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Enhanced Primary Tumor Penetration Facilitates Nanoparticle Draining into Lymph Nodes after Systemic Injection for Tumor Metastasis Inhibition Jing Liu,†,§ Hong-Jun Li,*,†,‡,⊥ Ying-Li Luo,†,# Cong-Fei Xu,‡,⊥ Xiao-Jiao Du,‡,⊥ Jin-Zhi Du,*,‡,⊥,¶ and Jun Wang†,∥,⊥,¶,△ †

School of Biomedical Sciences and Engineering, Guangzhou International Campus and ‡Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, P.R. China § School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China ∥ National Engineering Research Center for Tissue Restoration and Reconstruction, ⊥Key Laboratory of Biomedical Engineering of Guangdong Province, and Innovation Center for Tissue Restoration and Reconstruction, #Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou 510006, P.R. China ¶ Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou 510005, P.R. China △ Research Institute for Food Nutrition and Human Health, South China University of Technology, Guangzhou 510641, P.R. China S Supporting Information *

ABSTRACT: Lymph nodes (LNs) are normally the primary site of tumor metastasis, and effective delivery of chemotherapeutics into LNs through systemic administration is critical for metastatic cancer treatment. Here, we uncovered that improved perfusion in a primary tumor facilitates nanoparticle translocation to LNs for inhibiting tumor metastasis. On the basis of our finding that an iCluster platform, which undergoes size reduction from ∼100 nm to ∼5 nm at the tumor site, markedly improved particle perfusion in the interstitium of the primary tumor, we further revealed in the current study that such tumor-specific size transition promoted particle intravasation into tumor lymphatics and migration into LNs. Quantitative analysis indicated that the drug deposition in LNs after iCluster treatment was significantly higher in the presence of a primary tumor in comparison with that after primary tumor resection. Early intervention of metastatic 4T1 tumors with iCluster chemotherapy and subsequent surgical resection of the primary tumor resulted in significantly extending animal survival, with 4 out of the 10 mice remaining completely tumor-free for 110 days. Additionally, in the more clinical relevant late metastatic model, iCluster inhibited the metastatic colonies to the lungs and extended animal survival time. This finding provides insights into the design of more effective nanomedicines for treating metastatic cancer. KEYWORDS: nanomedicine, tumor perfusion, tumor metastasis, lymph nodes, drug delivery nodes (SLNs) as imaging agents.7−9 Then, SLN biopsy can be performed to prevent further cancer cell dissemination. However, this surgical procedure typically leads to long-term pain, lymphedema, and risk of infection.10,11 Moreover, local injection poses several disadvantages, notably “dye spillage”,9 which causes high background signals in the adjacent tissue and poses challenges to decision making. More importantly,

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ymph nodes (LNs) are normally the primary target of metastasis in a variety of epithelial-derived tumors, and a fraction of cancer cells that reside in LNs can disseminate to and colonize distant organs.1−4 Clinically, lymph node metastasis (LNM) in cancer patients is associated with poorer prognosis and partially dictates the treatment options.5,6 Therefore, information regarding the presence of LNM is important for the staging and management of cancers. To identify the spread of tumor, tracers such as dyes or isotopes are always injected into tumor mass or surrounding tumor parenchyma, so that they can drain into sentinel lymph © XXXX American Chemical Society

Received: May 6, 2019 Accepted: July 17, 2019

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DOI: 10.1021/acsnano.9b03472 ACS Nano XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of Tumor Acidity Triggered Size Change of iCluster and Its Translocation from Primary Tumor to LNs via Tumor Lymphatics To Inhibit Tumor Metastasisa

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The cisplatin prodrug-conjugated PAMAM dendrimer (PAMAM/Pt, 5 nm) was attached to a large nanoparticle (100 nm) via a tumor-acidityresponsive amide bond, forming iCluster/Pt. iCluster/Pt is stable in blood circulation but enables rapid release of PAMAM/Pt after docking at the tumor site. The small-sized PAMAM/Pt facilitates particle perfusion to the tumor interstitial space as well as subsequent intravasation into the tumor lymphatics, through which they translocate to the SLNs to inhibit tumor metastasis.

In our previous study, we invented an intelligent clustered nanoparticle delivery system which was denoted as iCluster, enabling size change from ∼100 nm to ∼5 nm in response to the tumor acidity to improve particle perfusion in the primary tumor.19 In the present work, capitalizing on this platform, we elaborate that small nanoparticles with robust penetration ability in primary tumors would intravasate into tumor lymphatics and access the LNs (Scheme 1). Quantitative analysis and fluorescent images clearly evidenced that the presence of a primary tumor benefits the deposition of both the fluorescence tracers and anticancer drugs in SLN for iCluster treatment. Based on this finding, we examined the efficacy of such a platform for the treatment of early and late LNM of breast cancer, in which improved therapeutic effects were observed.

such a local injection option has rarely been used for the delivery of chemotherapeutics to LNs. Systemic administration is an alternative route for LN delivery of drugs/tracers, which has already been exemplified in previous reports. For example, Kataoka and co-workers showed that small nanoparticles (∼30 nm) rather than larger ones (∼70−80 nm) can deliver chemotherapeutic drugs to LNM through the blood vascular system after systemic administration.12,13 Sunil and co-workers have shown that systemic administration of near-infrared tracers, such as indocyanine green (ICG), can identify metastatic LNs in both prostate cancer murine models and human patients by taking advantage of the enhanced permeability and retention (EPR) effect in combination with the second window ICG technique.14 These studies highlighted the importance of utilizing the feature of leaky tumor vasculature for LN drug delivery through systemic administration. However, recent studies suggest that angiogenesis is lacking during the formation of LNM, and the EPR effect might be absent in the early stage of LNM.15,16 Systemic chemotherapy may not likely benefit the treatment of early LNM. Therefore, the development of an alternative delivery strategy that is independent of the vasculature of LNs is required for the treatment of LNM, and more studies are urgently needed to discover innovative mechanisms for LN delivery. It has been reported that epithelial-derived tumors rely mainly on tumor lymphatics to metastasize to LNs from the primary tumors.3,4 This inspires us to propose that tumor lymphatics can be an alternative route to deliver tracers or drugs to LNs. This pathway has been validated by recent studies through intratumor injection of nanotherapeutics.17,18 However, whether this route still works for systemic nanoparticle injection is inconclusive because of the intrinsic conflicts in which nanoparticles after systemic injection mainly circulate in the blood vascular system. More direct and substantial evidence is needed to elaborate on how these particles enter lymphatic vessels and eventually drain into LNs.

RESULTS AND DISCUSSION iCluster Facilitated Cargo Draining into SLNs. The preparation and tumor-acidity-triggered size transition of iCluster and its nonresponsive counterpart Cluster were reported in our previous report19 (Figure S1). One important structural feature of iCluster is that the polyamidoamine (PAMAM, ∼5 nm) dendrimer was attached onto a large nanoparticle (∼100 nm) via a tumor-acidity-responsive amide bond.20−23 iCluster is stable in blood circulation but enables rapid release of PAMAM at the tumor site due to the amide bond cleavage from tumor acidity. The small-sized PAMAM facilitates particle perfusion to the tumor interstitial space as well as subsequent intravasation into the tumor lymphatics, through which they translocate to the SLNs to inhibit tumor metastasis (Scheme 1). In addition, the surface amino groups of PAMAM provide conjugation sites for fluorescent dyes (e.g., Rhodamine B (RhoB)) and chemotherapeutic drugs (e.g., prodrug of cisplatin). RhoB-labeled iCluster and Cluster (denoted as iClusterRhoB and ClusterRhoB) were used to visually examine their draining pathway into LNs. This was first tested in 4T1 tumors on the foot sole of Balb/c mice. iClusterRhoB or ClusterRhoB were injected intratumorally (Figure 1A), and 12 h B

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Figure 1. Schematic illustration (A) and stereoscopic fluorescence microscope image (B) showing PAMAM accumulation in SLN 12 h after intratumoral injection of Rhodamine B (red) labeled iClusterRhoB and ClusterRhoB (n = 3). Scale bar = 2 mm. (C) Quantitative analysis of Rhodamine B fluorescence in SLN with ImageJ. Schematic illustration (D) and in vivo images (E) of nanoparticle draining into SLNs via primary tumor lymphatic vessels after intravenous injection of iClusterRhoB after 4 and 12 h. The draining lymphatic vessel is indicated by a yellow arrow. The blood vessel is indicated by a white arrowhead. (F) Time-dependent PAMAM content in SLNs of ClusterRhoB and iClusterRhoB in the presence of primary tumor (solid) and without primary tumor (hollow). (G) Content of Pt drug in SLNs after 12 h injection of iCluster/Pt and Cluster/Pt with primary tumor (solid) and without primary tumor (hollow).

later, fluorescence images were taken on a stereoscopic microscope. Before imaging, fluorescein isothiocyanate (FITC)-labeled dextran (FITC-dextran) was injected to mark the tumor lymphatic drainage and the popliteal LNs.24,25 As indicated, iClusterRhoB could deliver more red signals to LNs than ClusterRhoB. In addition, the overlapping red and green signals in the lymphatic vessel (marked by arrow) indicated that iCluster passed through the lymphatic vessel to the sentinel lymph nodes (SLNs) (Figure S2). Ex vivo images further validated that iClusterRhoB treatment generated red fluorescent signals in the LNs more intense than those with the ClusterRhoB treatment (Figure 1B). Quantitative analysis showed that the fluorescence intensity resulting from iClusterRhoB treatment was ∼3.5-fold higher than that of ClusterRhoB treatment (Figure 1C). Whether iCluster could accumulate in the SLNs after systemic injection was examined in mice bearing 4T1

xenografts. iClusterRhoB or ClusterRhoB was injected intravenously into the mice tail vein (Figure 1D), whereas FITCdextran was injected locally to delineate the lymphatic vessels and SLNs. Fluorescence imaging showed clearly that the PAMAM (red) released from iClusterRhoB accumulated in the SLNs at 4 and 12 h post-nanoparticle injection (Figure 1E). More interestingly, the red fluorescent signals coexisted with the lymphatic vessels at 4 and 12 h (marked with yellow arrows), indicating that nanoparticle accumulation in the SLNs likely occurred through the afferent lymphatic vessels (Figure S3). Red fluorescence from ClusterRhoB was also detectable in the SLNs, although not as clearly as that from iClusterRhoB. However, a marked difference is that red fluorescence was more obvious in the blood vessels (marked with white arrowheads), whereas less fluorescence was detected in the lymphatic vessels, implying that the fluorescence of ClusterRhoB treatment might come from the vascular system (Figure S4). C

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Figure 2. iCluster facilitated the translocation of PAMAM from primary tumors to SLNs. (A,C) Microdistribution of RhoB-labeled PAMAM (red) in the primary tumor tissue 12 h post-intravenous injection of iClusterRhoB and ClusterRhoB. Colocalization of RhoB-labeled PAMAM with tumor blood vessels (A) and tumor lymphatic vessels (C). (B,D) Relative RhoB fluorescence intensity outside tumor blood vessels (B) and inside the lymphatic vessels (D). Data were analyzed with ImageJ. (E) Microdistribution of RhoB-labeled PAMAM (red) in SLNs after intravenous injection of ClusterRhoB (top), iClusterRhoB (middle) in the presence of primary tumor as well as iClusterRhoB with primary tumor resected before injection (bottom). (A,C,E) Blood vessels were marked with FITC anti-mouse PECAM-1 antibody (cyan), and lymphatic vessels were marked with eFluor 660 anti-mouse LYVE-1 antibody (green). (F) Pearson correlation coefficients of PAMAM with blood vessels and lymphatic vessels in SLNs. Data were analyzed with Image Pro Plus. (G) Quantitative analysis of relative RhoB fluorescence inside lymphatic vessels in (E). Data were analyzed with ImageJ. All data are represented as mean ± standard deviation and analyzed by Student’s ttest (*P < 0.05, **P < 0.01).

iCluster Facilitated the Translocation of PAMAM from the Primary Tumor to SLNs. iCluster was previously reported to facilitate perfusion of nanoparticles in pancreatic tumors.19 Here, we further demonstrated that iCluster could also facilitate PAMAM accumulation and perfusion in 4T1 breast tumor. As indicated in Figure 2A, strong PAMAM signals (red) were detected throughout the tumor tissue, and most of them were located outside the blood vessels that were labeled with FITC anti-mouse PECAM-1 (cyan).26 The PAMAM signal outside the blood vessels after iCluster treatment was approximately 17-fold higher than that after Cluster treatment (Figure 2B). More importantly, more than 89% of PAMAM enriched in tumor tissue after iCluster treatment was in the tumor interstitium, and only a small number of nanoparticles were internalized by cells in the tumor (Figure S7). The enhanced nanoparticle perfusion greatly increased the possibility of PAMAM entering lymphatic vessels in the primary tumor. To test this property, the lymphatic vessels were labeled with eFluor 660 anti-mouse LYVE-1.27 Intense PAMAM signals (red) appeared inside the lymphatic vessels in the iCluster group, whereas the Cluster group showed markedly less red fluorescence (Figure 2C and Figure S8). Quantitative analysis indicated that the PAMAM signal

Quantitative analysis of PAMAM and Pt−drug deposition in SLNs was performed with and without the primary tumor. As indicated, iClusterRhoB treatment resulted in significantly higher PAMAM deposition in SLNs than in ClusterRhoB treatment 12 h post-injection (Figure 1F, P < 0.05). However, after the primary tumor was resected, no difference could be observed in PAMAM accumulation in SLNs between iClusterRhoB and Cluster RhoB (Figure 1F). Such an improved PAMAM accumulation was further validated on a stroma-rich Panc02 pancreatic tumor model (Figure S5). An interesting phenomenon is that the PAMAM accumulation of the Cluster group in SLNs remained similar regardless of the presence or absence of the primary tumor. In contrast, the accumulation of iCluster in SLNs was highly dependent on the primary tumor. The abilities of these two systems to deliver cisplatin to LNs with or without the primary tumor were quantitatively measured by inductively coupled plasma mass spectrometry (ICP-MS) (Figure 1G). Consistent with above results, iCluster treatment in the presence of primary tumors showed significantly higher Pt deposition in LNs compared with all other treatments. More interestingly, iCluster was much more powerful than Cluster in drug delivery to LNs through tumor lymphatics (Figure S6). All of these results suggest that iCluster can deliver more therapeutics to SLNs. D

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Figure 3. Antimetastasis efficacy for early stage metastatic tumor through combination of chemotherapy and surgical resection of the primary tumor. (A) Images showing time-dependent tumor metastasis to SLNs and the lungs in luciferase-expressing metastatic 4T1 tumor model. (B) Survival curves of mice bearing luciferase-expressing 4T1 tumor after intravenous administration of PBS, cisplatin, Cluster/Pt, and iCluster/Pt with the dosage of platinum at 4 mg per kg mouse body weight on days 9 and 11 after tumor cell inoculation, with the primary tumor excision on day 13 (n = 10). (C) Detection of luciferase expression in main organs including SLN, heart, lung, spleen, kidney, brain, liver, and bone at end of the study.

colocalizing with the lymphatic vessels. iClusterRhoB treatment produced PAMAM signals inside the lymphatic vessels more than 3-fold higher than that with ClusterRhoB. However, if the primary tumor was resected before treatment, the overall PAMAM signals of iClusterRhoB were significantly diminished to a comparable level of ClusterRhoB. In addition, its microdistribution patterns, including the colocalization ratios with blood vessels and lymphatic vessels, became similar to those of ClusterRhoB (Figure 2E−G). Noticeably, both the Cluster and iCluster could barely ooze from blood vessels after resection of the primary tumor (Figure S10). These results strongly suggest that the primary tumor may act as a hub for nanoparticle translocation to SLNs when the iCluster platform was used as the delivery system. Antimetastasis Effect of iCluster in Metastatic Tumor Models. Our results suggest that the iCluster platform behaved better in terms of facilitating SLN drug deposition when the primary tumor was present than after it was resected.

inside lymphatic vessels for iCluster was approximately 4.6-fold higher than that for Cluster treatment (Figure 2D). We have demonstrated that iCluster could facilitate the deposition of PAMAM in SLNs. However, the microdistribution of PAMAM in SLNs has not been revealed. Thus, we compared the nanoparticle microdistribution in SLNs with and without primary tumor presence. In the presence of the primary tumor, both iClusterRhoB and ClusterRhoB exhibited visible PAMAM signals in SLNs 12 h after intravenous injection, but iCluster RhoB treatment generated an overall fluorescence 3-fold higher than that of ClusterRhoB (Figure 2E and Figure S9). Furthermore, their microdistribution patterns differed. For ClusterRhoB, the PAMAM signals mainly colocalized with the blood vessels, and only sporadic PAMAM signals could be found in the lymphatic vessels with a fairly low colocalization ratio (Figure 2F). In contrast, iClusterRhoB treatment led to a very large portion of PAMAM leaking out of the blood vessels and E

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Figure 4. Antimetastasis efficacy for late-stage metastatic tumor. (A) Optical images, H&E staining (B), and relative luciferase activity of SLNs (C) after the treatment on day 34. (D) Optical images (top) and H&E staining (bottom) of the lungs after the treatment on day 34 (arrows indicate the metastatic nodules), (E) tumor nodules on the surface of the lungs and relative luciferase activity in the lungs (F) after the treatment. (G) Survival curves of mice bearing luciferase-expressing 4T1 tumor after intravenous administration of PBS, cisplatin, PAMAM/Pt, Cluster/Pt, and iCluster/Pt with the dosage of platinum at 4 mg per kg mouse body weight on days 14, 17, and 20 after tumor inoculation (n = 10). All data are represented as mean ± standard deviation and analyzed by Student’s t test (*P < 0.05, **P < 0.01).

in the smallest SLN size, with an average weight significantly lower than that with other treatments (Figure 4A and Figure S11). Histopathological study also showed a significant reduction in tumor lesions in iCluster/Pt-treated LNs (Figure 4B). Quantitative analysis demonstrated that iCluster/Pt dramatically reduced the luciferase expression level in SLNs compared with those in other groups (Figure 4C). More apoptosis cells were observed in LNs after iCluster/Pt treatment (Figure S12). As shown by the appearance and pathological results of the lungs, iCluster/Pt treatment showed significantly reduced metastatic nodules and tumor cells compared with their numbers in other groups (Figure 4D). The mean number of visible metastatic nodules after iCluster/ Pt treatment was 5 versus 28 for Cluster/Pt, 34 for PAMAM/ Pt, 38 for cisplatin, and 50 for PBS treatment (Figure 4E). The luciferase activity, which indicates the presence of 4T1 tumor cells in the lungs, was further quantitatively analyzed. Approximately 12% of the control luciferase expression was detected in the lungs after iCluster/Pt treatment, which was substantially decreased in comparison with the PBS (100%), cisplatin (78%), PAMAM/Pt (75%), and Cluster/Pt (55%) treatments (Figure 4F). We confirmed that iCluster can accumulate more efficiently in the lungs with metastatic foci in the 4T1-GFP tumor model (Figure S13), which was responsible for substantially reduced lung metastasis in iCluster/Pt. It should be mentioned that PAMAM/Pt and cisplatin showed mild therapeutic efficacy due to their rapid

Then, we evaluate the potential of our strategy for the antimetastasis effect in an early metastatic tumor model. Using a stable luciferase-expressing metastatic 4T1 tumor model, we monitored that the cancer cells disseminated to SLNs and the lungs on proximately day 10 and day 16 after cancer cell inoculation (Figure 3A). Inspired by our results as well as the clinical practice of using neoadjuvant chemotherapy for the treatment of breast cancer, we first treated the mice with iCluster chemotherapy before SLN metastasis (here is day 9 and day 11) and subsequent surgical resection of the primary tumor on day 13, and then the survival of these mice was monitored. As shown in Figure 3B, all mice died within 51 days for the placebo treatment, whereas slight survival improvement was observed for the Cluster/Pt treatment, with all mice dead within 65 days. In contrast, 40% of the mice survived up to 110 days with iCluster/Pt treatment, which was a significant improvement in comparison with the control treatments. In addition, no luciferase signal could be detected in the major target organs of metastasis, such as the SLNs, lungs, brain, liver, and bones of the living mice, demonstrating that metastasis was completely inhibited by iCluster treatment (Figure 3C). The antimetastasis effect of iCluster was also tested in a late metastatic tumor model, which is defined as the cancer cells having disseminated to the lungs. This model is more clinically relevant and can provide more information on its efficacy. After three consecutive intravenous injections, iCluster/Pt resulted F

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the iCluster robustly inhibits the spread of tumor cells. This experiment was in accordance with the philosophy of neoadjuvant chemotherapy of breast cancer in clinical practice, implying the great potential of our strategy for future use. In the late metastatic model, which is more clinically relevant, iCluster treatment greatly inhibited cancer cell dissemination to the lungs and extended survival time of the mice. Tumordraining LNs are key orchestrators of tumor immune responses. Therefore, we further studied the influence of iCluster/Pt treatment on the immune system by analyzing important subsets of immune cells in the LNs. We can see that the systemic iCluster/Pt treatment did not lead to immunodepletion in the LNs. On the contrary, iCluster/Pt treatment caused an increase in T lymphocytes, especially cytotoxic T lymphocytes, as well as an increase in macrophages and dendritic cells. It is known that nanotechnology-based drug delivery systems have been envisioned as a powerful strategy to treat cancer metastasis.28−31 A variety of nanoparticles including nanoparticle generators,32 supramolecular assemblies,33 polymeric micelles,12,34 and cell membrane mimetic nanocarriers35−37 have been developed. Improved overall efficacies in treating metastatic cancers have been achieved by these drug delivery systems. However, the behind intervention mechanisms, especially how these particles interact with LNs, have yet been fully depicted. Our study provides a perspective for the possible underlying mechanism of particle draining into LNs after systemic administration, which is helpful for the design of more effective nanomedicine for treating metastatic cancers. It should be mentioned that particles of 100 nm size have poor lymphatic uptake, and it would be more comprehensive if nanoparticles with sizes other than 100 and 5 nm can be included in the study.

blood clearance and limited tumor accumulation according to our previous study.19 As a comprehensive effect, iCluster/Pt showed significantly prolonged mouse survival with a median survival time of 56 days versus 49.5, 47, 46.5, and 44.5 days for the Cluster/Pt, PAMAM/Pt, cisplatin, and PBS treatments, respectively (Figure 4G).

CONCLUSIONS Delivery of tracers or drugs into LNs is critically important for delineating the location of LNs and possible intervention of LN disease. However, the mechanism of nanoparticle draining into LNs after systemic injection has not been well understood. It is known that cancer cells of epithelial-derived tumors such as breast cancers rely mainly on tumor lymphatics spreading from the primary tumor to LNs and then to distant organs. Thus, we proposed that the route from primary tumor to LNs via tumor lymphatics might be a viable approach for LN cargo delivery. However, nanoparticles receiving systemic administration mainly circulate in the vascular system and hardly leak out from the blood vessels of normal tissues. Where and how these nanoparticles access the lymphatic system from blood circulation is inconclusive. In the present study, we demonstrated that the primary tumor can work as a transit to mediate the translocation of nanoparticle with proper size to LNs. In our study, both the intratumor and intravenous injection indicated that the tumor lymphatics can readily mediate the translocation of size-changeable iCluster other than size-fixed Cluster nanoparticles into LNs. Detailed microdistribution analysis of the primary tumor and LNs using fluorescence staining revealed that tumor-aciditytriggered size transition of iCluster improved particle perfusion inside the primary tumor, so that they were prone to diffusion into the interstitial fluid and then had more chance to drain into tumor lymphatics and the LNs. In contrast, in the Cluster group, the particles were mainly restricted within or nearby the blood vessels. It should also be noted that both the blood vasculature and tumor lymphatics contribute to the particle accumulation in the LNs. However, our imaging data indicated that nanoparticles transporting through the vasculature system could not effectively extravasate from blood vessels within LNs probably due to their tight endothelial structure. Nanoparticles draining via the tumor lymphatics can leak out and distribute more evenly inside LNs presumably because the lymphatic capillaries lack tight interendothelial junctions and the surrounding layers of pericytes. Quantitative analysis demonstrated that iCluster treatment resulted in significantly higher (∼1.7-fold) Pt−drug deposition in the LNs of mice with primary tumors compared with that after primary tumor resection. Nanoparticle microdistribution in LNs observed by confocal laser scanning microscopy further showed that the effective accumulation of nanoparticles within lymphatics receiving iCluster treatment was 3-fold higher than that after primary tumor resection. Based on these findings, we hypothesized that iCluster nanomedicine can be used for the inhibition of tumor metastasis. We tested our hypothesis in two models of different metastatic stages: roughly early stage and late stage. In the early metastatic model, iCluster was first administered in the presence of the primary tumor, and then the primary tumor was surgically removed. Following this protocol, we found that the mice receiving iCluster treatment showed markedly extended survival. More excitingly, 4 out of the 10 mice with iCluster treatment remained completely tumor-free until 110 days, which strongly demonstrates that

METHODS Preparation of iCluster. The platinum prodrug contained iCluster/Pt, as well as the Cluster/Pt, was prepared as previously reported,19 and the platinum drug content in these particles was 2.5% (mass ratio). Briefly, PCL-CDMPAMAM/Pt, PEG-b-PCL, and PCL were dissolved in DMSO at a concentration of 10 mg/mL, and then these three polymers were mixed with a predetermined dose and further stirred for 10 min. Five volumes of Milli-Q water was slowly added into the mixture and stirred for another 30 min. Subsequently, the nanoparticle solution was dialyzed against water to remove organic solvent. The control cluster nanoparticles (Cluster/Pt) were prepared following the same procedure by replacing PCL-CDMPAMAM/Pt with PCL−PAMAM/Pt. For the RhoB-labeled iClusterRhoB and ClusterRhoB preparation, the RhoB (Sigma-Aldrich, St. Louis, MO, USA)-conjugated PAMAM (Dendritech Inc., Michigan, USA) was first synthesized through the amidation reaction (molar ratio of rhodamine B/PAMAM was 1/1), and then the RhoBconjugated PAMAM was further modified and finally assembled into iCluster and Cluster with a method similar to that we previously reported.32 Characterizations. Particle size and zeta-potential measurements were conducted using a zeta-potential analyzer with dynamic lightscattering capability with a Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., England), a He−Ne laser (633 nm), and 90° collecting optics. Data were analyzed using Malvern Dispersion Technology Software 7.02. Morphology of the cluster nanoparticles was examined by a JEOL-2010 transmission electron microscope (JEOL Co., Ltd., Tokyo, Japan) at an accelerating voltage of 200 kV. The sample was prepared by pipetting a drop of the aqueous solution of nanoparticles (0.5 mg/mL) onto a 230 mesh copper grid coated with carbon. G

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ACS Nano PAMAM Release from Cluster Nanoparticles. iClusterRhoB and ClusterRhoB (2 mL, 2 mg/mL) in a dialysis bag (MWCO = 100000 Da) was immersed in phosphate buffer (PB) at pH 6.8 (0.02 M, 18 mL), and the external PB buffer was collected at predetermined times. The released PAMAM was quantified by high-performance liquid chromatography with a Waters 2475 fluorescence detector. Interaction of Clustered Nanoparticles with Plasma. The stability of clustered nanoparticles was characterized by size change in the presence of plasma. The nanoparticles (1 mg/mL, 1 mL) were incubated with plasma (100 μL) at 37 °C for predetermined time intervals and subjected to dynamic light scattering to measure size change. Cell Culture. Mouse mammary tumor cell line of 4T1 cells was purchased from the Cell Bank of Chinese Academy of Sciences. The 4T1 cells with stable luciferase expression (4T1-luc cells) were a generous gift from Prof. Ya-ping Li (Shanghai Institute of Materia Medica, China). Murine pancreatic tumor cell line of Panc02 cell was a generous gift from Prof. Zhi-gang Zhang (Shanghai Cancer Institute, China). These cells were cultured in a RMPI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). These cells were maintained in an incubator under a humidified atmosphere containing 5% CO2 at 37 °C. Tumor Models and Tumor Inoculation. BALB/c mice (female, 5 weeks old) and C57BL/6 mice (female, 6 weeks old) were purchased from Beijing Vital River Laboratory, Animal Technology Co. (Beijing, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of South China University of Technology. To evaluate whether iCluster facilitated PAMAM draining into sentinel lymph nodes after local administration, 5 × 105 4T1 cells in 10 μL of phosphate-buffered saline (PBS) were injected subcutaneously into right hind foot sole of BALB/c mice to generate the footpad tumor model. Thus, the SLN in this model was the popliteal lymph node. The tumor volume was 100−150 mm3 before the experiments. To evaluate the possibility that iCluster facilitated PAMAM draining into SLNs after system injection, 5 × 105 4T1 cancer cells in 50 μL of PBS were injected into the right fifth mammary fat pads to prepare the 4T1 xenograft tumor model, and 1 × 106 Panc02 pancreatic cells in 50 μL of PBS were injected into the right flank of C57BL/6 mice to prepare the Panc02 xenograft tumor model. Thus, the SLN in these tumor models were the inguinal lymph node. The tumor volume was 300−400 mm3 before the experiments. For the in vivo therapeutic study, immunofluorescence study, as well as drug accumulation study, the 4T1 xenograft tumor model was established by implanting 5 × 105 4T1 or 4T1-luc cancer cells in 50 μL of PBS into the right second mammary fat pads of the BALB/c mice. The axillary lymph node was the SLN in this tumor model. The initial tumor volume was 50−100 mm3 for the in vivo therapeutic study, and the tumor volume was 300−400 mm3 before the immunofluorescence study. Antimetastasis Efficacy for Late-Stage Metastatic Tumor. For the tumor suppression study, the mice were randomly divided into five groups (n = 5 per group). The mice bearing 4T1-luc tumors were treated with PBS, free cisplatin, PAMAM/Pt, Cluster/Pt, and iCluster/Pt by intravenous injection of an equivalent dose of 4.0 mg platinum per kg of mouse body weight. The mice received injections on days 14, 17, and 20 after tumor cell implantation. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. Tumor volume (mm3) was calculated as V = l × w2/2, in which l and w indicate the length and width of the tumor. All mice were sacrificed on day 34. All lung, sentinel lymph nodes and primary tumors were collected for imaging and histological examination. The macroscopic metastatic nodules in the lungs were counted. The mice survival of each treatment was monitored and recorded every day. Analysis of Immune Cells in SLNs. Mice were sacrificed, and SLNs were excised at the end of tumor suppression study in late-stage

metastatic tumor, and then SLNs were transferred to a dish and teased apart into a single cell suspension by pressing with the plunger. The single cell suspensions were filtered by a 200 mesh sieve and then collected by centrifugation at 1500 rpm for 10 min at 4 °C. To block nonspecific binding, cell suspensions were incubated with FcBlock (TruStain FcX antimouse CD16/32, clone 93, BioLegend) for 20 min. Multiparameter staining was performed by utilizing different combinations of fluorophore-conjugated antibodies for 40 min at 4 °C. The following immune cell subpopulations were investigated, using multichannel gating: (i) B cells (CD45+CD3−CD19+), (ii) CD4+ T cells (CD45+CD19−CD3+CD4+CD8−), (iii) CD8+ T cells (CD45+CD19−CD3+CD4−CD8+), (iv) macrophages (CD45+CD11b+F4/80+), (v) dendritic cells (CD45+CD11c+I-A/IE+). Anti-mouse antibody sources are as follows: CD45-Alexa Fluor 700 (30-F11), CD19-APC-Cy7 (6D5), CD3-BV785 (145-2C11), CD4-BV510 (RM4-5), CD8-PE-Cy7 (53-6.7), CD11b-BV650 (M1/ 70), F4/80-PE (BM8), CD11c-PerCP-Cy5.5 (N418), I-A/I-E-BV785 (M5/114.15.2). Above mentioned antibodies were purchased from BioLegend, Inc. (San Diego, CA, USA). Tissue Luciferase Activity. The lungs and lymph nodes were homogenized in PBS and lysed with 1× reporter lysis buffer (Promega, USA) and frozen overnight at −80 °C. Samples were then subjected to three freezes−thaw cycles and centrifuged at 14 000 rpm for 10 min. Supernatants were analyzed for protein quantity using the BCA protein assay kit (lot 23250, Thermo, USA). Luciferase activity was measured using a luminometer Veritas (Turner BioSystems, USA) and the Luciferase Assay System (Promega, USA). Histological Analysis. The lungs and SLNs were harvested and fixed with 4% paraformaldehyde phosphate buffer and embedded in paraffin for slicing. Paraffin-embedded 5 μm tissue sections were obtained for hematoxylin and eosin (H&E) staining. The samples of SLNs with H&E staining were observed on a microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany). The samples of lungs with H&E staining were imaged with AXIO Zoom.V16 macroscope (Carl Zeiss Co. Ltd., Jena, Germany). In Vivo Fluorescence Imaging in the Footpad Tumor Model. 4T1 cells were injected subcutaneously into the right hind foot sole of BALB/c mice to develop the footpad tumor model. The accumulation of nanoparticles in popliteal lymph node was assessed through fluorescence imaging by using RhoB-labeled clustered nanoparticle: 0.2 mg of iClusterRhoB and ClusterRhoB in 10 μL of PBS was intratumorally injected to the primary tumor at day 10 after tumor implantation. Tumor lymphatic drainage was tracked by intratumoral injection of 10 μL of FITC-dextran (12.5 mg/mL, MW = 500 000; Sigma) 30 min before the mice were sacrificed. Lymphangiography with FITC-dextran revealed the network of lymphatic capillaries, draining to a larger vessel at the primary tumor and subsequently to the popliteal lymph node. Mice were anaesthetized for in vivo observation 12 h after the nanoparticles were injected. The draining of the fluorescent signal between the primary tumor, tumor draining lymphatic vessels, and popliteal lymph nodes was imaged with AXIO Zoom.V16 macroscope (Carl Zeiss Co. Ltd., Jena, Germany). After that, the popliteal lymph nodes were taken out for further fluorescence imaging, and the amount of fluorescence signals in popliteal lymph nodes was calculated by ImageJ. Ex Vivo Fluorescence Imaging in the Inguinal Lymph Node Metastasis Tumor Model. The in vivo spontaneous metastasis model was developed by subcutaneously injecting 5 × 105 4T1 cells into the right fifth mammary fat pad of the BALB/c mice. The fifth mammary gland was selected to generate a tumor because it is more convenient than other places for the following fluorescent imaging observation. Mice were intravenously injected with 2 mg of iClusterRhoB and ClusterRhoB in PBS at day 15 after tumor inoculation. Tumor lymphatic drainage was tracked by intratumoral injection of 10 μL of FITC-dextran 30 min before the mice were sacrificed. Tumorbearing mice were dissected to expose the place between the primary tumor and inguinal lymph node after being injected with the nanoparticles for 4, 12, 18, and 24 h. The dissected mice were separately imaged at each time point with an AXIO Zoom.V16 macroscope (Carl Zeiss Co. Ltd., Jena, Germany) to observe the H

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ACS Nano draining of the fluorescent signal in tumor-draining lymphatic vessels and inguinal lymph nodes. Accumulation of Clustered Nanoparticles in SLNs in Pancreatic Tumor. The pancreatic tumor model was developed by subcutaneously injecting 1 × 106 Panc02 cells into the C57BL/6 mice. Mice were intravenously injected with 2 mg of iClusterRhoB and ClusterRhoB in PBS at day 20 after tumor inoculation. The mice were sacrificed after 12 h. SLNs were excised and imaged using the Bruker In Vivo Xtreme imaging system. Then the samples were washed with cold saline, dried on filter paper, weighed, and put into 2 mL FastPrep lysing matrix tubes with three stainless steel beads (2 mm), to which 200 μL of methanol was added. The tissue was then homogenized with a FastPrep system at a speed of 4 m/s for 40 s. After centrifugation (10 000g, 5 min), PAMAM in the supernatant was examined with a fluorescence spectrophotometer (Cytation 5, BioTek, Winooski, VT, USA). Pt Content Determination in SLNs. iCluster/Pt and Cluster/Pt were administered intravenously at an equivalent dose of 60 μg of platinum per mouse bearing 4T1 xenografts or with their primary tumors resected (n = 3 per group). The mice were sacrificed after 12 h. SLNs were excised, washed with cold saline, dried on filter paper, weighed, and dissolved in hot HNO3. Then, 1 mL of aqua regia solution (mixture of 30% HCl and 65% HNO3 in a volume ratio of 3:1) and 1 mL of H2O were added, and the Pt concentrations were measured by ICP-MS. Pt Content Determination in Tumors. Cispaltin, iCluster/Pt, Cluster/Pt, and PAMAM/Pt were administered intravenously at an equivalent dose of 60 μg of platinum per mouse bearing a 4T1 xenograft tumor (n = 3 per group). The mice were sacrificed after 4, 12, and 24 h. Tumors were excised, washed with cold saline, dried on filter paper, weighed, and dissolved in hot HNO3. Then, 1 mL of aqua regia solution (mixture of 30% HCl and 65% HNO3 in a volume ratio of 3:1) and 1 mL of H2O were added, and the Pt concentrations were measured by ICP-MS. Pt Content Determination in the Lungs. Cispaltin, iCluster/Pt, Cluster/Pt, and PAMAM/Pt were administered intravenously at an equivalent dose of 60 μg of platinum per mouse bearing a 4T1 xenograft tumor after primary tumor resected (n = 3 per group). The mice were sacrificed after 12 h. Lungs were excised, washed with cold saline, dried on filter paper, weighed, and dissolved in hot HNO3. Then, 1 mL of aqua regia solution (mixture of 30% HCl and 65% HNO3 in a volume ratio of 3:1) and 1 mL of H2O were added, and the Pt concentrations were measured by ICP-MS. Microdistribution of Nanoparticles in SLNs. The distribution of fluorescent clustered nanoparticles in sentinel lymph nodes was studied in tissue sections. Mice bearing 4T1 xenografts or with their primary tumors resected were intravenously injected with 2 mg of iClusterRhoB and ClusterRhoB at day 15 after tumor implantation. The mice were sacrificed, and SLNs were excised after the nanoparticles were injected for 12 h. The samples were sectioned into slides of 8 μm thickness in a cryostat and incubated with eFluor 660 anti-mouse LYVE-1 (eBioscience, USA) and FITC anti-mouse PECAM-1 (eBioscience, USA). Following the PBS washes, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Samples were observed under a Zeiss LSM710 confocal microscope with a 20× objective. RhoB-labeled PAMAM in iClusterRhoBand ClusterRhoB was detected using 543/590 nm excitation/emission filters. FITC and eFluor 660 were detected using 488/520 and 633/660 nm, respectively. Microdistribution of Nanoparticles in Tumors. For the immunofluorescence assay, mice bearing 4T1 xenografts were intravenously injected with iClusterRhoBand ClusterRhoB at day 15 after tumor inoculation. Mice were sacrificed, and tumors were excised 12 h postinjection. The microdistribution of iClusterRhoB and ClusterRhoB in tumors was studied. The samples were sectioned into slides of 8 μm thickness with a cryostat microtome (Leica CM1950; Leica Microsystems GmbH, Wetzlar, Germany) and incubated with eFluor 660 anti-mouse LYVE-1 (eBioscience, USA) and FITC antimouse PECAM-1 (eBioscience, USA). Following the PBS washes, the cell nuclei were stained with DAPI. Samples were examined under a

Zeiss LSM710 confocal microscope with a 20× objective. RhoBlabeled PAMAM in iClusterRhoB and ClusterRhoB was detected using 543/590 nm excitation/emission filters. FITC and eFluor 660 were detected using 488/520 and 633/660 nm, respectively. Microdistribution of Nanoparticles in the Lungs. BALB/c mice with 4T1-GFP metastatic tumors were injected intravenously with iClusterRhoB and ClusterRhoB after the primary tumor was resected. Mice were euthanized 12 h postinjection. The microdistribution of iClusterRhoB and ClusterRhoB in metastatic lungs was studied. The samples were sectioned into slides of 8 μm thickness with a cryostat microtome (Leica CM1950; Leica Microsystems GmbH, Wetzlar, Germany). Following the PBS washes, the cell nuclei were stained with DAPI. Samples were examined under a Zeiss LSM710 confocal microscope with a 20× objective. RhoB-labeled PAMAM in iClusterRhoB and ClusterRhoB was detected using 543/590 nm excitation/emission filters. GFP was detected using 488/520 nm excitation/emission filters. Determination of iCluster in Intratumoral Interstitium and Tissue Cells. Mice bearing 4T1 tumors were intravenously injected with 2 mg of iClusterRhoB. After administration of iCluster for 12 h, tumor masses were removed, minced, and digested with mixed enzyme digestion solution (1 mg/mL collagenase I, 1 mg/mL collagenase IV, and 0.1 mg/mL hyaluronidase, in RPMI 1640 medium) at 37 °C for 1 h. Next, the cell suspension solution was centrifuged at 350g for 5 min. The supernatant and the residual were separated as tumor interstitium and tissue cells, respectively. Fluorescence was measured in 96-well black fluorescence plates with Cytation5 with an excitation/emission setting of 540/590 nm. Accumulation of Nanoparticles in Sentinel Lymph Nodes. Mice bearing 4T1 xenografts or having primary tumors resected were treated with 2 mg of iClusterRhoB and ClusterRhoB in PBS at predetermined time points (1, 4, 8, 12, 16, 24, and 48 h). The axillary lymph nodes were harvested at each time point to analyze the accumulations of released PAMAM in tumor SLNs. The axillary lymph nodes were excised, washed with cold saline, dried over filter paper, weighed, and put into 2 mL of FastPrep lysing matrix tubes with three stainless steel beads (2 mm), to which 200 μL of methanol was added. The tissue was then homogenized with a FastPrep system at a speed of 4 m/s for 40 s. After centrifugation (10 000g, 5 min), RhoB-labeled PAMAM in the supernatant was analyzed with an ultraperformance liquid chromatography (UPLC, Waters ACQUITY, Waters Corp., Milford, MA) equipped with a Waters FLR detector, a Waters ACQUITY UPLC BEH C18 column using 1/1 (v/v) methanol/water as the mobile phase, with a 30 °C column temperature and a flow rate of 0.3 mL/min. The eluent was excited with a 540 nm laser and monitored at 590 nm. Bioluminescence Imaging of Metastatic Tumor Progression. BALB/c mice were inoculated with 5 × 105 4T1-luc cells into the right second mammary fat pads to develop the 4T1 xenograft tumor model. The tumor progression was monitored from day 6. The axillary lymph nodes and the lungs were taken out and immersed into 5 mg/mL D-luciferin in PBS for bioluminescence imaging daily. Tissues were imaged by a Xenogen IVIS Lumina system (Caliper Life Sciences, USA). The results were analyzed using Living Image 3.1 software (Caliper Life Sciences, USA). Antimetastasis Efficacy for Early Stage Metastatic Tumor Combined with Surgery Treatment. For the antimetastasis combination with surgery treatment study, the mice were randomly divided into four groups (n = 10 per group). The mice bearing 4T1luc tumors were treated with PBS, free cisplatin, Cluster/Pt, and iCluster/Pt by intravenous injection of an equivalent dose of 4.0 mg of platinum per kg of mouse body weight. The mice received injections on days 9 and 11 after tumor cell implantation. Surgical removal of the primary tumors was performed on day 13. Before surgery, mice were shaved around the area of the tumor and received direct oral feeding of meloxicam (2 mg/kg) as preoperative analgesia. The tumor mass was gently removed and the incision closed with a sterile suture. All surgical procedures were done under general anesthesia with pentobarbital sodium (50 mg/kg) administered by intraperitonial injection. The mice were kept under a veterinary I

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ACS Nano temperature monitor until they awakened. Mice received meloxicam (2 mg/kg) subcutaneously once daily for up to 3 days in the scruff as postoperative analgesia. The survival of each group was monitored and recorded every day. The remaining survivors were dissected on day 110 to detect the tumor metastasis. The main tissues including sentinel lymph node, heart, liver, spleen, lung, brain, and bone were obtained and immersed into 5 mg/mL D-luciferin (GoldBio, St. Louis, MO, USA) in PBS for bioluminescence imaging. Tissues were imaged by a Xenogen IVIS Lumina system (Caliper Life Sciences, USA). The results were analyzed using Living Image 3.1 software (Caliper Life Sciences, USA). Statistical Analysis. Comparisons between two groups were assessed by Student’s t test. Comparisons of tumor-growth curves were assessed by analysis of variance. P values lower than 0.05 were considered significant. Survival studies were assessed by Kaplan− Meier curves. Data were analyzed with Prism 7 software (GraphPad). Data were expressed as mean ± SD.

Program of Guangzhou (201902020018), and the Fundamental Research Funds for the Central Universities and 111 project.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b03472. Additional characterizations and results, including particle size, PAMAM release, images of particle draining into LNs, PAMAM and drug accumulation in primary tumor and LNs, weight and TUNEL-stained section of LNs after treatment, penetration of clustered nanoparticles in the metastatic lungs, and immune cell analyses in LNs (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hong-Jun Li: 0000-0002-1765-8445 Jin-Zhi Du: 0000-0003-4037-1212 Jun Wang: 0000-0001-9957-9208 Author Contributions

J.L., H.-J.L., Y.-L.L., C.-F.X., and X.-J.D. performed the experiments. H.-J.L. and J.-Z.D. conceived the project, designed the experiments, interpreted the data, and wrote the manuscript. J.W. supervised the project. All the authors discussed the results and reviewed the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2017YFA0205600), the National Natural Science Foundation of China (31771091 and 51633008), Guangdong Natural Science Funds for Distinguished Young Scholar (2017A030306018), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S054), Guangdong Provincial Pearl River Talents Program (2017GC010304, 2017GC010713, and 2017GC010482), Outstanding Scholar Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110102001), Young Elite Scientists Sponsorship Program by CAST (2018QNRC001), China Postdoctoral Science F oundat ion Grants (20 17M62 267 3 and 2018T110862), the Natural Science Foundation of Guangdong Province (2018A030313993), Science and Technology J

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DOI: 10.1021/acsnano.9b03472 ACS Nano XXXX, XXX, XXX−XXX