Inhibition of Metastatic Tumor Growth and Metastasis via Targeting

Feb 21, 2014 - Metastatic Breast Cancer by Chlorotoxin-Modified Liposomes ... had targeting ability to metastatic breast cancer in addition to brain c...
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Inhibition of Metastatic Tumor Growth and Metastasis via Targeting Metastatic Breast Cancer by Chlorotoxin-Modified Liposomes Chao Qin,†,‡ Bing He,‡ Wenbing Dai,‡ Hua Zhang,‡ Xueqing Wang,‡ Jiancheng Wang,‡ Xuan Zhang,‡ Guangji Wang,*,† Lifang Yin,*,†,‡ and Qiang Zhang*,‡ †

School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China

‡

ABSTRACT: A liposome system modified with chlorotoxin (ClTx), a scorpion venom peptide previously utilized for targeting brain tumors, was established. Its targeting efficiency and antimetastasis behavior against metastatic breast cancer highly expressed MMP-2, the receptor of ClTx, were investigated. 4T1, a metastatic breast cancer cell line derived from a murine breast tumor, was selected as the cell model. As results, the ClTx-modified liposomes displayed specific binding to 4T1 as determined by flow cytometry and confocal imaging. The cytotoxicity assay revealed that the ClTx modification increased the toxicity compared with nonmodified liposomes. In addition, the modified liposomes also exhibited high in vivo targeting efficiency in the BALB/c mice bearing 4T1 tumors. Importantly, this system inhibited the growth of metastatic tumor and prevented the incidence of lung metastasis in mice bearing 4T1 tumors with only low systemic toxicity. The data obtained from the in vitro and in vivo studies confirmed that the ClTx-modified liposomes increased the drug delivery to metastatic breast cancers. This study proved that the ClTx-modified liposomes had targeting ability to metastatic breast cancer in addition to brain cancer, and displayed an obvious antimetastasis effect. Generally, it may provide a promising strategy for metastatic breast cancer therapy. KEYWORDS: chlorotoxin, modified liposomes, targeting delivery, metastatic breast cancer, antimetastasis

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INTRODUCTION Breast cancer is the most common cancer and the second leading cause of cancer death in women in the United States.1 Metastasis is the most deadly aspect of cancer because of the difficult treatment and the spread of the cancer to the lung, liver, brain and other key organs. For metastatic breast cancer, the five-year survival rate is only 26%.2 Therefore, effective progress in the treatment of metastatic breast cancer is expected to have great benefits for women. Tumor metastasis is a multistep process, and cancer cells require some functional proteins and methods to escape from the primary tumor, migrate, invade surrounding tissues, enter the vasculature, circulate, reach secondary sites and establish metastatic foci.3,4 Among the functional proteins, matrix metalloproteinases (MMPs) are critical factors for metastasis. Among the MMPs, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are different from the others because of their ability to degrade gelatin and type IV collagen, the main © XXXX American Chemical Society

component of extracellular matrix (ECM), which is the main barrier separating in situ and invasive carcinoma.5 Namely, MMP-2 is an important protein in tumor invasion, a necessary step of metastasis. Chlorotoxin (ClTx), a peptide containing 36 amino acids and 4 disulfide bonds with a relative molecular mass of 3996, was originally isolated from Leiurus quinquestriatus scorpion venom. ClTx binds selectively to glioma cells and other tumors of neuroectodermal origin but not to nontransformed cells, such as human neurons, astrocytes and fibroblasts, as well as 15 types of normal human tissues.6,7 In initial studies, ClTx Special Issue: Recent Molecular Pharmaceutical Development in China Received: November 15, 2013 Revised: February 9, 2014 Accepted: February 21, 2014

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clonal antibody to MMP-2 was kindly provided by Bioworld (St. Louis Park, MN, USA). Other reagents were all analytical or HPLC grade. Cell Culture and Animals. The murine breast cancer cell line 4T1 was acquired from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China), and grown in RPMI 1640 with Lglutamine and 25 mM HEPES medium containing a final concentration of 10% (volume percentage) fetal bovine serum (FBS) and 1% antibiotics (penicillin, 100 unit/mL plus streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. The medium and the antibiotics were bought from Macgene Biotech (Beijing, China), and the FBS was from TianHang Biotech (Zhejiang, China). Female BALB/c mice of 6−8 weeks old were purchased from the Vital River Company and kept under SPF conditions in the Laboratory Animal Unit of Peking University Health Science Center with free access to standard food and water during the experiment. All animal experiments were performed under the guidelines of the Ethics Committee of Peking University. Synthesis of DSPE-PEG-ClTx and Preparation of Liposomes. The synthesis method for the targeting material DSPE-PEG-ClTx has been previously reported by our research group.19 Briefly, the mixture of ClTx peptide and DSPE-PEGNHS (1:5, molar ratio) in anhydrous DMF, adjusted to pH 8.5 with triethylamine, was stirred for 120 h in an ice bath. After dialysis (Mw cutoff 3500 Da) with deionized water, the material was lyophilized and stored at −20 °C. The conjugation efficiency was monitored by reverse high performance liquid chromatography (HPLC), and the targeting material was confirmed by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF; Bruker Daltonics, USA). The Dox-loading liposomes modified with ClTx (Dox-SSLClTx; HSPC:Chol:DSPE-PEG (2000):DSPE-PEG-ClTx = 20:10:1.9:0.1, molar ratio) and nonmodified liposomes loading Dox (Dox-SSL; HSPC:Chol:DSPE-PEG (2000) = 20:10:2, molar ratio) were prepared by thin film hydration followed by the ammonium sulfate transmembrane gradient method.19 The liposomes loading DiR (DiR-SSL-ClTx and DiR-SSL) for in vivo distribution were directly prepared by thin film hydration as previously described.19 Characterization of Liposomes. The particle size and potential of liposomes were measured with a Malvern Zetasizer Nano ZS (Malvern; Worcestershire, U.K.) by dynamic light scattering method. The morphological shapes of liposomes were confirmed by cryogenic transmission electron microscopy (cryo-TEM; Philips, The Netherlands). The encapsulation efficiency (EE) and the drug loading rate (DR) were calculated using the following calculations: EE (%) = drug loaded/total drug × 100%; DR (%) = drug loaded/total materials × 100%. The concentration of Dox was measured by UV−vis spectrophotometer (TU1900; Pepsee, China) at 480 nm after dissolving the liposomes with methanol. In Vitro Leakage. The leakage of Dox from liposomes was evaluated by the dialysis method27 to ensure that the results of cellular uptake and cytotoxicity could indicate the behaviors of the dosage forms and facilitate the avoidance of the effects of free drug released from the liposomes. For this evaluation, 1 mL of Dox-loading liposome solution was mixed with 4 mL of 1640 medium containing 10% FBS in the dialysis bag (Mw cutoff 12000−14000 Da) dialyzed against 15 mL, pH 7.4 PBS. The dialysis process was performed at 37 °C in a gas bath

blocked a unique chloride channel specifically expressed on human astrocytoma and glioma cells as well as acute slices of human gliomas.8−10 Further, using affinity purification followed by mass spectrometry, MMP-2 was demonstrated to be the receptor of ClTx.11 MMP-2 is a key factor involved in the process of tumor metastasis, and ClTx has been reported to inhibit the migration and invasion of glioma cells.12,13 In addition, ClTx is highly toxic to invertebrates but nontoxic to mammals.14 Generally, ClTx is a potential tool for tumor targeting therapy and diagnosis because of its specific binding and other natural properties. ClTx has been used in glioma imaging of animals bearing xenografted tumors by tagging Cy5.5 or iodine-131 to ClTx,14,15 and iodine-131-tagged ClTx reliably detected glioma cells in patient biopsies.16 In addition, ClTx-modified nanoparticles also have demonstrated great potential in gene therapy and glioma chemical therapy.17−19 However, all the studies of ClTx have focused on brain tumors, regardless of the area of tumor diagnosis, gene delivery method and chemical drug delivery method. It remains an open question whether a ClTxmodified drug delivery system could specifically bind to other tumor cells besides gliomas since the ClTx receptor is MMP-2, which is also highly expressed in metastatic breast cancers, metastatic lung cancers and so on.5,20−22 Previously, ClTx and a ClTx-modified nanoparticle system were found to effectively inhibit invasion and migration of glioma cells in vitro due to the interaction between ClTx and MMP-2.12,11 Thus, it is necessary to evaluate whether the ClTx-modified drug delivery system has an antimetastasis effect on breast cancer since MMP-2 plays an important role in the metastasis of carcinoma including glioma cancer, breast cancer and so on. Xenograft models, such as nude mice bearing human tumor models, are limited in the ability to properly represent the metastatic nature of tumors in the clinic because the tumor is mostly confined in the primary inoculation site.23−25 4T1, an aggressive metastatic breast cancer cell line derived from a BALB/c mouse mammary tumor, highly expresses MMP-2, and the 4T1 based metastatic breast model is well-established.24,26 Therefore, it is a desirable tumor model for our research. Based on all of the above, this paper reports the development of ClTx-modified liposomes loaded with doxorubicin hydrochloride (Dox) and their targeting to metastatic breast tumors. We found that the modified liposomes increased the uptake of Dox in 4T1 cells in vitro by MMP-2 mediation, and possessed higher targeting ability in vivo compared with nonmodified liposomes. The antitumor efficacy, inhibition of metastasis and the systemic toxicity were also investigated with BALB/c mice bearing 4T1 tumors.

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EXPERIMENTAL SECTION Materials. DSPE-PEG (2000)-NHS and DSPE-PEG (2000) were purchased from NOF Corporation (Tokyo, Japan). Chlorotoxin (ClTx) was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). Hydrogenated soybean phospholipid (HSPC) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (Chol), Sephadex G-50, trichloroacetic acid (TCA), sulforhodamine B (SRB) and anhydrous N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris was purchased from Amresco (Solon, OH, USA). Doxorubicin hydrochloride (Dox) was kindly provided by Hisun Pharm (Zhejiang, China). Fluorescent probe DiR and Hoechst 33258 were purchased from Life Technologies (Eugene, OR, USA). Rabbit monoB

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Figure 1. Characterization of DSPE-PEG-ClTx and liposomes. A: MALDI-TOF results for the DSPE-PEG-ClTx. B: Particle size distribution of DoxSSL-ClTx by intensity. C: Cryo-TEM image of Dox-SSL-ClTx. D: In vitro leakage of the Dox from liposomes in a medium containing 10% FBS and free Dox release out of the dialysis bag under the same conditions.

nucleic acid stain with Hoechst 33258. The fluorescence of the cells was imaged with a laser scanning confocal microscope (Leica, Heidelberg, Germany). Cytotoxicity Assay. The 4T1 cells were cultured in a 96well cell culture plate at a density of 5 × 103 cells per well for 24 h. Then, the cells were in incubated with Dox-SSL, Dox-SSLClTx and free Dox alone. The concentrations of Dox and the equal concentrations of Dox loaded in the liposomes were from 0.1 to 20 μg/mL. Then, 48 h later, the medium containing the drug was removed by aspiration, and each well was washed twice with PBS. The SRB assay was utilized to evaluate the cytotoxicity of free Dox and different liposomes.28 Briefly, the cells were fixed with 10% TCA for 1 h at 4 °C, followed by being washed and air-dried. The fixed cells were stained with 0.4% SRB at room temperature for a half-hour and washed with 1% acetic acid. The absorbance of each well was measured in a microplate reader (Multiscan FC, ThermoFisher Scientific, MA, USA) at the wavelength of 540 nm after the dye was dissolved in 10 mM Tris base solution. The survival rates of the cells were calculated. Competitive Inhibition Assay. The 4T1 cells were seeded in a 12-well cell culture plate as mentioned in In Vitro Cellular Uptake. After 24 h, the cells were preincubated with antibody to MMP2 (4 μg/mL, diluted with medium free of FBS) for 2 h. Then the Dox-SSL or Dox-SSL-ClTx was added into the medium, and the following operations were the same as for the in vitro cellular uptake with flow cytometry. Meanwhile, the

thermostatic oscillator (ZHWY-103B; ZhiCheng, China) at 100 rpm. As a control group, the release of free Dox solution from the dialysis bag was evaluated under the same conditions. At predetermined time intervals, 1 mL aliquots outside the dialysis bag were withdrawn for HPLC assay and replaced with an equal volume of fresh PBS. The cumulated release percentage of Dox was calculated with the concentration as the denominator after rupture of the liposomes by replacing with 1 mL of 10% Triton-X100 and sonication. The mobile phase of the Dox assay contained acetonitrile, water and acetic acid (50:45:5, v/v/v) at 233 nm. In Vitro Cellular Uptake. The 4T1 cells were seeded in a 12-well cell culture plate (Corning, NY, USA) at a density of 3 × 105 cells per well and allowed to attach for 24 h at 37 °C. Then, the cells were incubated with free Dox solution, Dox-SSL and Dox-SSL-ClTx (containing Dox 30 μg/mL) at 37 °C in darkness. In addition, the cells treated with medium were used as blank control. After 3 h, the cells were washed with cold PBS, trypsinized, harvested, washed 3 times by centrifugation and resuspended in PBS. The mean fluorescence intensity of Dox in cells was measured using a FACScan flow cytometer (Becton Dickinson FACS Calibur, USA). For confocal imaging tests, the cells seeded on 12 mm round glass coverslips were treated with Dox-SSL and Dox-SSL-ClTx (containing Dox 30 μg/mL) at 37 °C for 3 h. Then, after being rinsed twice with cold PBS, the cells were fixed with 4% paraformaldehyde for 15 min followed by a PBS wash and C

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Table 1. Characteristics of Two Dox-Loading Liposome Systems liposomes

particle size (nm)

PDI

zeta potential (mV)

Eencapsulation efficiency (%)

drug loading rate (%)

Dox-SSL Dox-SSL-ClTx

98.5 ± 4.1 103.1 ± 5.7

0.180 ± 0.013 0.196 ± 0.007

−3.4 ± 0.21 −4.1 ± 0.13

96.3 ± 1.3 93.2 ± 2.9

9.3 ± 0.08 9.0 ± 0.10

Figure 2. Cellular uptake of the two liposome systems. A: Flow cytometry analysis of the 4T1 cells treated with free Dox, Dox-SSL and Dox-SSLClTx using a medium free of Dox as control. B: Quantitative results of flow cytometry analysis (*p < 0.05). C: Laser scanning confocal microscopy images of the 4T1 cells incubated with Dox-SSL and Dox-SSL-ClTx.

4T1 cells using the method mentioned in section of in vivo targeting evaluation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated as V (mm3) = 0.5 × (the longest diameter) × (the shortest diameter)2. When the tumor volume reached approximately 60 mm3 (5 days after inoculation), the mice were randomly divided into 4 groups: saline, free Dox, Dox-SSL and Dox-SSLClTx. Then, 200 μL of Dox solution and Dox-loading liposomes were injected via the tail vein at a dose of 3 mg/ kg 5 times at 3-day intervals (days 5, 8, 11, 14, 17). The mice in the saline group were injected with 200 μL of saline as a control. The tumor sizes and the weights of mice were recorded every 3 days. After the final evaluation, the mice were sacrificed by cervical vertebra dislocation, the tumors were excised and weighed and the major tissues were also excised. The tumors

cells without treatment with the antibody were used as comparisons. In Vivo Targeting Evaluation. The BALB/c mice bearing tumors were prepared by injecting 1 × 106 cells suspended in 100 μL of culture medium without FBS into the subcutaneous dorsa of the mice. Then, 200 μL of DiR-loading liposomes or DiR solution was injected into the tail vein at a dose of 15 μg/ kg. Mice were anesthetized with isoflurane, and the distribution of DiR-loading liposomes and DiR were imaged using an in vivo imaging system (Carestream Molecular Imaging, New Haven, CT, USA) at predetermined time intervals. When the mice were sacrificed, the tumor and the major organs were excised for ex vivo images after the final time point. In Vivo Inhibition of Tumor Growth, Metastasis and Toxicity. The BALB/c mice were subcutaneously injected with D

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and tissues excised from 2 mice randomly selected in each group were fixed in 4% formalin, embedded in paraffin and sectioned. Finally, the sections were stained with hematoxylin and eosin (H&E) and observed using an optical microscope. The lungs obtained from the other 6 mice in each group were fixed in Bouin’s fixative. After 24 h, the tumor burdens on the lungs were observed and recorded. Statistical Analysis. All the experiments were repeated at least three times. Data are shown as the means ± standard deviation (SD). Student’s t test was used to determine the difference. P values less than 0.05 were considered statistically significant.

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RESULTS AND DISCUSSION Characterization of DSPE-PEG-ClTx and Liposomes. Using the reaction between the NHS group of DPSE-PEG-

Figure 4. The results of competitive inhibition assay with antibody of MMP-2. A: Flow cytometry analysis of the 4T1 cells incubated with Dox-SSL and Dox-SSL-ClTx, and the cells in the competitive group were pretreated with antibody of MMP-2 for 2 h. B: Quantitative results of flow cytometry analysis (*p < 0.05). Figure 3. Cytotoxicity of free Dox, Dox-SSL and Dox-SSL-ClTx at different concentration against 4T1 cells for 48 h (*p < 0.05).

containing 10% FBS and the Dox released from the dialysis bag completely at 4 h in the control group (Figure 1D). Less than 10% of Dox was leaked out of the liposomes at 48 h, indicating that the drug was mainly entrapped in the liposomes during the cellular uptake study and cytotoxicity assay. In addition, it was revealed that ClTx modification did not alter the leakage character of the liposomes significantly. Cellular Uptake. The cellular fluorescence intensity of the Dox reflected the cellular uptake behavior of the liposomes, as it was demonstrated that the Dox leakage from the liposomes only accounted for less than 2% of total drug after 4 h in the medium. As shown in Figure 2A and Figure 2B, the intracellular Dox in the Dox-SSL-ClTx was higher than that of the Dox-SSL by flow cytometry. In detail, the fluorescence intensity of the Dox-SSL-ClTx was 1.8 times as high as that of Dox-SSL. In addition, the uptake of free Dox was the highest due to the direct diffusion into the cells. The images acquired by confocal microscopy confirmed the flow cytometry data (Figure 2C). In detail, the fluorescence of the Dox-SSL-ClTx in the cytoplasm and in the nucleus areas was higher than that of the Dox-SSL. The results of flow cytometry and confocal imaging indicated that ClTx modification could increase the uptake of liposomes in this cell line of metastatic breast tumor. Cytotoxicity Assay. After being incubated with Dox, DoxSSL and Dox-SSL-ClTx for 48 h, the 4T1 cell viability was obviously inhibited (Figure 3). The inhibition increased as the concentration of Dox or the equivalent concentration of Dox loaded in the liposomes increased. The viability of cells treated with free Dox (IC50 = 0.75 μg/mL) decreased sharply because Dox molecule diffused directly into cells. The endocytosis of

NHS and the terminal amino group of ClTx, the targeting material DSPE-PEG-ClTx was prepared. The MALDI-TOF data of the targeting material is shown in Figure 1A. In detail, the desired peak of 7000 Mw was clear, and the peak of 3996 (the original Mw of ClTx) was almost absent, indicating that the targeting material was synthesized with high conjugation efficiency (>95% confirmed by the results of HPLC). Meanwhile, the NHS group of unreacted DSPE-PEG-NHS was hydrolyzed during the dialysis process and the DSPE-PEG (the peak of MW 3056) was used as normal DSPE-PEG (2000) in the preparation of liposomes.29−31 As shown in Table 1 and Figure 1B, the particle sizes of different liposome systems were similar, approximately 100 nm with a low particle disperse index (PDI). The morphology of Dox-SSL-ClTx imaged in cryo-TEM (Figure 1C) was consistent with the results of DLS, and the aggregate of Dox was observed in the core of the liposomes.32 The similar particle size indicated that the ClTx conjugation did not significantly affect the physical property of the liposomes, and the difference in the following biological studies did not result from the different particle sizes. All the liposome formulations had slightly negative surface charges. More than 90% of the Dox could be loaded in the liposomes, and the drug loading rate was more than 9%. With the high drug loading rate, it was easy to meet the requirements for drug dosage in the antitumor efficacy study. There was no significant difference in the leakage profiles of Dox from the Dox-SSL and Dox-SSL-ClTx in the medium E

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Figure 5. In vivo targeting evaluation of DiR-SSL and DiR-SSL-ClTx in BALB/c mice bearing a 4T1 tumor (DiR dose 15 μg/kg). A: In vivo nearinfrared fluorescence images of mice after intravenous administration of DiR-SSL or DiR-SSL-ClTx at different time points. B: Ex vivo image of tumors and main organs after the tumor-bearing mice were sacrificed at 48 h.

Figure 6. Antitumor efficacy of free Dox solution, Dox-SSL and Dox-SSL-ClTx in the BALB/c mice bearing 4T1 tumors at a dose of Dox 3 mg/kg; each formulation was intravenously administered every 3 days for a total of 5 times. A: The changes of tumor volume after injection of saline, free Dox solution, Dox-SSL and Dox-SSL-ClTx (the arrows indicate the injection dates). B: Quantitative results of tumor weight excised from the tumorbearing mice sacrificed on day 22. C: The picture of the tumors in different groups at the end of the experiment (*p < 0.05).

Table 2. Incidence Rates of Metastasis and Numbers of Tumor Nodules on Lungs Excised from the 4T1-Bearing BALB/c Mice at the End of the Antitumor Study incidence rates of metastasis numbers of tumor nodules a

a

control

Dox

Dox-SSL

Dox-SSL-ClTx

6/6 37.8 ± 9.8

3/6 12.7 ± 5.0

4/6 12.3 ± 7.1

0/6

The data are presented as the number of metastasis cases/the number of lungs investigated.

liposomes was slower than the cellular uptake of free Dox, so at the same concentration, free Dox produced higher cytotoxicity. In addition, compared with the Dox-SSL (IC50 = 6.49 μg/mL), Dox-SSL-ClTx (IC50 = 1.58 μg/mL) displayed higher growth inhibition at concentrations of 0.5 to 20 μg/mL. ClTx modification could increase the cytotoxicity of liposomes likely due to the high cellular uptake of Dox, consistent with the flow cytometry data and the confocal imaging. Competitive Inhibition Assay. Competitive inhibition on the cellular uptake with antibody of MMP-2 was used to confirm whether the uptake of Dox-SSL-ClTx was mediated by the ligand−receptor interaction. It was obvious that the

addition of antibody of MMP-2 decreased the uptake of DoxSSL-ClTx but had no effect on that of Dox-SSL as shown in Figure 4, indicating that MMP-2 was the receptor of ClTx in Dox-SSL-ClTx. Generally, the interaction between ClTx modified on the surface of liposomes and the MMP-2 on the surface of cells increases the targeting ability, and this is the reason that ClTx modification increases cellular uptake and cytotoxicity of liposomes compared with Dox-SSL. In Vivo Targeting Evaluation. DiR, a near-infrared fluorescent dye, was used to evaluate the biodistribution of the liposomes. The fluorescent signals of mice treated with DiR-SSL-ClTx at the tumor site were much stronger compared F

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Figure 7. A: The cancer burden condition in the lungs excised from the BALB/c mice bearing 4T1 tumors treated with saline, Dox, Dox-SSL and Dox-SSL-ClTx. B: The results of histopathologic examination of the main organs after treatment with saline, Dox, and liposome formulations. The tumor metastases (marked with black outlines) were found in the lungs and livers of groups treated with saline, Dox and Dox-SSL (scale bar: 100 μm).

In Vivo Inhibition of Tumor Growth. Figure 6A shows the in vivo antitumor efficacy of different formulations against BALB/c mice bearing 4T1 tumors. The tumor-bearing mice were treated 5 times with free Dox solution, Dox-SSL or DoxSSL-Dox at a dose of 3 mg/kg at intervals of 3 days (days 5, 8, 11, 14, 17). Compared with the group treated with saline as the control, all three formulations exhibited obvious inhibition after the third treatment, and the modified liposomes displayed a stronger inhibition efficacy than the others (p < 0.05) since then. There was no significant difference between the antitumor efficacy of the Dox and Dox-SSL (p > 0.05) except of the last observation, indicating that the nonmodified liposome system had a superior therapeutic effect compared with free Dox, but this advantage is only apparent after multiple dosing over a long period of time. Twenty-two days after inoculation of the 4T1 cells, the tumors were excised, weighed and imaged. In Figure 6B and Figure 6C, it was indicated that

with DiR-SSL from 2 h postinjection to the end of the test, 48 h (Figure 5). The signal of the DiR-SSL-ClTx increased with time during the first 12 h. The liposomes accumulated in the tumor because of the enhanced permeation and retention (EPR) effect,33 and ClTx modification further increased the drug accumulation at the tumor site on the basis of the EPR effect. In addition, the signals of all liposomes lasted 48 h because of the long circulation time resulting from the PEG group on the surface of the liposomes.34,35 In the ex vivo images (Figure 5B), the high accumulation of liposomes was observed in reticuloendothelial system (RES) organs, including liver and spleen, possibly caused by the clearance of the RES.36 The signal of mice injected with DiR solution was too low to observe (data not shown). Generally, the ClTx-modified liposomes displayed superior in vivo targeting ability compared with the nonmodified liposomes. G

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Dox was lower than that of free Dox. In detail, at day 7 and day 13, Dox-SSL displayed significantly better safety than Dox (p < 0.05), and the Dox-SSL-ClTx showed lower toxicity than Dox (p < 0.05) at all recorded time points except day 10. In addition, there was no significant difference in the body weight changes between the nonmodified and modified liposome systems. The results of the histopathologic examination of the heart (Figure 7B) indicated that the free Dox caused obvious cardiotoxicity with cardiac muscle cell swelling. The liposome systems groups did not present significant differences compared with the saline-treated group.

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CONCLUSIONS The ClTx-modified liposomes increased the cellular uptake and cytotoxicity in vitro in a cell line of murine metastatic breast cancer, 4T1. Desirable antitumor effects and antimetastasis efficacy were confirmed in the mice bearing 4T1 tumors with low systemic toxicity. The in vitro and in vivo data indicated that the ClTx-modified drug delivery system is capable of significant targeting to metastatic breast tumor, in addition to its previously demonstrated brain tumor targeting capability. Importantly, this targeted system has the potential to prevent breast tumor metastasis, the main cause of death among the cancer patients.

Figure 8. Body weight changes of BALB/c mice bearing 4T1 tumors during the treatment with free Dox, Dox-SSL and Dox-SSL-ClTx (the arrows indicate the injection dates) (*p < 0.05 between the Dox and Dox-SSL-ClTx groups, +p < 0.05 between the Dox and Dox-SSL groups).

the liposome system had superior antitumor efficacy compared with free Dox. Moreover, the ClTx modification increased the efficacy because of the higher accumulation in the tumor site, as proven in the in vivo targeting study. Although the cytotoxicity of free Dox was higher than that of the liposomes in vitro, the liposome systems displayed higher in vivo antitumor efficacy against 4T1 tumor probably because of the EPR effect, long circulation and receptor mediated targeting. Inhibition of Tumor Metastasis. Metastasis usually occurs at a later stage of cancer, and 4T1, as a metastatic breast cancer, primarily metastasizes to the lung and secondarily to the liver. Table 2 and Figure 7A show that the modified liposomes inhibited the metastasis of 4T1 to the lung significantly. There were abundant tumor nodules in the lung of the control group treated with saline, and metastasis nodules labeled with arrows were also visible in some lungs of the mice treated with Dox and Dox-SSL. In the group treated with Dox-SSL-ClTx, there was no significant tumor nodule. The metastasis was also examined with histopathology, as shown in Figure 7B. In the saline, Dox and Dox-SSL treated groups, the metastatic tumors (marked by black lines) were found to have spread into the lungs and livers. Generally, the modified liposomes effectively inhibited tumor metastasis. The Dox and nonmodified liposomes also had antimetastatic effects, but their efficacies were low, with a metastasis incidence rate of 50% and 33% respectively, as shown in Table 2. ClTx modification increased the antimetastasis effect probably because of the specific targeting to the primary tumor cells as well as the cells spreading in the blood or lymphatic routes. In addition, ClTx modification of liposomes could prevent the invasion of tumor cells most likely resulting from the inhibition of MMP-2.12,13 Systemic Toxicity. For any drug delivery system, systemic toxicity should be considered to ensure safety even if the system has a good therapy effect. Body weight change was utilized to evaluate the systemic toxicity of the three preparations as shown in Figure 8. Compared with saline, Dox, Dox-SSL and Dox-SSL-ClTx displayed various degrees of systemic toxicity (Figure 8). Notable growth was observed in the control group during the whole period, and the free Dox exhibited serious side effects with 15% body weight loss due to nonspecific distribution in the body. The toxicity of liposomal

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AUTHOR INFORMATION

Corresponding Authors

*G.W.: School of Pharmacy, China Pharmaceutical University, Tongjia Xiang, Nanjing, China; tel/fax, +86-25-83271018; email, [email protected]. *L.Y.: tel/fax, +86-25-86185258; e-mail, Lifangyin@hotmail. com. *Q.Z.: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road, Beijing, China; tel/fax, +86-10-82802791; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81130059), the National Research Fund for Fundamental Key Project (2009CB930300) and the Innovation Team of Ministry of Education (No. BMU20110263).

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REFERENCES

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dx.doi.org/10.1021/mp400691z | Mol. Pharmaceutics XXXX, XXX, XXX−XXX