Multistep Targeted Nano Drug Delivery System Aiming at Leukemic

May 6, 2013 - Proofs. Multistep Targeted Nano Drug Delivery System Aiming at Leukemic Stem Cells and Minimal Residual Disease. Citing Articles; Relate...
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Multistep Targeted Nano Drug Delivery System Aiming at Leukemic Stem Cells and Minimal Residual Disease Yongping Shi, Zhigui Su, Sai Li, Yinan Chen, Xi Chen, Yanyu Xiao, Minjie Sun, Qineng Ping,* and Li Zong* Department of Pharmaceutics, Key Lab of State Natural Medicine, China Pharmaceutical University, Nanjing 210009, P. R. China S Supporting Information *

ABSTRACT: Refractory leukemia remains the most common therapeutic problem in clinical treatment of leukemia. The key therapy of refractory leukemia is to kill, thoroughly, the minimal residual disease and leukemia stem cells in the highly vascularized red marrow areas. In this study, two new conjugates, alendronate-polyethylene glycol (100) monostearate and folatepolyethylene glycol (100) monostearate, were synthesized to develop a multistep targeting nanostructured lipid carriers by enhancing drug transport to the high bone turnover areas adjacent to the red marrow and targeting the minimal residual disease and leukemia stem cells. This dual targeting system demonstrated a great binding affinity to hydroxyapatite, a model component of bone minerals, and higher cell uptake (in the form of carriers but not drug) and cytotoxicity in the K562 cell line, a leukemia cell line with overexpressed folate receptors, were observed in vitro compared to unmodified carriers, especially when the cells were pretreated and the receptors were up-regulated by all-trans retinoic acid. The comodel test of K562 cells and HA showed that this dual targeting system could desorb from bone surface and be taken up by leukemia cells. For the in vivo study, this dual targeting system exhibited a significant increase in plasma half-life and could specifically accumulate in the bone tissue of rats or mice after intravenous injection. Ex vivo imaging of mice femurs and confocal laser scanning microscope imaging of mice femur slices further confirmed that this dual targeting system could favorably deposit to the osteoblast-enriched areas of high bone turnover in regions of trabecular bone surrounded by red marrow. In vivo antitumor activity in K562/BALB/c-nu leukemia mice showed that the treatment of this dual targeting system significantly reduced the white blood cell (WBC) number in peripheral blood and bone marrow to the normal level. In conclusion, this dual targeting system could precisely target to the regions where the minimal residual disease and leukemia stem cells are located and then be specifically uptaken in large amounts, which is a valuable target for refractory leukemia therapy. KEYWORDS: refractory leukemia, minimal residual disease, leukemic stem cell, multistep targeting delivery

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INTRODUCTION

LSC targeting therapy may be presented as a curable treatment for leukemia.3,4 Like all bisphosphonates, alendronate (ALN) exhibits an exceptionally high affinity for the bone mineral hydroxyapatite (HA). This unique feature of ALN makes it a good candidate for the bone targeting of antineoplastic compounds, radionucleotides, and nucleoside analogues. Wang and colleagues disclosed their design of a bisphosphonate-based poly(ethylene glycol) (PEG) delivery system targeting to bone. In the design, one terminus of the PEG chain was modified with bisphosphonate and the other with fluorescein isothiocyanate (FITC).5 Realizing the low drug-loading limitation and molecule-specificity of these bisphosphonate conjugates based on water-soluble polymers, bone-targeting nanoparticles (e.g., polymeric or micellar) or nanostructured lipid carriers (NLCs)

For decades, hematologists have known that many leukemia patients who appear to respond adequately to the initial courses of chemotherapy, as judged by the absence of morphologically identifiable leukemic cells in bone marrow smears, subsequently relapse. The remission induced by chemotherapy means that, after treatment with chemotherapy, the leukemic cells are less than 5% with bone marrow morphology examination. The residual leukemic cells, which are the components of an important clinical problem termed “minimal residual leukemia (MRL)”, may be the root of recurrence after complete remission. Thus, the search for and elimination of MRL cells are critical for leukemia therapy.1,2 Meanwhile, the cause of leukemia formation, growth, metastasis, and relapse, is now commonly referred to as the leukemia stem cells (LSCs) homing to and engrafting within the osteoblast-rich areas of the bone marrow in vivo of patients. Traditional treatment can only kill the most differentiated cancer cells and hardly affect the tumor stem cells. Thus, the © XXXX American Chemical Society

Received: March 4, 2013 Revised: April 11, 2013 Accepted: May 6, 2013

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are likely to be truly “generic”, in that the designed system should be able to deliver a range of therapeutic agents. It was reported that the expression level of the folate receptor (FR) in tumors was 100−300 times higher than that observed in normal tissue.6,7 Meanwhile, the current standard treatment for myeloid leukemia consists of all-trans retinoic acid (ATRA) and anthracyclines with or without cytarabine. ATRA can upregulate the FR-β in various myelogenous leukemia but without the terminal differentiation of cells and inhibition of cell proliferation. Pan and colleagues claimed that, in FR(+) KG-1 cell lines, selective FR-β mediated binding was obtained using folate-coated liposomes encapsulating doxorubicin, and treatment with ATRA further increased this specificity.8 Therefore, the combination of FR-β targeted therapy and ATRA therapy may be a more effective approach for myelocytic leukemia treatment. Herein, we present mitoxantrone (MTO, a routine drug in the treatment of leukemia) as the model anticancer drug to prepare the multistep targeting nano drug delivery system in which alendronate and folate are used as the stepped targeting ligands of the nanostructured lipid carriers (NLCs).9 ALN has a specific combination capacity with HA, which distributes mainly in the osteoblast-enriched areas of high bone turnover. Compared to other bone-targeting moieties, such as poly(aspartic acid) and poly(glutamic acids), ALN can mediate the accumulation of its conjugates to the osteoblast-enriched areas in regions of trabecular bone surrounded by highly vascularized red marrow after the intravenous administration.10 At the same time, it is noteworthy that the osteoblast-enriched areas account for only 5% of the endosteum. Considerable evidence has accumulated to indicate that LSCs are immobile, in intimate contact with osteoblasts beside the endosteum of the cancellous bone.11−13 Meanwhile, it is well-known that MRL distribute chiefly in the red bone marrow locating in the cancellous bone. Considering that most of the intraosseous vessels in bone distribute in the cancellous bone, all of these phenomena may significantly increase ALN’s targeting efficiency for the MRL, especially for the LSCs. By binding the folate-modified NLCs with the folate receptor expressed on these cells, the NLCs would be taken up by endocytosis in large amounts and then release more drug; therefore, the kill effect on LSCs and MDR would be increased significantly (Figure 1). To the best of our knowledge, this is the first example of the drug carrier to improve the therapy of refractory leukemia aiming at its major pathophysiologic bases. In this study, alendronate-PEG (100) monostearate (ALNS100) and folate-PEG (100) monostearate (FOL-S100) were synthesized as targeting materials, and 1H NMR was used to confirm their structure. The MTO-loaded NLCs (MTONLCs) were prepared by using a film-evaporation method. The size, surface charge, and morphology of NLCs were detected by dynamic light scattering (DLS) and an atomic force microscope (AFM). Several methods were employed to identify the dual-targeting effect, including the HA binding test, K562 cell uptake, cytotoxicity, and their combination model with HA in vitro, the study of pharmacokinetics in rats and biodistribution in mice, ex vivo imaging of mice, CLSM imaging of mice femur slices, and in vivo antitumor activity in K562/BALB/c-nu leukemia mice.

Figure 1. Schematic diagram of the multistep targeted nanodrug delivery system.

Biotechnology Co., Ltd. (Anhui, China). N-Hydroxy succinimide (NHS) was from China National Medicine Group Shanghai Chemical Reagent Company (Shanghai, China). 4Nitrophenyl chloroformate was obtained from Suzhou TimeChem Technologies Co., Ltd. (Jiangsu, China). Poly(ethylene glycol) (100) monostearate (S100) with PEG molecular weight 4400 g/mol was purchased from Sigma (Milwaukee, WI, USA). FOL and ALN were purchased from Shanghai Aladdin Co., Ltd. (Shanghai, China). Soybean phospholipids (PC, purity 90%) was a kind gift from Evonik Degussa China Co., Ltd. (Shanghai, China). Cholesterol was purchased from Huixing Biochemical Reagent Co., Ltd. (Shanghai, China). Mitoxantrone dihydrochloride (purity, 99.1%) was obtained from Sichuan Shenghe Pharmaceutical Co., Ltd. (Sichuan, China). Trilaurin and coumarin-6 were purchased from Tokyo Chemical Industry (Tokyo, Japan). 2-[2-[2-Chloro-3-[2-(1,3dihydro-1,1,3-trimethyl-2H-benzo[e]-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,1,3-trimethyl-1H-benzo[e]indolium 4-methylbenzenesulfonate (NIRD) was provided by Huahai-Lanfan Chemical Technology Co., Ltd. (Liaoning, China). The other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell Cultures. K562 cells (human erythroleukemia cells) was obtained from the cell bank of the Chinese Academy of Sciences. The cells were cultured in folate-free RPMI 1640 containing 10% (v:v) FBS, 100 U/mL penicillin, and 100 mg/ mL streptomycin in an incubator (Thermo Scientific, USA) at 37 °C under an atmosphere of 5% CO2 and 90% relative humidity and were subcultured approximately every 3 days. Animals. SD rats, ICR mice, and BALB/c-nu mice were obtained from the Shanghai Silaike Laboratory Animal Co., Ltd. (Shanghai, China). All animal experiments were performed in accordance with protocols evaluated and approved by the ethics committee of China Pharmaceutical University. Synthesis of FOL-S100 and ALN-S100. First, 0.883 g of FOL (2 mmol), 4.684 g of S100 (1 mmol), 0.192 g of EDC (1 mmol), and 0.138 g of NHS (1.2 mmol) were dissolved in 250 mL of dimethyl sulfoxide (DMSO) and stirred at room temperature for 24 h. The resultant solution was placed into a preswelled dialysis bag (3.5 kDa MW cutoff, Sigma, USA) and dialyzed in 20 L of NaHCO3 buffer (pH 9.0) for 12 h (change the buffer every 3 h) and then in 20 L of water for 24 h (change

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MATERIALS AND METHODS Materials. 1-Ethyl-3-(3-dimethylamino) propyl carbodiimide hydrochloride (EDC·HCl) was from Hefei Bomei B

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Additionally, AFM (Veeco diNanoScope V, USA) was also used to characterize the morphology. The samples of AFM were made according to the following description: the NLCs suspension was properly diluted with water and placed on the surface of a clean silicon wafer. After air-dried at room temperature, the sample was observed by AFM with a 5 μm scanner in contact mode. The concentration of MTO in NLCs was determined by Shimadzu 10A vp HPLC system (Tokyo, Japan).19 The stationary phase, Diamonsil C18 column (150 mm × 4.6 mm, 5 μm), was kept at 40 °C. The mobile phase was a mixture of methanol and 0.2 mol/L ammonium acetate buffer (48:52, v/v, pH = 2.8) adjusted with acetic acid. The flow rate was 1.0 mL/min, and effluent was monitored at 658 nm. The entrapment efficiency (EE%) was determined by ultrafiltration (100 kDa MW cutoff, Pall, USA, centrifuging at 8000 rpm for 5 min) and calculated as follows:

the water every 4 h) to remove reaction materials and byproducts. Then it was lyophilized to remove water and trace amounts of organic solvent. The synthetic product was further purified by column chromatography on silica gel H using chloroform/methanol (12:1, v/v) as an eluent.14 A known amount of dried FOL-S100 was dissolved in DMSO, and an UV absorbance value at 365 nm was measured to determine the concentration of conjugated FOL. Serially diluted concentrations of FOL in DMSO were used to construct a calibration curve.15 ALN-S100 was synthesized in a two-step procedure. First, 9.368 g of S100 (2 mmol) was dissolved in 9 mL of anhydrous dichloromethane and supplemented with 0.45 mL of triethylamine (3 mmol). Then, 0.702 g of 4-nitrophenyl chloroformate (3 mmol) dissolved in 1 mL of anhydrous dichloromethane was added to the mixture, and the sample was incubated overnight at room temperature with stirring. After filtering and removing the precipitated triethylamine hydrochloride, the organic solvents were removed using a rotary evaporator, and the residues were recrystallized in chilled diethyl ether and purified to form the new amphiphilic intermediate, p-nitrophenyl carbonyl-PEG (100) monostearate (pNP-S100). Then, 0.480 g of pNP-S100 (0.10 mmol) was dissolved in dichloromethane (15 mL) followed by the evaporation of organic solvent until complete dryness under vacuum and formation of a dried film. Then, 0.813 g of alendronate sodium (0.15 mmol) dissolved in 50 mL of 0.2 mol/L NaCO3−NaHCO3 buffer (pH 9.5) was added to the dried film, homogenized by sonication for 1 min to obtain suspension. The mixture was incubated for 12 h at room temperature and dialyzed in 20 L of water for 24 h (3.5 kDa MW cutoff, change the water every 4 h) to remove free ALN. Eventually, ALN-S100 solution was lyophilized and stored at 4 °C until further use.16 The conjugation percentage of ALN to S100 was determined and calculated on the basis of the chromophoric complex formed between ALN and Fe3+ ions in perchloric acid and against a calibration graph for ALN.17 Preparation of MTO-Loaded NLCs (MTO-NLCs). In a 0.25 L round-bottomed flask, 250 mg of soybean phospholipids, 25 mg of trilaurin, 75 mg of cholesterol, 10 mg of mitoxantrone hydrochloride (MTO·HCl), and 10 mg of sodium dodecyl sulfate (SDS) were dissolved in 30 mL of methanol/dichloromethane solvent mixture (1:1, v/v). The organic solvent was evaporated under vacuum at 37 °C for 3h, and then the flask was placed in desiccators under vacuum overnight. The dried lipid film was hydrated with 10 mL of Cremophor EL solution (2%, w/w) at 37 °C for 30 min and ultrasonicated at 300 W for 5 min. MTO·HCl and SDS could form an ion pair, which could be partitioned to a greater extent into the hydrophobic core of NLCs than the ion form MTO·HCl due to its hydrophobicity. For the preparation of modified NLCs, ALN-S100 (2 μmol/ mL), FOL-S100 (0.4 μmol/mL), or both of them (2 μmol/mL and 0.4 μmol/mL, respectively) were added into the lipid matrix and followed the above procedures.18 Based on the modification of agents, the NLCs were named in order as ALNMTO-NLCs, FOL-MTO-NLCs, and ALN-FOL-MTO-NLCs, respectively. For the preparation of FOL-MTO-NLCs and ALN-FOL-MTO-NLCs, NaHCO 3 was added into the hydration medium at the concentration of 1 mg/mL. Characterization of NLCs. Particle size, its distribution indicated by polydispersity index (PI) and zeta potential of NLCs (diluted 50 times with deionized water) were determined by Zetaplus (Brookhaven Instruments, UK).

EE% = (Wf /Wt) × 100%

where Wf is the analyzed amount of free MTO in the filtrate after ultrafiltration and Wt is the analyzed amount of total drug. In Vitro HA Binding Study. In order to evaluate the binding ability of the MTO loaded NLCs to bone mineral through ALN moiety, HA was used as a model component to mimic bone mineral tissue. NLCs (5 mL, MTO concentration 0.1 mg/mL) were incubated with HA (200 mg) for 60 min at room temperature under mild stirring.20 After filtration by microporous membrane (0.45 μm), the MTO concentration of filtrate and NLCs was measured by spectrophotometry at 658 nm. Quantitative Study of Cells Uptake and Cytotoxicity Study. Coumarin-6 (C6) NLCs was prepared according to the preparation of MTO-loaded NLCs, but MTO and SDS were replaced with C6. An in vitro release study showed that less than 0.5% of incorporated C6 leached out from C6-NLCs, FOL-C6-NLCs, and ALN-FOL-C6-NLCs in pH 7.4 PBS over 24 h. NLCs samples with different concentrations were prepared (diluted by folate-free RPMI 1640 media). K562 cells were seeded in 24-well plates (Costar, IL, USA) at a density of 105 cells/well and incubated in complete cell-culture medium for 24 h. Then, the medium was replaced with 0.5 mL of NLCs samples in the concentration range of 15−200 ng/mL, and the plates were incubated at 4 or 37 °C for 4 h. At the end of the incubation period, NLCs samples were removed, and the wells were washed with ice-cold PBS for three times. The quantitative analysis of the cellular uptake was carried out. Cellular uptake was also determined with cells exposed to 1 μM ATRA for 5 days.21 The uptake index (UI) of drug expressed as the amount of C6 (ng) uptaken per μg cell protein was calculated from the equation: UI(ng/μg) = C /P

where C is the intracellular concentration of C6 (ng/mL) and P is the concentration of cellular protein (μg/mL). The cytotoxicity of MTO formulations was evaluated by determining the cell viability using MTT assays. The cells (K562 cells) were seeded in 96-well plates (Costar, IL, USA) at a density of 5 × 103 cells/well 1 day before the experiment. Then, the medium was replaced by MTO-INJ or MTO-NLCs samples containing 0.01−20 μg/mL MTO diluted in folate-free RPMI 1640 and incubated for another 48 h at 37 °C. After incubation, the cells were washed with PBS and cultured in C

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Figure 2. 1H NMR spectra of FOL-S100 (A) and ALN-S100 (B).

folate-free RPMI-1640. Then, 20 μL of MTT (5 mg/mL in pH 7.4 PBS) was added and incubated for 4 h in incubator. 150 μL of DMSO was added to replace the medium in order to solubilize the formazan crystals. The UV absorbance intensity was measured by Microplate Reader (Thermo Electron Corporation) at 570 nm. Each point was performed in triplicate. The cell viability was calculated by the following equation:

The tissue distribution of MTO-INJ, FOL-MTO-NLCs, and ALN-FOL-MTO-NLCs were determined in male ICR mice (mean body weight, 20 g). For the free drug and both NLCs formulations, single intravenous doses were administered via tail vein at a dose of 10 mg/kg (n = 3). The organs of interest were sampled and whole organs collected where possible at 0.5, 1, 2, 6, 12, and 24 h after administration. The samples were rinsed in saline, blotted dry, weighed, and then frozen at −20 °C until assay. The concentrations (expressed as μg MTO/g) of MTO in different organisms at different times were detected by HPLC and analyzed by calculating AUC. Ex Vivo Imaging and CLSM Imaging (Healthy Mice). In this study, near-infrared dyes (NIRD) NLCs was prepared according to the preparation of MTO-NLCs, but MTO and SDS were replaced with NIRD. Male ICR mice were used for the investigation of deposition of NIRD- NLCs, FOL-NIRDNLCs and ALN-FOL-NIRD-NLCs in bone. All the preparations were administered into the bloodstream of the subject mice through tail vein injection at a dose of 0.1 mg/kg. The mice were sacrificed at 0.5, 2, 4, 8, 12, and 24 h, and femurs were separated and washed by saline for ex vivo fluorescence imaging. Fluorescence imaging was acquired with NIR imaging system (ex 720 nm and em 790 nm). To assist in the identification of different functional domains on bone surfaces, tetracycline (a fluorochrome marker of bone formation) was administered intraperitoneally (40 mg/kg) 3 days prior to the administration of NLCs.22 The tetracycline marker labeled bone formation surfaces permitted easier

cell viability(%) = ODs /ODc × 100%

where ODs stands for the absorbance intensity of the cells treated with MTO, while ODc stands for the absorbance intensity of the cells incubated with culture medium. Pharmacokinetic and Biodistribution Studies in Healthy Animals. The pharmacokinetics of MTO injection (MTO-INJ), MTO-NLCs, FOL-MTO-NLCs, ALN-MTONLCs, and ALN-FOL-MTO-NLCs were determined in male SD rats (mean body weight, 200 g). All of the preparations were administered intravenously into the tail vein at a dose of 2 mg/kg (n = 6), and blood samples were taken with a heparinized syringe at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Once drawn, the sample was centrifuged at 4000 rpm for 10 min at 4 °C. The plasma was frozen at −20 °C until assay. The concentrations of MTO in the blood were expressed as μg MTO/mL. The plasma concentrations versus time data were analyzed by Kinetica 4.4 (Thermo Electron Corporation, Waltham, MA, USA). D

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Table 1. Physicochemical Properties of NLCsa preparations MTO-NLCs FOL-MTO-NLCs ALN-MTO-NLCs ALN-FOL-MTO-NLCs a

size (nm) 52.3 50.5 45.3 45.9

± ± ± ±

2.6 2.0 2.9 2.7

PI 0.19 0.17 0.16 0.18

± 0.02 ± 0.02 + 0.03 ± 0.03

zeta potential (mV) −3.38 ± 1.25 −3.04 ± 1.57 −17.85 ± 2.06 −16.78 ± 2.17

EE (%) 99.5 99.3 99.6 99.7

± ± ± ±

0.3 0.2 0.2 0.1

Data are represented as mean ± SD (n = 3).

administered via the tail vein at a dose of 10 mg/kg every 3 days, 3 times. At day 18, all mice were sacrificed, and cell morphology of peripheral blood and bone marrow with WrightGiemsa stain were observed. Tissue HE staining were performed for detecting potential pathological changes and infiltration of leukemic cells (n = 3). K562/BALB/c-nu leukemia mice were also used for the tissue distribution study of NIRD-NLCs, FOL-NIRD-NLCs, and ALN-FOL-NIRDNLCs by the method as described above.

identification of areas of high bone turnover. Two hours after administration of NIRD-NLCs, FOL-NIRD-NLCs, or ALNFOL-NIRD-NLCs, the mice (three/group) were euthanized. Femurs were isolated, fixed, and processed for undecalcified histomorphometric analysis. Bone samples were fixed and dehydrated with acetone, embedded in paraffin, and sliced to the thickness of 200 μm for observation under a confocal laser scanning microscope (CLSM, Olympus FV300-BX Carl Zeiss LSM-410, Tokyo, Japan). All images were taken under same microscope settings. Dual-Targeting Effects Study. In order to analyze the potential dual-targeting effect, a K562 cells/HA comodel was established for simulating the situation in vivo. K562 cells was seeded on the upper compartment of the Transwell inserts at a density of 105 cells/compartment. After incubation for 2 days, the inserts were placed in Petri dishes (one insert per dish, n = 3). ALN-FOL-C6-NLCs were incubated with HA for 1 h at room temperature under mild stirring. After filtration, the trapped HA with NLCs adsorbing on the surface, HA and Hank’s solution were used to prepare the ALN-FOL-C6NLCs/HA mixed system at a concentration of 700 ng C6/mL. These system was added into the Petri dish and stirred mildly for 6h at room temperature. As a control, the ALN-FOL-C6NLCs was diluted to the same concentration by Hank’s solution and added into another three Petri dishes the same as above. At the end of the incubation period, the inserts were washed three times with ice-cold PBS. The quantitative analysis of the uptake of NLCs was carried out, and the relative uptake index (RUI%) was calculated as follows:

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RESULTS AND DISCUSSION Characterization of FOL-S100 and ALN-S100. 1H NMR spectroscopy measurements were carried out to identify the conjugation of FOL onto S100 (Figure 2). The hydrogen signals at δ (ppm) 0.8−1.4, δ (ppm) 3.5−3.9, and δ (ppm) 6.0−8.0 were assigned to the aliphatic hydrocarbons, PEG and FOL, respectively. The results primarily indicated that FOL was successfully attached to S100. The conjugation percentage of FOL to S100 was 85.6% on a molar ratio basis which was calculated by determining the amount of FOL conjugated in FOL-S100. The formation of ALN-S100 was monitored and confirmed by 1H NMR spectrum (Figure 2). The spectrum of ALN-S100 exhibited typical peaks of ALN at δ (ppm) 3.0.23 The results primarily indicated that ALN was successfully attached to S100. The conjugation percentage of ALN to S100 was 94.8% on a molar ratio basis. Characterization of NLCs. The particle size, polydispersity index (PI), zeta potential, and EE% are listed in Table 1. Figure 3 shows the AFM of MTO-NLCs and ALN-FOL-MTO-NLCs. Some NLCs showed irregular shapes, which might be caused by lipid spreading or flattening of NLCs onto the mica surface and fusion process during sample preparation.24,25 The modification of ALN-S100 led to the increasing of zeta potential due to the ionized ALN on the surface of NLCs. At the same time, the modification of FOL-S100 or ALN-S100 resulted in a decrease of the particle size as compared with unmodified NLCs, which might be due to the increase of surface-active effect with mixing surfactant.26 In addition, the PEG chains of these systems could act as a tighter net on the outside of vesicles, thus limiting the increase of vesicle size.27 For the preparation of FOL-MTONLCs and ALN-FOL-MTO-NLCs, NaHCO3 was added into the hydration medium. Valencia and colleagues prepared PLGA-PEG-FOL nanoparticles and found that only 20% of the FOL was present on the nanoparticle surface, while the rest remained presumably buried in the PLGA nanoparticle core due to hydrophobic interactions of PLGA and FOL.28 NaHCO3 could increase the hydrophilic property of FOL and the distribution on the NLC surface due to the higher solubility of FOL in weak basic solution. In this study, the size of MTO loaded NLCs was set at about 50 nm; some of the reasons could be briefly stated as follows. The size of the nanoparticles was shown to have a substantial effect on the protein absorption. It has been consistently shown that

RUI% = (UIs /UIc) × 100%

where UIs stands for the uptake index of the cells treated with mixed system, while UIc stands for the uptake index of the cells treated with NLCs. The ALN-FOL-C6-NLC/HA mixed system was filtrated by microporous membrane (0.45 μm) at the end of the incubation period (6 h), the desorption ratio of NLCs was calculated. The relative uptake index and desorption ratio were determinated the same as above at concentrations of 400, 200, 100, and 50 ng C6/mL, respectively. Ex Vivo Imaging (Leukemia Mice) and in Vivo Antitumor Activity. K562/BALB/c-nu leukemia mouse model was established to investigate the in vivo antitumor activity of ALN-FOL-MTO-NLCs. BALB/c-nu mice pretreated with cyclophosphamide (100 mg/kg) were inoculated subcutaneously with K562 cells (5 × 106 cells/mouse); then the local K562 tumor was taken out and the tumor tissues without necrosis were selected for preparing single cell suspension which was inoculated into BALB/c-nu mice pretreated with cyclophosphamide by intraperitoneal injection (5 × 106 cells/ mouse). On day 9 (the day of tumor inoculated was assigned as day 0), the mice were randomly divided into four groups (n = 3). The control saline or preparations (MTO-INJ, FOL-MTONLCs, and ALN-FOL-MTO-NLCs) were intravenously E

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Figure 4. Binding ability of differently modified NLCs to HA (ALNS100 0−2 μmol/mL, FOL-S100 0−0.4 μmol/mL, n = 3).

flexibility of the PEG chain of FOL-S100 and ALN-S100, which laid a good foundation for the expected dual-targeting efficacies. Quantitative Study of Cells Uptake and Cytotoxicity Study. As shown in Figure 5A, the UI of FOL-C6-NLCs was

Figure 3. AFM images of MTO-NLCs (A) and ALN-FOL-MTONLCs (B). Both of the particles were prepared with similar loading efficiencies of MTO.

pegylated nanoparticles smaller than 100 nm have reduced plasma protein adsorption on their surface and also have reduced the uptake of liver and spleen. Furthermore, although the maximum size of particles that can penetrate through the endothelial fenestrations of renal peritubular capillaries is not exactly known, the pore size seems to be around 5.0−5.5 nm.29 It is also noteworthy that there are fenestrated capillaries or sinusoids in the bone with pore size of 80−100 nm; the extravasation of the bone-targeting NLCs described here into the bone fluid should not be hampered. Therefore, MTO loaded NLCs in this study could pass through the leaky capillary wall in the bone and avoid renal excretion effectively. Finally, folate receptors showed a distinct clustered pattern in “uncoated pits” termed caveolae. Previous studies have shown that nanoparticles smaller than 60 nm can be endocytosed as covalent conjugates of folate at these uncoated pits easily.30 In this research, we chose S100 with PEG molecular weight 4400 g/mol to connect NLCs and targeting moieties. The length and flexibility of the PEG spacer may permit the formation of multiple tethers between a nanoparticle and a folate receptor cluster and the affinity of such multivalent attachments can be orders of magnitude higher than the association of monovalent folate with a single receptor.30 Furthermore, due to the micropore structure of bone surface which poses topographical barriers to NLCs binding, long tethers between ALN and nanoparticle could facilitate the binding to apatite in micropores.31 In Vitro HA Binding Study. The results of the binding of MTO-NLCs modified with different amounts of ALN-S100 (0, 1, 1.5, and 2 μmol/mL) to HA are shown in Figure 4. The efficiency of binding was expressed in % of the original amount of NLCs bound to HA in dependence of time (h). All of the bone-targeted NLCs showed good binding ability to HA, whereas unmodified NLCs yielded only nonspecific binding to HA. As expected, a higher content of ALN-S100 resulted in higher binding than the low content. As shown in Figure 4, the modification of FOL-S100 did not influence the binding ability of ALN-FOL-MTO-NLCs greatly due to the length and

Figure 5. Quantitative study of the uptake by K562 cells. (A) Uptake of C6-NLCs, FOL-C6-NLCs, and ALN-FOL-C6-NLCs without the introduction of ATRA (37 or 4 °C, C6 15−200 ng/mL). (B) Uptake of C6-NLCs, FOL-C6-NLCs, and ALN-FOL-C6-NLCs before or after the introduction of ATRA (37 °C, C6 100 ng/mL). **p < 0.05.

much higher than that of C6-NLCs at both 37 and 4 °C, which might be owing to the FR-selective uptake of K562 cells. At 37 °C,the UI of FOL-C6-NLCs was 3.4 times more than that of C6-NLCs at 100 ng/mL. Cellular uptake efficiency of NLCs was concentration-dependent within 4 h, and the equilibrium was almost achieved when the concentration increased to 100 ng/mL. The weak cellular uptake under 4 °C indicated that cellular uptake of the two NLCs was also energy-dependent, as 4 °C was a low energy incubation condition. It is noted that PEG modification not only reduces the rate of MPS uptake but also weakens the interaction between the nanoparticles and the target cells, which often causes inefficient intracellular delivery.32 As shown in the results, after conjugation with targeting ligands such as FOL, the tumoral uptake of FOL-C6NLCs could be greatly enhanced (p < 0.05). Due to the flexibility of the long PEG chain of S100, as shown in Figure 5A, the modification of ALN-S100 did not influence the F

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selective uptake of ALN-FOL-C6-NLCs, which laid a good foundation for the expected dual-targeting efficacies. Myeloid leukemia cells have previously been reported to up-regulate FRβ in response to ATRA. FOL-C6-NLCs and ALN-FOL-C6NLCs uptake were further increased 3.2-fold and 3.1-fold respectively in K562 cells cultured for 5 days in media containing 1 μM ATRA, presumably because of FR-β induction (Figure 5B). Based on all of these results, the combined administration of ATRA might further enhance the dualtargeting efficacies of ALN-FOL-NLCs. The cytotoxicity of MTO formulations to K562 cells was evaluated by MTT assays (Figure 6). Folate-targeted NLCs

Figure 7. Concentration−time curve of MTO in rat plasma after intravenous administration of MTO-INJ, MTO-NLCs, FOL-MTONLCs, ALN-MTO-NLCs, and ALN-FOL-MTO-NLCs at a dose of 2 mg/kg (n = 6).

Enhanced circulation lifetimes could result in greater tumor sequestration of drugs and, in turn, necessarily result in a corresponding increase in drug efficacy. Figure 8 presents the mean concentrations of MTO in tissues of healthy mice after intravenous injection. The AUC of the bone PK profile of the drug formulated in ALN-FOL-MTONLCs was 3.7-fold and 5.0-fold higher than that of FOL-NLCs and MTO-INJ, respectively. This indicated the accumulation and prolonged residence of ALN-FOL-MTO-NLCs in bone in comparison with FOL-MTO-NLCs and MTO-INJ (p < 0.05). For very small particles, including PEG modified nanoparticles, they can easily penetrate into the bone but can also be easily pushed out from the bone into the blood. Therefore, small particles have good permeability but poor retention.32,33 After conjugation with ALN, the retention of MTO loaded NLCs in the bone could be greatly enhanced. Compared with FOL-MTO-NLCs, ALN-FOL-MTO-NLCs displayed a reduction in drug toxicity because of the reduced accumulation in liver, spleen, heart, and kidney. In turn, this allows administration of larger and more efficacious drug doses. It is known that a high degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the liposomes, which often results in instability of the liposomes and accelerates the drug leakage due to the detergent-like properties of materials such as DSPE-PEG. In this study, trilaurin, cholesterol, and the ion-pair formed by MTO and SDS could form a compact core, which was coated with lipid bilayers. Prophase research has proven that amphiphilic derivatives, such as ALN-S100 or FOL-S100, could insert into the outer leaflet of the inner hydrophobic cores of core−shell structured nanoparticles, resulting in sufficient ALN-S100 or FOL-S100 presenting on the particle surface firmly. This might lead to the abolishment of liver and spleen uptake.32,33 Ex Vivo Imaging and CLSM Imaging (Healthy Mice). Ex vivo fluorescence imaging (Figure 9) of bones suggests that NIRD-NLCs, FOL-NIRD-NLCs, and ALN-FOL-NIRD-NLCs all located mainly in the proximal and distal metaphysis but not the diaphysis. Several important factors related to bone physiology that could influence the success of a pharmacological treatment include heterogeneity in bone remodeling activities throughout the skeleton, differences in blood supply and local vascularization, and the blood−bone barrier.34 Most of the intraosseous vessels in bone distribute in the cancellous bone, so all of the above NLCs located mainly in the epiphysis and metaphysis but not in the diaphysis. As shown in Figure 9, compared to NIRD-NLCs and FOLNIRD-NLCs, the modification of ALN-S100 significantly

Figure 6. In vitro cytotoxicity test (K562 cells) of MTO-INJ and MTO-NLCs with different concentrations (n = 3). (A) Cells untreated with ATRA and (B) cells pretreated with ATRA.

showed significantly higher cytotoxicity than nonfolate-targeted NLCs and MTO-INJ, especially when the cells were pretreated and the receptors were up-regulated by ATRA, which corresponded to their cellular uptake rate. In uninduced K562 cells, the IC50 of MTO-INJ, MTO-NLCs, FOL-MTONLCs, and ALN-FOL-MTO-NLCs were 13.75 μg/mL, 10.79 μg/mL, 3.76 μg/mL and 4.99 μg/mL, respectively. In introduced K562 cells, the IC50 of MTO-INJ, MTO-NLCs, FOL-MTO-NLCs and ALN-FOL-MTO-NLCs were 11.08 μg/ mL, 8.355 μg/mL, 0.88 μg/mL, and 1.05 μg/mL, respectively. Pharmacokinetic and Biodistribution Studies in Healthy Animals. Figure 7 depicts the plasma concentration−time profiles after intravenous injection in rats. The AUC of the blood PK profile of the drug formulated in MTONLCs, FOL-MTO-NLCs, ALN-MTO-NLCs, and ALN-FOLMTO-NLCs were 12.5, 18.7, 46.2, and 63.1 times that of the free drug. The MRT of the blood PK profile of the drug formulated in MTO-NLCs, FOL-MTO-NLCs, ALN-MTONLCs, and ALN-FOL-MTO-NLCs were 2.31, 3.59, 5.88, and 6.45 times that of the free drug (Table 2). Comparing the pharmacokinetic parameters of NLCs and modified NLCs, an even higher MRT and AUC were observed for the latter, confirming the protective effects of FOL-S100 and ALN-S100. G

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Table 2. Main Pharmacokinetic Parameters of MTO in Rats after iv Administration of MTO-INJ, MTO-NLCs, FOL-MTONLCs, ALN-MTO-NLCs, and ALN-FOL-MTO-NLCs (n = 6)a

a

preparations

MTO-INJ

MTO-NLCs

FOL-MTO-NLCs

ALN-MTO-NLCs

ALN-FOL-MTO-NLCs

AUC (mg/mL·h) t1/2α (h) t1/2β (h) CL (mL/h·10−2) MRT (h) Vc (L/kg·10−2)

1.18 ± 0.15 0.11 ± 0.02 1.53 ± 0.20 33.85 ± 1.67 1.66 ± 0.18 17.22 ± 2.16

14.71 ± 0.64* 0.71 ± 0.07* 9.04 ± 1.12* 2.72 ± 0.11* 3.83 ± 0.25* 3.54 ± 0.51*

22.03 ± 1.11* 1.09 ± 0.07* 13.13 ± 1.15* 1.81 ± 0.09* 5.96 ± 0.62* 3.47 ± 0.18*

54.55 ± 5.19** 1.54 ± 0.17** 14.41 ± 1.15* 0.65 ± 0.05** 9.79 ± 0.51** 2.31 ± 0.13**

74.46 ± 3.95** 1.68 ± 0.16** 14.53 ± 1.22* 0.54 ± 0.03** 10.77 ± 1.07** 2.19 ± 0.10**

Data are represented as mean ± SD (n = 6). *P < 0.05, versus MTO-INJ; **P < 0.05, versus MTO-INJ, MTO-NLCs, and FOL-MTO-NLCs.

Figure 8. Biodistribution profile of MTO-INJ, FOL-MTO-NLCs, and ALN-FOL-MTO-NLCs after intravenous injection at a dose of 10 mg/kg (healthy mice). *p < 0.05.

bone surrounded by highly vascularized red marrow compared with bone adjacent to less vascularized yellow (fatty) marrow because osteoblasts at bone sites with yellow marrow may receive a relatively poor nutrient supply. At the same time, we found that the distribution of NIRD was much more in the proximal metaphysis than in the distal metaphysis. At birth, all marrow is hematopoietic. With aging, there is progressive fatty replacement. In the long bones, the replacement starts first in the diaphyses with relative preservation of metaphyseal hematopoiesis. At the end of adolescence, red marrow remains in the proximal metaphysis of the femur and hemerus as well as in the vertebral bodies, sternum, ribs, and skull,36 so ALN-FOLNIRD-NLCs distributed differently between the proximal metaphysis and the distal metaphysis. Tetracycline exhibits an specific affinity to the bone calcium and can deposited at the mineralization front. Figure 10 shows that both tetracycline (yellow) and ALN-FOL-NIRD-NLCs (blue) distributed on areas of high bone turnover, but NIRDNLCs and FOL-NIRD-NLCs (blue) had no specific distribution on these areas. All of these results further confirmed that ALN-FOL-NLCs could favorably deposit to the osteoblastenriched areas of high bone turnover in regions of trabecular bone surrounded by red marrow. Considering that a lot of evidence have accumulated to indicate that MRL distribute chiefly in the red bone marrow and LSCs in the high calcium turnover areas, ALN-FOL-NLCs loading anticancer drug,

Figure 9. Ex vivo imaging of healthy mice femurs at different time after intravenous administration of NIRD-NLCs, FOL-NIRD-NLCs, and ALN-FOL-NIRD-NLCs at a dose of 0.1 mg/kg.

increased the distribution of NLCs in the proximal and distal metaphysis where red marrow is located, and the origins can be interpreted as follows. Intrabone distribution of bisphosphonates is not homogeneous. Generally, bisphosphonates tends to preferentially distribute in the part of bones where mineral density or calcium turnover rates are higher.35 Differences in local vasculature, such as that existing between red and yellow marrow, can have a profound influence on local bone metabolism. Bone turnover is higher in regions of trabecular H

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could implement the accumulation in the target tissue and selective uptake by target cells. For various bone diseases, previous researches mainly aimed at the direct modification of drugs by bone-seeking compounds. It is regrettable that most of these bone-targeting carriers to date do not include a drug release mechanism. After adsorption to bone, these carriers may remain for a long time and may even be embedded into newly formed bone. In this study, ALN-FOL-NLCs could desorb from the bone surface moderately due to their heavy weight, the desorption of ALN from bone surface or the detachment of ALN-S100 from NLCs and the encapsulated drug in the interior of NLCs could be released freely.37 Ex Vivo Imaging (Leukemia Mice) and in Vivo Antitumor Activity. Ex vivo fluorescence imaging of tissues (Figure 12) suggests that, in contrast with NIRD-NLCs, FOLNIRD-NLCs displayed an increasing accumulation in bone because of the folate receptor-mediated specific endocytosis.

Figure 10. Different in vivo cancellous bone-binding abilities of NIRDNLCs (A), FOL-NIRD-NLCs (B), and ALN-FOL-NIRD-NLCs (C). Yellow and blue fluorescence stood for tetracycline and NIRD loaded NLCs, respectively (healthy mice, bar = 500 μm).

especially drug having stronger toxicity to MRD and LSCs such as bortezomib, should achieve a much better effect compared to normal NLCs. Dual-Targeting Effects Study. A K562 cells/HA model was used for evaluating the dual-targeting effects with the results shown in Figure 11. For a given content of HA, 14−24%

Figure 12. Ex vivo imaging of leukemia mice tissues 2 h after intravenous administration of NIRD-NLCs, FOL-NIRD-NLCs, and ALN-FOL-NIRD-NLCs at a dose of 0.1 mg/kg.

Figure 11. Relative uptake index and desorption ratio of ALN-FOLC6-NLCs (n = 3).

Compared to NIRD-NLCs, FOL-NIRD-NLCs and ALNFOL-NIRD-NLCs displayed a reduced distribution in heart and kidney and an increasing accumulation in liver and spleen due to the selective infiltration of leukemia cells in liver and spleen. As is shown in Figure 13, in vivo antitumor activity confirmed the better tumor inhibition efficacy of ALN-FOL-MTO-NLCs. In the control saline group, the count of peripheral blood white blood cells (WBCs) was 6−7 times as much as that before inoculation and the bone marrow WBCs accounted to 70−80% on average. Histopathology examination revealed a large number of leukemia cell infiltration in liver and spleen. Compared with MTO-INJ and FOL-MTO-NLCs, the treatment of ALN-FOL-MTO-NLCs could further eliminate the residual leukemia cells in peripheral blood and bone marrow and significantly reduced the WBCs number to a normal level. At the same time, in the ALN-FOL-MTO-NLCs group, histopathology HE staining showed a slight infiltration of leukemic cells in liver and spleen. This improved antitumor efficacy was due to its great binding affinity to hydroxyapatite and higher cytotoxicity in K562 cells.

of the NLCs could desorb from HA powder, the RUI% decreased with the decreasing of the C6 content and still maintained a considerable level at the lowest C6 content. The experiment tried to simulate the process of ALN-FOL-C6NLCs content decreasing with the desorption from bone surface and the uptake by leukemia cells until exhausted. Considering that extracellular fluid in the marrow was flowable and the uptake of leukemia cells could facilitate the desorption of ALN-FOL-NLCs, in vivo, most of the ALN-FOL-NLCs binding on bone inner surface could desorb and be taken up by leukemia cells. This assay indicated that ALN-FOL-NLCs exhibited dualtargeting effects, and the likely mechanisms could be explained by the following three steps: (1) as a major grade I targeting ligand, ALN mediated the binding of ALN-FOL-NLCs on the bone surface; (2) ALN-FOL-NLCs desorbed from the bone surface; (3) FOL initiated the endocytosis of ALN-FOL-NLCs via recognition of the folate receptor on the surface of leukemia cells as a major grade II targeting ligand. The method suggested by us is a cascade targeting delivery system and could be referred to as “multistage rockets”. These multistage rockets I

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Figure 13. Blood and bone marrow smears and tissue sections of BALB/c-nu mice after intravenous application of different preparations. Purple (blood and bone marrow) or deep purple (liver and spleen) stood for WBCs. (A) Untreated healthy mice, (B) leukemia mice treated with saline, (C) leukemia mice treated with MTO-INJ, (D) leukemia mice treated with FOL-MTO-NLCs, (E) leukemia mice treated with ALN-FOL-MTONLCs (n = 3).

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CONCLUSIONS

ACKNOWLEDGMENTS We thank Dr. Sulieman Eltayeb for writing improvement. This work was financially supported by National Natural Science Foundation of China (No. 81273467).

MTO loaded nanocarrier with ALN and FOL as dual-targeting ligands on the periphery was successfully developed in a simple manner. With two targeting ligands on the surface, ALN-FOLNLCs could achieve bone transport and leukemia cells targeting in turn. The length and flexibility of the PEG chains connecting NLCs and targeting moieties ensured the dualtargeting efficiency without mutual interference. All of the undergoing work revealed a novel and promising therapeutic method for the leukemia and relapsed leukmia, which might be very interesting to the patients who suffered from the disease. The design of step targeting and the results might be beneficial not only to the research of drug delivery but also to the area of leukemia research.

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ASSOCIATED CONTENT

* Supporting Information S

Schemes for the synthesis of FOL-S100 and ALN-S100, figure of quantitative study of the uptake of DOX-NLCs, FOL-DOXNLCs, and ALN-FOL-DOX-NLCs, figure of in vitro release of MTO from MTO-NLCs and ALN-FOL-MTO-NLCs in serum, figure of the FT-IR spectra of S100, FOL, ALN, and FOL-S100. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Q.P.: Tel./fax: +86 25 83271092; e-mail address: [email protected]. L.Z.: Tel./fax: +86 25 83271317; email address: [email protected]. Notes

The authors declare no competing financial interest. J

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