Microwave responsive nanoplatform via P-selectin mediated drug

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Microwave responsive nanoplatform via P-selectin mediated drug delivery for treatment of hepatocellular carcinoma with distant metastasis Jinshun Xu, Xueqing Cheng, Longfei Tan, Changhui Fu, Muneeb Ahmed, Jie Tian, Jianping Dou, Qunfang Zhou, Xiangling Ren, Qiong Wu, Shunsong Tang, Hongqiao Zhou, Xianwei Meng, Jie Yu, and Ping Liang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b05202 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Microwave Responsive Nanoplatform via P-selectin Mediated Drug Delivery for Treatment of Hepatocellular Carcinoma with Distant Metastasis

Jinshun Xu†,‡,⫪, Xueqing Cheng§, Longfei Tan‡, Changhui Fu‡, Muneeb Ahmed&, Jie Tian¶, Jianping Dou†, Qunfang Zhou†,Xiangling Ren‡, Qiong Wu‡, Shunsong Tang‡, Hongqiao Zhou‡, Xianwei Meng*,‡, Jie Yu*,†,⫪, Ping Liang*,†,⫪

†Department

of Interventional Ultrasound, Chinese PLA General Hospital, Beijing, 100853,

China ‡CAS

Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese

Academy of Sciences, Beijing, 100190, China ⫪State

Key Laboratory of Kidney Disease, Chinese PLA General Hospital, Beijing, 100853,

China &Laboratory

for Minimally Invasive Tumor Therapies, Department of Radiology, Beth Israel

Deaconess Medical Center/Harvard Medical School, Boston, MA 02215, USA §Department ¶CAS

of Ultrasound, Sichuan Provincial Cancer Hospital, Sichuan, 610041, China

Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of

Sciences, Beijing 100190, China

Corresponding Authors Ping Liang ([email protected], Tel. +86 010-66937981, Fax. +86 010-66939530) Jie Yu ([email protected], Tel. +86 010-66937981, Fax +86 010-66939530) Xianwei Meng ([email protected], Tel. +86 010-82543521, Fax +86 010-82543521)

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ABSTRACT Hepatocellular carcinoma (HCC) with metastatic disease is associated with a low survival in clinical practice. Many curative options including liver resection, transplantation, and thermal ablation are effective in local but limited for patients with distant metastasis. This study, the efficacy, specificity and safety of P-selectin targeted delivery and microwave (MW) responsive drug release is investigated for development of HCC therapy. By encapsulating doxorubicin (DOX) and MW sensitizer (1-butyl-3-methylimidazolium-L-lactate, BML) into fucoidan conjugated liposomal nanoparticles (TBP@DOX), specific accumulation and prominent release of DOX in orthotopic HCC and lung metastasis are achieved with adjuvant MW exposure. This results in orthotopic HCC growth inhibition that is not only 1.95-fold higher than found for non-targeted BP@DOX and 1.6-fold higher than non-stimuli responsive TP@DOX but is also equivalent to treatment with free DOX at a 10-fold higher dose. Furthermore, the optimum anticancer efficacy against distant lung metastasis and effective prevention of widespread dissemination with a prolonged survival is described. In addition, no adverse metabolic events are identified using the TBP@DOX nanodelivery system despite these events being commonly observed with traditional DOX chemotherapy. Therefore, administering TBP@DOX with MW exposure could potentially enhance the therapeutic efficacy of thermal-chemotherapy of HCC, especially those in the advanced stages.

KEYWORDS Nanoplatform, P-selectin, microwave, hepatocellular carcinoma, metastasis


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Worldwide, liver cancer is one of the most common causes of death and approximately 90% of these are hepatocellular carcinomas (HCCs) with a considerably poor 5-year survival rate,1-3 since the majority of HCC patients frequently progress into an advanced stage when extensive metastases have already occurred and where curative treatments (e.g. resection, ablation and transplantation) are not an option, especially for those with distant lung metastasis.3-5 At present, sorafenib is one of the most representative options for evidence-based systemic therapy of advanced HCC.3, 5 However, the median overall survival was determined as only 10.7 months in a high-quality, large-sample, multicenter, randomized controlled trail.6 Moreover, treatment with sorafenib is accompanied by severe adverse events, including diarrhea, weight loss, hand-foot skin reaction, and hypophosphatemia.7-8 Thereby, more efficient strategies are highly desirable for the simultaneous treatment of both localized HCC and distant metastasis. Chemotherapeutic agents encapsulated in nanoparticles is a promising way to overcome limitations with conventional chemotherapy, including increasing circulation time and reducing systemic side effects.9-12 Yet, challenges with nanoparticle encapsulated drugs remain with insufficient targeting and limited intratumoral drug release, particularly in use with metastatic multifocal HCC.4,

13-14

Several potential solutions include targeting specific

molecules associated with HCC metastases and developing strategies for increased intratumoral drug release. Specifically, P-selectin expression has been associated with vascular metastatic dissemination in HCC through mediation of adhesion of circulating tumor cells with distant vascular endothelium.15-16 Intratumoral acidic HCC environments can also be used to facilitate drug payload release from nanoparticles.17-18 Finally, use of external energy application (such as with photodynamic therapy or ultrasound) with combination of specific adjuvant stimuli has been used to facilitate nanoparticle intratumoral drug release.19-20 Based on this, in the present study, a P-selectin targeted liposome nanoparticle was developed with co-encapsulation of both doxorubicin (DOX) and a microwave energy field sensitizing agent, 1-butyl-3-methylimidazolium-L-lactate (BML) to create a single nanodrug. The BML encapsulated into nanoparticles would be a robust sensitizer for MW-heating conversion based on the ionic confinement mechanisms by our previous reported.21-23 This was then studied to establish 1) in vitro properties with respect to stability and biocompatibility, 2) in vitro nanoparticle drug release in acidic and MW energy-exposed conditions, 3) in vitro Pselectin targeting and cellular uptake, 4) in vivo targeting and biodistribution, payload delivery and release, and treatment efficacy for intrahepatic HCC and extrahepatic HCC metastases in two representative HCC models, and 5) overall safety profile of the nanoparticle agent. The 3 ACS Paragon Plus Environment

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purpose of this study is to demonstrate the advantages of a selective P-selectin-specific and MW sensitive agent will yield greater treatment efficacy and overall safety for intrahepatic and metastatic HCC over comparative alternatives. The developed targeted nanoplatform (TP) incorporated with BML and DOX is hereinafter referred to as TBP@DOX. Firstly, with a nanomolar affinity for P-selectin,24-25 fucoidan was used to achieve specific nanoparticle accumulations of both orthotopic and metastatic HCCs. Secondly, as a MW sensitizer, BML ensured thorough DOX release under the combined stimuli of both acidic environments and adjuvant MW exposure in the targets. Thirdly, MWrelated hyperthermia was augmented by TBP@DOX to further minimize the common drug dosage for mitigating dose-limiting toxicity. More importantly, this safely designed nanoplatform would be completely metabolized in the blood and organs, with no remaining acute or chronic metabolic side effects. Therefore, the tumor-specific P-selectin-dependent accumulation and ultra-efficient stimuli responsive release could significantly suppress cancer growth in intrahepatic and metastatic HCC under adjuvant MW exposure, without emergence of metabolic adverse events after systemic nanoagent injection. The overall treatment strategy for fucoidan-based, MW stimuli responsive TBP@DOX nanoparticles was illustrated in the schema (Figure 1). To prepare TBP@DOX, both DOX and BML were co-encapsulated by a mixture of lipids with surface crosslinking by EDC-activated fucoidan (Figure 2a and Figure S1a), which contained 1,2-dipalmitoyl-sn-glycero-3phosphocholine (polyethylene

(DPPC), glycol)-2000]

1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[amino [DSPE-PEG(2000)Amine],

cholesterol,

and

fucoidan

polysaccharide at a 4:2:1:1 molar ratio, affording core-shell TBP@DOX nanoparticles. The self-assembled liposome nanoparticles contained DOX for chemotherapy; BML as a MW sensitizer for MW hyperthermia therapy; activated fucoidan as a target to achieve specific accumulation; stimuli-responsive DPPC and DSPE as a lipid bilayer to accurately control the endogenous and exogenous drug release;26 cholesterol as a lipid excipient to order, condense, and stabilize the lipid bilayer structure; PEG(2000) to minimize mononuclear phagocyte system (MPS) uptake and prolong blood circulation properties (Figure 2b). In order to determine the successful synthesis of TBP@DOX, a fourier transform infrared spectrometer (FTIR) was applied to examine the composition of the as-made nanoplatforms (Figure S1b). The stretching vibration absorption of the aromatic ring from 1620 cm-1 to 1438 cm-1 as the characteristic peak confirmed the presence of DOX. The characteristic absorption peak of the imidazole ring stretching vibration at 1169 cm-1 confirmed the presence of BML. The strong absorption band between 1255 cm-1 and 1240 cm-1 (S=O stretching) confirmed a 4 ACS Paragon Plus Environment

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significant amount of fucoidan polysaccharides in the as-made samples. These results indicated that DOX, BML, and fucoidan were successfully loaded into the liposome nanoparticles, demonstrating the P-selectin targeted, MW stimuli-responsive nanoplatforms were successfully obtained. Because the nanomolar affinity of fucoidan towards P-selectin is crucial for P-selectin mediated targeting in TBP@DOX, it was further validated by energy dispersive spectrum (EDS) (Figure S1c), transmission electron microscope (TEM) and dynamic light scattering (DLS) (Figure S1d). Before linking fucoidan, the surfaces of nanoparticles without targeting were smooth and the zeta potential was positive (13.3 ± 3.6 mV). After coating with negative fucoidan to form TBP@DOX, the surfaces became obviously rough and the zeta potential rapidly reversed (-15.9 ± 5.2 mV), indicating that fucoidan with targeting capacity was loaded in TBP@DOX. Simultaneously, the black dots in the TEM inset at the top right corner revealed that DOX and BML had been encapsulated in the core of the nanoparticles (Figure S1d_i), highly consistent with the FTIR results. The final as-synthesized TBP@DOX nanoparticles were uniformly spherical in shape with good dispersity as observed by TEM (Figure 2c). DLS measurements provided a series of nanoparticle properties for different solvents at 25 °C or 37 °C, including number-average diameters of 92.1 ± 12.5 or 96.4 ± 7.8 nm, 100.6 ± 14.7 or 102.3 ± 13.3 nm, 102.4 ± 12.8 or 107.2 ± 10.4 nm; polydispersity indices (PDI) of 0.19 ± 0.02 or 0.21 ± 0.03, 0.20 ± 0.03 or 0.22 ± 0.03, 0.21 ± 0.02 or 0.20 ± 0.02; and zeta potentials of -15.2 ± 3.6 or -14.4 ± 3.4 mV, 16.7 ± 2.8 or -14.4 ± 2.6 mV, -13.8 ± 2.5 or -15.5 ± 3.1 mV, in deionized water, culture medium and blood plasma, respectively (Figure 2d-e). No significant differences (P > 0.05) detected among these physical and physiological properties were indicative of the highly favorable structural stability of TBP@DOX. Considering the duration interval in blood circulation, the stability and targeting conjugation were further assessed through incubating TBP@DOX in plasma at 37 °C for up to 24 h with a shaker. As a result, the consistent findings of nanosize, PDI and sulfur content in TBP@DOX were evidenced by DLS and ICP-OES (Figure 2f-g)., respectively, exhibiting the excellent stability of nanoparticles and strong conjugation capability for targeting. To evaluate the potential biocompatibility of TBP@DOX, the hemolysis test was performed. As shown in Figure 1h, hemolysis ratios induced by a large range of TBP@DOX concentrations were all less than 2% and the maximum value was only 0.9% ± 0.3, indicating that TBP@DOX had low toxicity towards red blood cells. Compared with the obvious hemolysis in the deionized water (DW) as a positive control (inset of Figure 2h), no visual hemolysis was found with TBP@DOX, even at the extremely high concentration of 16 mg/ml, showing great comparability with the negative control (phosphate-buffered saline, 5 ACS Paragon Plus Environment

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PBS). These results demonstrated that the as-fabricated TBP@DOX nanoparticles exhibited excellent stability, strong targeting capability, and good biocompatibility for further in vivo experiments. In addition, we also prepared BP@DOX (non-targeted nanoplatform loaded with DOX and BML), TP@DOX (targeted nanoplatform loaded with DOX only), and TBP (targeted nanoplatform loaded with BML only) as negative controls. These control nanoparticles were fabricated using the same procedures and exhibited comparable physicochemical properties to those of TBP@DOX (Figure S2). Next, MW energy (450 MHz, 1.8 W/cm2) exposure on 1 ml of TBP@DOX nanoparticles at different concentrations (1, 2, 5 mg/ml) was used to investigate the influence of TBP@DOX with BML encapsulation on MW-heat conversion. As shown in the data graph (Figure 3a) with corresponding infrared thermal images (Figure 3b), the MW heating effect was rapidly raised with increasing TBP@DOX concentration, despite no remarkable temperature variances (only ~ 2 °C of up-fluctuation) incurred in two controls of both DW and TP@DOX (1 mg/ml). After exposing for 5 min, the temperature changes at the concentrations of 1, 2, 5 mg/ml were 18.2, 20.7, and 28.4 °C respectively, indicative of TBP@DOX nanoparticles harboring a valid MWheat conversion capacity. Subsequently, the effect of MW power on the temperature changes in TBP@DOX suspensions (1 ml, 5 mg/ml) was also evaluated (Figure 3c). Following 5 min exposure with an increase in MW power from 0.6 to 1.2 and 1.8 W/cm2, the values of temperature rose from 36.3 to 49.5 and 56.5 °C respectively, suggesting the MW-heating conversion enhanced with the power increase. These results demonstrated that TBP@DOX was an effective adjuvant for hyperthermia therapy. In the TBP@DOX nanoparticles, the BML loading content was 8.64 ± 2.1% as determined by thermogravimetric analysis (TGA, Figure 3d) and fucoidan loading content was 1.06 ± 0.27% as measured by ICP. The encapsulated DOX showed a high encapsulating efficiency (EE) of 85.4 ± 8.3% with a good loading capacity (LC) of 18.1 ± 4.5% as determined using ultravioletvisible (UV-Vis) spectrophotometer (Figure 3e). To evaluate the MW- and pH-responsive capabilities of TBP@DOX, the release profiles of DOX were investigated at 37 °C over a long time period. The initial DOX release at pH 7.4 was slow and sustained, with only 3.3 ± 0.5% and 23.7 ± 1.7% released within 2 and 50 h respectively, suggesting that TBP@DOX nanoparticles can effectively protect drug bioactivity to prevent premature drug release. Meanwhile, a greater release was exhibited at pH 5.5 with 56.5 ± 2.4% released over 50 h, demonstrating the specific drug release in the acidic environment (Figure 3f), which was consistent with the published literatures.13, 27-28 However, taking into account that the in vivo 6 ACS Paragon Plus Environment

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basal temperature (37 °C) is much higher than room temperature (25 °C), a low dosage of MW energy (450 MHz, 1.2 W/cm2) was applied for 3 min to simulate temperatures closer to natural body temperature. After being irradiated three times at 4, 8, and 25 h, a significant higher amount (70.3 ± 3.0%) of DOX was released at pH 7.4 over 50 h, resulting in ~124% greater drug release compared to an acidic environment alone of pH 5.5 (Figure 3g). Furthermore, when combining both MW energy and low pH 5.5 environment, the drug release was further accelerated to 93.7 ± 1.9% over 50 h, almost implementing the absolute release of DOX from the nano-delivery vehicle. In addition, drug release was controlled precisely with the dose (i.e., time and power) of MW energy (Figure 3h-i), consistent with the corresponding temperature changes of the nanoparticles under MW exposure (Figure 3c). In sum, MW- and pHresponsive TBP@DOX was fabricated for the efficient encapsulation of DOX and BML, and the ultra-efficient release of encapsulated drugs was prominently controlled by MW energy dosing. Subsequently, the in vitro anticancer capability of TBP@DOX nanoparticles was investigated using the two HCC cell lines, human HepG2 and murine H22. As incubating cells firstly with the targeted nanoparticles incorporated BML only (TBP), the cytotoxicity of nanoparticles with no drug was negligible (Figure S3a-b), consistent with the preceding hemolysis assay (Figure 2h), demonstrating the high biocompatibility of the as-fabricated delivery nanoplatform. After MW exposure for 3 min at 1.2 W/cm2, the cytotoxicity of TBP towards both HepG2 and H22 cells was greatly enhanced (Figure S3c-d), consistent with prior MW-heat conversion evaluation results (Figure 2a-c), demonstrating the enhanced cytotoxicity attributed to the hyperthermia effect for HCC cells treatment. Additionally, no significant cytotoxicity resulted in the MW exposure only as control (Figure S3c-d), indicating that the used dose of MW energy was sufficiently secure without damage to cells. Since DOX has to bind to DNA in the nuclei to induce cytotoxicity, the cellular uptake of DOX with MW exposure was evaluated via confocal images (Figure 4a and Figure S3e). When 2 h incubation HepG2 cells with nanoparticles, only a small amount of DOX was transported into cells in TBP@DOX and TP@DOX groups without MW exposure. However, a significant enhancement of cellular uptake occurred in the TBP@DOX group after MW exposure (450 MHz, 1.2 W/cm2) for 3 min. In comparison, TP@DOX group still resulted in a little cellular uptake despite the combination with MW energy. The underlying rationale may attribute to: Firstly, a large amount of DOX was released from TBP@DOX under MW stimuli. Secondly, via external MW irradiation, electronic vibrations on cell membranes were performed to result in the change of membrane water partitioning, leading to the migration and transformation of 7 ACS Paragon Plus Environment

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released drugs nearby.21, 29-30 Additionally, with the increase of TBP@DOX concentrations (from 20 to 40 µg/ml) under MW exposure for the co-incubation, more red DOX covered the blue nucleus, demonstrating an enhanced efficiency on the cellular uptake for anticancer treatment. As a result, in the cell viability assays, TBP@DOX combined with MW exposure at a wide range of DOX concentrations (2, 4, 20, 40 µg/ml) can most effectively kill the two HCC cells (HepG2 and H22) among all groups including TP@DOX and TBP@DOX with and without MW exposure (P < 0.0001) (Figure 4b-c), with an IC50 (DOX concentration, µg/ml) of 0.21 (95% confidence intervals, 0.11 to 0.37) and 0.32 (95% confidence intervals, 0.23 to 0.44) respectively, dropping by 3.5-fold and 3.0-fold compared with free DOX controls (Table 1). Before investigating the targeting capability of TBP@DOX, P-selectin expression in HCC cells was firstly determined using 42 clinical samples, including 24 orthotopic HCCs and 18 lung metastases by immunohistochemistry (Figure 4d-e). Findings of abundant expression of P-selectin were observed on samples of both orthotopic HCCs (83%) and lung metastases (78%) (Figure 4e). Compared to scarce expression of P-selectin in adjacent normal tissues as expected, the high prevalence of P-selectin expression was not only in the surrounding vascular endothelium but also on tumor cells and extracellular matrix (ECM) (Figure 4d). In addition, we observed that both HepG2 and H22 cell line-based tumors exhibited strong staining for Pselectin, based on assessing the microvasculature of subcutaneous tumors in the xenograft nude mice models (Figure 4f). These results demonstrated the high prevalence of P-selectin expression in the liver cancers, facilitating the targeting delivery of TBP@DOX via P-selectin mediation for treatment of advanced HCC with distant metastases. Next, the binding affinity of fucoidan-based nanoparticles to P-selectin was determined using human umbilical vein endothelial cells (HUVECs) under inflammatory conditions (Figure 4g-h and Figure S4). With stimulation of tumor necrosis factor 𝑎 (TNF𝑎), both TBP@DOX and TP@DOX bound to HUVECs, as validated by obvious red signal in fluorescence from the cells. In comparison, BP@DOX without fucoidan conjugation virtually showed no signal as the same as TNF𝑎 negative controls. After incubated with excess Pselectin antibody, the targeting capability of nanoparticles were suppressive, resulting in rare red fluorescence around the cells. To investigate the P-selectin-mediated targeting ability and dynamic biodistribution of TBP@DOX in vivo, the subcutaneous H22 tumor models in nude mice were used. After prepared with Cy 5.5 fluorescence dye, TBP@DOX or BP@DOX were intravenously (i.v.) administrated at the same dose, respectively. After administration, the average fluorescence intensity of TBP@DOX in tumor reached 1.8-fold, 2.2-fold, and 2.7-fold 8 ACS Paragon Plus Environment

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than that of passively targeted BP@DOX at 12 h, 24 h, and 48 h post-injection, respectively (Figure S5a-b). Subsequently, the biodistribution measured by fluorescence at 48 h postadministration showed a gathering preferential accumulation of TBP@DOX in the tumor over other organs (liver, spleen, heart, kidney, and lung), yielding a total of 60.1 ± 11.2% accumulation (Figure 5a and Figure S5c). In comparison, the same calculation made for BP@DOX nanoparticles was only 21.8 ± 5.3%, suggesting a superior tumor targeting efficacy of TBP@DOX with a 3.2-fold improvement over passive targeting mechanisms. When nude mice were intratumorally pre-injected with a P-selectin blocking antibody, the positive targeting accumulation of TBP@DOX in tumor was abrogated, showing the parallel biodistribution in the tumor models with BP@DOX (Figure 5a and Figure S5a-c). The pharmacokinetics (PK) study on subcutaneous H22 tumor-bearing mice was determined by UV-Vis based on the absorbance of DOX after i.v. administration. Owning to PEG2000 modification, TBP@DOX persisted in blood circulation for greater than 25 h with a half-life of 12.8 ± 2.4 h (Figure 5b), exhibiting its longer circulation than BP@DOX without PEG2000 with a half-life of 8.2 ± 1.2 h (P < 0.05). Both nanoparticles showed liver accumulation indicated that TBP@DOX and BP@DOX were mostly uptake in the MPS organs (Figure 5c and Figure S5d). However, compared to BP@DOX with high distribution in liver (34.1 ± 1.7%) and spleen (16.7 ± 1.2%) at 48 h after administration, TBP@DOX with distributions of 23.6 ± 2.2% in liver and 9.8 ± 0.8% in spleen exhibited lower efficiency in MPS uptake (P < 0.01, Figure 5d-e). Thus, the prolonged PK in blood circulation and low MPS capability contributed to a higher tumor accumulation of TBP@DOX (38.5 ± 5.2%, P < 0.01 versus 18.8 ± 3.3% of BP@DOX) (Figure 5f), revealing that the targeting ability of P-selectin can dramatically change the nanoparticle distribution for enhancing specific drug delivery. To further determine the P-selectin targeting ability in orthotopic HCC and distant metastasis, models of in situ intrahepatic HCC and distant HCC metastases were established. For intrahepatic HCC, H22 tumors in the liver lobe of mice were established by open laparotomy according to the previously reported procedures (Figure S5e).31 To simulate a model of the distant HCC metastasis, lung metastases were established through i.v. injection of H22 cells at 2-3 weeks (Figure S5f). As shown in Figure 5g and Figure S5g-h, the DOX fluorescence in tumors from TBP@DOX nanoparticle-treated mice was significantly stronger than that of mice treated with BP@DOX nanoparticles (P < 0.01), resulting in specific targeting accumulation of TBP@DOX in the orthotopic HCC and distant lung metastasis. All results revealed that the P-selectin mediated targeting ability can dramatically change the distribution of TBP@DOX, increasing the total accumulation of drugs in both orthotopic and metastatic 9 ACS Paragon Plus Environment

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tumors to a significantly greater extent than the passive targeting achieved with BP@DOX. More importantly, the presence of DOX in the nuclei was more evident with TBP@DOX combined with MW energy than that with TBP@DOX alone (P < 0.05), consistent with in vitro data (Figure 4a and Figure S3c), indicating exogenous MW stimuli can be used to precisely control drug release and synergistically enhance the entrance of drugs into the cell nucleus in vivo. To evaluate TBP@DOX anticancer activity under MW energy exposure, two orthotopic HCC (H22 and HepG2) models engrafted in mice liver were treated with either i.v. TP@DOX, BP@DOX, or TBP@DOX, respectively, at a DOX dose of 4 mg/kg per day for a total of three treatments (Figure 6a and Figure S6A). After 24 h each injection, MW energy (1.2 W/cm2, 3 min; 450 MHz) was performed outside of the liver but near the tumor with ultrasound guidance. The control arm consisted of i.v. PBS injections followed by MW energy application. As exhibited in Figure 6b-d and Figure S7a, MW exposure itself failed to delay HCC progression in both H22 and HepG2 models, consistent with Figure S4c-d, indicating that the MW energy output could not induce cancer cell necrosis and the MW exposure strategy used demonstrated a good in vivo safety. In contrast, the TBP@DOX/MW almost eradicated the orthotopic HepG2 cancers with a 99% reduction in tumor volume compared to the non-targeted BP@DOX/MW with 54% reduction and the non-stimuli responsive TP@DOX/MW with 65% reduction (Figure 6c). Notably, similar findings were observed in H22 engrafted mouse models, resulting in 2.1-fold and 1.7-fold greater cancer growth inhibition in the TBP@DOX/MW (99% reduction) compared with BP@DOX/MW (46%) and TP@DOX/MW (56%) (Figure 6d). Consistent with these results, the corresponding infrared thermal images of MW exposure zone visually documented the process of temperature variance among different arms (Figure 5e-f and Figure S7b-c), showing the highest temperature rise with TBP@DOX (P < 0.05 in all), revealing that the prominent accumulation of TBP@DOX nanoparticles with high MW sensitivity resulted in significant HCC growth inhibition. Whether TBP@DOX with MW energy exposure could enhance therapeutic efficiency over free DOX was also evaluated. Mice were randomized to four arms: PBS administration as a vehicle control; high-dose free DOX administered at 6 mg/kg per day for five days (total of 30 mg/kg); low-dose free DOX administered at 1 mg/kg per day for three days (total of 3 mg/kg); and TBP@DOX administered at a DOX does of 1 mg/kg every day for three days (total of 3 mg/kg) with MW energy exposure (1.2 W/cm2, 3 min; 450 MHz) at 24 h each post-injection (Figure 6g and Figure S8). As shown in Figure 6h-i, the TBP@DOX/MW resulted in a significant decrease in tumor burden, which was comparable to the results obtained with a 1010 ACS Paragon Plus Environment

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fold higher dose of free DOX at a total of 30 mg/kg (P > 0.05). In comparison, the equivalent dose of free DOX administrated at 1 mg/kg per day elicited no perceptible anticancer activity in HepG2-bearing mice (Figure 6h), whereas in H22 orthotopic HCC models it induced the transient inhibition of cancer growth followed by an acquired insensitivity to the treatment (Figure 6i). Furthermore, the median survival of HepG2-bearing mice treated with TBP@DOX/MW was significantly higher (46 days, with 50% complete and partial responses) than the vehicle control (15.5 days), low-dose free DOX (18 days), and high-dose free DOX (35 days, with 16.7% partial responses and no complete response) (Figure 6j; P < 0.0001). Similarly, TBP@DOX/MW treatment resulted in prolonged survival in H22 orthotopic HCC mouse models (Figure S7d; P < 0.0001). To investigate the anticancer activity of TBP@DOX nanoparticles against metastatic extrahepatic HCC in mice, two lung metastasis models were established with i.v. injection of HepG2 and H22 cells. The experimental protocol was shown in Figure 7a and Figure S9. Animals were allocated to one-time treatment that included free DOX (6 mg/kg) without MW energy exposure; TP@DOX, BP@DOX, and TBP@DOX at an equivalent DOX dose (6 mg/kg) with MW exposure; TBP nanoparticles (no DOX) without MW exposure at an equivalent dose with TBP@DOX as the vehicle control. To study the potential for dose escalation due to reduced systemic exposure, TBP@DOX at the uppermost dose (30 mg/kg) with single administration with equivalent MW exposure was also performed. The MW exposure (1.2 W/cm2, 3 min; 450 MHz) was used outside of the lung metastases but around the chest at supine position of mice. On CT imaging follow-up at day 15 post-treatment, all DOX loaded nanoparticles showed more effective anticancer activity than free DOX (P < 0.01) (Figure 7bd and Figure S10). At the low dose (6 mg/kg), TBP@DOX/MW exhibited greater anticancer efficacy than either TP@DOX/MW or BP@DOX/MW as well (P < 0.05). No difference was observed between free DOX and non-DOX vehicle control (P > 0.05). More importantly, the most distinct anticancer effect was observed at the high dose TBP@DOX/MW (30 mg/kg), with significantly fewer number and size of lung metastases compared to all other arms (P < 0.01). These results demonstrated that TBP@DOX nanoparticles with MW exposure reached the optimum anticancer efficacy for treatment of metastasis in lung and also provided an alternative to the use of ultra-high drug doses for treatment of advanced-stage HCC with widespread metastases. As known, the onset and progression of tumor metastasis is a multistep process that involves migration and invasion from primary tumor cells, permeation into blood or lymphatic vessels, transportation in the circulation system, extravasation into parenchyma of distant 11 ACS Paragon Plus Environment

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tissues, and proliferation to generate metastases.32-33 Therefore, treatment of satellite lesions or disseminated tumors apart from successful imaging surveillance is particularly pivotal for elevated therapeutic efficiency and prolonged overall survival. In this study, the extent of disseminated nodules in four different sites (lung, pleura, liver, and peritoneum) was evaluated by gross examination of tissues after 35 days treatment to assess whether TBP@DOX with MW exposure could inhibit the growth of satellite lesions for evasion from widespread dissemination. As shown in Table 2, free DOX (6 mg/kg) with extrapulmonary metastases in all (8 of 8 mice) showed little effect in reducing tumor burden of satellite lesions without prevention of widespread dissemination. In comparison, extrapulmonary metastases occurred in 1 of 8 mice with 6 mg/kg P-selectin-specific TP@DOX/MW treatment in both HepG2 and H22 models. The proportion of mice with extrapulmonary metastases was less with 6 mg/kg BP@DOX/MW treatment without P-selectin targeting (3 of 8 mice in HepG2, and 2 of 8 mice in H22). Most interestingly, no extrapulmonary metastases were evident after 6 mg/kg TBP@DOX/MW treatment and all of the perceptible metastases disappeared with 30 mg/kg TBP@DOX/MW treatment, demonstrating that TBP@DOX combined with MW exposure was the most effective at prevention of widespread dissemination. The results of both CT surveillance and gross examination corresponded to the survival analysis (Figure 7e-f). For both HepG2 or H22 mouse models, all of the DOX-loaded nanoparticles with MW treatment resulted in decreased tumor burden and prolonged survival. Both free DOX and TBP only as the controls provided no survival benefit. The median survival of HepG2 bearing mice treated with TBP@DOX/MW was 87.5 days (30 mg/kg) and 65 days (6 mg/kg), both significantly higher (Figure 7e; P < 0.0001) than that of TP@DOX/MW (43 days) and BP@DOX/MW (42.5 days). Similar efficacy was observed in corresponding studies in the H22 lung metastasis model (Figure 6f; P < 0.0001) with median survivals of 85 days for TBP@DOX/MW (30 mg/kg), 62 days for TBP@DOX/MW (6 mg/kg), 40.5 days for TP@DOX/MW (6 mg/kg), and 42.5 days for BP@DOX/MW (6 mg/kg). Thus, in both models, significant control of metastatic HCC was achieved with TBP@DOX combined with MW energy. The safety assessment was investigated according to the protocol in Figure 7g and Figure S11. Compared to PBS administration following MW energy exposure (the control arm), no metabolic side effects were observed for TBP@DOX combined with standardized MW over a wide range of DOX concentrations at 6, 12, 30 mg/kg (P > 0.05), respectively, as measured by body weight (Figure 7h), complete blood count (Table S1), as well as hematoxylin and eosin (H&E) staining (Figure S12). In combination, this demonstrated an absence of systemic 12 ACS Paragon Plus Environment

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toxicity from i.v. administration of TBP@DOX nanoparticles in vivo. In comparison, free DOX at the maximum tolerated dose (6 mg/kg per day for a total of 30 mg/kg) resulted in distinct weight loss (Figure 7h; P < 0.01) and intense myelosuppression at 16 days treatment, including a rapid decrease in both platelets (PLT, P < 0.0001) and white blood cells (WBC, P < 0.001) (Figure 7i-j and Table S1), although no morphologic damage to major organs was observed by H&E staining (Figure S12). However, even when an equivalent single high-dose TBP@DOX (30 mg/kg) was combined with MW, no significant drop in weight or decreases in both WBC and PLT from the normal range were observed. Collectively, the data indicated the superior safety of TBP@DOX over free DOX for in vivo. As a significant amount of HCC patients die in the advanced stages of the disease with distant metastasis, it is critical to develop effective treatment strategies that not only eradicate orthotopic liver cancers but also suppress metastatic nodules and prevent widespread dissemination. In the present study, we explore the efficacy of P-selectin-mediated delivery of DOX, using the fucoidan-based nanoparticles with entrapped BML in the mouse models of orthotopic HCC and lung metastasis. The goal of this work is to investigate whether the specific accumulation and controlled release of DOX with MW energy exposure in the orthotopic and metastatic HCC environments is sufficient to achieve significant anticancer effects while sparing healthy tissues from systemic exposure and related toxicities. The results demonstrate that TBP@DOX nanoparticles have the excellent structural stability, high biocompatibility, prolonged circulatory half-time, significant tumor targeting and high MW susceptibility. All of these traits result in outstanding anticancer efficacy without metabolic toxicity in both orthotopic and metastatic tumors in two separate and representative HCC models. The underlying rationale of TBP@DOX nanoparticles combined with MW energy exposure for simultaneous treatment of both orthotopic and metastatic HCCs may be related to the P-selectin-mediated nanodelivery. Since the cell adhesion molecule P-selectin is responsible for leukocyte recruitment and platelet binding,34 as a vital step in the metastasis progression, it is available to translocate fucoidan-based nanoparticles from intrahepatic HCC cells to distant metastases via P-selectin-mediated adhesion of circulating tumor cells15-16 and increase the chance for TBP@DOX to travel through the endothelial layer and reach to the target zone by activating extracellular signal-regulated kinase and p38 mitogen-activated protein kinase,35 which has been validated by the previous literature.36 Therefore, the prominent accumulation of TBP@DOX can be found in both orthotopic and metastatic HCCs. After using MW exposure on the targets, insufficient drug release can be significantly enhanced due to the sensitizing effect of BML. Then, the released DOX can effectively migrate and 13 ACS Paragon Plus Environment

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permeate into the nucleus to enhance drug efficiency as a result of the electronic vibrations generated in cell membranes under MW application.30 Simultaneously, the related hyperthermia could be augmented by the mediation of TBP@DOX as a MW sensitizer for the process. Ultimately, the optimum anticancer efficacy in both orthotopic HCCs and lung metastasis can be realized with effective prevention of widespread dissemination without the observation of metabolic side effects, highly translatable to clinical applications. In order to overcome the barrier of insufficient drug release from endogenous stimuli in targeted tumor regions, the combined exogenous responsive strategies conventionally used at present include near-infrared light37-39 and ultrasound.20, 40 However, owning to the limited optical-penetration depth and the severe interference of ribs or pulmonary gases to sonic propagation,14, 41-42 it is difficult to achieve valid drug release into the orthotopic liver tumors and distant lung metastasis. By comparison, the specific merits of microwaves, such as high MW-heat conversion efficiency, deep penetration of tissues, and limited interference from ribs and pulmonary gases,21, 43 could provide a more advantageous alternative for targeted drug release. Key limitations of this study include the deficiency of mechanism investigation as a result of technical limits, the use of small tumor-bearing nude mice models, the single chemotherapeutic drug encapsulation, and the simple MW energy exposure method. Even though both HepG2 and H22 hepatic cancer cells were chosen based on their wide applications, it is possible that results may vary in larger animal species. Because of the intrinsic immunodeficiency of nude mice, large investigation should be explored in the bodies of immune animals. Additionally, the external MW energy exposure used in the present study implements good safety but without sufficient precision, development of a clinically-relevant exogenous application platform is required. Given these concerns, caution should be taken in the extrapolation of the presented strategy to clinical applications. In conclusion, we have developed P-selectin-targeted and MW-responsive TBP@DOX nanoparticles for the effective treatment of orthotopic and metastatic HCCs in the tumorbearing mice models. After systemic administration, TBP@DOX exhibits prolonged blood circulation, satisfactory biocompatibility, excellent tumor accumulation, and prominently controlled release with MW energy exposure. As a result, the highly efficient growth inhibition of both intrahepatic HCC and extrahepatic lung metastasis, as well as significant prevention of widespread dissemination, was achieved in this study. Therefore, we suggest that this novel strategy holds potential to be exploited for the treatment of HCC patients with advanced malignancy. 14 ACS Paragon Plus Environment

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Figures and Tables

Figure 1. Schematic illustration of the overall treatment strategy with fucoidan-based microwave (MW) stimuli responsive TBP@DOX.


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Figure 2. Characterization of TBP@DOX Nanoparticles. (a) Chemical scheme of fucoidan conjugation with EDC in the surface of liposome. (b) Schematic presentation of the TBP@DOX structure and correlative integration of three treatment strategies. (c) Transmission electron microscopy image of TBP@DOX, showing the spherical and monodispersed morphology. Scale bar, 200 nm. (d) Number-average diameters and (e) polydispersity index (PDI) and zeta potential of TBP@DOX at 25 °C and 37 °C in deionized water, medium, and blood plasma, respectively. #P > 0.05 by Two-way ANOVA, Tukey’s multiple comparisons test. (f) Time-dependent particle size and PDI variances of TBP@DOX in blood plasma at 37 °C. (g) Quantitative analysis of S content in TBP@DOX in plasma at 37 °C for up to 24 h with a shaker by ICP-OES. (h) Biocompatibility of the as-fabricated TBP@DOX at different concentrations of 2, 4, 8, 16 mg/ml via in vitro hemolysis test. Picture inset showing the corresponded hemolysis images of positive control (deionized water, DW), TBP@DOX at


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different concentrations (2, 4, 8, 16 mg/ml), and negative control (phosphate-buffered saline, PBS), respectively. Data in (e-h) were expressed as means ± SD (n = 3).

Figure 3. MW heating properties and controlled release of DOX from stimuli responsive TBP@DOX. (a) Heating curves of the dispersed nanoparticles in deionized water (DW) with different concentrations for MW exposure (450 MHz, 1.8 W/cm2) for 5 min. (b) Infrared thermal imaging pictures corresponded with (a) were taken once per minute. (c) MW-heat conversion of TBP@DOX (1 ml, 5 mg/ml) under exposure at different power for 5 min (450 MHz; 0.6, 1.2, 1.8 W/cm2). (d) TGA curves of targeted nanoparticles (TP), TP@DOX and TBP@DOX in the temperature range from room temperature to 600 °C with a heating rate of 10 °C per minute in the nitrogen flow. (e) Encapsulating efficiency (EE) and loading capacity (LC) of DOX in TBP@DOX nanoparticles detected by UV-Vis. (f-g) DOX release from TBP@DOX nanoparticles is pH dependent and can be precisely controlled with MW exposure. The arrows in (g) indicated MW exposure (450 MHz, 1.2 W/cm2) for 3 min at three different times. Data in (e-g) were expressed as means ± SD (n = 3). (h-i) UV-Vis absorbance of the supernatant of TBP@DOX solutions (pH 7.4) at 37 °C after (h) MW exposure at 1.2 W/cm2 for 1, 2 and 3 min, and (i) MW exposure for 3 min at 0.6, 0.9, and 1.2 W/cm2. The same TBP@DOX solution without MW exposure as a control. 18 ACS Paragon Plus Environment

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Figure 4. Cytotoxicity, cellular uptake and targeting of nanoparticles. (a) Confocal laserscanning images of HepG2 cells treated in different groups. Nanoparticles concentration was 20 µg/ml in the former four groups. A high dosage of 40 µg/ml was performed in the last group. Scale bar, 40 μm. (b-c) Cell viability incubated with TBP@DOX at different concentrations (µg/ml) with and without MW exposure. TP@DOX nanoparticles without BML incorporation as control. MW exposure in (a-c) was performed for 3 min (450 MHz, 1.2 W/cm2). (d) Immunochemistry images of human specimen stained with P-selectin antibody. Scale bar, 100 μm. (e) Percentage of clinical samples positively stained with P-selectin antibody. (f) Representative images of immunofluorescence staining for CD-31 (green) and P-selectin (red) in HepG2 and H22 xenograft tumor models. DAPI nuclear stain (blue). Scale bar, 50 μm. (g) Merged fluorescence images of human umbilical vein endothelial cells (HUVECs) incubated with BP@DOX, TP@DOX, and TBP@DOX with and without TNFα and (or) P-selectin antibody. Red, DOX in nanoparticles; green, Calcein AM cytoplasm stain; blue, Hoechst 19 ACS Paragon Plus Environment

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nuclear stain. Scale bar, 40 μm. (h) Quantification of red fluorescence efficiency of HUVECs shown in g. Data in (b,c,h) were expressed as means ± SD (n = 3). ****P < 0.0001 by Twoway ANOVA with Tukey's multiple comparisons test.

Figure 5. TBP@DOX targeting and biodistribution. (a) Fluorescent images of tumors and major organs at 48 h i.v. post-administration of TBP@DOX, BP@DOX (5 mg/kg at a DOX dose,100 µl), and pre-treated with anti-P-selectin antibody (Ab). (b) Blood concentration of TBP@DOX and BP@DOX over time. ID: injection dosage. (c) Biodistribution and tumor uptake of TBP@DOX over time measured by fluorescence. (d) Pie charts of relative contribution of each organ to total signal over time in biodistribution and tumor uptake of TBP@DOX and BP@DOX. (e-f) Relative distribution of TBP@DOX or BP@DOX in the main MPS organs (e) and tumors (f) to the total over time, measured by fluorescence. ****P < 0.0001, **P < 0.01 (Two-way ANOVA in e, Sidak's multiple comparisons test). **P < 0.01 (Unpaired t test in f, two-tailed). Data were expressed as means ± S.D. (n = 3). (g) Fluorescence images of DOX in orthotopic HCC and lung metastasis of mice at 48 h post-administration 20 ACS Paragon Plus Environment

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with BP@DOX or TBP@DOX, with and without MW exposure (450 MHz) for 3 min at 1.2 W/cm2 before mice euthanasia. MW exposure accelerated DOX release from nanoparticles and strengthened DOX entrance into the nuclei of cancer cells in vivo. Otherwise, the minor released DOX mostly stayed in the cytoplasm with minimal cytotoxicity. Scale bar, 50 μm.

Figure 6. TBP@DOX nanoparticles treatment of orthotopic HCC mouse models. (a) Study design on evaluation of TBP@DOX anticancer activity to orthotopic HCC under MW energy exposure in comparison with controlled nanoparticles. (b) Ultrasonography surveillance of HepG2 liver cancers in different treatment arms before and after treatment 4, 8, 12, 16 days. Scale bar: 5 mm. (c-d) Tumor growth curves of HepG2 xenografts (c) and H22 xenografts (d) after treatment with i.v. administration of TBP@DOX, TP@DOX, and BP@DOX at the DOX dose of 4 mg/kg/day for three treatments with MW exposure (1.2 W/cm2, 450 MHz; 3 min) at 21 ACS Paragon Plus Environment

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24 h post-injection (n = 6). (e-f) In vivo infrared thermal imaging (e) and responding heating curves (f) recorded of HepG2 orthotopic tumors in liver in each arm irradiated by MW (1.2 W/cm2, 450 MHz) for 3 min (n = 3). (g) Study design on evaluation of TBP@DOX anticancer activity with MW energy exposure over free DOX. (h-i) Tumor growth curves of HepG2 xenografts (h) and H22 xenografts (i) treated with i.v. administration of free DOX at either 6 mg/kg/day for 5 days or 1 mg/kg/day for 3 days, or i.v. administration of TBP@DOX at 1 mg/kg/day for 3 days with MW exposure (1.2 W/cm2, 450 MHz; 3 min) at 24 h post-injection (n = 6). (j) Survival analysis of mice engrafted with orthotopic HepG2 xenografts treated with i.v. administration of free DOX at either 6 mg/kg/day for 5 days or 1 mg/kg/day for 3 days, or i.v. administration of TBP@DOX at 1 mg/kg/day for 3 days with MW exposure (1.2 W/cm2, 450 MHz; 3 min) at 24 h post-injection (n = 6). In c, d, f, h, i, data were expressed as means ± S.D. #P > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001, by using one-way ANOVA with Tukey's multiple comparisons test. In j, P < 0.0001, calculated by using the log-rank test.


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Figure 7. Anticancer efficacy in lung metastases and safety evaluation. (a) Study design on evaluation of TBP@DOX anticancer activity to lung metastases under MW exposure. (b) Representative CT imaging of HepG2 lung metastases on day 15 post-treatment. From top to bottom: TBP as vehicle control, free DOX (6 mg/kg), TP@DOX (+) (6 mg/kg), BP@DOX (+) (6 mg/kg), TBP@DOX (+) (6 mg/kg), TBP@DOX (+) (30 mg/kg). Scale bar: 5 mm. (c-d) The numbers of metastatic nodules counted in the lungs of HepG2 (c) or H22 (d) models, n = 6. #P > 0.05 versus free DOX at 6 mg/kg, **P < 0.01 versus all DOX loaded nanoparticles, *P < 0.05 between TP@DOX (+) (6 mg/kg) and TBP@DOX (+) (6 mg/kg), **P < 0.01 between BP@DOX (+) (6 mg/kg) and TBP@DOX (+) (6 mg/kg). (e-f) Survival assessment of mice incubated with HepG2 (e) or H22 (f) lung metastasis treated with i.v. administration of TBP (vehicle control), free DOX (6 mg/kg), TP@DOX (+) (6 mg/kg), BP@DOX (+) (6 mg/kg), TBP@DOX (+) (6 mg/kg), TBP@DOX (+) (30 mg/kg), n = 6. (g) Study design on safe 23 ACS Paragon Plus Environment

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assessment of the strategy of TBP@DOX combined with MW exposure. (h-j) Body weight, PLT and WBC count of the orthotopic HepG2 HCC mice on day 16 post-treatment among different groups. n = 4. #P > 0.05 between all TBP@DOX arms and vehicle control. **P < 0.01, ****P < 0.0001, ***P < 0.001 between free DOX (30 mg/kg) and vehicle control. The arm of free DOX (30 mg/kg) meant i.v. administration 6 mg/kg per day for 5 days. The other arms meant one-time treatment with listed DOX concentrations with or without encapsulation. “(+)” refers to treatment with MW exposure (1.2 W/cm2, 450 MHz; 3 min) at 24 h postinjection. Data were expressed as means ± S.D. In c, d, h-j, statistical analysis by using oneway ANOVA with Tukey's multiple comparisons test. In e-f, all P < 0.0001, calculated by using the log-rank test. Table 1. IC50 values and 95% confidence intervals (CI) in HepG2 and H22 cells treated with various formulations with and without MW exposure (µg/ml, DOX concentration) DOX

TP@DOX

TBP@DOX

TP@DOX+MW

TBP@DOX+MW

IC50

CI

IC50

CI

IC50

CI

IC50

CI

IC50

CI

HepG2

0.73

0.37 to 1.41

6.55

3.48 to 12.33

10.82

6.48 to 18.0

7.31

5.17 to 10.3

0.21

0.11 to 0.37

H22

0.96

0.57 to 1.6

6.81

3.71 to 12.50

13.49

5.31 to 34.24

8.94

4.88 to 16.36

0.32

0.23 to 0.44

MW exposure at 450 MHz, 1.2 W/cm2 for 3 min. Table 2. HCC metastases in HepG2 and H22 mice models among different arms during a 35-day observation post-treatment (n = 8). Lung Hep G2 Arms

TBP@DOX (+) 30 mg/kg TBP@DOX (+) 6 mg/kg TP@DOX (+) 6mg/kg BP@DOX (+) 6 mg/kg DOX 6 mg/kg Vehicle control

Lung H22

Total No. of metastatic mice

No. with metastases in lung

No. with metastases in pleura

No. with metastases in liver

No. with metastases in peritoneum

Total No. of metastatic mice

No. with metastases in lung

No. with metastases in pleura

No. with metastases in liver

No. with metastases in peritoneum

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

2

2

0

0

0

3

3

1

0

0

3

3

1

0

0

4

4

2

1

0

3

3

1

1

0

6 (2 death)

4

3

2

0

7 (2 death)

5

5

2

0

8 (4 death)

4

4

4

2

8 (3 death)

5

5

5

3

“(+)” in arms referred to treatment with MW application. All death cases died in widespread metastases.


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AUTHOR INFORMATION Notes: All authors declare no conflict of interest. ACKNOWLEDGMENTS General: The authors would like to gratefully acknowledge Dan long, Qianqian Chai and Zheng Li from CAS Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China for their work in the laboratory. The authors would like to express their gratitude of Wenting Shang and Kun Su from CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, China for their assistance with providing the IVIS Spectrum Imaging and Tomographic Imaging System in the experiment. Funding: National Key R&D Program of China (No. 2017YFC0112000); National Natural Science Foundation of China (NSFC) (No. 81627803, 91859201, 81622024, 81701797); and Beijing Natural Science Foundation (No. JQ18021, 7192200, 4161003). ASSOCIATED CONTENT Supporting Information: Detailed experimental materials and methods, additional results of preparation and characterization of TBP@DOX, cytotoxicity and cellular uptake study, targeting and pharmacokinetic profile, experimental protocol in vivo, evaluations of treatment efficacy of orthotopic HCC and lung metastases, relevant HE staining and blood assay are available in the supporting information. The supporting information file is available free of charge.


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Microwave responsive nanoplatform via P-selectin mediated drug

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