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Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx – xxx nanomedjournal.com
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Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles Xin Li, MS b , Yang Yu, MS b , Qian Ji, BA b , Liyan Qiu, PhD a,⁎
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Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China b College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Received 11 May 2014; accepted 29 August 2014
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Abstract
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Aptamers are single-stranded RNA or DNA ligands that can specifically bind to various molecular targets with high affinity. Owing to this unique character, they have become increasingly attractive in the field of drug delivery. In this study, we developed a multifunctional composite micelle (CM) with surface modification of aptamer AS1411 (Ap) for targeted delivery of doxorubicin (DOX) to human breast tumors. This binary mixed system consisting of AS1411 modified Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-bpolylactide could enhance DOX-loading capacity and increase micelle stability. Cellular uptake of CM-Ap was found to be higher than that of untargeted CM due to the nucleolin-mediated endocytosis effect. In vivo study in MCF-7 tumor-bearing mice demonstrated that the AS1411-functionalized composite micelles showed prolonged circulation time in blood, enhanced accumulation in tumor, improved antitumor activity, and decreased cardiotoxicity. In conclusion, aptamer-conjugated multifunctional composite micelles could be a potential delivery vehicle for cancer therapy. © 2014 Elsevier Inc. All rights reserved.
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Key words: Aptamer; Composite micelles; Cyclodextrin; Pluronic F127; Doxorubicin
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In the last few decades, there have been advances in nanotechnology for cancer treatment; however, various challenges remain pertaining to its application in clinical settings. First, the physicochemical properties of nanoparticles are far from satisfactory, such as complicated preparation process, 1 low drug loading, and poor stability. Second, although nanoparticles are expected to reach tumors by the effect of enhanced permeability and retention (EPR), their nonspecific uptake by mononuclear phagocytic cells that are present in the liver, spleen, and lungs may limit the effectiveness of therapeutic drugs and cause severe side effects. 2–4 Third, anticancer agents should be effectively transported into tumor cells to exhibit their pharmacological activity. However, in some cases, completion
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Conflict of interest statement: No conflict of interest is reported by the authors of this paper. Funding sources: This work was supported by National Natural Science Funds for Excellent Young Scholar (81222047), the Ph.D. Program Foundation of Ministry of Education of China (J20110026), and the Ministry of Education Program for New Century Excellent Talents (NCET-11-0454). ⁎Corresponding author. E-mail address: [email protected] (L. Qiu).
of this endocytosis process is difficult, resulting in an insufficient intracellular drug accumulation and a poor treatment efficacy. In this study, we designed a multifunctional drug delivery system to overcome these limitations. 5 We chose Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-b-polylactide block copolymers (β-CD-PELA) as co-carriers for anticancer drug doxorubicin (DOX), and meanwhile developed an aptamer (AS1411)-functionalized composite micelle (DOX-CM-Ap) to accomplish targeted therapy of human breast cancer (Figure 1). Pluronic F127 is an amphiphilic polymer, which has been extensively used in nano-drug delivery systems. 6 It tends to self-assemble into micelles to solubilize some hydrophobic drugs. In addition, the terminal hydroxyl groups of Pluronic F127 can be easily functionalized for bioconjugation purposes. 7 The key drawbacks of Pluronic F127 are unfortunately associated with poor drug-loading capacity and physical stability of its micelle formulation. Recently, to enhance micelle stability, binary mixed systems consisting of Pluronic F127 and other copolymers, for example Pluronic P123, 8 have been developed. Interestingly, our group previously designed a series of β-CD-linked PELA block copolymers, which can form selfassembled micelles with low critical micelle concentration
http://dx.doi.org/10.1016/j.nano.2014.08.013 1549-9634/© 2014 Elsevier Inc. All rights reserved. Please cite this article as: Li X., et al., Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles. Nanomedicine: NBM 2014;xx:1-10, http://dx.doi.org/10.1016/j.nano.2014.08.013
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In this study, we also performed a systematic evaluation of the antitumor efficacy of AS1411-functionalized Pluronic F127/βCD-PELA mixed micelles encapsulating DOX at the cellular and animal levels by intracellular accumulation, in vitro cytotoxicity, DOX-CM-Ap retention in circulation, in vivo real-time imaging, and in vivo tumor inhibition study in MCF-7 tumor-bearing mice.
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Methods
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Materials and reagents
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Pluronic F127 (MW 12,500) was kindly supplied by BASF China Ltd. (Shanghai, China). Monomethoxy poly (ethylene glycol) (MPEG, Mn 5000) and tin (II) 2-ethylhexanoate (Sn(Oct)2) were purchased from Sigma Aldrich. D,L-lactide was obtained from Shandong Institute of Medical Instruments (Shandong, China). Beta cyclodextrin (β-CD) was purchased from Shanghai Chemical Reagents Co. Ltd. (Shanghai, China) and recrystallized before use. Rhodamine B (RhB) was purchased from Aladin Ltd. (Shanghai, China). AS1411 aptamer (Ap sequence: 5′-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3′, 28 bp) and AS1411 aptamer(mut) (Ap sequence: 5′TTCCTCCTCCTCCTTCTCCTCCTCCTCC-3′, 28 bp, a scrambled aptamer) were synthesized by Sangon (Shanghai, China). Doxorubicin hydrochloride (DOX · HCl) was purchased from HaiKou Manfangyuan Chemical Company (Haikou, China). Indocyanine green (ICG) was purchased from Tokyo Chemical Industry (TCI) Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS), RPMI-1640 medium (RPMI), and trypsin-EDTA
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(CMC) and obviously enhance DOX-loading capacity as a result of the combined effect of hydrophobic inner cavity of β-CD and hydrophobic PLA blocks. 9 Therefore such a hybrid system consisting of Pluronic F127 and β-CD-PELA was supposed to improve micelle stability and drug-loading capacity if they could mix homogenously. Aptamers are single-stranded RNA or DNA ligands that can specifically bind to various molecular targets. They are randomly synthesized based on nucleic acid libraries by a process called SELEX. 10,11 Compared to peptides and small molecules ligands, aptamers have higher affinity and selectivity to their targets with dissociation constant values in the nanomolar range. 12 In addition, in comparison with antibodies, aptamer preparation process is much faster, cheaper, and more versatile. Owing to these unique characteristics, aptamers have been widely used to modify drugs and nanoparticles for cancer therapy. 13 For instance, AS1411, a 26-mer DNA aptamer, has been confirmed to selectively bind to nucleolin. 14 Nucleolin is normally a predominantly nuclear and cytoplasmic phosphoprotein, which plays key roles in diverse biological processes. Recent researches have shown that nucleolin is also present in the plasma membranes of some kinds of cancer cells with different expression levels such as lung cancer, breast cancer, and renal cell carcinoma. 15 Notably, aptamer AS1411 has already entered Phase II clinical trials for acute myeloid leukaemia therapy and the results suggested that addition of AS1411 to cytarabine may enhance anti-leukemic activity with an acceptable safety profile in patients. 16 Therefore, AS1411 modification on nanoparticles would facilitate specific recognition by tumor cells and subsequent endocytosis via AS1411 and nucleolin interaction.
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Figure 1. Schematic diagram of the multifunctional composite micelles modified by aptamer AS1411 and the mechanism of interaction between nucleolin receptors and aptamer AS1411 ligands.
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Synthesis and characterization of polymers
Preparation and characterization of micelles
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Aptamer-modified Pluronic F127 (Pluronic F127-Ap) was synthesized by the reaction of carboxylated Pluronic F127 with the amino groups at the ends of aptamers at the different molar ratios (MRs) (Figure 2, A (a)). Pluronic F127-Ap(mut) containing a scrambled aptamer was obtained by the same method. To confirm the conjugation and determine the maximum conjugation content of AS1411 on Pluronic F127 (the maximum actual molar ratio of Ap (nmol) conjugated with 1 μmol Pluronic F127), urea polyacrylamide gel electrophoresis was carried out. The samples including free AS1411, Pluronic F127, and various Pluronic F127-Aps with or without 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) used in the conguation reaction were separately subjected to 2% TAE Urea PAGE. Then the electrophoresis was performed at 90 V for 30 min, and the base pair (bp) band on the gel was displayed by 4S green nucleic acid stain. Rhodamine B-labeled
A co-solvent evaporation method was used for micelle preparation. 17 Briefly, β-CD-PELA and Pluronic F127-Ap (1:3, 2:2, 3:1) or Pluronic F127-Ap(mut) were co-dissolved in 2 mL acetone, followed by addition of 2 mL of distilled water. After being stirred for 0.5 h, the acetone was removed by rotary evaporation at 37 °C, and finally the composite micellar CM-Ap or CM-Ap(mut) solutions were obtained. For preparation of DOX-loaded micelles (DOX-CM-Ap and DOX-CM-Ap(mut), DOX was first neutralized with 2 mol excess of triethylamine in acetone followed by added into the polymer solution. To investigate the effect of different aptamer content on the targeting ability of the aptamer-conjugated micelles to cancer cells, a series of RhB-labeled micelles (RhB-CM) with different aptamer contents was obtained by mixing Pluronic F127-Ap, β-CD-PELA, and Pluronic F127-RhB at different weight ratios (1:3:0.05, 2:2:0.05, and 3:1:0.05).
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Pluronic F127 (Pluronic F127-RhB) was synthesized by coupling RhB to the hydroxyl groups of Pluronic F127 (Figure 2, A (b)). Methoxy poly(ethylene glycol)-poly(lactide) (PELA) amphiphilic block copolymers were synthesized by a ring-opening polymerization, followed by conjugation with the carboxylated β-CD to give β-CD-PELA (Figure 2, A (c)). The detailed synthesis procedure, chemical compositions, and characterization of the materials were given in Supplementary Information.
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were purchased from Ji Nuo Biotechnology Company (Hangzhou, China). The human breast tumor MCF-7 cells were kindly provided by the Cancer Research Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang, China and were routinely cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 100 IU/mL penicillin, 100 IU/mL streptomycin at 37 °C, 5% CO2, and 95% humidity.
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Figure 2. Synthesis and confirmation of polymers. (A) Synthesis route of (a) Pluronic F127-Ap, (b) Pluronic F127-RhB, and (c) β-CD-PELA. (B) Determination of the conjugation of aptamer to Pluronic F127 via urea polyacrylamide gel electrophoresis (− EDC means that the conjugation reaction was performed without EDC and NHS).
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MCF-7 cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well, allowing for attachment for 24 h. The cells were then treated with different RhB-labeled micelles at 37 °C. After incubation for 60 min, the cells were washed with cold PBS three times, harvested using trypsin-EDTA and fixed by 1% of paraformaldehyde PBS solution. This cell suspension was measured by flow cytometry using internal fluorescence of RhB. The effect of incubation time (5, 30, and 60 min) on cellular uptake of micelles was also investigated. Furthermore, the abilities of cellular internalization of aptamer-functionalized micelles loaded with DOX were studied in the same way. The DOX concentration used for this study was 5 μg/mL.
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In vitro anti-proliferation assay
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The cytotoxicity of DOX-loaded CM was evaluated in MCF-7 cells by MTT assay. Briefly, MCF-7 cells were seeded in 96-well plates at a density of 2 × 10 3 cells/well, allowed for attachment of 24 h before exposure to a series of different concentrations (from 0.0625 to 2 μg/mL) of DOX-loaded CM for 48 h. Thereafter, cell viability was determined by the MTT method. Cells without exposure to the DOX formulations were used as control.
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Animal models
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Five-to-six-week-old female BALB/c nude mice (20 ± 2 g) (Shanghai SLAC Laboratory Animal Co. Ltd, China) were implanted with xenograft fragments of MCF-7 human breast cancer subcutaneously in the right flank using 12 gauge trocar needles. When tumor volume reached 50-100 mm 3, the mice were randomly divided into several groups for the following investigations. Tumor volume was estimated as width 2 × length × 0.5. All animal experiments were conducted according to the Regulation on Experimental Animals of Zhejiang University.
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In vivo real-time imaging and biodistribution studies
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For in vivo real-time imaging study, ICG-loaded micelles (ICG-CM-Ap and ICG-CM-Ap(mut) were prepared by the same method as used to load DOX into composite micelles. ICG-CM-Ap(mut) and ICG-CM-Ap were intravenously administrated via the tail vein at an equivalent dosage of 2 mg/kg ICG. After injection, the whole-body fluorescence of ICG was detected at predetermined time points (0.5, 4, 6, 8, 24, and 48 h). At 48 h post-injection, the nude mice were sacrificed and the major organs (heart, liver, spleen, lung, and kidney) and the tumors were harvested for fluorescent imaging. All images were acquired via a CRI in vivo imaging system and the fluorescence intensity was measured and analyzed using commercial software (Maestro software).
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In vivo tumor inhibition study
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The therapeutic efficacy of free DOX and DOX-loaded micelles was evaluated in MCF-7 tumor-bearing mice. The mice were randomly divided into four groups (n = 5 per group). Free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap were intravenously injected via tail vein on days 0, 3, and 6 at an equivalent dosage of 5 mg/kg DOX. The control group was treated with normal saline solution. Tumor size and animal body weight were recorded every three days during the experiment. After day 27 day, the nude mice were sacrificed and their hearts were harvested for histological analysis. The percent tumor growth inhibition (%TGI) was calculated using the formula 100 − %T/C (mean relative tumor volume of treated group/mean relative tumor volume of the control group).
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Statistical analysis
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All data were presented as mean ± SD and analyzed using the GraphPad Prism Software (version 5.0, GraphPad Software). The difference between treatment groups was determined by using student's t test for pairs of groups and one-way analysis of
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The heterotopic MCF-7 implanted nude mice tumor model was used to validate the pharmacokinetics studies. Free DOX, DOX-CM-Ap(mut) and DOX-CM-Ap were intravenously administrated via the tail vein at an equivalent dose of 5 mg/kg DOX. At predetermined time points of 5, 10, 15, 30 min, 1, 2, 4, and 6 h post injection, blood samples were collected into heparinized polyethylene tubes from the periorbital vein and centrifuged immediately at 6000 rpm for 10 min to obtain plasma. The concentrations of DOX in plasma were assayed by a spectrofluorometric method previously described in the literature. 19,20 Briefly, 100 μL of plasma was diluted with 200 μL of ethanol (0.3 N HCl in isopropanol) and incubated at 4 °C overnight. The sample was then centrifuged for 10 min at 10,000 rpm and the supernatant was assayed in a spectrofluorometer (SpectraMax M2, Molecular Devices, CA) with an excitation wavelength of 488 nm and emission wavelength of 580 nm. Pharmacokinetic parameters of the DOX formulations were calculated by the Thermo Kinetica 4.4.1 software.
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The amount of DOX loaded in micelles was determined by the absorption at 480 nm using UV-Vis spectrophotometer (PuXi General Instrument Co., China). The drug entrapment efficiency (EE) was defined as the weight percentage of DOX in micelles relative to the initial feeding amount of DOX. The drug loading content (LC) was calculated from the mass of incorporated DOX divided by the weight of DOX-loaded micelles. The CMC of the aptamer-conjugated composite polymers was measured by fluorescent spectroscopy using pyrene as a probe. 18 The particle size and zeta potential of DOX-loaded micelles were characterized by a zeta sizer (Malvern Nano-ZS 90, UK). Transmission electron microscopy (TEM) images were obtained using a JEM-1230 (JEOL, Japan). The release behavior of DOX from micelles was investigated under various pH conditions (pH 7.4, 6.5 and 5.0). 1 mL of DOX-loaded micelle solutions was transferred into a dialysis bag (Mw cut-off of 3000 Da), placed in 10 mL PBS with various pH values and kept in an air bath shaker (100 rpm) under 37 °C. Samples were collected at designated times (1, 3, 5, 7, 9, 12, 24, 48, and 72 h) and determined by UV-Vis spectroscopy at 480 nm.
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EE (%)
Theoretical Experimental value data Pluronic F127-Ap β-CD-PELA Pluronic F127-Ap:β-CD-PELA (1:3) Pluronic F127-Ap:β-CD-PELA (2:2) Pluronic F127-Ap:β-CD-PELA (3:1)
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variance (ANOVA) for multiple groups. Statistical significance was determined at a value P b 0.05.
Results
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Synthesis and characterization of polymers
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The aptamer-conjugated Pluronic F127 (Pluronic F127-Ap) was obtained by the reaction of 5’-NH2-AS1411 with dicarboxylated Pluronic F127. From the urea PAGE images (Figure 2, B), we could observe that the free aptamer had a clear migration on the gel and the Pluronic F127 had no band on the PAGE gel. The Pluronic F127-Ap (MR = 2.84) showed a clear band at the origin site, and no band at the free aptamer site. The Pluronic F127-Ap (MR = 5.69) showed a clearer and brighter band at the origin site, and no band at the free Ap site either. When the molar ratio was increased to 7.11, a light band appeared at the free Ap site, indicating the saturation of Ap conjugation. Thus, we chose MR = 5.69 as an optimized Ap ratio for the synthesis of Pluronic F127-Ap to ensure the maximal Ap conjugation without any free Ap. However, the aptamer cannot be linked to the Pluronic F127 without EDC. The β-CD-PELA conjugate was obtained from the reaction of carboxylated β-CD with PELA and the related polymer characterization was detailed in Supplementary Information (Figure S1).
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Characterization of DOX-loaded micelles
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The effect of polymer composition of the composite micelles on drug LC and EE was examined and the results were shown in Table 1. The micelles prepared from pure Pluronic F127-Ap had a rather low EE for DOX, while those from pure β-CD-PELA had a high EE (N 90%), demonstrating the excellent loading capacity of β-CD-PELA for DOX. For the composite micelles, when the weight ratio of Pluronic F127-Ap to β-CD-PELA decreased from 3:1 to 1:3, the EE increased from 76.75% ± 1.39% to 92.54% ± 4.15% (1.5% theoretical LC), further confirming the predominant role of β-CD-PELA for drug loading. Taking Ap content and drug loading two factors into account, the optimal mixed ratio of Pluronic F127-Ap to β-CD-PELA was set as 2:2 for the following study. CMC is a quite predominant parameter for micelles to reflect their physical stability. In this study, the CMC of Pluronic F127-Ap/β-CD-PELA binary mixture with weight ratio of 2:2
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In vitro cellular uptake
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Fluorescent markers are frequently applied in cellular uptake studies by flow cytometry. In our case, an additional marker RhB linked on polymers and the entrapped DOX were used as fluorescent probes to measure cellular uptake, respectively. Pluronic F127-RhB was successfully synthesized by the esterification reaction (Figure 1, A (b)) and characterized by 1 H NMR and UV-visible spectroscopy (Figure S2). The effect of different aptamer contents on the targeting ability of the aptamer-conjugated micelles to MCF-7 cells was investigated through RhB-labeled micelles. When the ratio of Pluronic F127-Ap to β-CD-PELA increased from 1:3 to 2:2, a 1.97-fold increase in micelle uptake was obtained. Further increasing the ratio to 3:1, however, resulted in a slight increase in micelle uptake (Figure 4, A). This result further confirmed it was appropriate to set the weight ratio of Pluronic F127-Ap to β-CD-PELA as 2:2 in the study. In addition, it was found that the cellular uptake of aptamer-modified micelles was timedependent and increased with the increase of incubation time (Figure 4, B). As presented in Figure 4, C, the RhB-CM-Ap showed about 3.31 times higher fluorescence intensity in cells than RhB-CM-Ap(mut). Furthermore, for DOX-loaded micelles, the cellular uptake of DOX-CM-Ap was still 3.36-fold higher than that of DOX-CM-Ap(mut) (Figure 4, D). These results indicated that the aptamer-functionalized micelles were prone to undergo internalization by cancer cells, presumably due to the selective affinity between the aptamer AS1411 and the nucleolin.
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In vitro anti-proliferation assay
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To verify the pharmacological activity of DOX-loaded CM-Ap(mut) and CM-Ap, the in vitro cytotoxicity tests against MCF-7 cells were conducted. The DOX-CM-Ap showed enhanced antiproliferative activity relative to the DOX-CM-Ap(mut), as illustrated by the apparent IC50 values of 309.8 and 442.8 ng/mL, respectively. This enhanced cytotoxicity of DOX-CM-Ap
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Micelle composition
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was 0.0033 g/L, which was a little higher than that of β-CD-PELA (0.0023 g/L) but much lower than that of pure Pluronic F127 (0.039 g/L). 9 This result validated the homogenous formation of composite micelles by mixing Pluronic F127 with β-CD-PELA due to their good compatibility. As shown in Figure 3, A and B, the diameters of DOX-loaded CM-Ap(mut) and CM-Ap micelles were similar, about 39.15 nm and 38.23 nm respectively. Accordingly, the TEM images proved that both micelles were spheroidal shape and homogeneous in size. The zeta potentials were approximately − 9.86 mV and − 10.06 mV for DOX-CM-Ap(mut) and DOX-CM-Ap, respectively. The in vitro drug release study was performed at various pH values including those simulating the physiological environment (pH 7.4), tumor microenvironment (pH 6.5), and endosomal compartments (pH 5.0). The pH-dependent release profiles of DOX from micelles are shown in Figure 3, C and D. For DOX-CM-Ap(mut), the DOX release rate was faster in the first 9 h and reached a plateau in 24 h. In addition, the released amount of DOX from micelles increased as pH values of the medium decreased due to its increased solubility. Similar DOX release behavior was observed for DOX-CM-Ap.
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Table 1 Drug loading content (LC) and drug encapsulation efficiency (EE) of DOXloaded micelles (mean ± SD, n = 3).
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Figure 3. Characterization of DOX-loaded micelles. Particle size distribution and TEM images of (A) DOX-CM-Ap(mut) micelles and (B) DOX-CM-Ap micelles. In vitro DOX release from (C) DOX-CM-Ap(mut) micelles and (D) DOX-CM-Ap micelles. All data represent mean ± SD.
Figure 4. In vitro cellular uptake of micelles by MCF-7 cells. (A) RhB-labeled micelles (RhB-CM-Ap) with different aptamer content (Pluronic F127-Ap, β-CD-PELA, and Pluronic F127-RhB with different weight ratios of 1:3:0.05, 2:2:0.05, and 3:1:0.05) after 60 min incubation with cells. (B) RhB-CM-Ap after 5, 30, and 60 min incubation with cells, respectively. (C) RhB-CM-Ap(mut) and RhB-CM-Ap after 60 min incubation with cells, respectively. (D) DOXCM-Ap(mut) and DOX-CM-Ap after 60 min incubation with cells, respectively. All data represent mean ± SD.
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Figure 5. Comparison analysis of pharmacokinetics and biodistribution in MCF-7 tumor-bearing mice. (A) Pharmacokinetic profiles of DOX solution, DOX-CM-Ap(mut), and DOX-CM-Ap at 5 mg/kg DOX. (B) In vivo real-time imaging of mice at 0.5, 4, 6, 8, 24, and 48 h post-injection of ICG-CM-Ap(mut) and ICG-CM-Ap, respectively. (C) Average fluorescence signals determined at major organs and tumors at 48 h post-injection of ICG-CM-Ap(mut) and ICG-CM-Ap, respectively. Statistical significance: *P b 0.05. All data represent mean ± SD.
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A pharmacokinetic study in MCF-7 tumor-bearing mice was performed to determine the drug circulation in blood. The plasma concentration–time curves for DOX, DOX-CM-Ap(mut), and DOX-CM-Ap were all fitted to the two-compartment model. As shown in Figure 5, A, DOX-CM-Ap achieved a longer half-life and larger AUC in comparison with free DOX, whereas free DOX was quickly eliminated from circulation. Moreover, compartmental analysis of plasma concentrations exhibited significant differences in pharmacokinetic parameters for DOX in aptamer-functionalized micelles compared to free DOX (Table 2). For DOX-CM-Ap micelles, the half-life (t1/2β) was 1.94-fold longer, the AUC was 17.2-fold larger, the mean residence time (MRT) was 2.94-fold longer, and total body clearance (CL) was shorter than that of free DOX. No significant difference was observed in pharmacokinetic behavior between DOX-CM-Ap group and DOX-CM-Ap(mut) group.
micelles in vivo via the non-invasive and real-time near-infrared fluorescence imaging. Figure 5, B showed the fluorescence images at different time intervals after intravenously injection of ICG-loaded CM-Ap(mut) and CM-Ap. It can be seen that both micelles were prone to accumulate in liver and intestine for the initial 4 h, which may be attributed to the rapid recognition and uptake of nanoparticles by the cells of RES. Although the fluorescence signal occurred at the tumor site at 4 h post-injection of both micelles, the signal intensity of ICG-CM-Ap was much stronger than that of ICG-CM-Ap(mut) during the whole experimental period, suggesting that the aptamer-conjugated micelles had better tumor targeting potency than the non-targeted ones. The ex vivo fluorescence images at 48 h post-injection were obtained for the tumor tissue as well as for major organs such as heart, liver, spleen, lung, and kidney. As shown in Figure 5, C, the strong fluorescence was mainly observed in tumor tissue for both micelles, while other tissues such as heart showed negligible fluorescence signal. Furthermore, it was found that the fluorescence signal of the Ap-conjugated micelles in tumor tissue is significantly higher than that of the Ap(mut)-conjugated micelles.
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In vivo tumor growth inhibition and histopathology
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To evaluate whether the aptamer-modified micelles can indeed specifically target tumors, we monitored the fate of the
The in vivo antitumor efficacy was evaluated using different DOX formulations on mice bearing subcutaneous MCF-7
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against MCF-7 cells in culture may owe to the improved cellular uptake of micelles.
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Table 2 Pharmacokinetic parameters in mice after single intravenous administration of free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap at 5 mg/kg DOX (mean ± SD, n = 3).
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0.14 3.09 4.49 3.99 88.89 22.27
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0.06 0.45 0.77 0.58 7.23 2.25
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Discussion
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tumors. Figure 6, A shows the growth curves of MCF-7 tumors of mice after the treatment. It can be seen that the growth rate of the DOX-CM-Ap group was remarkably delayed when compared with the DOX-CM-Ap(mut) group. The group treated with free DOX showed a tumor growth curve similar to that of the DOX-CM-Ap(mut) group. At the end of experiment, the tumor volume of the DOX-CM-Ap group was much smaller than other groups, about 46% of the DOX-CM-Ap(mut) group and 54% of the free DOX group. The tumor growth inhibition (TGI) was 77.6% for DOX-CM-Ap, 51.2% for DOX-CM-Ap(mut), and 58.0% for free DOX, demonstrating significantly superior antitumor efficacy of DOX-CM-Ap. Multiple administration of DOX solution could cause toxicity, resulting in weight loss and cardiotoxicity. Figure 6, B presents the changes in the body weight of each group over time as an indication of overall systemic toxicity of different formulations. The free DOX group showed a sharp decrease in body weight during the initial 9 days and recovery after this period. This strongly suggests serious toxicity of free DOX at the given dose. However, no weight loss was observed for the micelle-treated groups. In addition, cardiotoxicity is the main drawback of DOX use, which ultimately restraints effective DOX application in patients. As shown in Figure 6, C, multiple administration of free DOX induced noticeable cardiac tissue degeneration and necrosis. However, there was no evidence of cardiotoxicity after either DOX-CM-Ap or DOX-CM-Ap(mut) therapy compared to the control group. These results indicated that the toxic effects of DOX could be alleviated by micelles, which may ascribe to the reduced accumulation of DOX in heart.
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Nowadays, ligand-modified nanoparticles have been considered to effectively improve the anti-tumor efficacy based on enhanced accumulation at tumor tissues via EPR effect and subsequent endocytosis of tumor cells via ligand–receptor interaction. As one of the most excellent targeting ligands, aptamers can selectively bind to a variety of targets ranging from small molecules to whole cells due to their three-dimensional structures. In the present study, we constructed a binary composite micelles composed of AS1411-modified Pluronic F127 and β-CD-PELA to load DOX. In vivo tumor inhibition study revealed that the AS1411-modified DOX-CM obtained significantly stronger tumor inhibition on MCF-7 tumor when
compared with untargeted DOX-CM and didn’t induce any cardiotoxicity when compared with free DOX. These expected results could be attributed to the following factors. Firstly, the physicochemical properties of AS1411-modified CM were fully developed. To ameliorate the weak drug loading capacity of Pluronic F127, structural modifications are often investigated (e.g., Pluronic F127-PLA, Pluronic F127-chitosan, etc.). 21,22 However, this paper overcome this drawback of Pluronic F127 micelle by the addition of β-CD-PELA. As shown in Table 1, DOX displayed stronger affinity to hydrophobic segments β-CD and PLA of β-CD-PELA than PPO of Pluronic copolymer, 23 so the DOX EE of β-CD-PELA micelles is about 2.4 times higher than that of Pluronic F127 micelles. The composite micelles at a weight ratio of 2:2 also had a 2.2-times-higher EE of DOX than pure micelles of Pluronic F127. β-CD has a toroidal shape containing a hydrophobic inner cavity, in which DOX could be act as the “guest” molecule and solubilizer. In addition, our previous work demonstrated that linking β-CD to PELA could make the amphiphilic copolymer easier to self-assemble in aqueous solution, as illustrated by the reduced CMC of 0.0023 g/L. As a result, this hybrid system achieved a low CMC of 0.0033 g/L and thus provided relatively high stability in solutions upon dilution. The dependency of drug release with pH may contribute to the increase of drug concentration in the tumor tissue. As revealed by Min et al, TRITC-loaded PEG-b-PAE micelles, a type of pH-dependent drug delivery systems, can successfully deliver TRITC to tumor site and release it there, resulting in a 11-folds-higher fluorescence in tumors than non-pH-sensitive micelles. 24 Secondly, DOX-CM-Ap micelles displayed long-circulation character in vivo with the longer half-life, larger AUC and shorter CL (Table 2) when compared with free DOX, which supplied more opportunities for DOX-CM-Ap micelles to leak into tumor tissues via EPR effect. Furthermore, the AS1411-modified CM tended to accumulate more efficiently within the tumor through the selective binding to receptors on MCF-7 cells than CMAp(mut) micelles. ICG is the only near-infrared dye approved by FDA at present. 25 In this study, ICG was encapsulated into the CM and acted as a fluorescence probe to trace the CM in the body of tumor-bearing mice. As shown in Figure 5, B, ICG could be delivered to tumors with the help of both CMs, but the accumulation degree of ICG-CM-Ap was significantly higher than that of ICG-CM-Ap(mut) during the entire experimental period. Thirdly, the specific interaction between aptamer and nucleolin significantly enhanced cellular uptake of AS1411modified CM in MCF-7 cells and increased the cytotoxicity of its payload. Nucleolin, present on the surface of various types of tumor cells, is a functional receptor for aptamers. The trafficking ability of nucleolin has been implicated in transporting aptamers as well as nanoparticle–aptamer bioconjugates from the membrane into the nucleus. 26,27 For example, Dam et al used a DNA aptamer (AS1411) by taking advantage of the shuttling properties of nucleolin to efficiently target gold nanoparticles into the nucleus of cancer cells. 26 On the basis of the current experimental data, we can propose the following chain of events to describe the in vivo behavior of DOX-CM-Ap. After intravenous administration to mice, the
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In this study, we successfully prepared binary mixed micellar system CM-Ap consisting of aptamer-conjugated Pluronic F127 and β-CD-PELA to load DOX with satisfactory drug loading capacity. The pharmacokinetic study demonstrated that CM-Ap could significantly extend the blood circulation time of DOX compared to free DOX. Moreover, in vivo real-time imaging study revealed that aptamer-conjugated micelles had better tumor targeting potency than non-targeted micelles. Based on a receptor-mediated endocytosis pathway and controllable cellular release of DOX, DOX-CM-Ap was validated to enhance the anti-tumor efficacy of DOX in vivo and prevent acute cardiotoxicity. Owing to these characteristics, this aptamerfunctionalized delivery system exhibited great potentials for cancer therapy.
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DOX-CM-Ap demonstrated a prolonged circulation time in blood, sufficient for their accumulation in the tumor via EPR effect. When they entered the tumor interstitial space, aptamer present on the micelle surface quickly recognized the tumor cells and bound with them (but not with normal tissues), followed by aptamer-mediated endocytosis of micelles. Thus, the combination of several advantages within this novel micellar system should allow for precise and effective tumor targeting and intracellular action, and consequently enhanced inhibition of tumor growth and alleviated side effects.
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Figure 6. Therapeutic efficacy of different DOX formulations in MCF-7 tumor-bearing mice (n = 5 per group). All data represent mean ± SD. (A) Growth curves of MCF-7 tumors of mice after the treatment of saline, free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap. (B) Changes in the body weight of each group. (C) In vivo cardiotoxicity of DOX-treated mice. Heart was evaluated by H&E staining after 27 days post-injection of each formulation. The original magnification is ×400. Statistical significance: *P b 0.05.
The authors thank Miss Yangmin Jin for animal experiments.
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2014.08.013.
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9. Qiu LY, Wang RJ, Zheng C, Jin Y, Jin LQ. β-Cyclodextrin-centered star-shaped amphiphilic polymers for doxorubicin delivery. Nanomedicine 2010;5:193-208. 10. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990;249:505-10. 11. Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990;346:818-22. 12. Tan WH, Wang H, Chen Y, Zhang XB, Zhu HZ, Yang CY, et al. Molecular aptamers for drug delivery. Trends Biotechnol 2011;29:634-40. 13. Li X, Zhao QH, Qiu LY. Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy. J Control Release 2013;171:152-62. 14. Hernandez FJ, Hernandez LI, Pinto A, Schäfer T, Ö zalp VC. Targeting cancer cells with controlled release nanocapsules based on a single aptamer. Chem Commun 2013;49:1285-7. 15. Bates PJ, Laber DA, Miller DM, Thomas SD, Trent JO. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp Mol Pathol 2009;86:151-64. 16. Rizzieri D, Stockerl-Goldstein K, Wei A, Herzig RH, Erlandsson F, Stuart RK. Long-term outcomes of responders in a randomized, controlled phase II trial of aptamer AS1411 in AML. J Clin Oncol 2010;28:6557. 17. Aliabadi HM, Elhasi S, Mahmud A, Gulamhusein R, Mahdipoor P, Lavasanifar A. Encapsulation of hydrophobic drugs in polymeric micelles through co-solvent evaporation: the effect of solvent composition on micellar properties and drug loading. Int J Pharm 2007;329:158-65. 18. Lee H, Ahn CH, Park TG. Poly[lactic-co-(glycolic acid)]-grafted hyaluronic acid copolymer micelle nanoparticles for target-specific delivery of doxorubicin. Macromol Biosci 2009;9:336-42.
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19. Hong RL, Huang CJ, Tseng YL, et al. Direct comparison of liposomal doxorubicin with or without polyethylene glycol coating in C-26 tumorbearing mice: is surface coating with polyethylene glycol beneficial? Clin Cancer Res 1999;5:3645-52. 20. Tagami T, Ernsting MJ, Li SD. Efficient tumor regression by a single and low dose treatment with a novel and enhanced formulation of thermosensitive liposomal doxorubicin. J Control Release 2011;152:303-9. 21. Zhang WJ, Gilstrap K, Wu LY, Bahadur KC Remant, Moss MA, Wang Q, et al. Synthesis and characterization of thermally responsive Pluronic F127 chitosan nanocapsules for controlled release and intracellular delivery of small molecules. ACS Nano 2010;4:6747-59. 22. Zhou Q, Guo X, Chen T, Zhang Z, Shao SJ, Luo C, et al. Target-specific cellular uptake of folate-decorated biodegradable polymer micelles. J Phys Chem B 2011;115:12662-70. 23. Allen C, Maysinger D, Eisenberg A. Nano-engineering block copolymer aggregates for drug delivery. Colloids Surf B Biointerfaces 1999;16:3-27. 24. Min KH, Kim JH, Bae SM, Shin H, Kim MS, Park SJ, et al. Tumoral acidic pH-responsive MPEG-poly(β-amino ester) polymeric micelles for cancer targeting therapy. J Control Release 2010;144:259-66. 25. Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19:316-7. 26. Chen X, Kube DM, Cooper MJ, Davis PB. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of PEGylated polylysine and DNA. Mol Ther 2007;16:333-42. 27. Dam DHM, Lee JH, Sisco PN, Co DT, Zhang W. Direct observation of nanoparticle–cancer cell nucleus interactions. ACS Nano 2012;6:3318-26.
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Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2014) xxx–xxx
Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles
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Xin Li, MS b, Yang Yu, MS b, Qian Ji, BA b, Liyan Qiu, PhD a,⁎ a
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A novel composite micelle (CM) with surface modification of aptamer AS1411 (Ap) was successfully developed for targeted delivery of doxorubicin (DOX) to human breast tumors. Mixing of two different kinds of polymers afforded the composite micelle-Ap (CM-Ap) various desirable physicochemical properties. In addition, the in vitro and in vivo studies demonstrated that the DOX-loaded CM-Ap could prolong blood circulation time, enhance cellular uptake and cytotoxicity, and consequently, improve tumor growth inhibition and minimize adverse effect.
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Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx – xxx nanomedjournal.com
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Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles Xin Li, MS b , Yang Yu, MS b , Qian Ji, BA b , Liyan Qiu, PhD a,⁎
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Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China b College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Received 11 May 2014; accepted 29 August 2014
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Aptamers are single-stranded RNA or DNA ligands that can specifically bind to various molecular targets with high affinity. Owing to this unique character, they have become increasingly attractive in the field of drug delivery. In this study, we developed a multifunctional composite micelle (CM) with surface modification of aptamer AS1411 (Ap) for targeted delivery of doxorubicin (DOX) to human breast tumors. This binary mixed system consisting of AS1411 modified Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-bpolylactide could enhance DOX-loading capacity and increase micelle stability. Cellular uptake of CM-Ap was found to be higher than that of untargeted CM due to the nucleolin-mediated endocytosis effect. In vivo study in MCF-7 tumor-bearing mice demonstrated that the AS1411-functionalized composite micelles showed prolonged circulation time in blood, enhanced accumulation in tumor, improved antitumor activity, and decreased cardiotoxicity. In conclusion, aptamer-conjugated multifunctional composite micelles could be a potential delivery vehicle for cancer therapy. © 2014 Elsevier Inc. All rights reserved.
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Key words: Aptamer; Composite micelles; Cyclodextrin; Pluronic F127; Doxorubicin
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In the last few decades, there have been advances in nanotechnology for cancer treatment; however, various challenges remain pertaining to its application in clinical settings. First, the physicochemical properties of nanoparticles are far from satisfactory, such as complicated preparation process, 1 low drug loading, and poor stability. Second, although nanoparticles are expected to reach tumors by the effect of enhanced permeability and retention (EPR), their nonspecific uptake by mononuclear phagocytic cells that are present in the liver, spleen, and lungs may limit the effectiveness of therapeutic drugs and cause severe side effects. 2–4 Third, anticancer agents should be effectively transported into tumor cells to exhibit their pharmacological activity. However, in some cases, completion
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Conflict of interest statement: No conflict of interest is reported by the authors of this paper. Funding sources: This work was supported by National Natural Science Funds for Excellent Young Scholar (81222047), the Ph.D. Program Foundation of Ministry of Education of China (J20110026), and the Ministry of Education Program for New Century Excellent Talents (NCET-11-0454). ⁎Corresponding author. E-mail address: [email protected] (L. Qiu).
of this endocytosis process is difficult, resulting in an insufficient intracellular drug accumulation and a poor treatment efficacy. In this study, we designed a multifunctional drug delivery system to overcome these limitations. 5 We chose Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-b-polylactide block copolymers (β-CD-PELA) as co-carriers for anticancer drug doxorubicin (DOX), and meanwhile developed an aptamer (AS1411)-functionalized composite micelle (DOX-CM-Ap) to accomplish targeted therapy of human breast cancer (Figure 1). Pluronic F127 is an amphiphilic polymer, which has been extensively used in nano-drug delivery systems. 6 It tends to self-assemble into micelles to solubilize some hydrophobic drugs. In addition, the terminal hydroxyl groups of Pluronic F127 can be easily functionalized for bioconjugation purposes. 7 The key drawbacks of Pluronic F127 are unfortunately associated with poor drug-loading capacity and physical stability of its micelle formulation. Recently, to enhance micelle stability, binary mixed systems consisting of Pluronic F127 and other copolymers, for example Pluronic P123, 8 have been developed. Interestingly, our group previously designed a series of β-CD-linked PELA block copolymers, which can form selfassembled micelles with low critical micelle concentration
http://dx.doi.org/10.1016/j.nano.2014.08.013 1549-9634/© 2014 Elsevier Inc. All rights reserved. Please cite this article as: Li X., et al., Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles. Nanomedicine: NBM 2014;xx:1-10, http://dx.doi.org/10.1016/j.nano.2014.08.013
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In this study, we also performed a systematic evaluation of the antitumor efficacy of AS1411-functionalized Pluronic F127/βCD-PELA mixed micelles encapsulating DOX at the cellular and animal levels by intracellular accumulation, in vitro cytotoxicity, DOX-CM-Ap retention in circulation, in vivo real-time imaging, and in vivo tumor inhibition study in MCF-7 tumor-bearing mice.
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Pluronic F127 (MW 12,500) was kindly supplied by BASF China Ltd. (Shanghai, China). Monomethoxy poly (ethylene glycol) (MPEG, Mn 5000) and tin (II) 2-ethylhexanoate (Sn(Oct)2) were purchased from Sigma Aldrich. D,L-lactide was obtained from Shandong Institute of Medical Instruments (Shandong, China). Beta cyclodextrin (β-CD) was purchased from Shanghai Chemical Reagents Co. Ltd. (Shanghai, China) and recrystallized before use. Rhodamine B (RhB) was purchased from Aladin Ltd. (Shanghai, China). AS1411 aptamer (Ap sequence: 5′-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3′, 28 bp) and AS1411 aptamer(mut) (Ap sequence: 5′TTCCTCCTCCTCCTTCTCCTCCTCCTCC-3′, 28 bp, a scrambled aptamer) were synthesized by Sangon (Shanghai, China). Doxorubicin hydrochloride (DOX · HCl) was purchased from HaiKou Manfangyuan Chemical Company (Haikou, China). Indocyanine green (ICG) was purchased from Tokyo Chemical Industry (TCI) Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS), RPMI-1640 medium (RPMI), and trypsin-EDTA
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(CMC) and obviously enhance DOX-loading capacity as a result of the combined effect of hydrophobic inner cavity of β-CD and hydrophobic PLA blocks. 9 Therefore such a hybrid system consisting of Pluronic F127 and β-CD-PELA was supposed to improve micelle stability and drug-loading capacity if they could mix homogenously. Aptamers are single-stranded RNA or DNA ligands that can specifically bind to various molecular targets. They are randomly synthesized based on nucleic acid libraries by a process called SELEX. 10,11 Compared to peptides and small molecules ligands, aptamers have higher affinity and selectivity to their targets with dissociation constant values in the nanomolar range. 12 In addition, in comparison with antibodies, aptamer preparation process is much faster, cheaper, and more versatile. Owing to these unique characteristics, aptamers have been widely used to modify drugs and nanoparticles for cancer therapy. 13 For instance, AS1411, a 26-mer DNA aptamer, has been confirmed to selectively bind to nucleolin. 14 Nucleolin is normally a predominantly nuclear and cytoplasmic phosphoprotein, which plays key roles in diverse biological processes. Recent researches have shown that nucleolin is also present in the plasma membranes of some kinds of cancer cells with different expression levels such as lung cancer, breast cancer, and renal cell carcinoma. 15 Notably, aptamer AS1411 has already entered Phase II clinical trials for acute myeloid leukaemia therapy and the results suggested that addition of AS1411 to cytarabine may enhance anti-leukemic activity with an acceptable safety profile in patients. 16 Therefore, AS1411 modification on nanoparticles would facilitate specific recognition by tumor cells and subsequent endocytosis via AS1411 and nucleolin interaction.
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Figure 1. Schematic diagram of the multifunctional composite micelles modified by aptamer AS1411 and the mechanism of interaction between nucleolin receptors and aptamer AS1411 ligands.
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Preparation and characterization of micelles
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Aptamer-modified Pluronic F127 (Pluronic F127-Ap) was synthesized by the reaction of carboxylated Pluronic F127 with the amino groups at the ends of aptamers at the different molar ratios (MRs) (Figure 2, A (a)). Pluronic F127-Ap(mut) containing a scrambled aptamer was obtained by the same method. To confirm the conjugation and determine the maximum conjugation content of AS1411 on Pluronic F127 (the maximum actual molar ratio of Ap (nmol) conjugated with 1 μmol Pluronic F127), urea polyacrylamide gel electrophoresis was carried out. The samples including free AS1411, Pluronic F127, and various Pluronic F127-Aps with or without 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) used in the conguation reaction were separately subjected to 2% TAE Urea PAGE. Then the electrophoresis was performed at 90 V for 30 min, and the base pair (bp) band on the gel was displayed by 4S green nucleic acid stain. Rhodamine B-labeled
A co-solvent evaporation method was used for micelle preparation. 17 Briefly, β-CD-PELA and Pluronic F127-Ap (1:3, 2:2, 3:1) or Pluronic F127-Ap(mut) were co-dissolved in 2 mL acetone, followed by addition of 2 mL of distilled water. After being stirred for 0.5 h, the acetone was removed by rotary evaporation at 37 °C, and finally the composite micellar CM-Ap or CM-Ap(mut) solutions were obtained. For preparation of DOX-loaded micelles (DOX-CM-Ap and DOX-CM-Ap(mut), DOX was first neutralized with 2 mol excess of triethylamine in acetone followed by added into the polymer solution. To investigate the effect of different aptamer content on the targeting ability of the aptamer-conjugated micelles to cancer cells, a series of RhB-labeled micelles (RhB-CM) with different aptamer contents was obtained by mixing Pluronic F127-Ap, β-CD-PELA, and Pluronic F127-RhB at different weight ratios (1:3:0.05, 2:2:0.05, and 3:1:0.05).
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were purchased from Ji Nuo Biotechnology Company (Hangzhou, China). The human breast tumor MCF-7 cells were kindly provided by the Cancer Research Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang, China and were routinely cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 100 IU/mL penicillin, 100 IU/mL streptomycin at 37 °C, 5% CO2, and 95% humidity.
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Figure 2. Synthesis and confirmation of polymers. (A) Synthesis route of (a) Pluronic F127-Ap, (b) Pluronic F127-RhB, and (c) β-CD-PELA. (B) Determination of the conjugation of aptamer to Pluronic F127 via urea polyacrylamide gel electrophoresis (− EDC means that the conjugation reaction was performed without EDC and NHS).
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MCF-7 cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well, allowing for attachment for 24 h. The cells were then treated with different RhB-labeled micelles at 37 °C. After incubation for 60 min, the cells were washed with cold PBS three times, harvested using trypsin-EDTA and fixed by 1% of paraformaldehyde PBS solution. This cell suspension was measured by flow cytometry using internal fluorescence of RhB. The effect of incubation time (5, 30, and 60 min) on cellular uptake of micelles was also investigated. Furthermore, the abilities of cellular internalization of aptamer-functionalized micelles loaded with DOX were studied in the same way. The DOX concentration used for this study was 5 μg/mL.
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The cytotoxicity of DOX-loaded CM was evaluated in MCF-7 cells by MTT assay. Briefly, MCF-7 cells were seeded in 96-well plates at a density of 2 × 10 3 cells/well, allowed for attachment of 24 h before exposure to a series of different concentrations (from 0.0625 to 2 μg/mL) of DOX-loaded CM for 48 h. Thereafter, cell viability was determined by the MTT method. Cells without exposure to the DOX formulations were used as control.
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Five-to-six-week-old female BALB/c nude mice (20 ± 2 g) (Shanghai SLAC Laboratory Animal Co. Ltd, China) were implanted with xenograft fragments of MCF-7 human breast cancer subcutaneously in the right flank using 12 gauge trocar needles. When tumor volume reached 50-100 mm 3, the mice were randomly divided into several groups for the following investigations. Tumor volume was estimated as width 2 × length × 0.5. All animal experiments were conducted according to the Regulation on Experimental Animals of Zhejiang University.
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For in vivo real-time imaging study, ICG-loaded micelles (ICG-CM-Ap and ICG-CM-Ap(mut) were prepared by the same method as used to load DOX into composite micelles. ICG-CM-Ap(mut) and ICG-CM-Ap were intravenously administrated via the tail vein at an equivalent dosage of 2 mg/kg ICG. After injection, the whole-body fluorescence of ICG was detected at predetermined time points (0.5, 4, 6, 8, 24, and 48 h). At 48 h post-injection, the nude mice were sacrificed and the major organs (heart, liver, spleen, lung, and kidney) and the tumors were harvested for fluorescent imaging. All images were acquired via a CRI in vivo imaging system and the fluorescence intensity was measured and analyzed using commercial software (Maestro software).
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The therapeutic efficacy of free DOX and DOX-loaded micelles was evaluated in MCF-7 tumor-bearing mice. The mice were randomly divided into four groups (n = 5 per group). Free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap were intravenously injected via tail vein on days 0, 3, and 6 at an equivalent dosage of 5 mg/kg DOX. The control group was treated with normal saline solution. Tumor size and animal body weight were recorded every three days during the experiment. After day 27 day, the nude mice were sacrificed and their hearts were harvested for histological analysis. The percent tumor growth inhibition (%TGI) was calculated using the formula 100 − %T/C (mean relative tumor volume of treated group/mean relative tumor volume of the control group).
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All data were presented as mean ± SD and analyzed using the GraphPad Prism Software (version 5.0, GraphPad Software). The difference between treatment groups was determined by using student's t test for pairs of groups and one-way analysis of
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The heterotopic MCF-7 implanted nude mice tumor model was used to validate the pharmacokinetics studies. Free DOX, DOX-CM-Ap(mut) and DOX-CM-Ap were intravenously administrated via the tail vein at an equivalent dose of 5 mg/kg DOX. At predetermined time points of 5, 10, 15, 30 min, 1, 2, 4, and 6 h post injection, blood samples were collected into heparinized polyethylene tubes from the periorbital vein and centrifuged immediately at 6000 rpm for 10 min to obtain plasma. The concentrations of DOX in plasma were assayed by a spectrofluorometric method previously described in the literature. 19,20 Briefly, 100 μL of plasma was diluted with 200 μL of ethanol (0.3 N HCl in isopropanol) and incubated at 4 °C overnight. The sample was then centrifuged for 10 min at 10,000 rpm and the supernatant was assayed in a spectrofluorometer (SpectraMax M2, Molecular Devices, CA) with an excitation wavelength of 488 nm and emission wavelength of 580 nm. Pharmacokinetic parameters of the DOX formulations were calculated by the Thermo Kinetica 4.4.1 software.
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The amount of DOX loaded in micelles was determined by the absorption at 480 nm using UV-Vis spectrophotometer (PuXi General Instrument Co., China). The drug entrapment efficiency (EE) was defined as the weight percentage of DOX in micelles relative to the initial feeding amount of DOX. The drug loading content (LC) was calculated from the mass of incorporated DOX divided by the weight of DOX-loaded micelles. The CMC of the aptamer-conjugated composite polymers was measured by fluorescent spectroscopy using pyrene as a probe. 18 The particle size and zeta potential of DOX-loaded micelles were characterized by a zeta sizer (Malvern Nano-ZS 90, UK). Transmission electron microscopy (TEM) images were obtained using a JEM-1230 (JEOL, Japan). The release behavior of DOX from micelles was investigated under various pH conditions (pH 7.4, 6.5 and 5.0). 1 mL of DOX-loaded micelle solutions was transferred into a dialysis bag (Mw cut-off of 3000 Da), placed in 10 mL PBS with various pH values and kept in an air bath shaker (100 rpm) under 37 °C. Samples were collected at designated times (1, 3, 5, 7, 9, 12, 24, 48, and 72 h) and determined by UV-Vis spectroscopy at 480 nm.
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EE (%)
Theoretical Experimental value data Pluronic F127-Ap β-CD-PELA Pluronic F127-Ap:β-CD-PELA (1:3) Pluronic F127-Ap:β-CD-PELA (2:2) Pluronic F127-Ap:β-CD-PELA (3:1)
1.50 1.50 1.50 1.50 1.50
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0.05 0.05 0.06 0.06 0.02
40.13 95.16 92.54 88.35 76.75
± ± ± ± ±
3.20 3.33 4.15 3.67 1.39
variance (ANOVA) for multiple groups. Statistical significance was determined at a value P b 0.05.
Results
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Synthesis and characterization of polymers
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The aptamer-conjugated Pluronic F127 (Pluronic F127-Ap) was obtained by the reaction of 5’-NH2-AS1411 with dicarboxylated Pluronic F127. From the urea PAGE images (Figure 2, B), we could observe that the free aptamer had a clear migration on the gel and the Pluronic F127 had no band on the PAGE gel. The Pluronic F127-Ap (MR = 2.84) showed a clear band at the origin site, and no band at the free aptamer site. The Pluronic F127-Ap (MR = 5.69) showed a clearer and brighter band at the origin site, and no band at the free Ap site either. When the molar ratio was increased to 7.11, a light band appeared at the free Ap site, indicating the saturation of Ap conjugation. Thus, we chose MR = 5.69 as an optimized Ap ratio for the synthesis of Pluronic F127-Ap to ensure the maximal Ap conjugation without any free Ap. However, the aptamer cannot be linked to the Pluronic F127 without EDC. The β-CD-PELA conjugate was obtained from the reaction of carboxylated β-CD with PELA and the related polymer characterization was detailed in Supplementary Information (Figure S1).
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Characterization of DOX-loaded micelles
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The effect of polymer composition of the composite micelles on drug LC and EE was examined and the results were shown in Table 1. The micelles prepared from pure Pluronic F127-Ap had a rather low EE for DOX, while those from pure β-CD-PELA had a high EE (N 90%), demonstrating the excellent loading capacity of β-CD-PELA for DOX. For the composite micelles, when the weight ratio of Pluronic F127-Ap to β-CD-PELA decreased from 3:1 to 1:3, the EE increased from 76.75% ± 1.39% to 92.54% ± 4.15% (1.5% theoretical LC), further confirming the predominant role of β-CD-PELA for drug loading. Taking Ap content and drug loading two factors into account, the optimal mixed ratio of Pluronic F127-Ap to β-CD-PELA was set as 2:2 for the following study. CMC is a quite predominant parameter for micelles to reflect their physical stability. In this study, the CMC of Pluronic F127-Ap/β-CD-PELA binary mixture with weight ratio of 2:2
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Fluorescent markers are frequently applied in cellular uptake studies by flow cytometry. In our case, an additional marker RhB linked on polymers and the entrapped DOX were used as fluorescent probes to measure cellular uptake, respectively. Pluronic F127-RhB was successfully synthesized by the esterification reaction (Figure 1, A (b)) and characterized by 1 H NMR and UV-visible spectroscopy (Figure S2). The effect of different aptamer contents on the targeting ability of the aptamer-conjugated micelles to MCF-7 cells was investigated through RhB-labeled micelles. When the ratio of Pluronic F127-Ap to β-CD-PELA increased from 1:3 to 2:2, a 1.97-fold increase in micelle uptake was obtained. Further increasing the ratio to 3:1, however, resulted in a slight increase in micelle uptake (Figure 4, A). This result further confirmed it was appropriate to set the weight ratio of Pluronic F127-Ap to β-CD-PELA as 2:2 in the study. In addition, it was found that the cellular uptake of aptamer-modified micelles was timedependent and increased with the increase of incubation time (Figure 4, B). As presented in Figure 4, C, the RhB-CM-Ap showed about 3.31 times higher fluorescence intensity in cells than RhB-CM-Ap(mut). Furthermore, for DOX-loaded micelles, the cellular uptake of DOX-CM-Ap was still 3.36-fold higher than that of DOX-CM-Ap(mut) (Figure 4, D). These results indicated that the aptamer-functionalized micelles were prone to undergo internalization by cancer cells, presumably due to the selective affinity between the aptamer AS1411 and the nucleolin.
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To verify the pharmacological activity of DOX-loaded CM-Ap(mut) and CM-Ap, the in vitro cytotoxicity tests against MCF-7 cells were conducted. The DOX-CM-Ap showed enhanced antiproliferative activity relative to the DOX-CM-Ap(mut), as illustrated by the apparent IC50 values of 309.8 and 442.8 ng/mL, respectively. This enhanced cytotoxicity of DOX-CM-Ap
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was 0.0033 g/L, which was a little higher than that of β-CD-PELA (0.0023 g/L) but much lower than that of pure Pluronic F127 (0.039 g/L). 9 This result validated the homogenous formation of composite micelles by mixing Pluronic F127 with β-CD-PELA due to their good compatibility. As shown in Figure 3, A and B, the diameters of DOX-loaded CM-Ap(mut) and CM-Ap micelles were similar, about 39.15 nm and 38.23 nm respectively. Accordingly, the TEM images proved that both micelles were spheroidal shape and homogeneous in size. The zeta potentials were approximately − 9.86 mV and − 10.06 mV for DOX-CM-Ap(mut) and DOX-CM-Ap, respectively. The in vitro drug release study was performed at various pH values including those simulating the physiological environment (pH 7.4), tumor microenvironment (pH 6.5), and endosomal compartments (pH 5.0). The pH-dependent release profiles of DOX from micelles are shown in Figure 3, C and D. For DOX-CM-Ap(mut), the DOX release rate was faster in the first 9 h and reached a plateau in 24 h. In addition, the released amount of DOX from micelles increased as pH values of the medium decreased due to its increased solubility. Similar DOX release behavior was observed for DOX-CM-Ap.
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Table 1 Drug loading content (LC) and drug encapsulation efficiency (EE) of DOXloaded micelles (mean ± SD, n = 3).
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Figure 3. Characterization of DOX-loaded micelles. Particle size distribution and TEM images of (A) DOX-CM-Ap(mut) micelles and (B) DOX-CM-Ap micelles. In vitro DOX release from (C) DOX-CM-Ap(mut) micelles and (D) DOX-CM-Ap micelles. All data represent mean ± SD.
Figure 4. In vitro cellular uptake of micelles by MCF-7 cells. (A) RhB-labeled micelles (RhB-CM-Ap) with different aptamer content (Pluronic F127-Ap, β-CD-PELA, and Pluronic F127-RhB with different weight ratios of 1:3:0.05, 2:2:0.05, and 3:1:0.05) after 60 min incubation with cells. (B) RhB-CM-Ap after 5, 30, and 60 min incubation with cells, respectively. (C) RhB-CM-Ap(mut) and RhB-CM-Ap after 60 min incubation with cells, respectively. (D) DOXCM-Ap(mut) and DOX-CM-Ap after 60 min incubation with cells, respectively. All data represent mean ± SD.
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Figure 5. Comparison analysis of pharmacokinetics and biodistribution in MCF-7 tumor-bearing mice. (A) Pharmacokinetic profiles of DOX solution, DOX-CM-Ap(mut), and DOX-CM-Ap at 5 mg/kg DOX. (B) In vivo real-time imaging of mice at 0.5, 4, 6, 8, 24, and 48 h post-injection of ICG-CM-Ap(mut) and ICG-CM-Ap, respectively. (C) Average fluorescence signals determined at major organs and tumors at 48 h post-injection of ICG-CM-Ap(mut) and ICG-CM-Ap, respectively. Statistical significance: *P b 0.05. All data represent mean ± SD.
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A pharmacokinetic study in MCF-7 tumor-bearing mice was performed to determine the drug circulation in blood. The plasma concentration–time curves for DOX, DOX-CM-Ap(mut), and DOX-CM-Ap were all fitted to the two-compartment model. As shown in Figure 5, A, DOX-CM-Ap achieved a longer half-life and larger AUC in comparison with free DOX, whereas free DOX was quickly eliminated from circulation. Moreover, compartmental analysis of plasma concentrations exhibited significant differences in pharmacokinetic parameters for DOX in aptamer-functionalized micelles compared to free DOX (Table 2). For DOX-CM-Ap micelles, the half-life (t1/2β) was 1.94-fold longer, the AUC was 17.2-fold larger, the mean residence time (MRT) was 2.94-fold longer, and total body clearance (CL) was shorter than that of free DOX. No significant difference was observed in pharmacokinetic behavior between DOX-CM-Ap group and DOX-CM-Ap(mut) group.
micelles in vivo via the non-invasive and real-time near-infrared fluorescence imaging. Figure 5, B showed the fluorescence images at different time intervals after intravenously injection of ICG-loaded CM-Ap(mut) and CM-Ap. It can be seen that both micelles were prone to accumulate in liver and intestine for the initial 4 h, which may be attributed to the rapid recognition and uptake of nanoparticles by the cells of RES. Although the fluorescence signal occurred at the tumor site at 4 h post-injection of both micelles, the signal intensity of ICG-CM-Ap was much stronger than that of ICG-CM-Ap(mut) during the whole experimental period, suggesting that the aptamer-conjugated micelles had better tumor targeting potency than the non-targeted ones. The ex vivo fluorescence images at 48 h post-injection were obtained for the tumor tissue as well as for major organs such as heart, liver, spleen, lung, and kidney. As shown in Figure 5, C, the strong fluorescence was mainly observed in tumor tissue for both micelles, while other tissues such as heart showed negligible fluorescence signal. Furthermore, it was found that the fluorescence signal of the Ap-conjugated micelles in tumor tissue is significantly higher than that of the Ap(mut)-conjugated micelles.
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In vivo tumor growth inhibition and histopathology
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To evaluate whether the aptamer-modified micelles can indeed specifically target tumors, we monitored the fate of the
The in vivo antitumor efficacy was evaluated using different DOX formulations on mice bearing subcutaneous MCF-7
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against MCF-7 cells in culture may owe to the improved cellular uptake of micelles.
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Table 2 Pharmacokinetic parameters in mice after single intravenous administration of free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap at 5 mg/kg DOX (mean ± SD, n = 3).
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0.08 1.67 0.26 1.39 542.78 391.07
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0.17 3.24 4.46 4.08 91.61 22.44
± ± ± ± ± ±
0.06 0.45 0.77 0.58 7.23 2.25
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tumors. Figure 6, A shows the growth curves of MCF-7 tumors of mice after the treatment. It can be seen that the growth rate of the DOX-CM-Ap group was remarkably delayed when compared with the DOX-CM-Ap(mut) group. The group treated with free DOX showed a tumor growth curve similar to that of the DOX-CM-Ap(mut) group. At the end of experiment, the tumor volume of the DOX-CM-Ap group was much smaller than other groups, about 46% of the DOX-CM-Ap(mut) group and 54% of the free DOX group. The tumor growth inhibition (TGI) was 77.6% for DOX-CM-Ap, 51.2% for DOX-CM-Ap(mut), and 58.0% for free DOX, demonstrating significantly superior antitumor efficacy of DOX-CM-Ap. Multiple administration of DOX solution could cause toxicity, resulting in weight loss and cardiotoxicity. Figure 6, B presents the changes in the body weight of each group over time as an indication of overall systemic toxicity of different formulations. The free DOX group showed a sharp decrease in body weight during the initial 9 days and recovery after this period. This strongly suggests serious toxicity of free DOX at the given dose. However, no weight loss was observed for the micelle-treated groups. In addition, cardiotoxicity is the main drawback of DOX use, which ultimately restraints effective DOX application in patients. As shown in Figure 6, C, multiple administration of free DOX induced noticeable cardiac tissue degeneration and necrosis. However, there was no evidence of cardiotoxicity after either DOX-CM-Ap or DOX-CM-Ap(mut) therapy compared to the control group. These results indicated that the toxic effects of DOX could be alleviated by micelles, which may ascribe to the reduced accumulation of DOX in heart.
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Nowadays, ligand-modified nanoparticles have been considered to effectively improve the anti-tumor efficacy based on enhanced accumulation at tumor tissues via EPR effect and subsequent endocytosis of tumor cells via ligand–receptor interaction. As one of the most excellent targeting ligands, aptamers can selectively bind to a variety of targets ranging from small molecules to whole cells due to their three-dimensional structures. In the present study, we constructed a binary composite micelles composed of AS1411-modified Pluronic F127 and β-CD-PELA to load DOX. In vivo tumor inhibition study revealed that the AS1411-modified DOX-CM obtained significantly stronger tumor inhibition on MCF-7 tumor when
compared with untargeted DOX-CM and didn’t induce any cardiotoxicity when compared with free DOX. These expected results could be attributed to the following factors. Firstly, the physicochemical properties of AS1411-modified CM were fully developed. To ameliorate the weak drug loading capacity of Pluronic F127, structural modifications are often investigated (e.g., Pluronic F127-PLA, Pluronic F127-chitosan, etc.). 21,22 However, this paper overcome this drawback of Pluronic F127 micelle by the addition of β-CD-PELA. As shown in Table 1, DOX displayed stronger affinity to hydrophobic segments β-CD and PLA of β-CD-PELA than PPO of Pluronic copolymer, 23 so the DOX EE of β-CD-PELA micelles is about 2.4 times higher than that of Pluronic F127 micelles. The composite micelles at a weight ratio of 2:2 also had a 2.2-times-higher EE of DOX than pure micelles of Pluronic F127. β-CD has a toroidal shape containing a hydrophobic inner cavity, in which DOX could be act as the “guest” molecule and solubilizer. In addition, our previous work demonstrated that linking β-CD to PELA could make the amphiphilic copolymer easier to self-assemble in aqueous solution, as illustrated by the reduced CMC of 0.0023 g/L. As a result, this hybrid system achieved a low CMC of 0.0033 g/L and thus provided relatively high stability in solutions upon dilution. The dependency of drug release with pH may contribute to the increase of drug concentration in the tumor tissue. As revealed by Min et al, TRITC-loaded PEG-b-PAE micelles, a type of pH-dependent drug delivery systems, can successfully deliver TRITC to tumor site and release it there, resulting in a 11-folds-higher fluorescence in tumors than non-pH-sensitive micelles. 24 Secondly, DOX-CM-Ap micelles displayed long-circulation character in vivo with the longer half-life, larger AUC and shorter CL (Table 2) when compared with free DOX, which supplied more opportunities for DOX-CM-Ap micelles to leak into tumor tissues via EPR effect. Furthermore, the AS1411-modified CM tended to accumulate more efficiently within the tumor through the selective binding to receptors on MCF-7 cells than CMAp(mut) micelles. ICG is the only near-infrared dye approved by FDA at present. 25 In this study, ICG was encapsulated into the CM and acted as a fluorescence probe to trace the CM in the body of tumor-bearing mice. As shown in Figure 5, B, ICG could be delivered to tumors with the help of both CMs, but the accumulation degree of ICG-CM-Ap was significantly higher than that of ICG-CM-Ap(mut) during the entire experimental period. Thirdly, the specific interaction between aptamer and nucleolin significantly enhanced cellular uptake of AS1411modified CM in MCF-7 cells and increased the cytotoxicity of its payload. Nucleolin, present on the surface of various types of tumor cells, is a functional receptor for aptamers. The trafficking ability of nucleolin has been implicated in transporting aptamers as well as nanoparticle–aptamer bioconjugates from the membrane into the nucleus. 26,27 For example, Dam et al used a DNA aptamer (AS1411) by taking advantage of the shuttling properties of nucleolin to efficiently target gold nanoparticles into the nucleus of cancer cells. 26 On the basis of the current experimental data, we can propose the following chain of events to describe the in vivo behavior of DOX-CM-Ap. After intravenous administration to mice, the
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In this study, we successfully prepared binary mixed micellar system CM-Ap consisting of aptamer-conjugated Pluronic F127 and β-CD-PELA to load DOX with satisfactory drug loading capacity. The pharmacokinetic study demonstrated that CM-Ap could significantly extend the blood circulation time of DOX compared to free DOX. Moreover, in vivo real-time imaging study revealed that aptamer-conjugated micelles had better tumor targeting potency than non-targeted micelles. Based on a receptor-mediated endocytosis pathway and controllable cellular release of DOX, DOX-CM-Ap was validated to enhance the anti-tumor efficacy of DOX in vivo and prevent acute cardiotoxicity. Owing to these characteristics, this aptamerfunctionalized delivery system exhibited great potentials for cancer therapy.
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DOX-CM-Ap demonstrated a prolonged circulation time in blood, sufficient for their accumulation in the tumor via EPR effect. When they entered the tumor interstitial space, aptamer present on the micelle surface quickly recognized the tumor cells and bound with them (but not with normal tissues), followed by aptamer-mediated endocytosis of micelles. Thus, the combination of several advantages within this novel micellar system should allow for precise and effective tumor targeting and intracellular action, and consequently enhanced inhibition of tumor growth and alleviated side effects.
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Figure 6. Therapeutic efficacy of different DOX formulations in MCF-7 tumor-bearing mice (n = 5 per group). All data represent mean ± SD. (A) Growth curves of MCF-7 tumors of mice after the treatment of saline, free DOX, DOX-CM-Ap(mut), and DOX-CM-Ap. (B) Changes in the body weight of each group. (C) In vivo cardiotoxicity of DOX-treated mice. Heart was evaluated by H&E staining after 27 days post-injection of each formulation. The original magnification is ×400. Statistical significance: *P b 0.05.
The authors thank Miss Yangmin Jin for animal experiments.
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2014.08.013.
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References
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Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2014) xxx–xxx
Targeted delivery of anticancer drugs by aptamer AS1411 mediated Pluronic F127/cyclodextrin-linked polymer composite micelles
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Xin Li, MS b, Yang Yu, MS b, Qian Ji, BA b, Liyan Qiu, PhD a,⁎ a
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A novel composite micelle (CM) with surface modification of aptamer AS1411 (Ap) was successfully developed for targeted delivery of doxorubicin (DOX) to human breast tumors. Mixing of two different kinds of polymers afforded the composite micelle-Ap (CM-Ap) various desirable physicochemical properties. In addition, the in vitro and in vivo studies demonstrated that the DOX-loaded CM-Ap could prolong blood circulation time, enhance cellular uptake and cytotoxicity, and consequently, improve tumor growth inhibition and minimize adverse effect.
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