using in vivo imaging (PerkinElmer IVIS Spectrum, MA, USA). Briefly, mice were injected intraperitoneally with 150 μl of XenoLight-d-luciferin (30 mg/ml) in saline. Fifteen minutes after the injection, mice were imaged using in vivo imaging. The average radiance (bioluminescence intensity) was evaluated using the software Living system. The animals were further monitored for their survival.
在早期转移模型中，治疗从接种后的第二天开始。在 6 天内进行了四次注射，即接种后的第 1、3、5 和 7 天。在接种后的第 6、8、10、12、18、23 和 31 天，使用活体成像（PerkinElmer IVIS Spectrum，MA，美国）对小鼠进行成像。简言之，小鼠腹腔注射 150 μl 含 30 mg/ml XenoLight-d-luciferin 的生理盐水。注射后 15 分钟，使用活体成像对小鼠进行成像。通过 Living 系统软件评估平均发光强度（生物发光强度）。此外，还对动物的存活情况进行了监测。
在早期转移模型中，治疗从接种后的第二天开始。在 6 天内进行了四次注射，即接种后的第 1、3、5 和 7 天。在接种后的第 6、8、10、12、18、23 和 31 天，使用活体成像（PerkinElmer IVIS Spectrum，MA，美国）对小鼠进行成像。简言之，小鼠腹腔注射 150 μl 含 30 mg/ml XenoLight-d-luciferin 的生理盐水。注射后 15 分钟，使用活体成像对小鼠进行成像。通过 Living 系统软件评估平均发光强度（生物发光强度）。此外，还对动物的存活情况进行了监测。
For the late-stage metastatic model, treatments were given 1 week after the inoculation. Four injections were given over 6 days, i.e., days 7, 9, 11, and 13 after the inoculation. The mice were imaged on days 6, 8, 10, 12, and 16 using in vivo imaging as described above. The average radiance was evaluated using the software Living system. On day 16, the mice were euthanized, and the lungs were excised and fixed using 10% formalin. The fixed lungs were used for counting of the surface nodules and H&E analysis. Survival in the late-stage model was evaluated by having the injection schedule as described above (n = 8 for the control and treatment groups).
在晚期转移模型中，治疗在接种后 1 周开始。共进行 4 次注射，间隔 6 天，即接种后的第 7、9、11 和 13 天。小鼠在第 6、8、10、12 和 16 天通过上述体内成像技术进行观察。平均辐射强度通过 Living 系统软件评估。第 16 天，小鼠被安乐死，肺部被切除并用 10%福尔马林固定。固定的肺组织用于表面结节计数和 H&E 染色分析。晚期模型的生存率评估依据上述注射计划进行（对照组和治疗组各 8 只小鼠）。
在晚期转移模型中，治疗在接种后 1 周开始。共进行 4 次注射，间隔 6 天，即接种后的第 7、9、11 和 13 天。小鼠在第 6、8、10、12 和 16 天通过上述体内成像技术进行观察。平均辐射强度通过 Living 系统软件评估。第 16 天，小鼠被安乐死，肺部被切除并用 10%福尔马林固定。固定的肺组织用于表面结节计数和 H&E 染色分析。晚期模型的生存率评估依据上述注射计划进行（对照组和治疗组各 8 只小鼠）。
Statistical analysis 统计分析
All data are presented as means ± SEM. Comparison between two groups was conducted using unpaired two-tailed Student’s t test. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) or Kruskal-Wallis test. Kruskal-Wallis tests were performed for data that were determined to be nonparametric by the normality test. All statistical analyses were carried out using GraphPad Prism 8 software. For the analysis of Kaplan-Meier survival curves, log-rank (Mantel-Cox) analysis was used. P values represent different levels of significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All the flow cytometry analyses were carried out using the FlowJo software.
所有数据以均值±标准误差表示。两组间的比较采用非配对的双尾学生 t 检验。多组间的比较采用单因素方差分析（ANOVA）或 Kruskal-Wallis 检验。对于经正态性检验确定为非参数的数据，进行 Kruskal-Wallis 检验。所有统计分析均使用 GraphPad Prism 8 软件进行。对于 Kaplan-Meier 生存曲线的分析，采用对数秩（Mantel-Cox）分析。P 值表示不同程度的显著性：*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。所有流式细胞术分析均使用 FlowJo 软件进行。
所有数据以均值±标准误差表示。两组间的比较采用非配对的双尾学生 t 检验。多组间的比较采用单因素方差分析（ANOVA）或 Kruskal-Wallis 检验。对于经正态性检验确定为非参数的数据，进行 Kruskal-Wallis 检验。所有统计分析均使用 GraphPad Prism 8 软件进行。对于 Kaplan-Meier 生存曲线的分析，采用对数秩（Mantel-Cox）分析。P 值表示不同程度的显著性：*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。所有流式细胞术分析均使用 FlowJo 软件进行。
Acknowledgments 致谢
Funding: This work was financially supported by Wyss Institute at Harvard University. We acknowledge funding from NIH (1R01HL143806-01). Author contributions: Z.Z., A.U., and S.M. conceived the project. Z.Z. and A.U. performed the experiments. Y.G. and J.K. helped with the LC-MS and histology analysis. Z.Z. and A.U. analyzed the data. Z.Z. prepared the graphs. Z.Z., A.U., and S.M. wrote the manuscript. All authors read and approved the manuscript. Competing interests: S.M., A.U., and Z.Z. are inventors on a patent application related to this work filed by Harvard University (no. 62/858,478, filed in June 2019). The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
资助：本研究由哈佛大学 Wyss 研究所提供资金支持。我们感谢 NIH 的资助（1R01HL143806-01）。作者贡献：Z.Z.、A.U.和 S.M.构思了项目。Z.Z.和 A.U.进行了实验。Y.G.和 J.K.协助进行了 LC-MS 和组织学分析。Z.Z.和 A.U.分析了数据。Z.Z.制作了图表。Z.Z.、A.U.和 S.M.撰写了手稿。所有作者阅读并批准了手稿。竞争利益：S.M.、A.U.和 Z.Z.是哈佛大学提交的相关专利申请的发明人（专利号 62/858,478，2019 年 6 月提交）。作者声明无其他竞争利益。数据与材料可用性：评估本文结论所需的所有数据均已在文中和/或补充材料中提供。与本文相关的其他数据可向作者索取。
资助：本研究由哈佛大学 Wyss 研究所提供资金支持。我们感谢 NIH 的资助（1R01HL143806-01）。作者贡献：Z.Z.、A.U.和 S.M.构思了项目。Z.Z.和 A.U.进行了实验。Y.G.和 J.K.协助进行了 LC-MS 和组织学分析。Z.Z.和 A.U.分析了数据。Z.Z.制作了图表。Z.Z.、A.U.和 S.M.撰写了手稿。所有作者阅读并批准了手稿。竞争利益：S.M.、A.U.和 Z.Z.是哈佛大学提交的相关专利申请的发明人（专利号 62/858,478，2019 年 6 月提交）。作者声明无其他竞争利益。数据与材料可用性：评估本文结论所需的所有数据均已在文中和/或补充材料中提供。与本文相关的其他数据可向作者索取。
Supplementary Material 补充材料
Summary 摘要
Supplementary Materials and Methods
补充材料与方法
补充材料与方法
Fig. S1. Representative H&E staining images of lungs of mice.
图 S1. 小鼠肺部的代表性 H&E 染色图像。
图 S1. 小鼠肺部的代表性 H&E 染色图像。
Fig. S2. Representative H&E staining images of organs of mice treated with different drug formulations.
图 S2. 不同药物配方处理小鼠器官的代表性 H&E 染色图像。
图 S2. 不同药物配方处理小鼠器官的代表性 H&E 染色图像。
Fig. S3. Size distribution of different chemotherapeutic agent–loaded biodegradable PLGA NPs.
图 S3. 不同化疗药物负载的可生物降解 PLGA 纳米粒的粒径分布。
图 S3. 不同化疗药物负载的可生物降解 PLGA 纳米粒的粒径分布。
Table S1. Physicochemical properties of different chemotherapeutic agent–loaded biodegradable PLGA NPs.
表 S1. 不同化疗药物负载的可生物降解 PLGA 纳米粒的物理化学性质。
表 S1. 不同化疗药物负载的可生物降解 PLGA 纳米粒的物理化学性质。
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Science Advances
Volume 5 | Issue 11
November 2019
November 2019
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Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
Submission history
Received: 5 May 2019
Accepted: 17 September 2019
Acknowledgments
Funding: This work was financially supported by Wyss Institute at Harvard University. We acknowledge funding from NIH (1R01HL143806-01). Author contributions: Z.Z., A.U., and S.M. conceived the project. Z.Z. and A.U. performed the experiments. Y.G. and J.K. helped with the LC-MS and histology analysis. Z.Z. and A.U. analyzed the data. Z.Z. prepared the graphs. Z.Z., A.U., and S.M. wrote the manuscript. All authors read and approved the manuscript. Competing interests: S.M., A.U., and Z.Z. are inventors on a patent application related to this work filed by Harvard University (no. 62/858,478, filed in June 2019). The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
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National Institutes of Health: 1R01HL143806-01
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Figures
Fig. 1 Schematic illustration of the ELeCt platform and characterization of drug (DOX)–loaded biodegradable PLGA NPs.
(A) Schematic illustration of the composition and mechanism of the biodegradable drug NP assembling on the erythrocyte platform (ELeCt) for lung metastasis treatment. (B to D) Average size (B), zeta potential (C), and drug loading contents (D) of plain and drug-loaded NPs. (E) SEM images showing the morphological features of the NPs. Scale bars, 200 nm. (F) Size distribution of plain and drug-loaded NPs. (G) Drug release kinetics from the biodegradable NPs in a complete medium (n = 4). (H and I) Flow cytometry histogram plots (H) and CLSM images (I) showing the interaction of drug-loaded NPs with B16F10-Luc melanoma cells. In (I), cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). (J and K) Dose-response curve (J) and median inhibitory concentration (IC50) values (K) of B16F10-Luc cells after being treated with different formulations for 24 hours (n = 6). n.s., not significantly different (Student’s t test).
Fig. 2 Doxorubicin-loaded biodegradable PLGA NPs efficiently assemble onto mouse and human erythrocytes.
(A) Flow cytometry analysis of assembly of DOX-loaded PLGA NPs to mouse erythrocytes at different NP-to-erythrocyte ratios (left to right: 0:1, 50:1, 200:1, 400:1, and 800:1). (B) Percentage of mouse erythrocytes carrying at least one NP. (C) Nanoparticle binding efficiency and (D) drug dose on mouse erythrocytes at different NP–to–mouse erythrocyte ratios. (E) CLSM and (F) SEM images of mouse erythrocytes with drug-loaded NPs assembled on them. Scale bars in (F), 2 μm. (G) CLSM and (H) SEM images of human erythrocytes with drug-loaded NPs assembled on them. Scale bars in (H), 2 μm. (I) Flow cytometry assay of the assembly of drug-loaded NPs to human erythrocytes at different NP-to-erythrocyte ratios (left to right: 0:1, 200:1, 800:1, and 1600:1). (J) Nanoparticle binding efficiency and (K) drug dose on human erythrocytes at different NP-to-erythrocyte ratios.
Fig. 3 The ELeCt platform enables enhanced and targeted delivery of NP drugs to the lungs bearing metastasis.
(A) Pharmacokinetics of intravenously administered DOX formulations. Extended blood circulation time of DOX was achieved by erythrocyte hitchhiking compared with using free drug or NPs alone (n = 3). Significantly different [one-way analysis of variance (ANOVA)]: *P < 0.05 and **P < 0.01. (B) Hitchhiked drug-loaded NPs could specifically detach from mouse and human erythrocytes under the lung-corresponding shear stress. Samples were sheared for 20 min (n = 3). Low shear indicates rotary shear (~1 Pa), while high shear was at 6 Pa. Significantly different (Student’s t test): ***P < 0.001. (C) Drug accumulation in the lungs of mice bearing B16F10-Luc lung metastasis at 20 min and 6 hours after intravenous administration of different DOX formulations (n = 3). Significantly different (one-way ANOVA): *P < 0.05 and ***P < 0.001. (D) Comparison of the drug concentration in the lungs of erythrocyte hitchhiking group to that of the free drug and NP-alone groups (n = 3). (E) Drug distribution in the diseased lungs 20 min after intravenous administration of DOX formulations. Dashed lines indicate the edge of metastasis nodules.
Fig. 4 The ELeCt platform inhibits lung metastasis progression and improves survival in the early-stage B16F10-Luc metastasis model.
(A) Schematic chart of the treatment schedule. (B) Bioluminescence images of lung metastasis at different time points. EXP indicates “Expired.” (C) Lung metastasis progression curve as depicted from in vivo bioluminescence signal intensity. (D) Quantification of lung metastasis burden at different time points (n = 7). (E) Scatter plot comparing the lung metastasis burden in different treatment groups as depicted from bioluminescence signal intensity on day 16 (n = 7). Significantly different (Kruskal-Wallis test): *P < 0.05, **P < 0.01, and ****P < 0.0001. (F) Scatter plot comparison of the lung metastasis burden on day 23 (n = 7). Significantly different (Kruskal-Wallis test): *P < 0.05, **P < 0.01, and ****P < 0.0001. (G) Body weight change of mice during the treatment period (n = 7). (H) Survival of mice under different treatments as displayed by Kaplan-Meier curves (n = 7). Significantly different (log-rank test): *P < 0.05 and ***P < 0.001.
Fig. 5 The ELeCt platform inhibits lung metastasis progression and extends survival in the late-stage B16F10-Luc metastasis model.
(A) Schematic illustration of the treatment schedule. (B) Bioluminescence images of lung metastasis progression at different time points. (C) Lung metastasis growth curve in mice treated with different DOX formulations. (D) Quantitative analysis of lung metastasis burden as depicted from bioluminescence signal intensity (n = 7). Significantly different (one-way ANOVA): *P < 0.05 and **P < 0.01. (E) Quantification of metastasis nodule numbers on excised lungs from mice in different treatment groups on day 16 (n = 7). Significantly different (one-way ANOVA): **P < 0.01 and ***P < 0.001. (F) Body weight change of mice during the treatment period (n = 7). (G) Kaplan-Meier survival curves of mice in different treatment groups. Significantly different (log-rank test): **P < 0.01 and ***P < 0.001.
Fig. 6 Other chemotherapeutic agent–loaded biodegradable NPs can efficiently bind to erythrocytes.
The tested chemotherapeutic agents include camptothecin, paclitaxel, docetaxel, 5-fluorouracil, gemcitabine, methotrexate, and the combination of 5-fluorouracil and methotrexate. Scale bars, 1 μm.
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