Liposomal Codelivery of Doxorubicin and Andrographolide Inhibits

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Liposomal Codelivery of Doxorubicin and Andrographolide Inhibits Breast Cancer Growth and Metastasis Xuejia Kang,†,‡,⊥ Zening Zheng,†,‡,⊥ Zehua Liu,†,‡ Huiyuan Wang,‡ Yuge Zhao,‡,§ Wenyuan Zhang,‡ Mingjie Shi,‡ Yang He,‡ Yang Cao,∥ Qin Xu,*,† Chengyuan Peng,‡ and Yongzhuo Huang*,‡ †

Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, China Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China § Nanchang University College of Pharmacy, 461 Bayi Rd, Nanchang 330006, China ∥ The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China ‡

ABSTRACT: Effective treatment of metastatic (stage IV) breast cancers remains a formidable challenge. To address this issue, a cell-penetrating peptideassisted liposomal system was developed for codelivery of doxorubicin and andrographolide. This nanomedicine-based combination therapy showed the ability to inhibit the in vitro migration and invasion of 4T1 cells through the wound healing and transwell invasion assays. Furthermore, this delivery system exhibited the enhanced accumulation in the tumor tissues and deep intratumoral penetration. The synergistic effect of doxorubicin and andrographolide led to an evident inhibition of tumor growth in an orthotopic breast tumor mouse model and efficient prevention of lung metastasis. The therapeutic mechanism was associated with the anti-angiogenesis effect. In conclusion, this nanomedicinebased combination therapy provides a potential method for overcoming metastatic breast cancers. KEYWORDS: cell-penetrating peptide, doxorubicin, andrographolide, liposome, angiogenesis, breast cancer

1. INTRODUCTION Breast cancer is the most commonly diagnosed carcinoma and the second leading cause of cancer-related deaths in women worldwide.1 Metastasis is the primary cause of mortality in breast cancer patients. Approximately 30% of breast cancer patients eventually develop to metastatic disease,2 and in addition, approximately 5% of patients are already at metastatic stage at the initial diagnosis.3 In spite of advancement in therapeutic options, metastatic (stage IV) breast cancers have a 5-year relative survival rate of a mere 22%.4 Anti-angiogenesis is an important treatment method. Because the diffusion limit of nutrients from the capillary is only at a range of 100−500 μm, tumor neovascularization is a key step for tumor expansion for the accessibility of nutrients.5 Moreover, angiogenesis is necessary for the migration of cancer cells into the bloodstream and for the development of metastatic colonies at distant sites.5 Anti-angiogenesis is a promising target for the inhibition of metastasis. Andrographolide (Andro) is an active compound isolated from andrographis paniculata that is a Chinese herb used for anti-inflammation for centuries. The anti-tumor activity of Andro has been well documented, of which the mechanisms include cell-cycle arrest, apoptosis induction, and immunostimulation.6 To our interest, the anti-angiogenesis effect of Andro has also been well elucidated, for example, via a mechanism of blocking VEGFR2 signaling.7 Moreover, a clinical trial (NCT01993472) has been ongoing for investigating Andro in combination with © XXXX American Chemical Society

chemotherapeutics to treat the locally advanced or recurrent or metastasis inoperable colon cancer. In this study, we developed a liposomal codelivery system for Andro/doxorubicin (DOX) combination therapy. A benefit of the codelivery method is that the combined drugs could sustain a relative identical in vivo fate by simultaneous action on the cancer cells.8 Cell-penetrating peptides (CPP) have been well demonstrated to overcome various biobarriers for assisting the delivery of nanomedicine.9 Our previous results have revealed that CPP can enhance intratumoral penetration and tumor cellular uptake, as well as intranuclear delivery.10−12 Therefore, a CPP-modified liposome system was prepared to codeliver Andro and DOX, and its anti-tumor efficacy and anti-metastasis effect were investigated in an orthotopic breast tumor mouse model.

2. MATERIALS AND METHODS 2.1. Materials. Soybean phosphatidylcholine (SPC), cholesterol, and DSPE-PEG2000-Mal were purchased from Advanced Vehicle Technology Co., LTD (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Received: December 27, 2017 Revised: February 1, 2018 Accepted: February 26, 2018

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DOI: 10.1021/acs.molpharmaceut.7b01164 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

2.6. In Vitro Stability and Drug Release Assay of Liposomes. The colloidal stability of the liposomes was evaluated for 15 d at 4 °C in phosphate-buffered saline (PBS) by measuring the changes of the particle size. The in vitro drug release was conducted using a dialysis method. The liposomes or the free drugs were placed into a dialysis tube (MWCO 14 kDa) and dialyzed in PBS (pH 7.4 or pH 6.5 containing 0.5% Tween 80) at 37 °C in a shaker (110 rpm). Drug concentrations were measured at varying time points. The experiments were performed in triplicate. 2.7. In Vitro Uptake, Penetration of Tumor Spheroid, and Intranuclear Delivery Studies. The cells were seeded in a 12-well plate at a density of 1 × 106 cells/well and cultured for 24 h. The cells were then treated with the coumarin6-labeled Lipo-PEG or Lipo-CPP for 1 h incubation at the same concentration. The cells were washed with PBS three times. Fluorescent images were obtained using a fluorescence microscope (Zeiss, Germany). In addition, the cellular uptake efficiency was determined using a flow cytometer (BD Pharmingen, USA). To investigate the tumor penetration ability of the liposomes, the tumor spheroid model was prepared using the 4T1 cells and L929 cells at a ratio of 1:3, which were seeded at a density of 2 × 103 cells per well in a 96-well plate pretreated with 1% (w/v) agarose gel and cultured for 7 d. The thus-formed spheroids were incubated with the coumarin-6 labeled liposomes for 6 h. The spheroids were then rinsed with PBS three times, transferred to a chambered covered slip, and analyzed using confocal microscopy (TCS-SP8, Leica, Germany). Intracellular drug delivery was studied by treating the 4T1 cells with Lipo-PEG or Lipo-CPP (2 μg/mL DOX) for 2 h. After a thorough wash, the cells were treated with DAPI for the nucleus staining, and the imaging was processed using confocal laser scanning microscopy (CLSM) (TCS-SP8, Leica, Germany). 2.8. In Vitro Cytotoxicity Studies. The cell viability was measured by a standard MTT assay. The 4T1 cells were plated into the 96-well plates at 5 × 103 cells per well for overnight incubation. The blank liposomes, free combo drugs, Lipo-PEG, and Lipo-CPP were added to the cells and cultured for 48 h. The cell viability was calculated using a formula as follows. A variable slope model (log(inhibitor) vs normalized response) with a 95% confidence interval (CI) was used for the fitting and IC50 calculation using GraphPad Prism.

(St. Louis, USA). An Annexin V-FITC/PI apoptosis detection kit was purchased from Beyotime Institute Biotechnology (Shanghai, China). Fetal bovine serum (FBS) and RPMI1640 medium (Gibco) were purchased from Thermo Fisher Scientific (Waltham, USA). Anti-VEGF antibody was obtained from Abcam (Cambridge, UK), and anti-VEGFR2 was from Cell Signaling Technology (Danvers, USA). β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, USA). DOX and Andro were obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). 2.2. Cell Lines and Animals. The murine breast cancer cell line 4T1 was obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics (100 μg/mL streptomycin and 100 μg/mL penicillin) at 37 °C in 90% relative humidity and 5% CO2. The female BALB/c nude mice (4 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. 2.3. Synthesis of DSPE-PEG2000-LMWP. DSPE-PEG2000LMWP was prepared using the thiol−maleimide coupling reaction. In brief, DSPE-PEG2000-Mal and low molecular weight protamine (LMWP, sequence: CVSRRRRRRGGRRRR) were dissolved in HEPES buffer (pH 7.2) with a molar ratio of 1:1.5, and they were incubated for 24 h at room temperature with gentle shaking. The final solution was dialyzed against ultrapure water for purification. The product was confirmed by 1H NMR spectroscopy. 2.4. Preparation of Liposomes. The liposomes were prepared by a thin-film hydration technique. Lipids (SPC/Chol/ DSPE-PEG2000-LMWP) at a molar ratio of 25:11:0.7 were dissolved in chloroform, and then they were mixed with the desalted form of DOX methanol solution and Andro ethanol solution. The ratio between DOX, Andro, and lipids was 0.1:1:20 (w/w). The organic solvents were removed on a rotary evaporator in a 42 °C water bath. The thus-formed lipid film was hydrated with 5% glucose solution. The suspensions were dispersed and extruded through a polycarbonate membrane (200 nm) using an extruder (Avanti Polar Lipids, Alabaster, USA). The un-encapsulated DOX and Andro were removed using a Sephadex G-50 column. The CPP-modified liposomes were termed as Lipo-CPP, while the DSPE-PEG2000-modified liposomes were used as a control, termed as Lipo-PEG. 2.5. Characterization of Liposomes. The particle size, polydispersity index, and ζ-potential of the liposomes were analyzed by ZetaSizer nano-ZS90 (Malvern, UK). The morphology of the liposomes was investigated using transmission electron microscopy (TEM) after the samples were deposited on a copper grid and stained with 1% acetic acid glaze. The DOX concentration was determined by a fluorometer (λex 485 nm and λem 590 nm). Meanwhile, the concentration of Andro was detected by HPLC equipped with a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm) (mobile phase: methanol/ water 45:55; flow rate: 1 mL/min; detection λ: 225 nm). The drug loading efficient (DL%) and encapsulation efficiency (EE%) of the liposomes were calculated by the equations:

Cell viability(%) = (ODtest − ODDMSO)/(ODcontrol − ODDMSO) × 100%

The cell apoptosis rate was measured by using flow cytometry. The cells were seeded in a 12-well plate and cultured for 24 h. The cells were exposed to the drugs (free combo drugs, and the liposomes at a dose equal to 0.5 μg/mL of DOX and 5 μg/mL of Andro) for 24 h. The cells were collected and washed with PBS three times and stained with an FITC-Annexin V apoptosis detection kit (BD Pharmingen, USA) according to the manufacturer’s protocol. The cells were subjected to apoptosis analysis. 2.9. Wound-Healing Assay. The wound healing assay was used to determine the directional cell migration. The 4T1 cells were seeded in a 24-well plate. After the cells formed a confluent monolayer, the monolayer in each well was scraped to produce a gap with a 10 μL pipet tip. The cells were then treated with the drugs at a dose equal to 0.5 μg/mL of DOX and 5 μg/mL of Andro for 24 h. The cells were photographed by a microscope (Zeiss, Germany) to evaluate the migration ability.

DL% = WDE/WL × 100%; EE% = WDE/WT × 100%

where WDE is the drug amount encapsulated in the liposomes, WL is the total weight of the liposomes, and WT is the total amount of drug. B


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(HUVEC) were seeded at a density of 4 × 104 per well onto a 24-well plate that was pretreated with 50 μL of 1% (w/v) Matrigel Matrix (Corning, USA) and treated with free DOX, free Andro, free combo drugs, and the liposomes (the same ratio as the cytotoxicity study) for 12 h. The endothelial cell tube formation was observed by a fluorescence microscope (CARL ZEISS, Germany) in bright field. Western Blot Assay. The HUVEC cells seeded in the 6-well plates were treated with the same drugs as above and incubated for 24 h. Then collected cells were treated with the RIPA lysate buffer (Beyotime, China). The expression of vascular endothelial growth factor receptor-2 (VEGFR2) was analyzed by Western blotting with the anti-VEGFR2 antibody. The tumor tissues dissected at the end point of the therapeutic experiment were lysed with RIPA buffer for protein extraction. After centrifugation (10 000 g for 15 min), the supernatant was collected for detection of VEGFR2 and VEGF using a standard Western blot procedure. The bands were detected with ChemiDoc*MP imaging system (Bio-Rad, Hercules, USA). The optical density of each protein band was analyzed using ImageJ software, and quantitative normalization was conducted by dividing the intensity of each target protein band by that of an internal loading control. 2.14. Statistical Analysis. Data analysis were performed using GraphPad Prism 6 software. All data were depicted as the mean ± SD from ≥3 experiments or samples. The Student’s

2.10. Cell Invasion Assay. To assess the capacity for invasion, the 4T1 cells (5 × 103) in the RPMI-1640 medium containing 1% FBS were added to the upper chambers that were precoated with Matrigel in the Transwell inserts (Corning, 0.8 μm membrane). The RPMI-1640 medium containing 10% FBS was added to the lower chamber. The cells were then treated with the drugs (equal to 0.5 μg/mL of DOX and 5 μg/mL of Andro) and incubated for 24 h. The cells that invaded to the bottom of the membrane were stained with crystal violet solution for 30 min and washed twice with PBS. The stained cells were visualized under a microscope (Olympus, Tokyo, Japan). 2.11. In Vivo Imaging. The 4T1 tumor-bearing mice were administered with free DIR or the DIR-labeled liposomes at 0.8 mg/kg DIR by tail vein injection. After the treatment, the mice were subjected to in vivo imaging at various time points using the IVIS imaging system (PE IVIS spectrum, USA). At 24 h postinjection, the mice were sacrificed and the major organs (heart, lung, spleen, kidney, and liver) and tumors were collected for ex vivo fluorescence imaging. The radiant efficiency of images was analyzed by Living Image software (IVIS, USA). The tumor tissues were then used for cryosection, and the sections were fixed in 4% formaldehyde and stained with DAPI. The slides were observed using a confocal fluorescence microscope (TCS-SP8, Leica, Germany). 2.12. In Vivo Therapeutic Efficacy on Tumor Growth and Lung Metastasis. The female BALB/c nude mice were xenografted with 5 × 105 4T1 cells/mouse in the breast to establish an orthotopic tumor model that was characterized by the spontaneous development of lung metastasis. The tumor volume (V) was calculated with the formula: Tumor volume (mm3) = (long diameter) × (short diameter)2 /2

When the mice had a tumor size of about 100 mm3, they were treated with drugs (PBS, free combo, Lipo-PEG, or Lipo-CPP) by intravenous injection at a dose of DOX 0.5 mg/kg and Andro 5 mg/kg every 2 d over a period of 20 d. Four mice were in each group. At the experimental end point, the mice were sacrificed, and the organs and tumors were excised for weight measurement and then processed for histological and immunohistochemical examination. Meanwhile, the metastasis nodules in the lungs were counted. To assess in vivo apoptosis, the TdT-mediated dUTP nick-end labeling technique (TUNEL) staining was performed. The inhibition rate of tumor growth was calculated using the tumor weight as the following formula. Tumor inhibition rate (%) = (WTumor control − WTumor test)/WTumor control × 100%

where WTumor control referred to the tumor weight of the PBS group, and WTumor test referred to the tumor weight of a drug treatment group. The organ coefficient was used to preliminarily evaluate the side toxicity in the organs. The organs of the mice were harvested at the end of the experiment to calculate the organ coefficient. The organ coefficient was calculated using the following formula. Organ coefficient (%) = Weight of the organ/Body weight × 100%

2.13. Anti-Angiogenesis Studies. Endothelial Cell Tube Formation Assay. The human umbilical vein endothelia cells

Figure 1. Characteristic 1H NMR spectra. C


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Figure 2. (A) Size of the Lipo-PEG. (B) Size of the Lipo-CPP. (C) Zeta potential of Lipo-PEG and Lipo-CPP. (D) The TEM of Lipo-PEG. (E) The TEM of Lipo-CPP. (F) The stability of liposomes. (G) In vitro release of DOX.

t test was used when two parameters were evaluated. P < 0.05, P < 0.01, and P < 0.001 were denoted by *, **, and ***, respectively.

Table 1. Drug Encapsulation and Drug-Loading Efficiency of the Liposomes Lipo-PEG EE (%)

3. RESULTS AND DISCUSSION 3.1. Characterization of DSPE-PEG2000-LMWP and Liposomes. The 1H NMR spectrum showed characteristic signals of DSPE-PEG2000-LMWP (Figure 1). The peaks at 1.0−1.5 ppm represent the methylene groups (−CH2−) in DSPE-PEG2000-Mal, and the peaks at 7.0−8.0 ppm represent LMWP. The mean diameters were 177 nm (PDI 0.06) for Lipo-PEG and 181 nm (PDI 0.07) for Lipo-CPP (Figure 2A,B). The zeta potential of Lipo-PEG was −17.4 mV, while that of Lipo-CPP showed positive 1.14 mV due to the modification of the cationic LMWP (Figure 2C). Transmission electron microscopy (TEM) was also used to visualize the size and morphology of Lipo-CPP as shown in Figure 2D,E. The EE% and DL% of the Lipo-PEG and Lipo-CPP were similar (Table 1). The EE% of Andro and DOX were greater than 70% in both liposomes, and the DL% of Andro and DOX was about 2.8−2.9% and 0.26−0.29%, respectively (Table 1). The liposomes remained stable, and the particle size showed a very minor change in a period of 15 days (Figure 2F). The DOX release from the liposomes was pH-dependent, showing a fast release in acidic

DL (%)

Andro DOX Andro DOX

76.02 72.03 2.81 0.26

± ± ± ±

5.12 6.23 0.19 0.02

Lipo-CPP 79.03 74.40 2.91 0.29

± ± ± ±

6.23 7.74 0.09 0.03

medium (Figure 2G), similar to our previous observation.11 This could be due to the fact that the desalted DOX (with a pKa ≤ 9.46) is poorly soluble in neutral PBS, but it is readily ionized and dissolved in an acidic medium. 3.2. Cell Uptake, Cell Spheroid Penetration, and Intranuclear Delivery of Liposomes. The coumarin6-labeled Lipo-CPP showed a higher cellular uptake efficiency than the Lipo-PEG, with an approximately 1.7 times enhancement (Figure 3A,B). LMWP is a cell-penetrating sequence identified from an enzymatically digestive product of protamine.9 The LMWP-mediated tumor penetration ability was evaluated in a 3-D cultured cell spheroid. The Lipo-CPP showed a deeper tumor infiltration than that of the Lipo-PEG (Figure 3C), as demonstrated by the confocal microscopic multilevel scanning images from the top of the spheroid. The results revealed that LMWP modification enhanced the uptake and penetration into D


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Figure 3. Cell uptake of the liposomes. (A) Fluorescence images of the cells treated with liposomes. (B) FACS analysis of the uptake efficiency of the liposomes. (C) Penetration into 4T1 spheroids treated with Lipo-PEG and Lipo-CPP. Z-axis continuous top-down scanning of the spheroids layers by confocal microscope (20 μm for each depth level). (D) Improved intranuclear delivery of Lipo-CPP in 4T1 cells (CLSM images). The nucleus was stained with DAPI (blue), while DOX shows red fluorescence.

Figure 4. Anti-tumor activity of the liposomes. (A) MTT of different groups in 4T1 cells. (B) Cytotoxicity test of blank liposomes. (C) Quantitative analysis of the total apoptosis rate. (D) Flow cytometry scatterplots of 4T1 cell in apoptosis assay.

3.4. In Vitro Wound-Healing Assay and Transwell Migration Assays. The wound healing assays indicated that the Lipo-CPP had a remarkably decreased migration ability of 4T1 cells compared with that of the other groups (Figure 5A). Consistently, a similar inhibition about the invasion of breast cancer cells in the transwell assay was found. The number of cancer cells passing through the transwell membrane filter was significantly reduced in the group of the Lipo-CPP (Figure 5B). These results suggested that the Lipo-CPP inhibited the migration and invasion abilities of the malignant 4T1 cells. 3.5. Biodistribution Study of Liposomes. The fluorescent intensity, indicating the liposome accumulation, was gradually increased at the tumor site, and it reached the maximum at 8 h. The real-time in vivo imaging showed the Lipo-CPP group had an accumulation at the tumor site higher than that of the Lipo-PEG (Figure 6A,B). The ex vivo imaging of the dissected tissues further confirmed the enhanced tumor deposit in the Lipo-CPP group (Figure 6C,D). The tumor slices showed that both of the liposomes were able to be distributed inside the tumor tissues, but the Lipo-CPP group had a better intratumoral infiltration (Figure 6E).

the 4T1 tumor spheroids, which was consistent with our previous findings of the ability of LMWP-mediated tumor infiltration in various xenograft models.11−13 Importantly, enhanced intranuclear drug accumulation was found in the 4T1 cells treated with the Lipo-CPP, suggesting a nuclear-targeting effect of LMWP (Figure 3D). It could account for LMWP’s cationic sequence with a function similar to a nuclear localization signal (NLS). On the basis of these results, we demonstrated LMWP’s potent ability of mediating tumor drug delivery. This synergistic multipronged delivery function may offer a promising method for overcoming 4T1 breast cancer. 3.3. In Vitro Anti-tumor Activity of Liposomes. The MTT assay showed that both of the liposomes had cytotoxicity higher than that of the free combo drugs (Figure 4A). The highest anti-tumor activity was seen in the Lipo-CPP with an IC50 of 0.12 μg/mL (95%CI 0.072−0.195), compared to 0.31 μg/mL (95%CI 0.246−0.395) of the Lipo-PEG. The blank liposomes exhibited no apparent cytotoxicity (Figure 4B). In line with the result above, the apoptosis rates were 39.9, 45.3, and 69% for free combo drugs, Lipo-PEG, and Lipo-CPP, respectively (Figure 4C). E


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evaluated in the orthotopic 4T1 breast tumor mouse model with spontaneous lung metastasis. At the experimental end point, the dissected tumors were weighed and displayed a similar trend to the tumor volume result. The Lipo-CPP displayed the best anti-tumor activity among all of the treatment groups with a statistical significance. The treatment of the Lipo-CPP and Lipo-PEG resulted in the significant shrinking of the tumors, and their inhibition rates of tumor growth were 80.1 and 60.2%, respectively, while that of the group receiving free combo drugs was 36.7% (Figure 7A−D). The TUNEL results showed the Lipo-CPP with the highest apoptosis rate in the tumor, and the Lipo-PEG displayed superiority over the free combo group (Figure 7E). Pulmonary metastasis often occurs in breast cancers. In the model established in our study, lung metastasis was readily developed, as shown in the control group (Figure 8A). However, the Lipo-CPP treatment efficiently arrested the metastasis with an inhibition rate of 71%, compared to that of the control group (Figure 8B,C) The H&E staining results suggested that there were no visible lesions in the major organs, which indicated there were no significant toxic side effects caused by drug treatment (Figure 9A). Moreover, there was no major body weight-loss and organ coefficient chances in the treatment (Figure 9B,C). 3.7. Preliminary Study of Anti-Angiogenesis Mechanism. Angiogenesis is a key driving factor for tumor proliferation and metastasis. VEGF is regarded as the most potent angiogenic factor and its receptor, VEGFR2, is the predominant mediator in angiogenic signaling.20 Neovessels not only support the tumor growth by supplying nutrients, but they also become an escape route for the cancer cells to migrate into the circulation to establish distant metastasis, and a high vascular density in the tumor generally leads to a great chance for the escape of tumor cells.5 Our results demonstrated that the HUVEC tube formation on the Matrigel Matrix was obviously inhibited with a different treatment (Figure 10A), and there was down-regulated expression of VEGFR2 after drug treatment (Figure 10B). Furthermore, in the tumor tissues, it was found that the Lipo-CPP decreased the expression of VEGF and its receptor VEGFR2 protein (Figure 10C). To further investigate the antiangiogenesis effect, we performed immunofluorescence and

Figure 5. Cell migration and invasion studies using 4T1 cells. (A) Wound-healing assays. (B) Transwell migration assays with staining by crystal violet. Scale bar, 100 μm.

Conventionally, the CPP-mediated delivery has been considered to go widely in vivo and lack tumor-specific distribution. This perception has gradually changed, and the CPP-binding receptors (i.e., neuropilin-1 and syndecan-4) have been found on the cancer cells.14,15 CPP also displays stronger electrostatic interactions with the cancer cells than with the normal cells because the former ones overexpress certain glycosaminoglycans and bear rich anionic lipids, thus rendering an increased negative charge.16 In addition, the delivery specificity could be further improved by the use of the charge-reversal strategy by responding to tumor microenvironments (e.g., ROS and enzymes).17−19 3.6. In Vivo Therapeutic Efficacy on Tumor Progression and Metastasis. The in vivo anti-tumor efficacy was

Figure 6. In vivo imaging of the liposomes biodistribution in the mice bearing 4T1 tumors. (A) Whole body imaging from 1 to 24 h. (B) In vivo radiant efficiency at the tumor sites. (C) Ex vivo imaging of the major organs dissected from the mice. (D) Ex vivo radiant efficiency of the tumors. (E) Tumor infiltration of the DIR-labeled liposomes. F


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Figure 7. Anti-tumor and anti-metastasis efficacy. (A) Tumor tissues. (B) Tumor growth curve. (C) Tumor weight at the end point. (D) Tumor weight inhibition rate. (E) TUNEL assay of apoptosis in tumors.

Figure 8. (A) The representative H&E sections of lung tissues from each group (blue arrow indicating the lung metastasis). (B)The lung metastasis (blue cycles). (C) Quantitative analysis of pulmonary metastatic nodules. G


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Figure 9. (A) H&E staining of major organs after treatment. (B) Changes in body weight during the course of treatment. (C) Organ coefficients.

Figure 10. In vitro anti-angiogenesis study. (A) The vascular endothelial cell tube formation on Matrigel Matrix. (B) VEGFR2 protein expression in HUVEC cells after treatment with different groups. (C) The VEGFR2 and VEGF protein expression of tumor tissues measured by Western blotting. The values represent the quantitative normalization using densitometry. (D) Confocal images of tumor slides stained by anti-CD31 antibody (red) and DAPI (blue). (E) Investigation of tumor-associated blood vessels using anti-CD31 immunohistochemical staining (brown color).

involve anti-angeogenesis. This study suggested the Lipo-CPP was a promising nanomedicine for controlling breast tumor growth and metastasis based on a combination therapy using Andro and DOX. The high druggability of liposomal formulations offers a potential for clinical translation.

immunohistochemical staining using anti-CD31 on the sections of the tumor tissue from the animals that received different treatments. Anti-CD31 staining showed that the vessel number in the Lipo-CPP group was obviously less than other groups (Figure 10D,E). The combination therapy exhibited synergistic anti-angiogenesis. It demonstrated that inhibition of angiogenesis was an anti-metastasis mechanism for this combination therapy.

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AUTHOR INFORMATION

Corresponding Authors

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

4. CONCLUSION In the present study, we developed a liposomal system for co-encapsulation of Andro and DOX with a CPP-assisted delivery strategy. The CPP can effectively increase the intratumoral infiltration, cellular uptake, and intranuclear accumulation. Both the in vitro and in vivo studies demonstrated that the Lipo-CPP significantly arrested the tumor growth and lung metastasis in an orthotopic breast tumor model. The mechanism could

ORCID

Yongzhuo Huang: 0000-0001-7067-8915 Author Contributions ⊥

X.K. and Z.Z. contributed to this work equally.

Notes

The authors declare no competing financial interest. H


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(17) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Liu, X.; Huang, Y.; Shen, Y. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Adv. Mater. 2016, 28, 1743−1752. (18) Qiu, N.; Liu, X.; Zhong, Y.; Zhou, Z.; Piao, Y.; Miao, L.; Zhang, Q.; Tang, J.; Huang, L.; Shen, Y. Esterase-Activated Charge-Reversal Polymer for Fibroblast-Exempt Cancer Gene Therapy. Adv. Mater. 2016, 28, 10613−10622. (19) Sun, Q.; Zhou, Z.; Qiu, N.; Shen, Y. Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv. Mater. 2017, 29, 1606628. (20) Zorko, M.; Langel, U. Cell-penetrating Peptides: Mechanism and Kinetics of Cargo Delivery. Adv. Drug Delivery Rev. 2005, 57, 529− 545.

ACKNOWLEDGMENTS We are thankful for the support of the 973 Program, China (2014CB931900), NFSC (81402883, 81422048, 81673382, and 81521005), the Strategic Priority Research Program of CAS (XDA12050307), Scientific Research and Equipment Development Project (YZ201437), SANOFI-SIBS Scholarship Program, Youth Innovation Promotion Association of CAS, and the Fudan-SIMM Joint Research Fund (FU-SIMM20174009). We also thank the technical assistance from the Molecular Imaging and Electron Microscopy Core Facilities, SIMM.

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