Article pubs.acs.org/molecularpharmaceutics
Development of Synthetic Peptide-Modified Liposomes with LDL Receptor Targeting Capacity and Improved Anticancer Activity Mei Liu,† Wei Li,† Caroline A. Larregieu,‡ Meng Cheng,† Bihan Yan,† Ting Chu,† Hui Li,§,∥ and Sheng-jun Mao*,† †
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education & West China School of Pharmacy, Sichuan University, No.17, Section 3, Southern Renmin Road, Chengdu 610041, P. R. China ‡ Department of Bioengineering and Therapeutic Sciences, University of CaliforniaSan Francisco, San Francisco, California 94143-0912, United States § Department of Hematology, Sichuan Academy of Medical Sciences & Sichuan Provincial People Hospital, Chengdu 610072, P. R. China ∥ Department of Hematology, West China Hospital & State Key Laboratory of Biotherapy, Sichuan University, Chengdu, Sichuan 610041, P. R. China
ABSTRACT: In this study, we report an active targeting liposomal formulation directed by a novel peptide (AA13) that specifically binds to the low density lipoprotein receptor (LDLR) overexpressed on acute myeloid leukemia (AML) cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of AA13-anchored liposomes on AML cells. AA13 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a postinsertion method. To study the effect of the peptide decoration and density on tumor cell targeting and internalization by AML cells (THP-1 and NB4), stealth liposomes bearing 3% (peptide/S100PC, molar ratio, LL) and 7% (peptide/S100PC, molar ratio, HL) AA13 were prepared, respectively. Higher uptake of LL (1.9- and 2.6-fold) and HL (2.3- and 3.6-fold) targeted liposomes occurred in THP-1 and NB4 cells, respectively, compared to untargeted liposomes. An LDLR inhibitor was used to confirm inhibition of the receptor-mediated cellular association of AA13 modified liposome in both cells. Daunorubicin (DNR) demonstrated a 2.2- and 3.5-fold higher cytotoxicity with the HL formulation and a 1.2- and 2.0-fold higher cytotoxicity with the LL formulation compared to the unmodified liposomal formulation in THP-1 and NB4 cells, respectively. Tumor drug accumulation of DNR-loaded HL was greater than that of the untargeted liposome in the biodistribution assay. The in vivo efficacy study in BALB/c nude mice bearing NB4 xenografts treated with DNR loaded HL also showed more tumor volume inhibition and a longer survival time compared to the untargeted formulation. In conclusion, the AA13-anchored liposomes demonstrated desirable potential as a promising vector for enhanced AML tumor drug targeting. KEYWORDS: low density lipoprotein receptor (LDLR), drug targeting, liposome, acute myeloid leukemia (AML), daunorubicin
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INTRODUCTION Acute myeloid leukemia (AML) is a type of hematological disease characterized by malignant proliferation, differentiation block, and apoptosis inhibition of abnormal hematopoietic stem and progenitor cells. Although daunorubicin (DNR)-based chemotherapy has been used extensively in the treatment of AML and proven to be one of the most effective induction treatments for over three decades, there is still concern of its severe side effects, relapse, and low cure rate.1,2 The nonspecific © 2014 American Chemical Society
drug distribution, which induces substantial toxicity in normal cells especially rapidly dividing cells such as bone marrow cells and hair follicles, is responsible for severe side effects such as myelosuppression and cardiotoxicity. To reduce these effects, Received: Revised: Accepted: Published: 2305
December 18, 2013 March 30, 2014 May 15, 2014 May 15, 2014 dx.doi.org/10.1021/mp400759d | Mol. Pharmaceutics 2014, 11, 2305−2312
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efforts have been made to load DNR into liposomal vehicles and provide evidence that liposomal DNR may be an attractive formulation for the clinical treatment in acute leukemia.3−5 It is well-known that stealth/PEGylated liposomes exhibit a passive targeting mechanism and accumulate into tumor sites via enhanced permeability and retention (EPR) effects. Most solid tumor diseases have special pathophysiological features such as high vascular density and permeability, poor structural integrity, and lymphatic reflux disorder. All these features are the prerequisites for the occurrence of the EPR phenomenon. However, AML is a hematologic malignancy where PEGylated liposomes may not work fully to achieve specific distribution. Therefore, to enhance selective delivery of encapsulated cargo to AML cells and reduce nonspecific interaction of drug carriers, an active targeting technique is ideal. Active targeting, which has recently gained growing popularity, is capable of improving the homing of drug carriers to tumor sites and enhancing the selective delivery of anticancer drugs to tumor cells. Active targeting technologies exploit the overexpression of surface receptors on cancer cells with which a targeting ligand can interact, and ligand (e.g., antibody, peptide, transferrin, and folate) modification further improves the therapeutic index of the capsulated anticancer drugs in turn.6−9 In targeting treatment for AML, Gemtuzumab Ozogamicin, which is a calicheamicin-conjugated humanized anti-CD33 monoclonal antibody, was developed to target the CD33 antigen expressed on approximately 90% of AML myeloblasts and received accelerated approval by the U.S. FDA. Unfortunately, Pfizer Inc. announced the voluntary withdrawal from the U.S. market at the request of the FDA due to concerns about the product’s safety.10−13 Though studies afterward have proven the safety and efficacy of Gemtuzumab Ozogamicin, it is still controversial to list it as a standard treatment of AML. Therefore, other targeting strategies for the treatment of AML are desirable.14,15 Early studies have shown that leukemia cells from patients with AML have an elevated uptake (3 to 100-fold higher) of low density lipoprotein (LDL) compared to normal white blood cells from healthy individuals.16,17 Neoplasm cells depend either on abundant endogenous LDL synthesis or receptor-mediated endocytosis of LDL from plasma.18 Researchers have confirmed that more than 90% of the cholesterol input is derived from receptor-mediated degradation of LDL in AML cells and that the expression of the LDL receptor (LDLR) on leukemia cells is much more than that of normal cells.19−22 Therefore, it appears that LDLR is a potential molecular target for the selective delivery of antitumor agents to AML cells. Great efforts have also been made to elucidate the mechanistic interaction between LDL and LDLR. Up until now, it is well-known that apolipoprotein B-100 (apoB) incorporated on the native LDL particle surface plays a crucial role in receptor recognition and binding. Further primary structure studies of apoB have revealed that the binding site for LDLR is located between the 3359−3369 residues.23,24 The feasibility of developing synthetic LDL particles using a lipid emulsion and LDLR-binding domain containing peptides as a replacement for serum in the U937 cell culture media has been examined.25,26 Nikanjam and co-workers have developed a synthetic nano-LDL bearing a bifunctional peptide, which is made up of 29 amino acids and confirmed its practicability of delivering paclitaxel oleate to glioblastoma multiforme cells via LDLR.27,28
Given the potential for synthetic peptide binding to LDLR, which is highly expressed on AML cells, and a liposomal formulation of DNR as an attractive candidate for the AML treatment, we engineered and propose a novel synthetic LDLRtargeting peptide-modified liposomal vehicle with a payload of DNR. We evaluated its targeting capacity against AML cells and in vivo therapeutic efficacy, which support that this novel formulation can provide a potential strategy for the targeting treatment of AML.
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EXPERIMENTAL SECTION Materials and Animals. Soybean phosphatidylcholine (S100PC) was purchased from Lipoid KG (Ludwigshafen, Germany). Cholesterol (CHOL), poly(ethylene glycol)-distearoylphosphoethanolamine (mPEG2000-DSPE), and maleimide-derivatized DSPE-PEG2000 (Mal-PEG-DSPE) were obtained from Avanti Polar Lipids (USA). AA13 Peptide (Cys-GlyThr-Thr-Arg-Leu-Thr-Arg-Lys-Arg-Gly-Leu-Lys-Leu, MW 1602.98) was synthesized by Chutai Biotechnology Co. Ltd. (Shanghai, China) with greater than 95% purity determined by HPLC. Daunorubicin was purchased from Shanghai Baili Biotechnology Co. Ltd. (Shanghai, China) with greater than 97% purity. Coumarin-6, streptomycin, and penicillin were purchased from Sigma-Aldrich Chemical Co. (USA). All other chemical reagents used were of analytical grade or better. Five-week-old BALB/c nude mice weighing 20−25 g were kindly provided by Laboratory Animal Center of Sichuan University (Sichuan, China) and housed in a pathogen free environment for 1 week before the commencement of experiments. All experiments were conducted according to protocols approved by the University Ethics Committee for the Use of Laboratory Animals and in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Cell Lines. The human acute promyelocytic leukemia cell line, NB4, and acute monocytic leukemia cell line, THP-1, which are subtypes of AML cells, were purchased from ATCC (Manassas, USA) and cultivated in RPMI 1640 solution with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 incubator at 37 °C. For most experiments, cells were also treated with a 24 h fetal bovine serum-free cultivation medium during the exponentially growing phase to approximately 70% confluence to activate LDLR activity in vitro. Vehicle Preparation. Synthesis of AA13-PEG-DSPE. The AA13-PEG-DSPE peptide−lipid conjugate was prepared as previously reported.29 Briefly, Mal-PEG-DSPE was dissolved in chloroform and evaporated at 60 °C under vacuum for several hours to form a thin lipid film. Two milliliters of peptide PBS solution (peptide/Mal-PEG-DSPE, 1:2, molar ratio) was added to the flask, shaken well, and incubated overnight at room temperature so that the terminal cysteine of the peptide chain could be covalently conjugated to the maleimide group of MalPEG-DSPE specifically and efficiently.30 Unreacted peptide was removed by dialyzing with distilled water for 24 h. The conjugation of the peptide to Mal-PEG-DSPE was confirmed by RP-HPLC. Detection was performed at 220 nm using a Kromasil 100−5C18 column. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (solution A) and 0.1% TFA in acetonitrile (solution B) and was performed at a flow rate of 1 mL/min. The mobile phase was programmed with the following four steps: (1) 15% solution A for 1 min, (2) linear gradient from 15% to 35% solution A in 25 min, (3) linear 2306
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to qualitatively study cellular uptake (CFM-500, Changfang Optical Instrument Co., Ltd. Shanghai, China). In Vitro Cytotoxicity Study. The cytotoxicity of various liposomal DNR formulations were examined in AML cells (NB4 and THP-1). Briefly, 100 μL cell suspensions (1× 105 cells/mL) were added to each well of a 96-well plate. Cells were exposed to either free DNR or nontargeted or targeted liposomes loaded with DNR and incubated at 37 °C. Liposomal samples were diluted with FBS-free medium and adjusted to serial concentrations of the entrapped DNR. The culture medium was removed at 4 h and replaced with equal volume of fresh medium. After 24 h, 20 μL of 5 mg/mL MTT solution was added to each well and further incubated for 4 h. Finally, 100 μL of formazan-dissolving buffer consisting of 10% sodium dodecyl sulfonate, 5% isobutanol, and 0.01 M hydrochloric acid was introduced to each well and the absorbance was read at 570 nm using a microplate reader (Bio-Rad, Richmond, CA, USA). In Vivo Therapeutic Efficacy and Tissue Biodistribution Assay. The AML tumor model was established as previously reported.31,32 Four hundred microliters of 5 × 106 NB4 cells in matrigel were subcutaneously injected into the back of mice, which were randomly assigned to three groups (saline group, untargeted liposome group, and HL group) with 6 to 8 mice per group. When tumor sizes were approximately 0.1 cm3 on day 10 postinoculation, the animals were treated with four doses of equivalent 2.5 mg/kg DNR concentrations by tail vein injection every 5 days. Tumor volumes were measured twice a week and monitored for up to 27 days since the first injection and calculated using the formula V = 0.5ab2, where a and b represent the longest and shortest diameters, respectively. To assay biodistribution, mice were treated with one dosage of equivalent 10 mg/kg DNR concentrations. Briefly, the tumor and tissues (heart, liver, spleen, kidney, and lung) were excised 24 h after injection and processed. Four hundred microliters of acetonitrile was added to 200 μL of tissue homogenate. The mixture was then vortexed and centrifuged at 3000g for 10 min. The supernatant was transferred to a new tube containing 3 mL of chloroform, vortexed, and then centrifuged at 3000g for 5 min. The chloroform layer was carefully collected and dried under nitrogen. Finally, the residue was reconstituted in 100 μL methanol and analyzed for DNR content via HPLC. A standard curve prepared using blank tissue spiked with known DNR concentrations was used for content determination. The HPLC conditions included a Diamonsil-C18 column (150 × 4.6 mm, 5 μm, Dikma Technology Co., Ltd. Beijing, China), a mobile phase composed of sodium dodecyl sulfate/acetonitrile/ methanol (47:47:6), a 1 mL/min flow rate, a 481 nm detection wavelength, a 20 μL injection volume, and a 30 °C column temperature.
gradient from 35% to 100% solution A in 5 min, and (4) 100% solution A for 5 min. The concentration of unreacted peptide was calculated based on a calibration curve of known concentrations of peptide solutions. Preparation of Liposomes. Daunorubicin (DNR)-loaded liposomes were prepared using an ammonium sulfate gradient method. Briefly, S100PC/CHOL/mPEG2000-DSPE (4:1:0.2, molar ratio) was dissolved in chloroform and evaporated at 60 °C under vacuum. The formed thin film was then hydrated with 0.25 M ammonium sulfate at 60 °C. The obtained large unilamellar vesicles were subjected to probe sonication and dialyzed against 9% sucrose solution for 24 h using a dialysis bag (MWCO, 7000). The resultant liposome was mixed with daunorubicin sucrose solution and incubated at 60 °C for 2 h. Untrapped drug was removed using a Sephadex G-50 column. Coumarin-6 loaded liposome was also prepared. S100PC/ CHOL/mPEG2000-DSPE (4:1:0.2, molar ratio) and coumarin-6 were dissolved in chloroform, evaporated, and hydrated with PBS. Particle size was adjusted by probe sonication, and the liposome was stored at 4 °C away from light. A postinsertion method was applied to prepare targeted liposomes of different peptide densities. For the preparation of 3% or 7% (peptide/S100PC, molar ratio, namely, LL or HL, respectively) AA13-modified liposomes, 2 mL of AA13-PEGDSPE stock solutions containing 1.0 or 2.4 mg/mL AA13 was added to equal volumes of preformed stealth liposomes, mixed gently, and placed at room temperature overnight. The peptidelipid conjugate was incorporated onto the liposomal surface via hydrophobic DSPE domains. Unattached peptide-PEG-DSPE was removed by passing the resultant dispersion through a Sephadex G-50 column with pH 7.4 phosphate buffer. The size of the liposomes was measured by dynamic laser scattering (DLS) (Malvern Zetasizer Nano ZS90, UK). The amount of trapped DNR within the liposomal formulation was determined by HPLC (Agilent 1100, USA). In Vitro Release Study. The in vitro release of DNR from each liposomal formulation was studied using the dialysis method. Unmodified liposomes, liposomes bearing 3% AA13 (peptide/S100PC, molar ratio, LL), and liposomes bearing 7% AA13 (peptide/S100PC, molar ratio, HL) containing equivalent 1.5 mg/mL DNR concentrations were placed in dialysis bags (MWCO, 6000). Dialysis bags containing 2 mL of liposomes were immersed in 600 mL pH 7.4 phosphate buffer at 37 °C and shaken at 150 rpm. At fixed time intervals, 1 mL of the dialysis medium was taken from the outside of dialysis bag and replaced with an equal volume of fresh medium. The collected samples were filtered through a 0.22 μm membrane, and the DNR contents were determined. In Vitro Cell Uptake Study. Cellular uptake studies for different liposomal formulations were carried out in AML cells (NB4 and THP-1). Cells at their exponentially growing phase were treated with fetal bovine serum-deficient medium for 24 h before the experiment and seeded into 16-well plates at a density of 5 × 106 cells per well. Liposomes (unmodified, LL, or HL) containing equivalent 2.5 μg/mL coumarin-6 concentrations were added to and incubated with cells for 4 h at 37 °C. For competitive binding studies, 10 μL of 5 mM suramin, an inhibitor of the LDL receptor, was added to each well 2 h before the addition of liposomes. At the conclusion of each uptake study, cells were collected and washed thrice with ice-cold PBS. The fluorescence intensity of cell-associated coumarin-6 was quantified using flow cytometry (BD FACSCalibur, America). A fluorescence microscope was used
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RESULTS AND DISCUSSION Synthesis of AA13-PEG-DSPE. AA13-PEG-DSPE was synthesized by reaction of AA13 peptide with Mal-PEG-DSPE in PBS (pH 7.4). The conjugation was confirmed by RP-HPLC. AA13 eluted at a retention time of 13.1 min. Free peptide was removed after the completion of the reaction by extensive dialysis. The AA13 conjugation efficiency was ∼91%. The high conjugation efficiency of the peptide to Mal-PEG-DSPE is likely due to the high reactivity between maleimide and cysteine.33 Physicochemical Properties of DNR-Loaded Liposomes. The particle size and polydispersity index (PDI) of 2307
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Table 1. Characteristics of the Liposomal DNR Formulations (n = 3) formulation
peptide content (% AA13/S100PC)
mean particle size (nm)
PDI value
encapsulation efficiency (EE)
unmodified liposomes AA13-modified liposomes (LL) AA13-modified liposomes (HL)
0 3 7
98 ± 3.5 95 ± 1.4 102 ± 1.9
0.179 ± 0.04 0.172 ± 0.01 0.158 ± 0.02
96.4 ± 0.26 95.3 ± 0.32 94.8 ± 0.41
liposomal samples were measured via a nanoparticle analyzer at a fixed 90° angle at 20 °C with results given in Table 1. Targeted liposomes (LL and HL) had a mean particle size about 100 nm, similar to that of the nontargeted liposome. All of the liposomal samples had a narrow PDI value, indicating homogeneity in their dispersion state. The encapsulation efficiency (EE) of DNR in the liposomes determined by HPLC ranged from 94% to 97%, indicating that peptide modification did not affect DNR loading into the liposome. In Vitro DNR Release Study. The release profile of DNR from nontargeted and targeted liposomal samples in pH 7.4 phosphate buffer showed that neither peptide modification nor density affected the rate and extent of DNR release from prepared liposomes (Figure 1). After 36 h, 32.5% and 34.2% of
Figure 2. Fluorescence images of the cellular uptake of coumarin-6 loaded liposomes by NB4 cells (a, nontargeted liposomes; b, LL; and c, HL) and THP-1 cells (d, nontargeted liposome; e, LL; and f, HL) without suramin after 4 h incubation. Abbreviations: LL, 3% AA13modified liposomes; HL, 7% AA13-modified liposomes.
Figure 1. Release profiles of DNR from different liposomal formulations in PBS (pH 7.4) at 37 °C. Each point represents mean ± SD (n = 3). Abbreviations: LL, 3% AA13-modified liposomes; HL, 7% AA13-modified liposomes.
HL were 2.6- and 3.6-fold higher, respectively, than that of nontargeted liposomes in NB4 cells, exhibiting AA13 densitydependent uptake. Increasing peptide density also increased the cellular association in THP-1 cells with 1.9- and 2.2-fold higher uptake of LL and HL than that of nontargeted liposome. We found that the uptake of targeted liposomes in NB4 cells was greater than that in THP-1 cells, which may be attributed to differences in LDLR expressed on different AML cells. When endogenous LDL binds to LDLR, they become internalized via endocytosis, and a drop in pH in endosome allows the receptor to dissociate from LDL. The receptor is recycled back to the surface of cells, while LDL becomes degraded by the lysosome.35 It has been reported that the more receptors expressed on AML cells, the less time is needed for LDLR to recycle back to cell surface and thus the greater the uptake of LDL by these cells.36 Our engineered liposome decorated with a LDLR-binding domain mimics plasma-derived LDL and is capable of targeting LDLR on AML cells. The greater uptake of LL and HL in NB4 cells than in THP-1 cells likely resulted from the greater expression of LDLR in NB4 cells than THP-1 cells. To confirm whether the increased cellular uptake of AA13modified liposomes was due to the presence of excess surface
the DNR content were released into dialysis medium from the LL and HL formulations, respectively, which were not significantly different from the nontargeted liposome that released 36.4% of its DNR content under the same conditions. The utilization of the ammonium sulfate gradient method may explain the low release rate of DNR from prepared liposomes. The ammonium sulfate gradient method has been shown to generate an aggregated gel-like state with DNR at the inner phase of liposomes, resulting in efficient DNR encapsulation and slow DNR dissolution and release.34 Cellular Association Study. In our study, NB4 and THP-1 cells could endocytose coumarin-6-loaded nontargeted and targeted liposomes with the density of the conjugated peptide being an important factor affecting liposomal uptake. Figure 2 depicts qualitatively the cellular association of the liposomes by NB4 and THP-1 cells. The fluorescence intensities of the AA13modified liposomes were greater than that of the nontargeted liposome, indicating enhanced accumulation by the peptide in vitro. Figure 3 provides quantitative results that are consistent with the qualitative results. The cellular association of LL and 2308
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Figure 3. Quantitative results of the cellular uptake of coumarin-6 loaded liposomes by NB4 cells (a, nontargeted liposomes; b, LL; and c, HL) and THP-1 cells (d, nontargeted liposome; e, LL; and f, HL) with (+) or without (−) suramin after 4 h incubation. (g,h) Summary of cellular association in NB4 and THP-1 cells, respectively. Each bar represents mean fluorescence intensity ± SD (n = 3). Abbreviations: LL, 3% AA13-modified liposomes; HL, 7% AA13-modified liposomes.
LDLR, a competitive binding experiment was conducted, in which the cellular association was evaluated in the presence of suramin, an LDLR inhibitor. Figure 3 reveals that the presence of the inhibitor significantly suppressed the cellular association of targeted liposomes in NB4 and THP-1 cells. The uptake of
nontargeted liposome was low irrespective of the presence of inhibitor. Both of these results confirm a LDLR-mediated endocytosis mechanism of AA13-decorated liposome. In Vitro Cytotoxicity Study. The effect of peptide decoration and content on liposomal DNR cytotoxicity was 2309
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evaluated in both NB4 and THP-1 cells after 24 h incubation using an MTT assay. The AA13-modified liposomes exhibit higher cytotoxicity (i.e., lower IC50) than the unmodified liposome (Table 2). Compared to unmodified liposome with Table 2. IC50 Values of Liposomal DNR Formulations in NB4 and THP-1 Cells after 24 h Incubation IC50 (μM) formulation unmodified liposomes AA13-modified liposomes (LL) AA13-modified liposomes (HL)
peptide content (% AA13/S100PC)
NB4
THP-1
0
0.646 ± 0.09
0.757 ± 0.13
3
0.329 ± 0.04
0.631 ± 0.07
7
0.186 ± 0.02
0.344 ± 0.11
Figure 4. Mean tumor volumes in BALB/c nude mice bearing NB4 xenografts treated for intravenous injections given on the 10th, 15th, 20th, and 25th day of 2.5 mg/kg DNR equivalent liposomal formulations or saline. Each point represents the mean tumor volume (n = 6−8) ± SD. * means that it is statistically significantly different from the unmodified group (ANOVA, p < 0.05). Abbreviations: HL, 7% AA13-modified liposomes.
DNR IC50 values of 0.757 ± 0.03 μM in THP-1 cells and 0.646 ± 0.04 μM in NB4 cells, targeted liposome bearing 7% AA13 (HL) showed 2.2- and 3.5-fold higer cytotoxicity in THP-1 and NB4 cells, respectively, while 3% AA13 modified liposome (LL) showed 1.2- and 2.0-fold higher DNR cytotoxicity, respectively. The cytotoxicity results are in agreement with the cellular association findings and validate the capacity of AA13 to exert active LDLR targeting. It is well-known that cancer targeted formulations, such as Herceptin and Mabthera, exploit biologically active targeting ligands. Apart from their active targeting properties, these ligands also possess intrinsic cytotoxicity and are capable of interfering with cell signaling pathways. For example, D-(KLAKLAK)2, which was originally designed as an antibacterial peptide, interrupts the negatively charged mitochondrial membrane and induces cell death through a mitochondrial-dependent apoptosis manner when internalized within cells.37 It is possible that the improved cytotoxicity of AA13-modified liposomal DNR may be attributed to the intense interaction between LDLR and AA13, which is of crucial significance to mediate sufficient receptor-dependent endocytosis and improve the cytotoxicity of liposomal DNR. In Vivo Therapeutic Efficacy and Tissue Biodistribution Assay. The in vivo anticancer activities of developed liposomal formulations following intravenous multiple dosing were assessed using NB4 subcutaneous xenografts in BALB/c nude mice. Even though the organ distribution of the disease differs as it is in humans, this model provides various advantages such as the formation of visible tumors, which allows for the evaluation of tumor growth inhibition and biodistribution. According to Figure 4, both AA13-modified and unmodified liposomes significantly decreased the tumor volume compared with control group (i.e., saline group). Mice treated with DNR liposome bearing 7% AA13 (HL) show better tumor volume inhibition than with their unmodified counterparts, especially on the last two measurements taken on the 24th and 27th day with a 0.46- and 0.52-fold higher reduction in the mean tumor volume. HL was also more efficacious than stealth liposome in extending the long-term survival rate (Figure 5). The median survival times for the three groups (saline, stealth liposome, and HL) were 26, 34, and at least 45 days, respectively. The extended survival time of HL is presumably due to the effective DNR delivery through LDLR-mediated tumor cell targeting. When developing active targeting/delivery nanoparticle systems, it is key to deliver therapeutic/cytotoxic molecules into cancer cells as much as possible rather than
Figure 5. Effect of AA13-modified liposomes on the survival of BALB/c nude mice with AML tumors. BALB/c nude mice inoculated subcutaneously with NB4 cells were treated with saline, unmodified liposomes, or 7% AA13-modified liposomes (HL). Animal survival was recorded starting from the day of tumor cell inoculation (n = 8). * means that it is statistically significantly different from the unmodified liposome group (long-rank test, p < 0.05).
accumulate them around tumor sites or outside cancer cells. Incorporating a targeting ligand in the nanoparticle surface is directly intended for actively binding nanoparticles to receptors specifically expressed on target cells and further triggering receptor-mediated endocytic pathways. In our study, the improved tumor inhibition and prolonged survival time were likely due to sufficient intracellular DNR content, resulting from the intense internalization of HL directed by AA13. To explore the tissue distribution and site accumulation, concentrations from modified and unmodified liposomal DNR formulations in tumors and major organs were measured 24 h following a 10 mg/kg equivalent dosage injection. Despite PEGylation, organs of the reticuloendothelial system (i.e., liver and spleen) trapped significant amounts of liposomal vehicles (Figure 6). Regarding tumor site accumulation, a higher content of DNR (7100 ± 490 ng/g tissue) was found in the 2310
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ACKNOWLEDGMENTS
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REFERENCES
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This work was supported by the grants from National Scientific and Technological Major Special Project of China “Key New Drugs Creation and Manufacturing” Grants (No. 2009ZX09310-002 and No. 2009ZX09503-020).
(1) Kimby, E.; Nygren, P.; Glimelius, B. A systematic overview of chemotherapy effects in acute myeloid leukaemia. Acta Oncol. 2001, 40, 231−252. (2) Dores, G. M.; Devesa, S. S.; Curtis, R. E.; Linet, M. S.; Morton, L. M. Acute leukemia incidence and patient survival among children and adults in the United States, 2001−2007. Blood 2012, 119, 33−34. (3) Ermacora, A.; Michieli, M.; Pea, F.; Visani, G.; Bucalossi, A.; Russo, D. Liposome encapsulated daunorubicin (daunoxome) for acute leukemia. Haematol. 2000, 85, 324−325. (4) Slingerland, M.; Guchelaar, H. J.; Gelderbiom, H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discovery Today 2012, 17, 160−166. (5) Tardi, P.; Johnstone, S.; Harasym, N.; Xie, S.; Harasym, T.; Zisman, N.; et al. In vivo maintenance of synergistic cytarabine: daunorubicin ratios greatly enhances therapeutic efficacy. Leuk. Res. 2009, 33, 129−139. (6) Bhattacharya, R.; Patra, C. R.; Earl, A.; Wang, S.; Katarya, A.; Lu, L.; et al. Attaching folic acid on gold nanoparticles using noncovalent interaction via different polyethylene glycol backbones and targeting of cancer cells. Nanomed.: Nanotechnol., Biol. Med. 2007, 3, 224−238. (7) Choi, C. H. J.; Alabi, C. A.; Webster, P.; Davis, M. E. Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1235−1240. (8) Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.; Josephson, L. Multivalent effects of RGD peptides obtained by nanoparticle display. J. Med. Chem. 2006, 49, 6087−6093. (9) Zhou, Y.; Drummond, D. C.; Zou, H.; Hayes, M. E.; Adams, G. P.; Kirpotin, D. B.; et al. Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptorexpressing tumor cells. J. Mol. Biol. 2007, 371, 934−947. (10) Bross, P. F.; Beitz, J.; Chen, G.; Chen, X. H.; Duffy, E.; Kieffer, L.; et al. Approval summary: Gemtuzumab Ozogamicin in relapsed acute myeloid leukemia. Clin. Cancer Res. 2001, 7, 1490−1496. (11) Petersdorf, S. H.; Kopecky, K. J.; Slovak, M.; Willman, C.; Nevill, T.; Brandwein, J.; et al. A phase 3 study of Gemtuzumab Ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood 2013, 121, 4854−4860. (12) Jensen, M. K. No evidence of survival benefit with addition of Gemtuzumab Ozogamicin to standard medical research council induction chemotherapy. J. Clin. Oncol. 2013, 31, 1700. (13) Castaigne, S. Why is it so difficult to use Gemtuzumab Ozogamicin? Blood 2013, 121, 4813−4814. (14) Rowe, J. M.; Löwenberg, B. Gemtuzumab Ozogamicin in acute myeloid leukemia: a remarkable saga about an active drug. Blood 2013, 121, 4838−4841. (15) O’Hear, C.; Inaba, H.; Pounds, S.; Shi, L.; Dahl, G.; Bowman, W. P.; et al. Gemtuzumab ozogamicin can reduce minimal residual disease in patients with childhood acute myeloid leukemia. Cancer 2013, 119, 4036−4043. (16) Tatidis, L.; Masquelier, M.; Vitols, S. Elevated uptake of low density lipoprotein by drug resistant human leukemic cell lines. Biochem. Pharmacol. 2002, 63, 2169−2180. (17) Vitols, S.; Angelin, B.; Ericsson, S.; Gahrton, G.; Juliusson, G.; Masquelier, M.; et al. Uptake of low density lipoproteins by human leukemic cells in vivo: relation to plasma lipoprotein levels and possible relevance for selective chemotherapy. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2598−2602. (18) Vitols, S.; Norgren, S.; Juliusson, G.; Tatidis, L.; Luthman, H. Multilevel regulation of low-density lipoprotein receptor and 3-
Figure 6. Tissue distribution of DNR following treatment. Three mice from the unmodified and 7% AA13-modified liposome (HL) groups were treated intravenously with equivalent 10 mg/kg of DNR concentrations. Mice were killed 24 h after treatment, and tissues were analyzed for DNR accumulation. Data shown as mean ± SD (n = 3).
HL group than its unmodified counterpart (4500 ± 470 ng/g tissue). Gu et al. have reported that increasing the aptamer density from 0.05% to 5% can increase uptake by 5-fold in targeted cells with the maximal tumor targeting attained using the 5% aptamer decorated formulation. Tumor retention significantly decreased when aptamer density was beyond 10%.38 In our study, the tumor targeting capacity of 7% AA13decorated liposomes was examined and a 1.58-fold higher tumor retention of DNR was found. Additionally, peptide decoration unexpectedly increased the retention of liposomes in the liver. As previously discussed by Shahin and coworkers,39 high ligand density on the carriers is not always preferred since it can increase their clearance by the liver and spleen, thereby endangering the targeting capability of the formulation. This should be taken into consideration when preparing ligand-decorated drug delivery systems. Determining an optimal targeting ligand density, which not only achieves effective targeting but also enables liposomes to bypass the reticuloendothelial system, is of vital significance. Below this threshold, targeted liposomes manifest an in vivo distribution behavior similar to that of the nontargeted vectors. Above this threshold, the tedious ligand on the PEG chain may exert more detrimental rather than advantageous effects on tumor targeting. Hence, further optimization of the AA13 density is preferred in our study.
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CONCLUSIONS In this study, a synthetic peptide-modified liposome was successfully developed. The AA13-decorated liposomes can selectively target LDLR and improve the cytotoxicity of DNR in AML cells. Our in vivo study in BALB/c nude mice bearing NB4 xenografts showed increased tumor accumulation, improved tumor volume inhibition, and prolonged survival times. Taking all of these findings into consideration, our novel AA13-modified liposomes with their actively targeting capacity and antitumor activity offer a promising strategy for targeting therapy for AML.
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AUTHOR INFORMATION
Corresponding Author
*(S.M.) Tel: +86-28-85501070. Fax: +86-28-85502775. E-mail: [email protected]. Notes
The authors declare no competing financial interest. 2311
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hydroxy-3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cells. Blood 1994, 84, 2689−2698. (19) Ho, Y.; Smith, R. G.; Brown, M. S.; Goldstein, J. L. Low-density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells. Blood 1978, 52, 1099−1114. (20) Vitols, S.; Gahrton, G.; Ost, A.; Peterson, C. Elevated low density lipoprotein receptor activity in leukemic cells with monocytic differentiation. Blood 1984, 63, 1186−1193. (21) Frostegård, J.; Hamsten, A.; Gidlund, M.; Nilsson, J. Low density lipoprotein-induced growth of U937 cells: a novel method to determine the receptor binding of low density lipoprotein. J. Lipid Res. 1990, 31, 37−44. (22) Gorfien, S.; Paul, B.; Walowitz, J.; Keem, R.; Biddle, W.; Jayme, D. Growth of NS0 cells in protein-free, chemically defined medium. Biotechnol. Prog. 2000, 16, 682−687. (23) Borén, J.; Lee, I.; Zhu, W.; Arnold, K.; Taylor, S.; Innerarity, T. L. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100. J. Clin. Invest. 1998, 101, 1084. (24) Segrest, J. P.; Jones, M. K.; De Loof, H.; Dashti, N. Structure of apolipoprotein B-100 in low density lipoproteins. J. Lipid Res. 2001, 42, 1346−1367. (25) Baillie, G.; Owens, M.; Halbert, G. A synthetic low density lipoprotein particle capable of supporting U937 proliferation in vitro. J. Lipid Res. 2002, 43, 69−73. (26) Hayavi, S.; Halbert, G. W. Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture. Biotechnol. Prog. 2005, 21, 1262−1268. (27) Nikanjam, M.; Gibbs, A. R.; Hunt, C. A.; Budinger, T. F.; Forte, T. M. Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme. Int. J. Pharm. 2007, 328, 86−94. (28) Nikanjam, M.; Gibbs, A. R.; Hunt, C. A.; Budinger, T. F.; Forte, T. M. Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme. J. Controlled Release 2007, 124, 163−171. (29) Lehtinen, J.; Magarkar, A.; Stepniewski, M.; Hakola, S.; Bergman, M.; Róg, T.; et al. Analysis of cause of failure of new targeting peptide in PEGylated liposome: molecular modeling as rational design tool for nanomedicine. Eur. J. Pharm. Sci. 2012, 46, 121−130. (30) Santoso, B.; Lam, S.; Murray, B. W.; Chen, G. A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library. Bioorg. Med. Chem. Lett. 2013, 23, 5680−5683. (31) Callens, C.; Coulon, S.; Naudin, J.; Radford-Weiss, I.; Boissel, N.; Raffoux, E.; et al. Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia. J. Exp. Med. 2010, 207, 731−750. (32) McCormack, E.; Mujić, M.; Osdal, T.; Bruserud, Ø.; Gjertsen, B. T. Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia. Blood 2013, 121, 34−42. (33) Ichihara, T.; Akada, J. K.; Kamei, S.; Ohshiro, S.; Sato, D.; Fujimoto, M.; et al. A novel approach of protein immobilization for protein chips using an oligo-cysteine tag. Int. J. Protein Res. 2006, 5, 2144−2151. (34) Gubernator, J. Active methods of drug loading into liposomes: recent strategies for stable drug entrapment and increased in vivo activity. Expert Opin. Drug Delivery 2011, 8, 565−580. (35) Goldstein, J. L.; Brown, M. S.; Anderson, R. G. W.; Russell, D. W.; Schneider, W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1985, 1, 1−39. (36) Sainte-Marie, J.; Vidal, M.; Sune, A.; Ravel, S.; Philippot, J. R.; Bienvenue, A. Modification of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fatal calf serum. Biochim. Biophys. Acta 1989, 982, 265−270. (37) Young, T. K.; Falcao, C.; Torchilin, V. P. Cationic liposomes loaded with proapoptotic peptide D-(KLAKLAK)2 and Bcl-2
antisense oligodeoxynucleotide G3139 for enhanced anticancer therapy. Mol. Pharmaceutics 2009, 6, 971−977. (38) Gu, F.; Zhang, L. F.; Teply, B. A.; Mann, N.; Wang, A.; RadovicMoreno, A. F. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586−2591. (39) Shahin, M.; Soudy, R.; Aliabadi, H. M.; Kneteman, N.; Kaur, K.; Lavasanifar, A. Engineered breast tumor targeting peptide ligand modified liposomal doxorubicin and the effect of peptide density on anticancer activity. Biomaterials 2013, 34, 4089−4097.
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Development of Synthetic Peptide-Modified Liposomes with LDL Receptor Targeting Capacity and Improved Anticancer Activity Mei Liu,† Wei Li,† Caroline A. Larregieu,‡ Meng Cheng,† Bihan Yan,† Ting Chu,† Hui Li,§,∥ and Sheng-jun Mao*,† †
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education & West China School of Pharmacy, Sichuan University, No.17, Section 3, Southern Renmin Road, Chengdu 610041, P. R. China ‡ Department of Bioengineering and Therapeutic Sciences, University of CaliforniaSan Francisco, San Francisco, California 94143-0912, United States § Department of Hematology, Sichuan Academy of Medical Sciences & Sichuan Provincial People Hospital, Chengdu 610072, P. R. China ∥ Department of Hematology, West China Hospital & State Key Laboratory of Biotherapy, Sichuan University, Chengdu, Sichuan 610041, P. R. China
ABSTRACT: In this study, we report an active targeting liposomal formulation directed by a novel peptide (AA13) that specifically binds to the low density lipoprotein receptor (LDLR) overexpressed on acute myeloid leukemia (AML) cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of AA13-anchored liposomes on AML cells. AA13 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a postinsertion method. To study the effect of the peptide decoration and density on tumor cell targeting and internalization by AML cells (THP-1 and NB4), stealth liposomes bearing 3% (peptide/S100PC, molar ratio, LL) and 7% (peptide/S100PC, molar ratio, HL) AA13 were prepared, respectively. Higher uptake of LL (1.9- and 2.6-fold) and HL (2.3- and 3.6-fold) targeted liposomes occurred in THP-1 and NB4 cells, respectively, compared to untargeted liposomes. An LDLR inhibitor was used to confirm inhibition of the receptor-mediated cellular association of AA13 modified liposome in both cells. Daunorubicin (DNR) demonstrated a 2.2- and 3.5-fold higher cytotoxicity with the HL formulation and a 1.2- and 2.0-fold higher cytotoxicity with the LL formulation compared to the unmodified liposomal formulation in THP-1 and NB4 cells, respectively. Tumor drug accumulation of DNR-loaded HL was greater than that of the untargeted liposome in the biodistribution assay. The in vivo efficacy study in BALB/c nude mice bearing NB4 xenografts treated with DNR loaded HL also showed more tumor volume inhibition and a longer survival time compared to the untargeted formulation. In conclusion, the AA13-anchored liposomes demonstrated desirable potential as a promising vector for enhanced AML tumor drug targeting. KEYWORDS: low density lipoprotein receptor (LDLR), drug targeting, liposome, acute myeloid leukemia (AML), daunorubicin
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INTRODUCTION Acute myeloid leukemia (AML) is a type of hematological disease characterized by malignant proliferation, differentiation block, and apoptosis inhibition of abnormal hematopoietic stem and progenitor cells. Although daunorubicin (DNR)-based chemotherapy has been used extensively in the treatment of AML and proven to be one of the most effective induction treatments for over three decades, there is still concern of its severe side effects, relapse, and low cure rate.1,2 The nonspecific © 2014 American Chemical Society
drug distribution, which induces substantial toxicity in normal cells especially rapidly dividing cells such as bone marrow cells and hair follicles, is responsible for severe side effects such as myelosuppression and cardiotoxicity. To reduce these effects, Received: Revised: Accepted: Published: 2305
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efforts have been made to load DNR into liposomal vehicles and provide evidence that liposomal DNR may be an attractive formulation for the clinical treatment in acute leukemia.3−5 It is well-known that stealth/PEGylated liposomes exhibit a passive targeting mechanism and accumulate into tumor sites via enhanced permeability and retention (EPR) effects. Most solid tumor diseases have special pathophysiological features such as high vascular density and permeability, poor structural integrity, and lymphatic reflux disorder. All these features are the prerequisites for the occurrence of the EPR phenomenon. However, AML is a hematologic malignancy where PEGylated liposomes may not work fully to achieve specific distribution. Therefore, to enhance selective delivery of encapsulated cargo to AML cells and reduce nonspecific interaction of drug carriers, an active targeting technique is ideal. Active targeting, which has recently gained growing popularity, is capable of improving the homing of drug carriers to tumor sites and enhancing the selective delivery of anticancer drugs to tumor cells. Active targeting technologies exploit the overexpression of surface receptors on cancer cells with which a targeting ligand can interact, and ligand (e.g., antibody, peptide, transferrin, and folate) modification further improves the therapeutic index of the capsulated anticancer drugs in turn.6−9 In targeting treatment for AML, Gemtuzumab Ozogamicin, which is a calicheamicin-conjugated humanized anti-CD33 monoclonal antibody, was developed to target the CD33 antigen expressed on approximately 90% of AML myeloblasts and received accelerated approval by the U.S. FDA. Unfortunately, Pfizer Inc. announced the voluntary withdrawal from the U.S. market at the request of the FDA due to concerns about the product’s safety.10−13 Though studies afterward have proven the safety and efficacy of Gemtuzumab Ozogamicin, it is still controversial to list it as a standard treatment of AML. Therefore, other targeting strategies for the treatment of AML are desirable.14,15 Early studies have shown that leukemia cells from patients with AML have an elevated uptake (3 to 100-fold higher) of low density lipoprotein (LDL) compared to normal white blood cells from healthy individuals.16,17 Neoplasm cells depend either on abundant endogenous LDL synthesis or receptor-mediated endocytosis of LDL from plasma.18 Researchers have confirmed that more than 90% of the cholesterol input is derived from receptor-mediated degradation of LDL in AML cells and that the expression of the LDL receptor (LDLR) on leukemia cells is much more than that of normal cells.19−22 Therefore, it appears that LDLR is a potential molecular target for the selective delivery of antitumor agents to AML cells. Great efforts have also been made to elucidate the mechanistic interaction between LDL and LDLR. Up until now, it is well-known that apolipoprotein B-100 (apoB) incorporated on the native LDL particle surface plays a crucial role in receptor recognition and binding. Further primary structure studies of apoB have revealed that the binding site for LDLR is located between the 3359−3369 residues.23,24 The feasibility of developing synthetic LDL particles using a lipid emulsion and LDLR-binding domain containing peptides as a replacement for serum in the U937 cell culture media has been examined.25,26 Nikanjam and co-workers have developed a synthetic nano-LDL bearing a bifunctional peptide, which is made up of 29 amino acids and confirmed its practicability of delivering paclitaxel oleate to glioblastoma multiforme cells via LDLR.27,28
Given the potential for synthetic peptide binding to LDLR, which is highly expressed on AML cells, and a liposomal formulation of DNR as an attractive candidate for the AML treatment, we engineered and propose a novel synthetic LDLRtargeting peptide-modified liposomal vehicle with a payload of DNR. We evaluated its targeting capacity against AML cells and in vivo therapeutic efficacy, which support that this novel formulation can provide a potential strategy for the targeting treatment of AML.
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EXPERIMENTAL SECTION Materials and Animals. Soybean phosphatidylcholine (S100PC) was purchased from Lipoid KG (Ludwigshafen, Germany). Cholesterol (CHOL), poly(ethylene glycol)-distearoylphosphoethanolamine (mPEG2000-DSPE), and maleimide-derivatized DSPE-PEG2000 (Mal-PEG-DSPE) were obtained from Avanti Polar Lipids (USA). AA13 Peptide (Cys-GlyThr-Thr-Arg-Leu-Thr-Arg-Lys-Arg-Gly-Leu-Lys-Leu, MW 1602.98) was synthesized by Chutai Biotechnology Co. Ltd. (Shanghai, China) with greater than 95% purity determined by HPLC. Daunorubicin was purchased from Shanghai Baili Biotechnology Co. Ltd. (Shanghai, China) with greater than 97% purity. Coumarin-6, streptomycin, and penicillin were purchased from Sigma-Aldrich Chemical Co. (USA). All other chemical reagents used were of analytical grade or better. Five-week-old BALB/c nude mice weighing 20−25 g were kindly provided by Laboratory Animal Center of Sichuan University (Sichuan, China) and housed in a pathogen free environment for 1 week before the commencement of experiments. All experiments were conducted according to protocols approved by the University Ethics Committee for the Use of Laboratory Animals and in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Cell Lines. The human acute promyelocytic leukemia cell line, NB4, and acute monocytic leukemia cell line, THP-1, which are subtypes of AML cells, were purchased from ATCC (Manassas, USA) and cultivated in RPMI 1640 solution with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 incubator at 37 °C. For most experiments, cells were also treated with a 24 h fetal bovine serum-free cultivation medium during the exponentially growing phase to approximately 70% confluence to activate LDLR activity in vitro. Vehicle Preparation. Synthesis of AA13-PEG-DSPE. The AA13-PEG-DSPE peptide−lipid conjugate was prepared as previously reported.29 Briefly, Mal-PEG-DSPE was dissolved in chloroform and evaporated at 60 °C under vacuum for several hours to form a thin lipid film. Two milliliters of peptide PBS solution (peptide/Mal-PEG-DSPE, 1:2, molar ratio) was added to the flask, shaken well, and incubated overnight at room temperature so that the terminal cysteine of the peptide chain could be covalently conjugated to the maleimide group of MalPEG-DSPE specifically and efficiently.30 Unreacted peptide was removed by dialyzing with distilled water for 24 h. The conjugation of the peptide to Mal-PEG-DSPE was confirmed by RP-HPLC. Detection was performed at 220 nm using a Kromasil 100−5C18 column. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (solution A) and 0.1% TFA in acetonitrile (solution B) and was performed at a flow rate of 1 mL/min. The mobile phase was programmed with the following four steps: (1) 15% solution A for 1 min, (2) linear gradient from 15% to 35% solution A in 25 min, (3) linear 2306
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to qualitatively study cellular uptake (CFM-500, Changfang Optical Instrument Co., Ltd. Shanghai, China). In Vitro Cytotoxicity Study. The cytotoxicity of various liposomal DNR formulations were examined in AML cells (NB4 and THP-1). Briefly, 100 μL cell suspensions (1× 105 cells/mL) were added to each well of a 96-well plate. Cells were exposed to either free DNR or nontargeted or targeted liposomes loaded with DNR and incubated at 37 °C. Liposomal samples were diluted with FBS-free medium and adjusted to serial concentrations of the entrapped DNR. The culture medium was removed at 4 h and replaced with equal volume of fresh medium. After 24 h, 20 μL of 5 mg/mL MTT solution was added to each well and further incubated for 4 h. Finally, 100 μL of formazan-dissolving buffer consisting of 10% sodium dodecyl sulfonate, 5% isobutanol, and 0.01 M hydrochloric acid was introduced to each well and the absorbance was read at 570 nm using a microplate reader (Bio-Rad, Richmond, CA, USA). In Vivo Therapeutic Efficacy and Tissue Biodistribution Assay. The AML tumor model was established as previously reported.31,32 Four hundred microliters of 5 × 106 NB4 cells in matrigel were subcutaneously injected into the back of mice, which were randomly assigned to three groups (saline group, untargeted liposome group, and HL group) with 6 to 8 mice per group. When tumor sizes were approximately 0.1 cm3 on day 10 postinoculation, the animals were treated with four doses of equivalent 2.5 mg/kg DNR concentrations by tail vein injection every 5 days. Tumor volumes were measured twice a week and monitored for up to 27 days since the first injection and calculated using the formula V = 0.5ab2, where a and b represent the longest and shortest diameters, respectively. To assay biodistribution, mice were treated with one dosage of equivalent 10 mg/kg DNR concentrations. Briefly, the tumor and tissues (heart, liver, spleen, kidney, and lung) were excised 24 h after injection and processed. Four hundred microliters of acetonitrile was added to 200 μL of tissue homogenate. The mixture was then vortexed and centrifuged at 3000g for 10 min. The supernatant was transferred to a new tube containing 3 mL of chloroform, vortexed, and then centrifuged at 3000g for 5 min. The chloroform layer was carefully collected and dried under nitrogen. Finally, the residue was reconstituted in 100 μL methanol and analyzed for DNR content via HPLC. A standard curve prepared using blank tissue spiked with known DNR concentrations was used for content determination. The HPLC conditions included a Diamonsil-C18 column (150 × 4.6 mm, 5 μm, Dikma Technology Co., Ltd. Beijing, China), a mobile phase composed of sodium dodecyl sulfate/acetonitrile/ methanol (47:47:6), a 1 mL/min flow rate, a 481 nm detection wavelength, a 20 μL injection volume, and a 30 °C column temperature.
gradient from 35% to 100% solution A in 5 min, and (4) 100% solution A for 5 min. The concentration of unreacted peptide was calculated based on a calibration curve of known concentrations of peptide solutions. Preparation of Liposomes. Daunorubicin (DNR)-loaded liposomes were prepared using an ammonium sulfate gradient method. Briefly, S100PC/CHOL/mPEG2000-DSPE (4:1:0.2, molar ratio) was dissolved in chloroform and evaporated at 60 °C under vacuum. The formed thin film was then hydrated with 0.25 M ammonium sulfate at 60 °C. The obtained large unilamellar vesicles were subjected to probe sonication and dialyzed against 9% sucrose solution for 24 h using a dialysis bag (MWCO, 7000). The resultant liposome was mixed with daunorubicin sucrose solution and incubated at 60 °C for 2 h. Untrapped drug was removed using a Sephadex G-50 column. Coumarin-6 loaded liposome was also prepared. S100PC/ CHOL/mPEG2000-DSPE (4:1:0.2, molar ratio) and coumarin-6 were dissolved in chloroform, evaporated, and hydrated with PBS. Particle size was adjusted by probe sonication, and the liposome was stored at 4 °C away from light. A postinsertion method was applied to prepare targeted liposomes of different peptide densities. For the preparation of 3% or 7% (peptide/S100PC, molar ratio, namely, LL or HL, respectively) AA13-modified liposomes, 2 mL of AA13-PEGDSPE stock solutions containing 1.0 or 2.4 mg/mL AA13 was added to equal volumes of preformed stealth liposomes, mixed gently, and placed at room temperature overnight. The peptidelipid conjugate was incorporated onto the liposomal surface via hydrophobic DSPE domains. Unattached peptide-PEG-DSPE was removed by passing the resultant dispersion through a Sephadex G-50 column with pH 7.4 phosphate buffer. The size of the liposomes was measured by dynamic laser scattering (DLS) (Malvern Zetasizer Nano ZS90, UK). The amount of trapped DNR within the liposomal formulation was determined by HPLC (Agilent 1100, USA). In Vitro Release Study. The in vitro release of DNR from each liposomal formulation was studied using the dialysis method. Unmodified liposomes, liposomes bearing 3% AA13 (peptide/S100PC, molar ratio, LL), and liposomes bearing 7% AA13 (peptide/S100PC, molar ratio, HL) containing equivalent 1.5 mg/mL DNR concentrations were placed in dialysis bags (MWCO, 6000). Dialysis bags containing 2 mL of liposomes were immersed in 600 mL pH 7.4 phosphate buffer at 37 °C and shaken at 150 rpm. At fixed time intervals, 1 mL of the dialysis medium was taken from the outside of dialysis bag and replaced with an equal volume of fresh medium. The collected samples were filtered through a 0.22 μm membrane, and the DNR contents were determined. In Vitro Cell Uptake Study. Cellular uptake studies for different liposomal formulations were carried out in AML cells (NB4 and THP-1). Cells at their exponentially growing phase were treated with fetal bovine serum-deficient medium for 24 h before the experiment and seeded into 16-well plates at a density of 5 × 106 cells per well. Liposomes (unmodified, LL, or HL) containing equivalent 2.5 μg/mL coumarin-6 concentrations were added to and incubated with cells for 4 h at 37 °C. For competitive binding studies, 10 μL of 5 mM suramin, an inhibitor of the LDL receptor, was added to each well 2 h before the addition of liposomes. At the conclusion of each uptake study, cells were collected and washed thrice with ice-cold PBS. The fluorescence intensity of cell-associated coumarin-6 was quantified using flow cytometry (BD FACSCalibur, America). A fluorescence microscope was used
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RESULTS AND DISCUSSION Synthesis of AA13-PEG-DSPE. AA13-PEG-DSPE was synthesized by reaction of AA13 peptide with Mal-PEG-DSPE in PBS (pH 7.4). The conjugation was confirmed by RP-HPLC. AA13 eluted at a retention time of 13.1 min. Free peptide was removed after the completion of the reaction by extensive dialysis. The AA13 conjugation efficiency was ∼91%. The high conjugation efficiency of the peptide to Mal-PEG-DSPE is likely due to the high reactivity between maleimide and cysteine.33 Physicochemical Properties of DNR-Loaded Liposomes. The particle size and polydispersity index (PDI) of 2307
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Table 1. Characteristics of the Liposomal DNR Formulations (n = 3) formulation
peptide content (% AA13/S100PC)
mean particle size (nm)
PDI value
encapsulation efficiency (EE)
unmodified liposomes AA13-modified liposomes (LL) AA13-modified liposomes (HL)
0 3 7
98 ± 3.5 95 ± 1.4 102 ± 1.9
0.179 ± 0.04 0.172 ± 0.01 0.158 ± 0.02
96.4 ± 0.26 95.3 ± 0.32 94.8 ± 0.41
liposomal samples were measured via a nanoparticle analyzer at a fixed 90° angle at 20 °C with results given in Table 1. Targeted liposomes (LL and HL) had a mean particle size about 100 nm, similar to that of the nontargeted liposome. All of the liposomal samples had a narrow PDI value, indicating homogeneity in their dispersion state. The encapsulation efficiency (EE) of DNR in the liposomes determined by HPLC ranged from 94% to 97%, indicating that peptide modification did not affect DNR loading into the liposome. In Vitro DNR Release Study. The release profile of DNR from nontargeted and targeted liposomal samples in pH 7.4 phosphate buffer showed that neither peptide modification nor density affected the rate and extent of DNR release from prepared liposomes (Figure 1). After 36 h, 32.5% and 34.2% of
Figure 2. Fluorescence images of the cellular uptake of coumarin-6 loaded liposomes by NB4 cells (a, nontargeted liposomes; b, LL; and c, HL) and THP-1 cells (d, nontargeted liposome; e, LL; and f, HL) without suramin after 4 h incubation. Abbreviations: LL, 3% AA13modified liposomes; HL, 7% AA13-modified liposomes.
Figure 1. Release profiles of DNR from different liposomal formulations in PBS (pH 7.4) at 37 °C. Each point represents mean ± SD (n = 3). Abbreviations: LL, 3% AA13-modified liposomes; HL, 7% AA13-modified liposomes.
HL were 2.6- and 3.6-fold higher, respectively, than that of nontargeted liposomes in NB4 cells, exhibiting AA13 densitydependent uptake. Increasing peptide density also increased the cellular association in THP-1 cells with 1.9- and 2.2-fold higher uptake of LL and HL than that of nontargeted liposome. We found that the uptake of targeted liposomes in NB4 cells was greater than that in THP-1 cells, which may be attributed to differences in LDLR expressed on different AML cells. When endogenous LDL binds to LDLR, they become internalized via endocytosis, and a drop in pH in endosome allows the receptor to dissociate from LDL. The receptor is recycled back to the surface of cells, while LDL becomes degraded by the lysosome.35 It has been reported that the more receptors expressed on AML cells, the less time is needed for LDLR to recycle back to cell surface and thus the greater the uptake of LDL by these cells.36 Our engineered liposome decorated with a LDLR-binding domain mimics plasma-derived LDL and is capable of targeting LDLR on AML cells. The greater uptake of LL and HL in NB4 cells than in THP-1 cells likely resulted from the greater expression of LDLR in NB4 cells than THP-1 cells. To confirm whether the increased cellular uptake of AA13modified liposomes was due to the presence of excess surface
the DNR content were released into dialysis medium from the LL and HL formulations, respectively, which were not significantly different from the nontargeted liposome that released 36.4% of its DNR content under the same conditions. The utilization of the ammonium sulfate gradient method may explain the low release rate of DNR from prepared liposomes. The ammonium sulfate gradient method has been shown to generate an aggregated gel-like state with DNR at the inner phase of liposomes, resulting in efficient DNR encapsulation and slow DNR dissolution and release.34 Cellular Association Study. In our study, NB4 and THP-1 cells could endocytose coumarin-6-loaded nontargeted and targeted liposomes with the density of the conjugated peptide being an important factor affecting liposomal uptake. Figure 2 depicts qualitatively the cellular association of the liposomes by NB4 and THP-1 cells. The fluorescence intensities of the AA13modified liposomes were greater than that of the nontargeted liposome, indicating enhanced accumulation by the peptide in vitro. Figure 3 provides quantitative results that are consistent with the qualitative results. The cellular association of LL and 2308
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Figure 3. Quantitative results of the cellular uptake of coumarin-6 loaded liposomes by NB4 cells (a, nontargeted liposomes; b, LL; and c, HL) and THP-1 cells (d, nontargeted liposome; e, LL; and f, HL) with (+) or without (−) suramin after 4 h incubation. (g,h) Summary of cellular association in NB4 and THP-1 cells, respectively. Each bar represents mean fluorescence intensity ± SD (n = 3). Abbreviations: LL, 3% AA13-modified liposomes; HL, 7% AA13-modified liposomes.
LDLR, a competitive binding experiment was conducted, in which the cellular association was evaluated in the presence of suramin, an LDLR inhibitor. Figure 3 reveals that the presence of the inhibitor significantly suppressed the cellular association of targeted liposomes in NB4 and THP-1 cells. The uptake of
nontargeted liposome was low irrespective of the presence of inhibitor. Both of these results confirm a LDLR-mediated endocytosis mechanism of AA13-decorated liposome. In Vitro Cytotoxicity Study. The effect of peptide decoration and content on liposomal DNR cytotoxicity was 2309
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evaluated in both NB4 and THP-1 cells after 24 h incubation using an MTT assay. The AA13-modified liposomes exhibit higher cytotoxicity (i.e., lower IC50) than the unmodified liposome (Table 2). Compared to unmodified liposome with Table 2. IC50 Values of Liposomal DNR Formulations in NB4 and THP-1 Cells after 24 h Incubation IC50 (μM) formulation unmodified liposomes AA13-modified liposomes (LL) AA13-modified liposomes (HL)
peptide content (% AA13/S100PC)
NB4
THP-1
0
0.646 ± 0.09
0.757 ± 0.13
3
0.329 ± 0.04
0.631 ± 0.07
7
0.186 ± 0.02
0.344 ± 0.11
Figure 4. Mean tumor volumes in BALB/c nude mice bearing NB4 xenografts treated for intravenous injections given on the 10th, 15th, 20th, and 25th day of 2.5 mg/kg DNR equivalent liposomal formulations or saline. Each point represents the mean tumor volume (n = 6−8) ± SD. * means that it is statistically significantly different from the unmodified group (ANOVA, p < 0.05). Abbreviations: HL, 7% AA13-modified liposomes.
DNR IC50 values of 0.757 ± 0.03 μM in THP-1 cells and 0.646 ± 0.04 μM in NB4 cells, targeted liposome bearing 7% AA13 (HL) showed 2.2- and 3.5-fold higer cytotoxicity in THP-1 and NB4 cells, respectively, while 3% AA13 modified liposome (LL) showed 1.2- and 2.0-fold higher DNR cytotoxicity, respectively. The cytotoxicity results are in agreement with the cellular association findings and validate the capacity of AA13 to exert active LDLR targeting. It is well-known that cancer targeted formulations, such as Herceptin and Mabthera, exploit biologically active targeting ligands. Apart from their active targeting properties, these ligands also possess intrinsic cytotoxicity and are capable of interfering with cell signaling pathways. For example, D-(KLAKLAK)2, which was originally designed as an antibacterial peptide, interrupts the negatively charged mitochondrial membrane and induces cell death through a mitochondrial-dependent apoptosis manner when internalized within cells.37 It is possible that the improved cytotoxicity of AA13-modified liposomal DNR may be attributed to the intense interaction between LDLR and AA13, which is of crucial significance to mediate sufficient receptor-dependent endocytosis and improve the cytotoxicity of liposomal DNR. In Vivo Therapeutic Efficacy and Tissue Biodistribution Assay. The in vivo anticancer activities of developed liposomal formulations following intravenous multiple dosing were assessed using NB4 subcutaneous xenografts in BALB/c nude mice. Even though the organ distribution of the disease differs as it is in humans, this model provides various advantages such as the formation of visible tumors, which allows for the evaluation of tumor growth inhibition and biodistribution. According to Figure 4, both AA13-modified and unmodified liposomes significantly decreased the tumor volume compared with control group (i.e., saline group). Mice treated with DNR liposome bearing 7% AA13 (HL) show better tumor volume inhibition than with their unmodified counterparts, especially on the last two measurements taken on the 24th and 27th day with a 0.46- and 0.52-fold higher reduction in the mean tumor volume. HL was also more efficacious than stealth liposome in extending the long-term survival rate (Figure 5). The median survival times for the three groups (saline, stealth liposome, and HL) were 26, 34, and at least 45 days, respectively. The extended survival time of HL is presumably due to the effective DNR delivery through LDLR-mediated tumor cell targeting. When developing active targeting/delivery nanoparticle systems, it is key to deliver therapeutic/cytotoxic molecules into cancer cells as much as possible rather than
Figure 5. Effect of AA13-modified liposomes on the survival of BALB/c nude mice with AML tumors. BALB/c nude mice inoculated subcutaneously with NB4 cells were treated with saline, unmodified liposomes, or 7% AA13-modified liposomes (HL). Animal survival was recorded starting from the day of tumor cell inoculation (n = 8). * means that it is statistically significantly different from the unmodified liposome group (long-rank test, p < 0.05).
accumulate them around tumor sites or outside cancer cells. Incorporating a targeting ligand in the nanoparticle surface is directly intended for actively binding nanoparticles to receptors specifically expressed on target cells and further triggering receptor-mediated endocytic pathways. In our study, the improved tumor inhibition and prolonged survival time were likely due to sufficient intracellular DNR content, resulting from the intense internalization of HL directed by AA13. To explore the tissue distribution and site accumulation, concentrations from modified and unmodified liposomal DNR formulations in tumors and major organs were measured 24 h following a 10 mg/kg equivalent dosage injection. Despite PEGylation, organs of the reticuloendothelial system (i.e., liver and spleen) trapped significant amounts of liposomal vehicles (Figure 6). Regarding tumor site accumulation, a higher content of DNR (7100 ± 490 ng/g tissue) was found in the 2310
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ACKNOWLEDGMENTS
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REFERENCES
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This work was supported by the grants from National Scientific and Technological Major Special Project of China “Key New Drugs Creation and Manufacturing” Grants (No. 2009ZX09310-002 and No. 2009ZX09503-020).
(1) Kimby, E.; Nygren, P.; Glimelius, B. A systematic overview of chemotherapy effects in acute myeloid leukaemia. Acta Oncol. 2001, 40, 231−252. (2) Dores, G. M.; Devesa, S. S.; Curtis, R. E.; Linet, M. S.; Morton, L. M. Acute leukemia incidence and patient survival among children and adults in the United States, 2001−2007. Blood 2012, 119, 33−34. (3) Ermacora, A.; Michieli, M.; Pea, F.; Visani, G.; Bucalossi, A.; Russo, D. Liposome encapsulated daunorubicin (daunoxome) for acute leukemia. Haematol. 2000, 85, 324−325. (4) Slingerland, M.; Guchelaar, H. J.; Gelderbiom, H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discovery Today 2012, 17, 160−166. (5) Tardi, P.; Johnstone, S.; Harasym, N.; Xie, S.; Harasym, T.; Zisman, N.; et al. In vivo maintenance of synergistic cytarabine: daunorubicin ratios greatly enhances therapeutic efficacy. Leuk. Res. 2009, 33, 129−139. (6) Bhattacharya, R.; Patra, C. R.; Earl, A.; Wang, S.; Katarya, A.; Lu, L.; et al. Attaching folic acid on gold nanoparticles using noncovalent interaction via different polyethylene glycol backbones and targeting of cancer cells. Nanomed.: Nanotechnol., Biol. Med. 2007, 3, 224−238. (7) Choi, C. H. J.; Alabi, C. A.; Webster, P.; Davis, M. E. Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1235−1240. (8) Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.; Josephson, L. Multivalent effects of RGD peptides obtained by nanoparticle display. J. Med. Chem. 2006, 49, 6087−6093. (9) Zhou, Y.; Drummond, D. C.; Zou, H.; Hayes, M. E.; Adams, G. P.; Kirpotin, D. B.; et al. Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptorexpressing tumor cells. J. Mol. Biol. 2007, 371, 934−947. (10) Bross, P. F.; Beitz, J.; Chen, G.; Chen, X. H.; Duffy, E.; Kieffer, L.; et al. Approval summary: Gemtuzumab Ozogamicin in relapsed acute myeloid leukemia. Clin. Cancer Res. 2001, 7, 1490−1496. (11) Petersdorf, S. H.; Kopecky, K. J.; Slovak, M.; Willman, C.; Nevill, T.; Brandwein, J.; et al. A phase 3 study of Gemtuzumab Ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood 2013, 121, 4854−4860. (12) Jensen, M. K. No evidence of survival benefit with addition of Gemtuzumab Ozogamicin to standard medical research council induction chemotherapy. J. Clin. Oncol. 2013, 31, 1700. (13) Castaigne, S. Why is it so difficult to use Gemtuzumab Ozogamicin? Blood 2013, 121, 4813−4814. (14) Rowe, J. M.; Löwenberg, B. Gemtuzumab Ozogamicin in acute myeloid leukemia: a remarkable saga about an active drug. Blood 2013, 121, 4838−4841. (15) O’Hear, C.; Inaba, H.; Pounds, S.; Shi, L.; Dahl, G.; Bowman, W. P.; et al. Gemtuzumab ozogamicin can reduce minimal residual disease in patients with childhood acute myeloid leukemia. Cancer 2013, 119, 4036−4043. (16) Tatidis, L.; Masquelier, M.; Vitols, S. Elevated uptake of low density lipoprotein by drug resistant human leukemic cell lines. Biochem. Pharmacol. 2002, 63, 2169−2180. (17) Vitols, S.; Angelin, B.; Ericsson, S.; Gahrton, G.; Juliusson, G.; Masquelier, M.; et al. Uptake of low density lipoproteins by human leukemic cells in vivo: relation to plasma lipoprotein levels and possible relevance for selective chemotherapy. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2598−2602. (18) Vitols, S.; Norgren, S.; Juliusson, G.; Tatidis, L.; Luthman, H. Multilevel regulation of low-density lipoprotein receptor and 3-
Figure 6. Tissue distribution of DNR following treatment. Three mice from the unmodified and 7% AA13-modified liposome (HL) groups were treated intravenously with equivalent 10 mg/kg of DNR concentrations. Mice were killed 24 h after treatment, and tissues were analyzed for DNR accumulation. Data shown as mean ± SD (n = 3).
HL group than its unmodified counterpart (4500 ± 470 ng/g tissue). Gu et al. have reported that increasing the aptamer density from 0.05% to 5% can increase uptake by 5-fold in targeted cells with the maximal tumor targeting attained using the 5% aptamer decorated formulation. Tumor retention significantly decreased when aptamer density was beyond 10%.38 In our study, the tumor targeting capacity of 7% AA13decorated liposomes was examined and a 1.58-fold higher tumor retention of DNR was found. Additionally, peptide decoration unexpectedly increased the retention of liposomes in the liver. As previously discussed by Shahin and coworkers,39 high ligand density on the carriers is not always preferred since it can increase their clearance by the liver and spleen, thereby endangering the targeting capability of the formulation. This should be taken into consideration when preparing ligand-decorated drug delivery systems. Determining an optimal targeting ligand density, which not only achieves effective targeting but also enables liposomes to bypass the reticuloendothelial system, is of vital significance. Below this threshold, targeted liposomes manifest an in vivo distribution behavior similar to that of the nontargeted vectors. Above this threshold, the tedious ligand on the PEG chain may exert more detrimental rather than advantageous effects on tumor targeting. Hence, further optimization of the AA13 density is preferred in our study.
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CONCLUSIONS In this study, a synthetic peptide-modified liposome was successfully developed. The AA13-decorated liposomes can selectively target LDLR and improve the cytotoxicity of DNR in AML cells. Our in vivo study in BALB/c nude mice bearing NB4 xenografts showed increased tumor accumulation, improved tumor volume inhibition, and prolonged survival times. Taking all of these findings into consideration, our novel AA13-modified liposomes with their actively targeting capacity and antitumor activity offer a promising strategy for targeting therapy for AML.
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AUTHOR INFORMATION
Corresponding Author
*(S.M.) Tel: +86-28-85501070. Fax: +86-28-85502775. E-mail: [email protected]. Notes
The authors declare no competing financial interest. 2311
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hydroxy-3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cells. Blood 1994, 84, 2689−2698. (19) Ho, Y.; Smith, R. G.; Brown, M. S.; Goldstein, J. L. Low-density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells. Blood 1978, 52, 1099−1114. (20) Vitols, S.; Gahrton, G.; Ost, A.; Peterson, C. Elevated low density lipoprotein receptor activity in leukemic cells with monocytic differentiation. Blood 1984, 63, 1186−1193. (21) Frostegård, J.; Hamsten, A.; Gidlund, M.; Nilsson, J. Low density lipoprotein-induced growth of U937 cells: a novel method to determine the receptor binding of low density lipoprotein. J. Lipid Res. 1990, 31, 37−44. (22) Gorfien, S.; Paul, B.; Walowitz, J.; Keem, R.; Biddle, W.; Jayme, D. Growth of NS0 cells in protein-free, chemically defined medium. Biotechnol. Prog. 2000, 16, 682−687. (23) Borén, J.; Lee, I.; Zhu, W.; Arnold, K.; Taylor, S.; Innerarity, T. L. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100. J. Clin. Invest. 1998, 101, 1084. (24) Segrest, J. P.; Jones, M. K.; De Loof, H.; Dashti, N. Structure of apolipoprotein B-100 in low density lipoproteins. J. Lipid Res. 2001, 42, 1346−1367. (25) Baillie, G.; Owens, M.; Halbert, G. A synthetic low density lipoprotein particle capable of supporting U937 proliferation in vitro. J. Lipid Res. 2002, 43, 69−73. (26) Hayavi, S.; Halbert, G. W. Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture. Biotechnol. Prog. 2005, 21, 1262−1268. (27) Nikanjam, M.; Gibbs, A. R.; Hunt, C. A.; Budinger, T. F.; Forte, T. M. Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme. Int. J. Pharm. 2007, 328, 86−94. (28) Nikanjam, M.; Gibbs, A. R.; Hunt, C. A.; Budinger, T. F.; Forte, T. M. Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme. J. Controlled Release 2007, 124, 163−171. (29) Lehtinen, J.; Magarkar, A.; Stepniewski, M.; Hakola, S.; Bergman, M.; Róg, T.; et al. Analysis of cause of failure of new targeting peptide in PEGylated liposome: molecular modeling as rational design tool for nanomedicine. Eur. J. Pharm. Sci. 2012, 46, 121−130. (30) Santoso, B.; Lam, S.; Murray, B. W.; Chen, G. A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library. Bioorg. Med. Chem. Lett. 2013, 23, 5680−5683. (31) Callens, C.; Coulon, S.; Naudin, J.; Radford-Weiss, I.; Boissel, N.; Raffoux, E.; et al. Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia. J. Exp. Med. 2010, 207, 731−750. (32) McCormack, E.; Mujić, M.; Osdal, T.; Bruserud, Ø.; Gjertsen, B. T. Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia. Blood 2013, 121, 34−42. (33) Ichihara, T.; Akada, J. K.; Kamei, S.; Ohshiro, S.; Sato, D.; Fujimoto, M.; et al. A novel approach of protein immobilization for protein chips using an oligo-cysteine tag. Int. J. Protein Res. 2006, 5, 2144−2151. (34) Gubernator, J. Active methods of drug loading into liposomes: recent strategies for stable drug entrapment and increased in vivo activity. Expert Opin. Drug Delivery 2011, 8, 565−580. (35) Goldstein, J. L.; Brown, M. S.; Anderson, R. G. W.; Russell, D. W.; Schneider, W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1985, 1, 1−39. (36) Sainte-Marie, J.; Vidal, M.; Sune, A.; Ravel, S.; Philippot, J. R.; Bienvenue, A. Modification of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fatal calf serum. Biochim. Biophys. Acta 1989, 982, 265−270. (37) Young, T. K.; Falcao, C.; Torchilin, V. P. Cationic liposomes loaded with proapoptotic peptide D-(KLAKLAK)2 and Bcl-2
antisense oligodeoxynucleotide G3139 for enhanced anticancer therapy. Mol. Pharmaceutics 2009, 6, 971−977. (38) Gu, F.; Zhang, L. F.; Teply, B. A.; Mann, N.; Wang, A.; RadovicMoreno, A. F. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586−2591. (39) Shahin, M.; Soudy, R.; Aliabadi, H. M.; Kneteman, N.; Kaur, K.; Lavasanifar, A. Engineered breast tumor targeting peptide ligand modified liposomal doxorubicin and the effect of peptide density on anticancer activity. Biomaterials 2013, 34, 4089−4097.
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