A specific peptide ligand-modified lipid nanoparticle carrier for the inhibition of tumor metastasis growth

Biomaterials 34 (2013) 756e764

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A specific peptide ligand-modified lipid nanoparticle carrier for the inhibition of tumor metastasis growth Zhaohui Wang a, b, Yang Yu a, b, Wenbing Dai b, Jingrong Cui c, Hounan Wu d, Lan Yuan d, Hua Zhang b, Xueqing Wang b, Jiancheng Wang b, Xuan Zhang b, Qiang Zhang a, b, * a

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China Department of Pharmaceutics, Peking University, Beijing, China Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China d Medical and Healthy Analytical Center, Peking University, Beijing, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 September 2012 Accepted 8 October 2012 Available online 30 October 2012

Tumor metastasis accounts for 90% of cancer-associated deaths and is almost inaccessible by chemotherapy, surgical operation or radiotherapy. Here, a tumor metastasis-specific nanocarrier system has been constructed by modification of stealth lipid nanoparticles with a specific peptide ligand. Highly metastatic breast cancer MDA-MB-231 that stably expressed luciferase (MDA-MB-231/Luc) was used as tumor cell model. The nanocarrier was very specific for highly metastatic cancer cells in vitro and could specifically target to cancer metastases foci following systemic administration in vivo by both fluorescence imaging and bioluminescence imaging compared to a passive-targeted system. It greatly facilitated the efficacy of doxorubicin loaded in inhibiting tumor metastasis growth and prolonging the survival time of mice. Importantly, this system was also found to prevent the initiation and progression of tumor metastasis. The tumor metastasis-targeted nanocarriers hold great potential in the treatment of cancer metastasis foci and even for the prevention of tumor metastasis. This study may also provide new strategy in the development of nanomedicine for diagnosis and therapy of tumor metastasis. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Tumor metastasis Targeting Nanomedicine Lipid nanoparticles Delivery

1. Introduction Cancer metastasis accounts for 90% of cancer-associated deaths [1,2]. Millions of people die every year from cancer metastasis and the 5-year survival rate of such patients is low [3e5]. The metastatic cancer cells may form distant microfoci and maintain a balance between proliferation and apoptosis without obvious clinical symptoms [6,7]. Therefore, the ability to detect and treat occult cancer metastasis, as well as prevent their spread and growth, will be of great clinical significance [8]. Nevertheless, there is still no cure for the most deadly cancer metastasis despite the tremendous efforts that have been made [9]. Although advances in nanomedicine have provided a promising strategy for cancertargeted drug delivery [10,11], passive-targeted delivery systems, like Doxil (liposomal doxorubicin) and Abraxane (paclitaxel nanoparticles), have provided only modest survival benefits [12e14].

* Corresponding author. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China. Tel./fax: þ86 10 82802791. E-mail address: [email protected] (Q. Zhang). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.10.018

Importantly, these drugs have little therapeutic effect on cancer metastasis because of their low accumulation in metastasis foci [15]. To increase the tumor-specific drug delivery, active targeting was exploited by functionalization of nanocarriers with ligands that interact with receptors overexpressed in the cancerous cells [16]. However, most of active delivery is developed to target primary tumors, not tumor metastasis. Due to the heterogeneity of cancer metastasis, the biomarkers related in primary tumors may be very different from that in their metastasis foci [17]. Besides, the small metastasis (<100 mm3 in volume) is poorly vascularized [18]. As a result, very limited success has been achieved in the active targeting of tumor metastasis [19]. While tumor metastasis-specific delivery is extremely important in cancer therapy it remains a very big challenge, and few investigations have been performed to date. One strategy to achieve cancer metastasis-targeted drug delivery is the use of unique molecular markers that are specifically expressed in cancer metastasis foci [20,21]. A cyclic ten-amino acid peptide (GCGNVVRQGC), referred to as tumor metastasis targeting (TMT) peptide, has been found to effectively target to tumor metastasis foci and specifically bind to a series of highly metastatic

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tumor in vitro and in vivo, but not to the non-metastatic cell lines [22]. This actually offers an excellent opportunity to construct a tumor metastasis-targeted drug delivery system. Based on our previous results, TMT modified liposomes achieve superior targeting and efficacy to highly metastatic primary tumor [23]. In this paper, we hypothesized that attachment of TMT peptide on the surface of nanocarriers like PEGylated liposomes may lead to their preferential uptake by cancer metastasis after intravenous administration, resulting in higher delivery of drug molecules to the target cells and superior efficacy against these malignances. Their specificity and efficacy to the metastasis foci related were further investigated in various cell lines in vitro and a series of tumor models in vivo and ex vivo. Besides, the possibility of preventing tumor metastasis was also evaluated. 2. Materials and methods 2.1. Materials N-hydroxysuccinimidyl-PEG2000-DSPE and DSPE-PEG2000 were purchased from NOF Corporation (Tokyo, Japan). Soybean phosphatidylcholine (SPC) was obtained from LIPOID (Germany). Cholesterol was supplied from SigmaeAldrich (St. Louis, MO, USA). TMT peptide (GCGNVVRQGC) was synthesized by GL Biochem Co., Ltd (Shanghai, China). Doxorubicin hydrochloride (DOX) was obtained from Hisun Pharmaceutical Co. Ltd (Zhejiang, China). Fluorescent probe Hoechst 33258 was from Molecular Probes Inc. (Oregon, USA), while, 10-dioctadecyl-3,3,30,30tetramethylindodicarbocyanine-4-chlorobenzene-sulfonate salt (DiD) was purchased from Biotium (Hayward, USA). Sephadex G-50 was from Pharmacia Biotech (Piscataway, NJ, USA). D-luciferin was bought from Synchem (Illinois, USA). Other reagents were all of analytical grade and used without further purification. The female BALB/c nude mice of 18e20 g were obtained from Peking University Health Science Center (Beijing, China), and kept under SPF condition for 1 week before the study, with free access to standard food and water. All studies in mice were performed in accordance with guidelines approved by the Ethics Committee of Peking University. 2.2. Preparation of TMT modified liposomes For tumor metastasis targeting peptide-modified liposomes (TMT-LS), TMT peptide was conjugated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[poly(ethylene glycol)-2000]-N-hydroxysuccinimidyl (DSPE-PEG2000-NHS) by the nucleophilic substitution reaction to obtain a targeting compound TMT-PEG2000DSPE [23,24]. The conjugation was confirmed by a matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, USA). TMT-LS were composed of soy phospholipids, cholesterol, PEG2000-DSPE, TMTPEG-DSPE (molar ratio ¼ 20:10:2:0.2) and prepared by lipid film hydration method as described previously [24]. Doxorubicin was loaded via an ammonium sulfate gradient [25]. For the in vivo fluorescence imaging investigation, the near-infrared fluorescent probe DiD was loaded into liposomes. The above lipids and DiD were co-dissolved and a thin film formed by evaporation of the solvents. Phosphate buffered saline (PBS, pH7.4) was added, followed by sonication for 30 min. The liposomal suspension was eluted using a Sephadex G-50 column to remove the free DiD. For the in vivo bioluminescence imaging investigation, D-luciferin functionalized liposomes were prepared using the same liposomal-forming materials. In brief, the lipids were dissolved, dried to form thin film, and hydrated with PBS (pH7.4) containing D-luciferin, followed by sonication for 30 min [26]. The free D-luciferin was separated by gel filtration on a Sephadex G-50 column.

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2.5. In vitro cellular uptake studies The cellular uptake of DOX-loaded liposomes was assessed by flow cytometry analysis. Briefly, MDA-MB-231/Luc cells were seeded into six-well plates and treated with free DOX, LS-DOX or TMT-LS-DOX (containing 20 mg/mL DOX) at 37  C. After 3 h incubation, the cells were washed three times with cold PBS and measured by a FAScan flow cytometer (Becton Dickinson FACS Calibur, USA). For confocal fluorescence imaging observation, cells were incubated with above DOX formulations (containing 10 mg/mL DOX) at 37  C for 3 h, and then rinsed three times with PBS, fixed with 4% paraformaldehyde in PBS, followed by staining the cell nuclei with Hoechst 33258 and imaging by confocal fluorescence microscope (Leica, Heidelberg, Germany). 2.6. Targeted delivery to metastasis foci by fluorescence and bioluminescence imaging For the lung metastasis model, 1  106 MDA-MB-231/Luc cells were inoculated via the tail vein of the nude mice and tumor metastasis foci formed in lungs can be monitored by bioluminescence imaging [27]. To assess the metastasis foci targeting of TMT-LS by fluorescence imaging, DiDloaded TMT-LS and LS were intravenously injected into mice bearing lung metastasis (50 mg/kg of DiD), respectively. After 24 h circulation in the body, the mice were anesthetized and the fluorescent images were detected using a molecular imaging system (Carestream, USA) [28]. During the scan, mice were maintained under gaseous anesthesia (1e2% isofluorane) at 37  C. The mice were sacrificed and lungs were collected, and imaged. For bioluminescence imagining, D-luciferin-loaded TMT-LS and LS were administered intravenously (50 mg/kg of D-luciferin) into the MDA-MB-231/Luc lung metastasis-bearing mice [26]. After 3.0 h circulation in the body, bioluminescence imagining was performed non-invasively using a molecular imaging system (Carestream, USA) with total exposure time of 5.0 min, bin 8 [27]. Due to the presence of luciferase in all the tumor cells, D-luciferin delivered to tumor could be converted by luciferase resulting in visible bioluminescent signal which could be imaged. 2.7. Growth inhibition of tumor metastasis foci in vivo The anti-metastasis activity of the TMT-LS-DOX was evaluated by bioluminescence imaging and survival time. In brief, mice bearing lung metastasis foci of MDAMB-231/Luc were treated with saline, free DOX, LS-DOX or TMT-LS-DOX intravenously at the dosage of 2.0 mg DOX/kg body weight every 2 days for 12 days, respectively. After the treatment, the mice were given the substrate D-luciferin (150 mg/kg in DPBS) by intraperitoneal injection. Bioluminescence imaging was initiated 10 min after the injection with total exposure time of 5 min, bin 8. Mice were received continuous exposure to 1e2% isoflurane to sustain sedation during imaging. The signal intensity of lung metastasis was quantified as the sum of all detected photon counts within the region of interest (ROI) after subtraction of background luminescence measured using the same ROI on a corresponding region from control mice [29]. Identical illumination settings were used for acquiring all images. Also, the survival rate of each group was recorded for 30 days. The end point of treatment was recorded as day 0th. 2.8. Specific prevention of tumor metastasis in vivo In the preventive protocol, the animal model and treatment were almost the same as above treatments, except that each treatment was started just on the next day of MDA-MB-231/Luc cells injected intravenously into mice. The dose of DOX was 2.0 mg/kg, totaling 3 treatments at a three-day interval via intravenous injection (Day 1, 4 and 7 post cell inoculation) [30]. On the 14-day after tumor inoculation, the tumor metastasis burden was investigated by bioluminescence imaging as described above. 2.9. Statistics analysis

2.3. Characterization of TMT modified liposomes and the controls related The particle size and zeta potential of liposomes were recorded by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25  C. The data for each sample were obtained in three measurements. The morphological shapes of liposomes were observed using a transmission electron microscope (TEM; JEM-1230, JEOL, Japan). Liposomes solution was placed on a carbon-coated copper grid, negatively stained with 1% uranyl acetate solution, and then air-dried.

Quantitative data were expressed as the mean values with standard deviation (SD). For the anti-metastasis efficacy in vivo, the metastasis burden was estimated using the ManneWhitney test for between-group differences. Survival was assessed using the KaplaneMeier method and log-rank test. For the others, ANOVA was performed to determine the significance among groups followed by Bonferroni’s post hoc test. All statistical tests were two-sided, and P values less than 0.05 were considered to be statistically significant.

2.4. Cell culture

3. Results and discussions

Highly metastatic breast cancer MDA-MB-231 that stably expressed luciferase (MDA-MB-231/Luc) was obtained from Peking University Medical and Healthy Analytical Center and grown in Leibovitz’s L15 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin in a 37  C humidified incubator.

3.1. Development and characterization of liposomal formulations A schematic representation of TMT peptide-modified liposomes is shown in Fig. 1A. TMT peptide was successfully conjugated to the

758 Z. Wang et al. / Biomaterials 34 (2013) 756e764 Fig. 1. The characterization of TMT modified liposomes. (A) Schematic illustration of TMT modified liposomes loaded with different contents (TMT-LS). (B) MALDI-TOF MS spectra of NHS-PEG-DSPE and TMT-PEG-DSPE. (C) Particle size of TMT-LS by dynamic light scattering analysis (average size, D ¼ 100 nm). (D) Transmission electron microscopy images of TMT-LS.

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Table 1 Particle sizes and zeta potentials of liposomes (n ¼ 3). Characterizations loaded contents

LS TMT-LS

Particle size (nm)

Zeta potential (mV)

DOX

DiD

D-luciferin

DOX

DiD

D-luciferin

91.43  2.03 94.41  0.70

93.39  1.32 96.30  2.02

91.33  1.62 93.04  0.98

26.5  1.0 27.2  0.5

31.0  1.3 29.6  0.9

28.3  0.7 27.1  1.7

DSPE-PEG-NHS, as the experimental Mw of DSPE-PEG-TMT at about 4000 was in accordance with its theoretical Mw, which confirmed that the targeting compound was in the resulting synthetic product (Fig. 1B). Both the drug and fluorescent probe-loaded liposomes have very similar average diameters at about 100 nm (Fig. 1C and Table 1), which may be an optimal size for tumor targeting by the enhanced permeability and retention (EPR) effect [31]. All

liposome systems exhibited a strong negative surface charge around 30 mV, which implies their good dispersion stability [32]. Transmission electron microscopy images showed the spherical shape of liposomes (Fig. 1D). These data showed that control and TMT modified liposomes have similar characters, demonstrating the ligand modification would not significantly bring the influence in the comparison of these two liposomal systems [33,34].

Fig. 2. TMT liposomes demonstrated specific delivery of DOX in highly metastatic cancer cells in vitro. (A) Flow cytometric measurement of DOX uptake by highly metastatic cancer cell MDA-MB-231 after incubated with LS-DOX, TMT-LS-DOX and free DOX for 3 h. Untreated cells served as negative control. (B) Laser scanning confocal microscopy (LSCM) images of the MDA-MB-231 cells incubated with free DOX, LS-DOX or TMT-LS-DOX (DOX ¼ 10 mg/mL) at 37  C for 3 h.

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3.2. Targeted delivery to highly metastatic cancer cells in vitro

precisely analyzed and these images showed that TMT-LS-DiD was localized and spread to whole lung organ, while only a weak signal was detected in the metastasis region in the LS-DiD group (Fig. 3A). Ex vivo images of excised lungs also exhibited much stronger signals of TMT-LS in lung metastasis than that in the LS group (Fig. 3B), confirmed the high accumulation of TMT-LS in tumor metastasis. More importantly, a comparison of the distribution was made here between the lungs with metastasis foci and normal lungs. This proved the preferential distribution of TMT-LS in the metastasized lungs rather than in the corresponding normal ones, while LS exhibited low fluorescence and no marked difference between these two tissues. Meanwhile, we explored the targeting capability and internalization of TMT-LS to metastasis foci by bioluminescence imagining. Consistent with the fluorescence imaging results, the targeted delivery of D-luciferin to the tumor metastasis foci was confirmed again in the TMT-LS group, as demonstrated by a highly intense luminescent signal imaged in the tumor metastasis region at 3.0 h (Fig. 3C), indicating effective release of the cargo from the targeted liposomes in vivo. Notably, TMT-LS produced a very strong optical signal of highly hidden cancer metastasis foci. Relatively, the lung luminescent signal of LS group in metastasis foci was rather weak. In fact, the luminescent intensity of the TMT-LS group in tumor metastasis foci was about 4-fold that of LS group, which demonstrating the superior tumor metastasis targeting of TMT-LS to LS. On the whole, the superiority of TMT-LS in tumor metastasis targeting over non-modified LS was confirmed by both fluorescence and bioluminescence imaging approaches. Besides, the specific distribution of TMT-LS to metastasis foci rather than the

We firstly evaluated the cellular uptake of LS and TMT-LS in metastasis cancer cell MDA-MB-231/Luc. From flow cytometry analysis, free DOX displayed the highest cellular uptake (Fig. 2A). Compared to LS-DOX, the cellular DOX in the TMT-LS-DOX group was significantly higher, demonstrating that TMT modification significantly increased the endocytosis of liposomes. Fig. 2B exhibits the intracellular uptake and distribution of DOX by laser confocal scanning analysis. Free DOX was showed the most intense intracellular fluorescence, attributing to its direct and rapid partition into the membrane without release from liposomes and its highly nucleophilic nature. TMT-LS-DOX showed brighter intracellular red fluorescence of DOX than LS-DOX. It was suggested here that the TMT could markedly improve the recognition and uptake of liposomes [23]. 3.3. Targeted delivery to tumor metastasis foci in vivo A nude mouse model with metastasis foci formed in the lung was established and the targeted delivery of TMT peptide-modified liposomes to tumor metastasis foci was studied by both fluorescence and bioluminescence imaging [26,27]. We employed MDA-MB-231/ Luc with expression of luciferase to visualize the metastasis foci. 14 days after injection of MDA-MB-231/Luc cells via tail vein, the tumor metastasis was successfully established in lung tissues. After intravenous administration of TMT-LS-DiD in tumor metastasis foci bearing mice, significant red fluorescence of DiD was observed in the tumor metastasized lungs, which could be

A TMT-LS-DiD

C

LS-DiD

Free luciferin

Free luciferin 20.00

8000.0

15.50 11.50

5000.0

7.25 over lay

3500.0

1.00 2 p/sec/cm /sr

2000.0

B

TMT-LS-DiD

TMT-LS

LS-DiD Lung metastasis

×10

6

6500.0

LS

4.00

Normal lung

2.50

×10

6

3.25

1.75 500.0 1625.0 2750.0 3875.0 5000.0

1.00 2 p/sec/cm /sr

Fig. 3. TMT liposomes demonstrated tumor metastasis-specific delivery in a lung metastasis model. In vivo (A) and ex vivo (B) fluorescence imaging of lung metastasis foci at 24 h post DiD-loaded LS and TMT-LS injection. (C) The targeted delivery of liposomes carrying D-luciferin into lung metastasis. Before liposomes administration, mice were given Dluciferin by intraperitoneal injection to test the formation of cancer metastasis foci. Then, the mice on the left were intravenously injected with D-luciferin-loaded TMT-LS, and on the right with D-luciferin-loaded LS, respectively. Luciferase activity is measured in photons per cm2 per second per steradian (p/s/cm2/sr).

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corresponding normal tissue was also demonstrated. So, it appears that such an improvement was also resulted from the interaction between TMT peptide and its receptor expressed on the tumor cells of metastasis foci. In addition, it is worthwhile to mention that early detection of unknown cancer metastasis is crucial in terms of controlling metastasis and elevating the survival rate [8]. However, targeted delivery to highly hidden metastasis foci is much more difficult than to primary solid tumor [18,19]. It was very important here that D-luciferin-loaded TMT-LS was able to produce a strong optical signal for occult metastasis and the diagnostic capabilities of lung metastasis-specific imaging of TMT-LS by bioluminescence imaging were therefore demonstrated. In this sense, our findings are especially meaningful and may be used for cancer metastasisspecific imaging and therapy.

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The above data suggest that TMT-LS seem to reach the tumor metastasis more efficiently, reinforcing the potential use of liposomes as a tumor metastasis-targeted drug delivery system. 3.4. Specific inhibition of tumor metastasis foci in vivo Having verified the tumor targeting of TMT modified liposomes in vivo, the anti-metastasis efficacy of TMT-LS loaded with DOX was further evaluated by direct monitoring tumor metastasis burden and the survival rate. Bioluminescence imaging was utilized here because it is able to provide a simultaneous and sensitive analysis for multiple tissues/organs, and it also involves more predictive and humane animal model of cancer, which is especially advantageous in evaluating the therapeutic efficacy of cancer metastasis [35,36].

Fig. 4. DOX-loaded TMT liposomes reduced the metastasis burden in vivo. (A) Representative in vivo bioluminescent images of mice (n ¼ 10) after treated with saline and various DOX formulations, red signal represents the highest level on the colorimetric scale. (B) Quantification of total tumor metastasis burden in mice over the indicated time course by bioluminescence imaging (n ¼ 10, *P < 0.001 ). Luciferase activity is measured in p/s/cm2/sr. (C) Survival rate of mice after treated with different DOX formulations above. The end point of treatments was recorded as day 0th. (*P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Generally, the relative level of bioluminescence of tissues correlates with the cancer burden [37]. Bioluminescent images of mice treated with saline or different DOX formulations (2.0 mg DOX/kg body weight every 2 days for 12 days) are shown in Fig. 4A. At the end of treatment, TMT-LS-DOX achieved a good control of tumor metastasis, as evidenced by the lowest luciferase activity in tumor metastasis foci and its tumor metastasis inhibitory rate was 89.21  3.48% compared with the control group (P < 0.05) (Fig. 4B). However, free DOX was not significantly different from the control (P ¼ 0.218). LS-DOX showed modest effect against tumor metastasis (P ¼ 0.009), while TMT

modification significantly improved the efficacy of DOX liposomes (P < 0.001, TMT-LS-DOX vs LS-DOX). In addition, the survival of mice carrying lung metastasis in response to the above treatments was investigated (Fig. 4C). The mean survival period of mice was 5 days, 9 days, 12 days and 23 days for the control, free DOX, LS-DOX and TMT-LS-DOX, respectively. TMT-LS-DOX significantly increased the life span of mice compared with LS-DOX (P < 0.001) or free DOX (P < 0.001), while LS-DOX and free DOX also prolonged the survival time of mice compared with the control group (P < 0.001 and P ¼ 0.047, respectively).

Fig. 5. DOX-loaded TMT liposomes decreased the incidence rate of tumor metastasis in vivo. (A) Representative in vivo bioluminescent images of mice in a preventive protocol treated with saline, free DOX, LS-DOX or TMT-LS-DOX (n ¼ 5). (B) The extent of tumor metastasis burden was quantified (n ¼ 5). (*P < 0.01).

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In agreement with the distribution studies in vivo, it was clearly indicated for the first time that TMT modified DOX liposomes demonstrated the strongest inhibitory effects against metastasis foci among all the DOX formulations at a relatively low dose (2.0 mg DOX/kg), which was far beyond our expectation. The enhanced anti-metastasis efficacy was probably attributing to the active targeting via receptor-mediated endocytosis besides passive targeting effect, more drug molecules could deliver to the metastasis foci, which was consistent well with the previous observations in the metastasis foci targeting studies. Free DOX exhibited a negligible inhibition of cancer metastasis, which might result from the short circulation time and poor distribution into metastasis foci, while LS-DOX improved the efficacy to some degree due to the long circulation and passive targeting effect of pegylation [38]. 3.5. Specific prevention of tumor metastasis in vivo Besides demonstrating the efficacy of TMT-LS-DOX on the tumor metastasis foci formed, we also wished to know if such system could work on the prevention of tumor metastasis. In this regard, a preventive protocol was designed here, in which drug administering was initiated on the next day of tumor cell inoculation. According to bioluminescence imagining analysis on 14th day, it was clear that TMT-LS-DOX markedly inhibited the incidence rate and progression of tumor metastasis (Fig. 5A and B). This is also very significant since there are very limited means available to prevent tumor metastasis, although it accounts for 90% of cancer-associated deaths. However, LS-DOX and free DOX only decreased the size of tumor metastasis, but did not possess the metastasis-preventing effect. More concretely, the incidence rate of tumor metastasis was found to be 60% for TMT-LS-DOX and 100% for all other groups. And the tumor metastasis burden in TMT-LS-DOX group was only about 10% that in the LS-DOX group (P ¼ 0.008) or free DOX group (P ¼ 0.008). Totally, it demonstrated here that TMT-LS-DOX could greatly inhibit the initiation and progression of highly metastatic malignancies. 4. Conclusions It has been demonstrated that the TMT peptide-modified lipid nanocarriers exhibited strong specificity to cancer metastasis in vitro and in vivo. They efficiently targeted to tumor metastasized tissues but not to the corresponding normal ones. Accordingly, the metastasis-specific delivery system greatly facilitated the efficacy of loaded cytotoxic agent in terms of suppressing metastasis foci burden and prolonging the mice survival time. It was also proved that TMT-LS-DOX largely inhibited the incidence of tumor metastasis. Generally, these studies prove the potential of TMT conjugated nanocarriers as a tumor metastasis-targeted drug delivery system in the treatment of cancer metastasis, as well as the prevention of tumor metastasis. We expect that these explorations can also provide a new strategy for the development of nanomedicine for both diagnosis and therapy of tumor metastasis. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 81130059) and the National Research Fund for Fundamental Key Project (No. 2009CB930300). The authors are grateful to Dr. David Jack for his kind editorial help in revising the English of this article. References [1] Parker B, Sukumar S. Distant metastasis in breast cancer: molecular mechanisms and therapeutic targets. Cancer Biol Ther 2003;2:14e21.

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