Article pubs.acs.org/molecularpharmaceutics
LyP‑1 Modification To Enhance Delivery of Artemisinin or Fluorescent Probe Loaded Polymeric Micelles to Highly Metastatic Tumor and Its Lymphatics Zhaohui Wang,† Yang Yu,† Jie Ma,‡ Haoran Zhang,† Hua Zhang,† Xueqing Wang,† Jiancheng Wang,† Xuan Zhang,† and Qiang Zhang*,† †
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China ‡ State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China ABSTRACT: Metastatic cancers are prone to form metastasis at a distance and acquire drug resistance, which are very common clinically and major obstacles to successful chemotherapy. Besides the tumor itself, the lymphatic system is increasingly emerging as a new target for anticancer therapy because it is an important route of tumor metastasis. To specifically deliver drug to both highly metastatic tumor and its lymphatics, tumor- and tumor lymphatics-homing peptide (LyP-1) conjugated PEG-PCL micelles (LyP-1PM) were first constructed. Artemisinin (ART), a natural product with potential anticancer and antilymphangiogenesis effects, was chosen as the model drug and associated into the micelles. Both PM and LyP-1-PM had similar physiochemical properties, about 30 nm in size with uniform distribution. Highly metastatic breast cancer MDA-MB-435S cells and lymphatic endothelial cells (LEC) were applied as cell models. Flow cytometry and confocal microscopy studies showed that LyP-1-PM exhibited its specificity to both cell lines evidenced by its higher cellular uptake than PM. LyP-1-PM-ART demonstrated higher inhibition effect than PM-ART against these two cell lines in cell apoptosis, cell cycle and cytotoxicity tests. Near-infrared imaging showed that LyP-1-PM was distributed more in orthotopic MDA-MB-435S tumor than PM. Further study by colocalization indicated that PM accumulated near blood vessels, while LyP-1-PM further homed to tumor lymphatic vessels. LyP-1-PM achieved higher antitumor efficacy than other ART formulations in vivo with low toxicity. Both in vitro and in vivo studies here proved that LyP-1 modification enhanced the specific delivery of ART or fluorescent probe loaded polymeric micelles to MDA-MB-435S and LEC. Therefore, LyP-1-PM might be promising in terms of specific delivery of therapeutic or imaging agents to both highly metastatic breast tumor and its lymphatics. KEYWORDS: highly metastatic breast cancer, tumor lymphatics, specific delivery, artemisinin, polymeric micelles
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INTRODUCTION Millions of people die from cancer every year.1 Metastatic cancers are more prone to form a metastasis focus at distant tissues and acquire drug resistance, which are very common in the clinic and become major obstacles to successful chemotherapy.2−4 Naturally, metastatic cancers are especially difficult to treat, and drug resistance often leads to inadequate drug effects, resulting in limited therapeutic efficacy or poor prognosis.5 In addition, cancer metastasis is responsible for 90% of cancer-associated deaths.6 Therefore, exploring the targeting delivery of drugs to treat metastatic tumor is emerging as a new strategy accordingly. The lymphatic system is an important route of tumor metastasis.7 Many cancers preferentially spread through the lymphatic system, and tumor lymphatics significantly facilitate the tumor metastasis from primary tumor to distant tissues.8,9 Recent surveys strongly suggest that the number of lymph vessels in a tumor and the expression of lymphangiogenic © 2012 American Chemical Society
growth factors are important determinants in tumor metastasis.10,11 Thus, it is very significant to specifically deliver therapeutic agents to tumor lymphatics for destruction. However, for some reasons, there is no tumor lymphatic cell line available currently for in vitro mechanism studies. Artemisinin (ART) is a natural product isolated from the Chinese herb Artemisia annua L.12 It has been used in humans for decades as an antimalarial drug, and the related pharmacology has been well studied.13 Further studies on ART have shown that it possessed potent anticancer activity, inhibiting cellular proliferation in a selective way and inducing cell apoptosis in breast, colon and many other cancers.14−16 Lately, a report revealed that ART could inhibit lymphangioReceived: Revised: Accepted: Published: 2646
April 16, 2012 June 25, 2012 August 1, 2012 August 1, 2012 dx.doi.org/10.1021/mp3002107 | Mol. Pharmaceutics 2012, 9, 2646−2657
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98%) was synthesized by the GL Biochem Co., Ltd. (Shanghai, China). Artemisinin (ART) was supplied by Huali Pharmaceutical Co., Ltd. (Chongqing, China). Fluorescent probes Hoechst 33258 and coumarin-6 were from Molecular Probes Inc. (Eugene, OR, USA), while 10-dioctadecyl-3,3,30,30tetramethylindodicarbocyanine-4-chlorobenzene-sulfonate salt (DiD) was purchased from Biotium (Hayward, CA, USA). Sulforhodamine B (SRB) and Tris base were from SigmaAldrich (St. Louis, MO, USA). Rabbit polyclonal to LYVE-1 (lymphatic vessel marker) and rabbit polyclonal to CD31 antibody (blood vessel marker) were from Abcam (Cambridge, MA, USA). DyLight 649-conjugated goat anti-rabbit IgG (H +L) polyclonal antibody was obtained from GeneTex (Irvine, CA, USA). All other chemicals and reagents used were of analytical or HPLC grade. Highly metastatic breast cancer cell line MDA-MB-435S was purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China), and grown in Leibovitz’s L-15 medium containing a final concentration of 10% fetal bovine serum (FBS). Human normal LEC was purchased from PriCells Biomedical Technology Co., Ltd. (Wuhan, China) and maintained in the endothelial cell medium (ScienCell, Carlsbad, CA, USA) consists of 500 mL of basal medium, 25 mL of FBS, 5 mL of endothelial cell growth supplement and 5 mL of penicillin/streptomycin solution. Female BALB/c nude mice of 18−20 g were obtained from Peking University Health Science Center (Beijing, China), and kept under SPF conditions for 1 week before the study, with free access to standard food and water. All care and handling of animals were performed with the approval of the Ethics Committee of Peking University. Synthesis of Targeting Compound. The LyP-1 peptide was first conjugated with NHS-PEG-PCL (1:2, molar ratio) in newly distilled DMF, adjusting to pH 8.0 with Nmethylmorpholine.24 After 48 h reaction at room temperature under moderate stirring, the resulting mixture was put into a dialysis bag with cutoff molecular weight (Mw) of 3,500 Da and dialyzed against deionized water for 48 h to remove unreacted peptide. Then the final solution was lyophilized and stored at −20 °C until use. The conjugation of LyP-1 peptide with PEG-PCL was confirmed by thin layer chromatography (TLC), and the conjugation efficiency was determined by reverse phase high performance liquid chromatography (RP-HPLC, Shimadzu, LC-10AT, Japan). The reaction mixture was sampled at different time points during the reaction, treated with a nitrogen stream to evaporate the solvent, and then redissolved in the mobile phase of acetonitrile−water (17:83, v/v) containing 0.1% trifluoroacetic acid. The peptide was detected with a UV detector at 220 nm. Preparation of Polymeric Micelles. ART-loaded polymeric micelles were prepared using the thin film hydration method.28 Briefly, PEG-PCL and ART at 1:1 molar ratio were codissolved in acetonitrile. Then, the organic solvent was evaporated on a rotary evaporator under reduced pressure at 37 °C. The dried polymer film was warmed to 60 °C, subsequently hydrated with phosphate buffered saline (PBS, pH 7.4) and then sonicated for 5 min. The resulting micellar solution was centrifugated at 12,000 g for 10 min and filtered through a 0.22 μm membrane filter to remove the ART precipitates. For the preparation of ART-loaded active targeted polymeric micelles (LyP-1-PM-ART), the same protocol was conducted except
genesis, suppressing the lymph node and lung metastasis in a lung carcinoma model.17 However, few studies on the ART delivery system were reported until now.18 Additionally, the bioavailability of ART is low since it is not or hardly watersoluble. Polymeric micelles made of amphiphilic block copolymer have emerged as powerful drug delivery vehicles with remarkable in vitro and in vivo success.19 They provide a hydrophobic inner core for accommodating lipophilic therapeutic drugs and hydrophilic outer shell as a brushlike protective corona that stabilizes micelles and protects the micelles from rapid clearance.20 Among the different graft polymers or copolymers, poly(ethylene glycol)-block-poly(3caprolactone) (PEG-PCL) has attracted much attention due to its excellent characteristics as a kind of biomaterial, including biodegradability, safety, high encapsulation for lipophilic drugs, and so on.21,22 Polymeric micelles may accumulate in tumors through either passive or active targeting mechanisms. Passive targeting results from the enhanced permeability and retention (EPR) effects of particle delivery system after intravenous administration,23 while active targeting can be based on the general receptor-mediated interaction between the ligand modified on the surface of micelles and the molecular markers specifically overexpressed in the cancer cells, such as folate receptors, integrins, epidermal growth factor receptors and so on.24 LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys), a cyclic nine-amino acid peptide identified by in vivo phage display technology, was proved to be able to specifically recognize and bind with its receptor (p32/gC1qR) in certain tumor cells (such as highly metastatic breast tumor cells MDA-MB-435S and MDA-MB-231), offering the potential for a drug delivery system to selectively target tumor.25,26 LyP-1 modified nanoparticles were reported for their good tumor targeting and enhanced inhibition effect of drugs loaded against tumors.27 Besides, LyP-1 peptide was also found to specifically bind with tumor lymphatic vessels, providing one possible avenue for tumor targeted therapy that can specifically destroy tumor lymph system simultaneously.7 Additionally, it remains a question if LyP-1 can also bind with normal lymphatic endothelial cells since there is no such report right now. Based on the above background, we hypothesized that the attachment of LyP-1 peptide on the surface of polymeric micelles may lead to their favorable uptake in highly metastatic tumor and lymphatic endothelial cells in vitro and their specific delivery to the tumor and tumor lymphatics in vivo. For the proof-of-concept, LyP-1 peptide modified PEG-PCL micelles were first constructed and characterized. Their targeting ability to highly metastatic breast cancer cells (MDA-MB-435S) and lymphatic endothelial cells (LEC) was investigated in vitro by cellular uptake and in vivo by near-infrared fluorescence imaging of tumor as well as the colocalization of LyP-1 conjugated micelles with lymphatic vessel biomarker. Their targeting properties were further confirmed by the antitumor efficacy studies in vitro including cell apoptosis, cell cycle and cytotoxicity, along with that in nude mice bearing MDA-MB435S orthotopic tumor.
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EXPERIMENTAL SECTION Materials. N-Hydroxysuccinimidyl-PEG 4000 -b-PCL 2500 (NHS-PEG-b-PCL, Mw/Mn = 1.30) and mPEG3000-b-PCL2500 (Mw/Mn = 1.09) were purchased from Advanced Polymer Materials Inc. (Montreal, Canada). The LyP-1 peptide (purity 2647
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In Vitro Cellular Uptake Studies by Flow Cytometry. The cellular uptake of polymeric micelles including PM and LyP-1-PM was studied by fluorescence detection.31 Briefly, MDA-MB-435S or LEC were seeded into six-well plates at a density of 5 × 105 cells/well and cultured at 37 °C for 24 h, respectively. Prior to the experiment, cells were washed twice with PBS (pH 7.4) to remove the remnant growth medium, and then incubated with various coumarin-6 formulations in serum-free medium. The drug-free culture medium was applied as the blank control. After 3 h incubation, the cells were washed, trypsinized and resuspended in PBS. The coumarin-6 fluorescence intensity of the cell suspension was measured by a FACScan flow cytometer (Becton Dickinson; San Jose, CA, USA). The events collected were 10,000. In competition experiments, excess free LyP-1 peptide (10 mM) was preincubated with the cells for 1 h, followed by the coincubation with LyP-1-PM for another 3 h. In Vitro Cellular Uptake Studies by Laser Confocal Microscopy. A confocal fluorescent microscopy was used to compare the cellular uptake of micelles and their cellular distribution. Typically, MDA-MB-435S or LEC were grown on coverslips to 50% confluence and incubated with coumarin-6 loaded PM or LyP-1-PM (containing 10 ng/mL coumarin-6) diluted with culture medium at 37 °C for 3 h. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min, followed by cell nuclei staining with Hoechst 33258 for 5 min. Fluorescent images of cells were analyzed using a laser scanning confocal microscope (Leica; Heidelberg, Germany). Cell Apoptosis Analysis by Flow Cytometry. The cell apoptosis was quantified by flow cytometry using an Annexin V-PI apoptosis detection kit (KeyGEN, China) in accordance with the manufacturer’s instructions. Briefly, MDA-MB-435S cells or LEC were seeded into six-well plates at a density of 5 × 105 cells/well and cultured at 37 °C for 24 h, respectively. Cells were treated with PM-ART or LyP-1-PM-ART for 24 h. Then the cells were washed twice with PBS and harvested by trypsin, followed by centrifugation at 1,000 rpm for 5 min. The medium was discarded, and the cells were suspended in the provided binding buffer, and 5 μL of FITC-conjugated Annexin V was added into the cell suspensions. The mixture was incubated at room temperature in the dark for 15 min, and then 5 μL of PI (propidium iodide) was added. The cell suspension was analyzed by a FACScan flow cytometer with 10,000 events collected. Untreated cells were used to set the background. Cells that stained positive for Annexin V−FITC and PI were designated as apoptotic, and unstained cells were designated as alive. Cell Life-Cycle Analysis by Flow Cytometry. For cell cycle analysis, MDA-MB-435S cells were seeded in 6-well plates at 5.0 × 105 cells/well and allowed to attach overnight.32 Then, the cells were treated with various ART formulations for 48 h. After incubation, the cells were harvested and fixed in ice cold 70% ethanol at 4 °C overnight. Following fixation, the cells were harvested by centrifugation at 1,000 rpm for 5 min and exposed to RNaseA (10 μg/mL) at 37 °C for 30 min. The recovered cells’ DNA was stained with PI (10 μg/mL) for 30 min in the dark. The cell cycle profiles were analyzed using a FACScan cytometer, with 10,000 events counted for each sample. In Vitro Cytotoxicity Studies. To further investigate the efficacy of the micelles in vitro, the cytotoxicity of various ART formulations against MDA-MB-435S and LEC was evaluated by
that 10% (mol ratio) of LyP-1-PEG-PCL was included during the thin film formation process. For the in vivo fluorescence imaging investigation, the nearinfrared fluorescent probe of DiD was loaded into polymeric micelles (PM-DiD). In the film formation, DiD (0.05% mol of copolymer) was incorporated and micelle was prepared by the same procedures as those for PM-ART. The preparation of coumarin-6-loaded polymeric micelles (PM-Cou) was exactly the same as that of PM-DiD, except that DiD was replaced by coumarin-6. Characterization of Polymeric Micelles. The mean particle size and zeta potential of polymeric micelles were recorded using a Malvern Zetasizer Nano ZS (Malvern, U.K.) at 25 °C. The data for each sample were obtained in three measurements. The morphological shapes of micelles were observed by transmission electron microscopy (TEM; JEM200CX, JEOL, Japan) and scanning electron microscopy (SEM; JSM-5600LV, JEOL, Japan). For TEM, micellar solution was placed on a carbon-coated copper grid, negatively stained with 1% uranyl acetate solution and then air-dried at room temperature, while for SEM analysis, samples were completely dried by lyophilization under reduced pressure and coated with gold. Powder X-ray diffraction was carried out in an X-ray diffractometer (XRD; Rigaku, Japan) by Ni-filtered Cu Kα radiation with a 4 deg/min scanning rate at room temperature. The encapsulation efficiency of ART in micelles was determined by the disintegration of micelles in organic solvent. In brief, micellar solution was diluted 10 times with acetonitrile. The ART concentrations in the micelle were quantified by HPLC with a precolumn derivatization method.29 Drug encapsulation efficiency was calculated using the following equations: encapsulation efficiency amount of loaded drug in mg = × 100% amount of added drug in mg
For fluorescent probe loaded micelles, after dilution with 10 times volume of acetonitrile, the level of encapsulated DiD or coumarin-6 in micellar solution was evaluated by fluorophotometer. In Vitro Release Kinetics of Coumarin-6 from Polymeric Micelles. Coumarin-6 was used as a hydrophobic fluorescent probe to study cellular uptake and tumor distribution of polymeric micelles.30 To evaluate if the fluorescence probe remained associated with the micelles during a 24 h incubation period, the in vitro release kinetics of coumarin-6 from micelles was determined using a dialysis method under sink conditions. Briefly, 1.0 mL of coumarin-6loaded micelles was suspended in 2.0 mL of PBS (pH 7.4 or 5.0) containing 10% fetal bovine serum, which represents the normal physiologic pH and the endolysosomal compartment, respectively. Then the diluted solution was transferred into the dialysis bag (Mw cutoff = 12,000−14,000 Da) and dialyzed against 22 mL of PBS buffer containing 10% FBS under continuous gentle stirring at 37 °C. At various time points, samples were withdrawn and replaced with an equal volume of fresh medium. Concentrations of coumarin-6 were measured by a fluorospectrophotometer with an excitation wavelength of 461 nm and an emission wavelength of 501 nm. The cumulative percentage of coumarin-6 release over the 24 h was calculated, and results were plotted against time. All measurements were performed in triplicate. 2648
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Figure 1. Characterization of drug-loaded micelles. (A) Schematic illustration of LyP-1-PM, (B) particle size distribution and (C) zeta potential of polymeric micelles by dynamic light scattering analysis. (D) Transmission electron microscopy and scanning electron microscopy (inset) images of LyP-1-PM. (E) X-ray diffraction patterns of ART powder, blank PEG-PCL micelles, physical mixture of ART and blank PEG-PCL micelles, and ART-loaded PEG-PCL micelles, respectively.
SRB assay.33 Briefly, MDA-MB-435S or LEC were plated in 96well plates at a density of 5,000 cells/well and cultured for 24 h to allow them to attach. Then, the cells were treated with serial concentrations of PM-ART or LyP-1-PM-ART at 37 °C for 48 h. At the end of incubation time, cells were fixed with cold trichloroacetic acid, washed and dried in the air. The fixed cells were then stained with 0.4% SRB for 30 min, and the excess dye was washed out by 1% acetic acid. The absorbance was measured at 540 nm using a Thermo scientific multiscan FC microplate photometer after bound dye dissolved in 10 mM Tris base solution. The data are expressed as the viability of cells compared to the survival of control group and presented as mean ± SD (n = 3). Animal Models. The mouse model of orthotopic breast cancer was established by inoculation of 2 × 106 MDA-MB435S cells into the mammary fat pad of 4-week old female BALB/c nude mice.34 In Vivo and Ex Vivo Imaging Investigations. The DiDloaded micelles were applied to investigate the tumor targeting efficacy in nude mice bearing orthotopic MDA-MB-435S breast tumors.35 Typically, DiD-loaded PM and LyP-1-PM were injected into the tail vein of mice at a dose of DiD 50 μg/kg, respectively. At a predetermined time, the anesthetized mice were put into the chamber and the fluorescent images were detected using an in vivo molecular imaging system (Carestream; Rochester, NY, USA) equipped with an excitation bandpass filter at 630 nm and an emission at 700 nm. During the imaging acquiring process, the 2% isoflurane anesthesia was
delivered via a nose cone system for maintaining anesthesia. After 96 h, the mice were sacrificed, and tumor and major organs (including heart, liver, spleen, lung and kidney) were removed and finally visualized. Distribution of Polymeric Micelles in Tumor. Immunofluorescence was carried out to examine the distribution of micelles in tumor after intravenous injection of the coumarin-6labeled micelles (50 mg of micelles/kg) into the tail vein of MDA-MB-435S breast tumor-bearing mice.7,27 The mice were killed 3 h after injection by cardiac perfusion with PBS through the heart under anesthesia, and tumors were excised and frozen in optimal cutting temperature (OCT) embedding medium. Sections about 5 μm were prepared and first incubated with 10% bovine serum albumin for 1 h at room temperature, followed by incubation with the primary antibody overnight at 4 °C. For labeling lymphatic and blood endothelial cells, the following primary antibodies were used: rabbit polyclonal antimouse LYVE-1 (10 μg/mL) and rabbit polyclonal anti-mouse CD31 (10 μg/mL). DyLight649-conjugated goat anti-rabbit IgG (H+L) polyclonal antibody was used as the secondary antibodies. Sections only with secondary antibodies were included as negative control in each staining experiment. Nuclei were counterstained with Hoechst 33258 (5 μg/mL). The sections were visualized under a Leica confocal microscope. In Vivo Efficacy Evaluations. The therapeutic efficacies were compared in MDA-MB-435S tumor bearing BALB/c mice for different treatments.36 When the tumor volume reached 2649
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Table 1. Particle Sizes and Zeta Potentials of Polymeric Micelles (n = 3) particle size (nm) PM LyP-1-PM a
zeta potential (mV)
ARTa
DiDa
coumarin-6a
ARTa
DiDa
coumarin-6a
30.39 ± 0.71 31.18 ± 0.51
34.91 ± 0.52 32.72 ± 0.37
31.21 ± 0.30 31.57 ± 0.49
0.15 ± 0.50 0.07 ± 0.09
0.41 ± 0.15 0.82 ± 0.14
0.80 ± 0.37 0.46 ± 0.39
Loaded contents.
50−100 mm3, mice were randomly divided into four groups (n = 6), and treated with one of the following formulations: group 1 for PBS solution, group 2 for PM-ART at 20 mg ART/kg, group 3 for LyP-1-PM-ART at 20 mg ART/kg and group 4 for free ART at 20 mg ART/kg, respectively. Free ART was prepared by dissolving in DMSO and diluting with PBS. Mice were administered the formulations every 3 days for 7 times via tail vein injection except that free ART was administered by intraperitoneal injection. Tumor volume (V) was recorded every 3 days with a caliper in two dimensions, and then calculated using the following formula: V = [length × (width)2]/2. The growth curve was plotted using the mean tumor volumes for each treatment group at every time point. The body weight of each group was also measured every three days during the treatment period. Statistical Analysis. All results were analyzed by one-way analysis of variance (ANOVA). Data were expressed as means ± standard deviation (SD). A P-value less than 0.05 was considered to be statistically significant, while less than 0.01 was highly significant.
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RESULTS AND DISCUSSION Preparation and Characterization of Targeting Compound. The targeting compound of LyP-1-PEG-PCL was synthesized by conjugation of the LyP-1 peptide with NHSPEG-PCL through the nucleophilic substitution reaction. According to the HPLC analysis, the retention time of LyP-1 peptide was about 10 min, and NHS-PEG-PCL did not interfere with its detection (data not shown). For the reaction between peptide and copolymer, the peak of LyP-1 peptide disappeared after 48 h reaction. The results of TLC observation were similar to HPLC and further confirmed that LyP-1 peptide was successfully conjugated to NHS-PEG-PCL via an amide bond. Characterization of Polymeric Micelles. A schematic representation of LyP-1 peptide modified polymeric micelles is shown in Figure 1A. The particle size and zeta potential of ART, DiD and coumarin-6 loaded micelles modified with LyP1 or not are summarized in Table 1. It was shown that polymeric micelles were about 30 nm with a narrow distribution (Figure 1B) and had almost no surface charge (Figure 1C). TEM images showed their uniform nanoscale particles, and SEM confirmed their spherical morphologies (Figure 1D). The encapsulation efficiency for ART was consistently greater than 90%, and that for DiD or coumarin6 was more than 95%. The XRD spectra of ART, PEG-PCL micelles, physical mixture of ART and PEG-PCL micelles, and ART-loaded PEGPCL micelles are presented in Figure 1E. It was indicated that ART powder showed its crystalline peak at 2θ (11.86°), and the drug peak was also detected in the physical mixture of ART and blank micelles. However, the XRD patterns between ARTloaded micelles and PEG-PCL micelles were almost identical, where ART peak was absent, revealing that ART was
Figure 2. In vitro release profile of coumarin-6 from micelles using a dialysis method. The coumarin-6 loaded micelles were incubated in PBS at pH 5.0 (A) and 7.4 (B) containing 10% FBS. At various time points, aliquots were withdrawn and the coumarin-6 was measured spectrophotometrically.
successfully loaded into the PEG-PCL micelles and might exist as the amorphous or molecular state.37 By the way, the stability of drug-loaded polymer micelles, especially in vivo, is always a key issue. The CMC value of PEG3000-PCL2500 we used is 1.5 × 10−7 M, which is very low, so it means that the stability of this kind of polymer micelles is relatively high in theory. In our in vitro and in vivo test, the calculated concentration of PEG3000-PCL2500 was higher than its CMC value, therefore the test micelles might be stable during the study. On the other hand, drug molecules may release or diffuse from the intact micelles due to the concentration gradient if the micelles are stable. Then, the release of drug loaded may be accelerated in vivo as the degradation of the copolymer. In summary, there was no significant difference found in particle size, zeta potential and encapsulation efficiency after peptide modification of the micelles, so these factors might not bring their influences into the following comparison study between the active and passive groups at the cellular level.38,39 In Vitro Release Kinetics of Coumarin-6 from Micelles. The results of the in vitro release study in different pH media 2650
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Figure 3. The cellular uptake of polymeric micelles by LEC. (A) Flow cytometric analysis of the association of micelles with LEC. LEC cell monolayer was incubated with free coumarin-6, PM or LyP-1-PM at a final concentration of 20 ng/mL at 37 °C for 3 h. In competition experiments, 10 mM excess free LyP-1 peptide was added to the cells 1 h prior to the addition of LyP-1-PM. (B) Laser scanning confocal microscopy (LSCM) images of LEC incubated with above coumarin-6 formulations at a final concentration of 10 ng/mL at 37 °C for 3 h. Green: fluorescence of coumarin-6. Blue: fluorescence of Hoechst 33258. All images were taken under identical instrumental conditions and presented at the same intensity scale.
LyP-1-PM at both pH values revealed that the ligand modification did not significantly change the release behavior of micelles. The Specificity of LyP-1-PM to Lymphatic Endothelial Cells in Vitro. We first compared the specific cellular uptake of PM and LyP-1-PM in LEC by flow cytometry analysis. As shown in Figure 3A, the cellular coumarin-6 level of LyP-1-PM was significantly higher than that of PM. Both micelle groups possess the similar properties and compositions except LyP-1
are presented in Figure 2. No more than 2% of coumarin-6 released from PM and LyP-1-PM after 24 h in both pH 5.0 (Figure 2A) and pH 7.4 (Figure 2B) release medium, namely, more than 95% of the encapsulated coumarin-6 was still retained in micelles. The less leakage observed here even in the presence of plasma demonstrated the good stability of micelles, guaranteeing that the fluorescence signal detected in the cellular uptake was mainly attributed to the coumarin-6 inside of the micelles.30 In addition, the similar release kinetics of PM and 2651
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Figure 4. The cellular uptake of polymeric micelles by highly metastatic cancer cells MDA-MB-435S. (A) Flow cytometry studies of cells after incubation with PM or LyP-1-PM micelles (coumarin-6 = 50 ng/mL) at 37 °C for 3 h. (B) LSCM images of cells incubated with above formulations at 37 °C for 3 h (coumarin-6 = 10 ng/mL). Cells were fixed with 4% paraformaldehyde and treated with Hoechst 33258 for nuclei staining (blue).
Figure 5. In vitro cell apoptosis evaluation of micelles against MDA-MB-435S (A) or LEC (B). After incubation with ART-loaded micelles, the cells were stained with Annexin V−FITC/PI and analyzed by FACS analysis. ART = 50 μg/mL.
modification, so it was supposed that higher uptake of ligandmodified micelles in LEC might result from receptor-mediated endocytosis. Additionally, competitive assay was conducted to confirm the role of LyP-1 peptide in the cellular uptake. It was clear that excess free LyP-1 peptide significantly inhibited the uptake of LyP-1-PM, demonstrating that uptake of LyP-1-PM was mostly mediated by the interaction between the peptide and its receptor on the surface of LEC. The positive control of free coumarin-6 showed the highest uptake due to direct act with cells without release process. The laser confocal scanning in Figure 3B indicated more evident and intense intracellular fluorescence of coumarin-6 in LyP-1-PM group compared to that in PM group, suggesting the higher uptake of active targeted micelles in the cell line again. Besides, the majority of visible fluorescence was distributed in the cytoplasm. In general, the studies here proved that LyP-1-PM greatly increased the drug delivery to the LEC mostly through receptor-mediated endocytosis. This finding is significant
since the free peptide of LyP-1 was found here to inhibit the uptake of LyP-1-PM by normal lymphatic endothelial cells, suggesting the possible expression of LyP-1 receptors in normal LEC besides in tumor lymphatics.26 The Specificity of LyP-1-PM to Highly Metastatic Tumor Cells in Vitro. Cellular uptake of micelles by MDAMB-435S and the distribution of micelles in this cell line were also studied by flow cytometry and laser confocal scanning, respectively. Similarly, LyP-1-PM demonstrated significantly higher cellular coumarin-6 level than PM (P < 0.05, n = 3), revealing the LyP-1 conjugation was favorable for the improved cellular uptake of micelles (Figure 4A). Also, more obvious coumarin-6 fluorescence was found in cells treated with LyP-1PM than that with PM, confirming LyP-1 modification enhanced the efficient intracellular delivery of polymeric micelles (Figure 4B). Moreover, most of the visible fluorescence was observed in the cytoplasm compartment and it seemed that micelles did not enter the nuclei, in accordance with our previous study.40 2652
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micelles by both highly metastatic breast tumor and lymphatic endothelial cells. Since the release of coumarin-6 from PM was so little in the test condition (Figure 2), the fluorescence signal in cells might imply the position or quantity of micelles related. The Effect of LyP-1-PM-ART on Cell Apoptosis. The apoptosis-inducing effects of LyP-1 modified micelles on MDAMB-435S and LEC are illustrated in Figure 5. After treatment with PM-ART and LyP-1-PM-ART for 24 h, the percentage of induced apoptotic cells in the MDA-MB-435S cell line was 28.68% and 50.50%, respectively (Figure 5A); and that in LEC was 8.20% and 14.18% (Figure 5B), respectively. Obviously, a higher ratio of apoptosis was induced in active targeted micelles than that in passive targeted group for the same cell line. ART is known to induce cancer cell apoptosis, however, the mechanism of its action is not well understood. The Effect of LyP-1-PM-ART on Cytotoxicity. The cell viability of LEC and MDA-MB-435S after treatment with two kinds of ART micelles is displayed in Figure 6, and a dosedependent effect was found for both formulations. For both LEC (A) and MDA-MB-435S (B), it was clear that LyP-1-PMART showed a significantly higher inhibitive effect on cell proliferation in comparison with PM-ART at a concentration of 5−50 μg/mL (P < 0.05). This revealed that the peptide modification could increase the delivery efficiency of ART, which was consistent with cellular uptake results in flow cytometry and confocal microscopy analysis above. Additionally, the blank polymer micelles (LyP-1-PM and PM) were also investigated in the cytotoxicity assay with MDAMB-435S (data not shown). As a result, the cell viability in each micelle group was all around 100% in various tested concentrations, revealing the biocompatibility of the polymeric micelles tested. The Effect of LyP-1-PM-ART on Cell Life-cycle. Furthermore, the effect of active targeted micelles on the cell cycle of highly metastatic tumor cells is shown in Figure 7. Quantitative analysis of the cell cycle distribution showed that 27.85%, 29.90% and 36.61% of cells were in the S phase after treatment with control, PM-ART, and LyP-1-PM-ART, respectively. Namely, more cell proliferation process was blocked in S phase when MDA-MB-435S cells were treated
Figure 6. In vitro cell toxicity tests of PM-ART and LyP-1-PM-ART against LEC (A) and MDA-MB-435S (B) at different concentrations. Following the 48 h treatment, the degree of cytotoxicity was determined using the SRB assay (n = 3). *P < 0.05 and **P < 0.01 for LyP-1-PM-ART versus PM-ART, respectively.
The above quantitative and qualitative studies consistently demonstrated that the LyP-1 modification could markedly improve the specific recognition and uptake of the polymeric
Figure 7. Cell cycle distribution of MDA-MB-435S cells incubated with ART-loaded PM and LyP-1-PM for 48 h. The cell cycle phases were analyzed using fluorescence-activated cell sorting (FACS). 2653
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Figure 8. The targeted delivery of LyP-1-PM to highly metastatic breast tumor in vivo. (A) In vivo near-infrared fluorescent images of mice after intravenous administration of DiD-loaded LyP-1-PM or PM at different time points and (B) ex vivo image of tumors and organs after the tumorbearing mice above were sacrificed at 96 h.
Figure 9. The targeted delivery of LyP-1-PM to tumor lymphatics in vivo by immunofluorescence technique. (A) Colocalization of LyP-1PM in MDA-MB-435S tumor tissue with the lymphatic endothelial marker (LYVE-1). (B) Colocalization of LyP-1-PM in the tumor tissue with blood vessel markers (CD31). (C) Colocalization of PM in the tumor tissue with LYVE-1. (D) Colocalization of PM in the tumor tissue with CD31. Nuclei were counterstained with Hoechst 33258 (blue).
with LyP-1-PM-ART. The difference between the two PM groups could explain the effect of LyP-1 mediated delivery on the antitumor efficacy. It was exhibited here that ART-loaded PM did show an inhibitory effect against both MDA-MB-435S and LEC. It was supposed that only the free drug molecules already released from micelles could show an anticancer effect. The delivery of ART into cancer cells could lead to growth inhibition and cell cycle arresting in S phase which in turn result in cell death through apoptosis.33 As demonstrated here, all antitumor studies in vitro were consistent with each other. In other words, LyP-1-PM-ART demonstrated better activity in the test cells
Figure 10. Antitumor efficacy and system toxicity of LyP-1-PM-ART in vivo. (A) Tumor volumes of different treatment groups in MDAMB-435S tumor-bearing mice. Mice were injected with PBS, free ART, PM-ART or LyP-1-PM-ART at a 20 mg/kg ART equivalent dose for 7 times at a 3 day interval. Arrows represent drug administration. Data represented as mean ± SD (n = 6). * P < 0.05 and ** P < 0.01 for LyP-1-PM-ART versus each of the other treatments. (B) Body weight changes were also recorded during efficacy study.
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overexpressed on the lymphatic endothelial cells in the tumor tissue.27 Taken together with the in vivo imaging tests, LyP-1PM could target not only highly metastatic breast tumor but also tumor-associated lymphatics, which might be very significant for tumor therapy and metastasis prevention. Antitumor Efficacy of LyP-1-PM-ART In Vivo. The in vivo therapeutic efficacy of three ART treatments against orthotopic metastatic breast tumor is summarized in Figure 10A. Compared to PBS, which showed the fastest tumor growth, all the ART formulations were effective in inhibiting tumor growth, while LyP-1-PM-ART exhibited the most effective suppression on tumor growth. As seen in Table 2, LyP-1-PM-ART had the highest inhibition ratio (50.9%) among all treatments at day 30, followed by PM-ART (26.4%) and free ART (14.4%). Significant difference was noticed between LyP-1-PM-ART and PM-ART (P < 0.01) or free ART (P < 0.01), however, no significant difference was observed between PM-ART and free ART (P > 0.01). On the other hand, an important consideration in nanocarriers is their potential toxicity, especially for the system administration.42 We therefore assessed the changes of body weight in the xenograft mouse model during the efficacy test. For all the treatments in this work, no significant loss of body weight was observed (Figure 10B). Besides, the animal status in each test group was similar to that in the control group. All these indicated a low toxicity at this dose for ART and the two micelle systems. ART was precipitated when diluted with PBS because of low solubility in aqueous medium even with the aid of DMSO and intravenous injection thus could not be performed. Therefore, intraperitoneal injection was given as an alternative, which was considered to possess similar bioavailability with intravenous administration.24 The free ART exhibited a negligible inhibition of tumor growth, which might result from its poor specificity to tumor mass and fast elimination in circulation. The LyP-1-PM-ART achieved superior antitumor effects to its controls, likely attributable to the combined processes of passive targeting via the EPR effect and the active targeting through receptor mediated endocytosis.43,44 The favorable efficacy of LyP-1-PMART was consistent well with the previous observation in cellular studies and in vivo fluorescent imaging. The efficacy study further confirmed the tumor targeting ability of LyP-1 modified polymeric micelles.
Table 2. The Inhibition Rates of Various ART Formulations on Highly Metastatic Tumor MDA-MB-435S Bearing Micea treatment group control PM LyP-1-PM free ART a
tumor vol (mm3)
inhibn rate (%)
± ± ± ±
26.4 50.9 14.4
1964.8 1445.7 965.5 1681.2
437.5 200.1 208.7 371.2
Data are presented as mean ± SD (n = 6).
than PM-ART. This result was in good accordance with the findings in cellular uptake, which could clarify the reason of better efficacy of active targeted micelles in vitro. Namely, through a receptor mediated endocytosis pathway, more LyP-1PM-ART could be internalized into these cell lines and exhibited better efficacy. In fact, these efficacy tests in vitro also indicated the specificity of active targeted micelles to the test cell lines related. By the way, the free ART was not used here as a control in antitumor effect study because of the solubility problem. It was found that ART could crystallize even in the presence of DMSO, while excessive DMSO may cause great cytotoxicity itself. The Targeted Delivery of LyP-1-PM to Highly Metastatic Tumor in Vivo. Figure 8 shows the in vivo and ex vivo near-infrared fluorescence imaging of in MDA-MB-435S bearing nude mice treated with DiD-labled micelles. As seen in Figure 8A, preferential accumulation of DiD fluorescence was observed in the tumor of LyP-1-PM-DiD group at all test points from 3 h to 96 h compared to that of PM-DiD group. Additionally, the ex vivo images of excised organs showed that a stronger signal was found in the mouse tumor of LyP-1-PMDiD group than that of PM-DiD group (P = 0.01, n = 3) (Figure 8B). In addition, it is worthwhile to mention that, in the PM group, the fluorescence signals in the major organs including heart, liver, spleen, lung and kidney were much weaker than that in tumor and this actually demonstrated the EPR effect of the passive targeted micelles.41 The higher fluorescence signals in the tumor of LyP-1-PM-DiD group than PM-DiD group suggest that better targeting effect can be achieved by peptide modification based on common micelle system. Generally, these two tests demonstrated the in vivo targeting delivery of peptide modified micelles to highly metastatic breast tumor, better than unmodified micelles, in good accordance with the specific cellular uptake and efficacy studies in vitro. The Targeted Delivery of LyP-1-PM to Tumor Lymphatics in Vivo. In MDA-MB-435S bearing nude mice, two micelle systems were investigated for their targeting to tumor lymphatics through the colocalization study in tumor sections and the results are shown in Figure 9. It was found that LyP-1-PM colocalized well with the lymphatic vessel marker LYVE-1 (Figure 9A) but not with the blood vessel marker CD31 (Figure 9B). Almost on the contrary, PM had a good colocalization with the blood vessel marker CD31 (Figure 9D) but not with the lymphatic vessel markers LYVE-1 (Figure 9C). This finding supports that the passive targeted micelles distribute into tumor tissue through the EPR effect, resulting in their accumulation near the blood vessels of the tumor. Meanwhile, the active targeted micelles further home to tumor lymphatic vessels through the targeting effect of LyP-1, after their distribution into tumor by EPR effect. The tumor lymphatics targeting of LyP-1-PM might be attributed to the special interaction of LyP-1 with its receptors
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CONCLUSIONS Herein, LyP-1 peptide modified micelles were successfully developed to specifically delivery of therapeutic agent to both highly metastatic breast cancer and its lymphatics. ART encapsulated polymeric micelles were prepared and studied in vitro and in vivo. Besides the targeting effect to tumor cells, it was demonstrated for the first time that modification of the peptide significantly enhanced cellular uptake of micelles in LEC via receptor-mediated endocytosis. Also, we proved here that ART-loaded micelles were able to inhibit the growth of MDA-MB-435S and LEC, and LyP-1 conjugation enhanced such effect. Moreover, LyP-1-PM was observed to distribute more in the tumor of nude mice than PM, and then further home to tumor lymphatic vessels within the tumor. Higher antitumor efficacy than PM and low toxicity of LyP-1-PM was also observed. All in vivo tests further confirmed the specificity of LyP-1-PM to metastatic breast tumor and its lymphatics. Collectively, both in vitro and in vivo studies here proved that 2655
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LyP-1 modified polymeric micelles might be promising in terms of specific delivery of therapeutic or imagining agents to both highly metastatic breast tumor and tumor lymphatics.
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AUTHOR INFORMATION
Corresponding Author
*Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing, 100191, China. E-mail:
[email protected]. cn. Tel/fax: 86-10-82802791. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by projects from National Science Foundation (No. 81130059) and Ministry of Science and Technology of China (No. 2009CB930300 and No. 2009ZX09310-001).
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