Functionalized Nanoscale Micelles Improve Drug Delivery for Cancer

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Letter pubs.acs.org/NanoLett

Functionalized Nanoscale Micelles Improve Drug Delivery for Cancer Therapy in Vitro and in Vivo Tuo Wei,† Juan Liu,† Huili Ma,† Qiang Cheng,§ Yuanyu Huang,§ Jing Zhao,† Shuaidong Huo,† Xiangdong Xue,† Zicai Liang,§ and Xing-Jie Liang*,† †

Chinese Academy of Sciences (CAS) Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, No. 11, First North Road, Zhongguancun, Beijing, China 100190 § Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, China 100871 S Supporting Information *

ABSTRACT: Poor penetration of therapeutic drugs into tumors is a major challenge in anticancer therapy, especially in solid tumors, leading to reduced therapeutic efficacy in vivo. In the study, we used a new tumor-penetrating peptide, CRGDK, to conjugate onto the surface of doxorubicin encapsulated nanoscale micelles. The CRGDK peptide triggered specific binding to neuropilin-1, leading to enhanced cellular uptake and cytotoxicity in vitro and highly accumulation and penetration in the tumors in vivo. KEYWORDS: Targeted drug delivery, neuropilin-1, tumor-penetrating peptide, micelle, doxorubicin

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including lung, breast, prostate, pancreas, and colon.18 Thus, we hypothesized that using a tumor-penetrating peptide as a target ligand to specially bind to Nrp-1 receptors may be a more effective way to overcome the poor penetration of drug delivery systems. Hence, we chose a novel CendR peptide ligand, CysArg-Gly-Asp-Lys (CRGDK), the corresponding receptor for which as Nrp-1, as demonstrated in our previous study.19 Systematic evaluation of this peptide as a target ligand has not been reported before. Nanocarriers provide an innovative platform for targeted delivery of small chemotherapeutic molecules, due to their pharmacokinetics and biodistribution behavior. Existing delivery vehicles include polymers,20 dendrimers,21 liposomes,22 micelles,23 nanotubes,24 and nanorods.25 1,2-Distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(polyethylene glycol) 2000] (DSPE-PEG2000) is an amphiphilic copolymer that easily forms micelles and has been FDA-certified for clinical application. DSPE-PEG2000 micelles, with their low critical micelle concentration and small size, have been widely used as nanocarriers for drug delivery.26 In this work, we coupled DSPEPEG2000 with CRGDK peptides and prepared nanomicelles encapsulating doxorubicin. The targeting and penetrating efficiencies of the CRGDK-modified nanomicelles were first systematically evaluated in vitro and in vivo. Preparation and Characterization of Nanomicelles. To prepare DSPE-PEG2000-CRGDK, activated DSPE-PEG2000-MAL was used to conjugate CRGDK to DSPE-PEG2000 (Scheme 1A).

argeted drug delivery has become increasingly attractive in cancer therapy since it can both improve drug efficacy and reduce the side effects of drugs on normal tissues.1 Ligands including aptamers,2,3 peptides,4,5 and antibodies,6 which have good targeting abilities in vitro, have been widely used to deliver drugs or macromolecules to tumor vasculature or tumor cells.7 However, targeted drug delivery in vivo has not met expectations, because most targeted drug delivery systems can only penetrate 3−5 cell diameters and mainly locate around tumor vessels, due to the dysfunctional structure of tumor vessels and high interstitial pressure in the tumor itself.8,9 Overcoming these drawbacks and increasing the penetration of drugs into tumor parenchyma remains the major challenge in developing efficient tumor chemotherapy. An optimal solution may be to find a receptor that is shared by vessels and cells in the tumor region and a corresponding ligand that has tumor targeting and penetrating properties. Neuropilin-1 (Nrp-1) is a transmembrane receptor glycoprotein that plays an essential role in angiogenesis and vascular permeability.10,11 It consists of a large extracellular region containing a1/a2, b1/b2, and c domains, a transmembrane domain, and an intracellular domain.12 Nrp-1 receptors bind peptides carrying a C-terminal R/KXXR/K motif, called the CendR motif, which is found in Semaphorin 3A, VEGF-A165, and the tumor-homing peptide iRGD.10,13,14 Semaphorin 3A interacts with the a1/a2 domains of Nrp-1, while VEGF-A165 interacts with b1/b2 domains, both using their CendR motifs.11,15,16 The interaction between the CendR motif and Nrp-1 is crucial for cell internalization and tissue penetration; for example, the HTLV-1 virus uses its CendR motif to bind to and enter immune cells.17 Nrp-1 receptors are overexpressed by tumor vessels and by a wide variety of human carcinoma cells, © XXXX American Chemical Society

Received: February 14, 2013 Revised: April 17, 2013

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Scheme 1. Schematic Illustrations of the Preparation of the Doxorubicin-Encapsulating Micelles M-Dox and TPFM-Dox. (A) DSPE-PEG2000-CRGDK Was Synthesized by Coupling the Thiol Group of CRGDK with the Maleimide Group of DSPE-PEG2000-MAL. (B) Schematic Representations of the Doxorubicin-Encapsulating Micelles M-Dox and TPFM-Dox

Figure 1. Characterization of the doxorubicin-encapsulating micelles MDox and TPFM-Dox. (A) Transmission electron microscopy (TEM) images of M-Dox and TPFM-Dox after staining with 1% uranyl acetate. Scale bar = 50 nm. (B) Time course of doxorubicin release from micelles at 37 °C at pH 5.0 or pH 7.4. Released doxorubicin was separated from M-Dox or TPFM-Dox by dialysis and quantified by a spectrophotometer.

CRGDK and DSPE-PEG2000-MAL (1:5, w/w) were dissolved in 50 mM HEPES buffer (pH 6.5), stirring continuously at room temperature for 48 h. In this reaction, the thiol group (−SH) of CRGDK is coupled to the maleimide group of DSPE-PEG2000MAL.7,27,28 MALDI-TOF-MS analysis showed that the peak was right-shifted after conjugation, indicating that CRGDK peptides had been successfully conjugated to DSPE-PEG2000-MAL (Figure S1, Supporting Information). Doxorubicin-encapsulated DSPE-PEG2000 micelles (M-Dox) and tumor-penetrating peptide-functionalized micelles (TPFMDox) were then prepared by a film dispersion method (Scheme 1B). To prepare TPFM-Dox, DSPE-PEG2000 lipid was replaced by a mixture of DSPE-PEG2000-CRGDK and DSPE-PEG2000 (1:4 w/w). The morphologies of M-Dox and TPFM-Dox were shown by TEM; both M-Dox and TPFM-Dox micelles were spherical in shape, with a size of around 10 nm, and had good dispersion (Figure 1A). The average hydrodynamic diameter and surface charge were characterized by measuring the size and zeta potential with dynamic light scattering (DLS), as shown in Table S1, Supporting Information. The diameter of M-Dox was 11.5 ± 0.3 nm, and the zeta potential was 0.2 ± 0.1 mV, while the diameter of TPFM-Dox was 13.1 ± 1.5 nm and the zeta potential was −6.9 ± 1.4 mV. Both M-Dox and TPFM-Dox had high encapsulation efficiencies (97.5% and 99.4%, respectively) and high drug loading capacities (16.3% and 15.3%, respectively). In Vitro Drug Release. Controlled and sustained drug release is very important for drug delivery systems. The pH of tumor tissue is much lower than normal, because of lactic acid produced due to hypoxia and acidic intracellular organelles.29,30 Therefore, the doxorubicin release profile from M-Dox or TPFM-Dox was evaluated by a dialysis method at pH 5.0 and 7.4 (Figure 1B). Drug release from M-Dox or TPFM-Dox was much lower at pH 7.4 (30% and 34%, respectively) than at pH 5.0 (79% and 77%, respectively). The release profiles of M-Dox and

TPFM-Dox were similar at each pH. The result was consistent with the previous report.31 The pH-sensitive drug release from the nanomicelles was related to the isoelectric points (PI) of doxorubicin and DSPE-PEG2000. When the pH was reduced from 7.4 to 5.0, dissociation of doxorubicin (PI = 9.06) was enhanced, and the electrostatic repulsion between drug molecules increased. Furthermore, the negatively charged DSPE-PEG2000 molecules (PI = 5.93) became positively charged and lost their electrostatic attraction to doxorubicin. Cytotoxicity Studies of M-Dox and TPFM-Dox. To evaluate the cytotoxicity of CRGDK modified nanomicelles, we chose two human breast cancer cell lines, MDA-MB-231 and MCF-7. We confirmed that Nrp-1 receptors were expressed at high levels on the surface of MDA-MB-231 cells, but at much lower levels on MCF-7 cells using immunofluorescence (Figure 2A). The expression of Nrp-1 receptors was also verified in mRNA level (Figure 2B) and protein level (Figure 2C) by RTPCR and WB. These results are consistent with reports that MDA-MB-231 cells overexpress Nrp-1 on their cell membrane.32 Next, we used these two cell lines to assess the efficacy of the Nrp-1 target ligand CRGDK. We incubated free doxorubicin (FDox), M-Dox, or TPFM-Dox at doxorubicin concentrations of 0.01−100 μg/mL for 24 h and evaluated the cell viability by MTT assay. Compared to F-Dox, M-Dox and TPFM-Dox were more toxic to both MDA-MB-231 and MCF-7 cells (Figure 2D). For MDA-MB-231 cells, M-Dox and TPFM-Dox showed a lower IC50 (2.08 μg/mL and 0.38 μg/mL, respectively) than F-Dox B

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Figure 2. Expression of neuropilin-1 on cell membranes and cytotoxicity of various doxorubicin formulations. (A) Confocal images of Nrp-1 expression, detected with FITC-labeled anti-Nrp-1 antibody (green), on the membranes of MDA-MB-231 and MCF-7 cells. The controls represent the cells are not pretreated with primary monoclonal Nrp-1 antibody, only treated by FITC-conjugated secondary antibody. Nuclei are stained with DAPI (blue). (B) RT-PCR and (C) Western blotting analysis of Nrp-1 expression in MDA-MB-231 and MCF-7 cells. (D) In vitro cytotoxicity, assessed by MTT assay, of F-Dox (free doxorubicin), M-Dox, and TPFM-Dox against MDA-MB-231 and MCF-7 cells.

(4.89 μg/mL). Furthermore, the IC50 of TPFM-Dox was much lower than that of M-Dox, which may be attributed to the improved targeting efficacy of the CRGDK-modified micelles. However, there was little difference in the IC50 when MCF-7 cells were treated with M-Dox or TPFM-Dox (1.52 μg/mL and 1.94 μg/mL, respectively), suggesting that the CRGDK ligand played a key role in enhanced cytotoxicity in vitro. In addition, we evaluated the cytotoxicity of empty DSPEPEG2000 micelles to avoid the cytotoxicity caused by DSPEPEG2000 micelles themselves. The result indicated that empty DSPE-PEG2000 micelles had little toxicity to both MCF-7 cells and MDA-MB-231 cells at the concentration from 0.05 to 500 μg/mL, corresponding to the concentration of doxorubicin (Figure S2, Supporting Information). Hence, lower IC50 of MDOX was not due to the toxic effect of DSPE-PEG2000 micelles themselves, mainly by enhanced cellular internalization of doxorubicin when encapsulated into micelles. Competition Assay. To address the specificity of TPFMDox for neuropilin-1 receptors, we performed a competition assay on MDA-MB-231 cells. Cells were pretreated with excess anti-Nrp-1 monoclonal antibody (dilution 1:50) for 15 min and then incubated with 10 μg/mL TPFM-Dox for 15 min.33,34 The internalization of TPFM-Dox into antibody-treated cells was greatly inhibited compared to untreated cells (Figure 3). This result suggests that CRGDK-modified nanomicelles could bind specifically to neuropilin-1 receptors.

Figure 3. In vitro competition assay to detect targeting of neuropilin-1 by TPFM-Dox in MDA-MB-231 cells. Cells were preincubated with (+) or without (−) anti Nrp-1 primary antibody for 15 min and then incubated with TPFM-Dox for 15 min. Intracellular doxorubicin (red) was observed by confocal microscopy.

Internalization and Distribution of Micelle-Encapsulated Doxorubicin in Vitro. We investigated the cellular uptake of CRGDK-modified micelles by MDA-MB-231 cells using laser confocal scanning microscopy. Cells were incubated with F-Dox, M-Dox, and TPFM-Dox at 37 °C for 0.5 or 1 h with C

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Figure 4. Cellular uptake of F-Dox, M-Dox, and TPFM-Dox by MDA-MB-231 cells. (A) Confocal images of cells treated with free doxorubicin (F-Dox), M-Dox, or TPFM-Dox at 10 μg/mL for 0.5 and 1 h. (B) Quantitative analysis of micelle uptake by flow cytometry. (C) Percentages of cells with increased fluorescence. (D) Mean fluorescence intensity of cells after 0.5 or 1 h. Control cells were untreated. The asterisk (**) represents data points that have a highly significant difference (p < 0.01; two-tailed Student’s t tests).

the doxorubicin concentration at 10 μg/mL. The CRGDK modification enhanced the cellular uptake of TPFM-Dox compared with F-Dox and M-Dox (Figure 4A). This result also indicated that micelles entered cells more quickly than free doxorubicin (F-Dox), which might be due to different endocytosis pathways. In addition, cellular uptake increased as the incubation time increased from 0.5 to 1 h. We next made quantitative measurements of the cellular uptake of various Dox formulations using flow cytometry (Figure 4B). MDA-MB-231 cells treated with TPFM-Dox showed a prominent right shift upon cytometric analysis, suggesting greater cellular uptake of the CRGDK-modified micelles. Almost all cells were able to internalize Dox formulations, since nearly 100% of cells had increased fluorescence except the control group (Figure 4C). However, there was variation of the mean fluorescence intensity (MFI) among different Dox formulations (Figure 4D). The intracellular fluorescence intensity increased when the treatment time was extended from 0.5 to 1 h. The mean intensity of TPFM-Dox was 2.6-fold higher than F-Dox (p < 0.01) and 1.6-fold higher than M-Dox (p < 0.01) after treatment for 0.5 h, indicating that CRGDK improves the target efficacy.

The difference of cellular uptake can be explained by their distinct uptake mechanisms. As shown in Figure S3, Supporting Information, cellular uptake of M-Dox and TPFM-Dox decreased markedly after treated with different endocytosis inhibitors, indicating that micelles entered cells by endocytosis. The cellular uptake inhibition to F-Dox was much lower than that to M-Dox and TPFM-Dox, indicating that F-Dox, a small molecule, mainly entered into cells by passive diffusion mechanism.35,36 The subcellular localization of F-Dox, M-Dox, and TPFM-Dox after 4 h treatment was examined by confocal laser scanning microscopy. Lysotracker Green was used to identify endosomes/ lysosomes. In cells treated with M-Dox and TPFM-Dox, doxorubicin fluorescence was localized in lysosomes and in cytoplasm. Less F-Dox was localized in lysosomes (Figure S4, Supporting Information). The doxorubicin encapsulated nanomicelles entered into endosomes (pH 5.0−6.0) or lysosomes (pH 4.0−5.0) of cancer cells, and doxorubicin could easily escape from micelles and enter the cytoplasm,37,38 because of the pH sensitivity property. This can explain why the doxorubicin located both in endosomes/lysosomes and in cytoplasm. D

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Figure 5. Evaluation of tumor targeting and penetrating efficiencies of nanomicelles in vivo. Mice with armpit tumors derived from MDA-MB-231 cells were given tail vein injections of micelles loaded with the fluorescent dye DiR (A−D) or doxorubicin (10 mg/kg; E,F). Physiological saline was administered as a control. (A) In vivo images of mice 1, 3, 8, 12, and 24 h after treatment with DiR-loaded micelles. (B) Average fluorescence signals of tumors 1, 3, 8, 12, and 24 h after DiR treatment. The asterisk (**) represents data points that have highly significant difference (p < 0.01; two-tailed Student’s t tests). (C, D) Ex vivo images of tumors and other tissues 24 h after DiR treatment (H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; B, brain; I, intestine). (E, F) Frozen sections of tumors removed 24 h after treatment with Dox-loaded micelles were stained with DAPI to label nuclei (E) or CD31 antibody to label tumor vessels (F). Red signal, doxorubicin; blue signal, DAPI; green signal, FITC-tagged CD31.

hemolysis).41 For blood smears, mouse blood was mixed with empty nanomicelles at the same concentration used in the in vivo experiments (1 mg/mL, in whole blood). The cells in the treated blood showed no obvious aggregation or morphological changes (Figure S6, Supporting Information). These results demonstrated that the nanomicelles had good blood compatibility and can be used for in vivo experiments. The tumor-targeting efficacy of CRGDK-modified micelles was then evaluated in mice bearing armpit tumors derived from human breast cancer MDA-MB-231 cells. Micelles loaded with the fluorescent dye DiR were injected intravenously at a dose corresponding to 200 ng/mL of DiR. Figure 5A showed the real-

Enhanced Tumor Accumulation and Penetration in Vivo. Before evaluating the tumor targeting and penetrating efficacy in mice, we used hemolysis analysis and blood smear text to evaluate the blood compatibility of empty DSPE-PEG2000 micelles. For hemolysis analysis, if erythrocytes are lysed, hemoglobin will be released and the supernatant will appear red and the hemoglobin in the supernatant can be measured by measuring the absorbance at 577 nm with a reference wavelength of 655 nm.39,40 As shown in Figure S5, Supporting Information, no visible hemolytic effects were seen even at the highest micelle concentration of 320 μg/mL in PBS, indicating that the empty DSPE-PEG2000 micelles had good hemocompatibility (