Antibody-Targeted Cyclodextrin-Based Nanoparticles for siRNA

Article pubs.acs.org/molecularpharmaceutics

Antibody-Targeted Cyclodextrin-Based Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukemia: Physicochemical Characteristics, in Vitro Mechanistic Studies, and ex Vivo Patient Derived Therapeutic Efficacy Jianfeng Guo,*,†,‡ Eileen G. Russell,§ Raphael Darcy,‡ Thomas G. Cotter,§ Sharon L. McKenna,∥ Mary R. Cahill,⊥ and Caitriona M. O’Driscoll*,‡ †

School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China Pharmacodelivery Group, School of Pharmacy, University College Cork, Cork, Ireland § Tumour Biology Laboratory, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland ∥ Cork Cancer Research Centre, University College Cork, Cork, Ireland ⊥ Department of Haematology, Cork University Hospital, Cork, Ireland ‡

S Supporting Information *

ABSTRACT: Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with high relapse rates. It is known that leukemia stem cells (LSCs), a very small subpopulation of the total number of leukemic cells, maintain the leukemia phenotype (∼80−90% of AML remain the same as at first diagnosis), display chemotherapy resistance, and contribute to disease regeneration. Therefore, targeting LSCs could control the relapse of AML. Small interfering RNA (siRNA), an effector of the RNA interference (RNAi) pathway, can selectively downregulate any gene implicated in the pathology of disease, presenting great potential for treatment of AML. In this study an antibody targeted cyclodextrin-based nanoparticle (NP) (CD.DSPE-PEG-Fab) was developed for siRNA delivery specifically to AML LSCs. The targeted CD.siRNA.DSPE-PEG-Fab formulation, where Fab specifically targets the IL-3 receptor α-chain (IL-3Rα, also known as CD123, a cell surface antigen for human AML LSCs), achieved antigen-mediated cellular uptake in KG1 cells (an AML leukemia stem and progenitor cell line). Efficient delivery of bromodomain-containing protein 4 (BRD4) siRNA using the targeted formulation resulted in downregulation of the corresponding mRNA and protein in KG1 cells and in ex vivo primary AML patient derived samples. The resulting silencing of BRD4 induced myeloid differentiation and triggered leukemia apoptosis. In addition, a synergistic therapeutic effect was detected when administered in combination with the chemotherapeutic, cytarabine (Ara-C). These results indicate the clinical potential of the antibody-tagged cyclodextrin NP for targeted delivery of therapeutic siRNA in the treatment of AML. KEYWORDS: nonviral nanoparticles, RNAi therapeutics, leukemia stem cells, cancer epigenetic gene therapy, personalized medicine

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INTRODUCTION

20% of older patients remain in remission 5 years after diagnosis.3 AML is a clonal disorder; since recent molecular-based knowledge in oncogenomics of AML has resulted in significant advances in the understanding of the molecular pathogenesis of this disease, it is now known that most patients have one or more detectable mutations relevant to hematopoiesis.4 The development and emergence of the founding clone is often accompanied by the development of subclones and clonal

Acute myeloid leukemia (AML) is a heterogeneous malignant blood cancer, characterized by maturational arrest and proliferation of myeloid blasts (marrow cells which fail to differentiate) in the bone marrow (BM), peripheral blood (PB), and spleen. In AML, the incidence increases and the prognosis worsens in older adults. In the US in 2015 there were an estimated 20,830 new cases and 10,460 deaths.1 Even with aggressive treatments, including high dose chemotherapy and bone marrow transplantation, the overall 5-year survival rate is less than 50%.2 In addition, relapse rates of AML patients are high, as only 40% of patients younger than 60 years and 10− © 2017 American Chemical Society

Received: December 21, 2016 Accepted: February 1, 2017 Published: February 1, 2017 940

DOI: 10.1021/acs.molpharmaceut.6b01150 Mol. Pharmaceutics 2017, 14, 940−952

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Molecular Pharmaceutics evolution of the established clone (or clones).5 Following conventional cytoreductive therapy, a 3 log reduction in detectable disease can be expected from a presentation count of 1011−1012 cells to hematological remission with less than 5% bone marrow blasts. Subsequent chemotherapy can be expected to produce a 1−2 log reduction in leukemia cells, with a predicted residual number of leukemia cells of approximately 104 −106 cells. From this small residual pool, relapse can be propagated by the rare population of leukemia stem cells (LSCs).6 As LSCs often spend most of the time in the nondividing G0 cell cycle state, they are substantially more resistant to standard chemotherapeutic regimens than bulk leukemia populations. This provides an insight into the reasons why conventional chemotherapy may reduce leukemia burden, but relapse often occurs at a later stage.7 For patients destined to relapse, targeting and eliminating the LSCs is logical. The expanding knowledge of the genetic basis of leukemia has presented potential for the utilization of RNA interference (RNAi) as a new generation of leukemia therapeutics.8−10 However, the clinical application of RNAi effectors [i.e., small interference RNA (siRNA)] is greatly retarded by several issues such as short plasma half-life, nonspecific tissue distribution, poor intracellular trafficking, and toxicity effects.11 Recently, multifunctional biomimetic nonviral nanoparticles (NPs) have been developed as gene delivery systems showing great potential to overcome these challenges.12 A wide range of cyclodextrin-based formulations for gene delivery have been previously synthesized in our laboratory and characterized using cell culture models13−15 and preclinical animal models of prostate cancer,16 Huntington’s disease,17 and acute colitis.18 Among these functionalized cyclodextrins, an amphiphilic cationic β-cyclodextrin, namely, “SC12-clickpropylamine-CD” (hereafter referred to as CD, Figure S1) has promoted siRNA delivery due to its high transfection capacity17,18 and low levels of cytotoxicity and immunological activation.19 However, incubation of cationic CD.siRNA formulation in salt- and serum-containing media can promote particulate aggregation,20 suggesting that a further modification of CD is required to facilitate its application to systemic disorders (i.e., leukemia). In this study we have developed an antibody targeted cyclodextrin-based siRNA delivery vector (CD.DSPE-PEGFab) by incorporating the fragment antigen-binding (Fab) of a monoclonal antibody onto CD through a PEGylated linker (DSPE-PEG-Maleimide) using a self-assembling process, namely, “postinsertion”.21,22 The presence of the CD enables binding of siRNA, the application of PEG is designed to stabilize NPs in physiological environments thereby prolonging the systemic circulation and reducing in vivo toxicity, and the incorporation of Fab is to target IL-3 receptor α-chain (IL-3Rα, highly expressed on AML LSCs) for selective targeting of AML. Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extraterminal (BET) family that bind to acetylated histones to influence transcription, has been recently identified as a therapeutic target in AML.23 Consequently, complexes of the antibody-targeted cyclodextrin with BRD4 siRNA were investigated here for their therapeutic potential in the treatment of AML.

the NPs, the CD was dissolved in chloroform and evaporated under a stream of gaseous nitrogen. The CD was then rehydrated in sterilized deionized water (DIW) and sonicated for 1 h. For CD.siRNA complexation (Figure 1), the CD was mixed with siRNA solutions and incubated at room temperature (rt) for 30 min.

Figure 1. Antibody-targeted cyclodextrin for siRNA delivery into AML LSCs. (a) The targeted CD.siRNA.DSPE-PEG-Fab formulation was achieved by incorporating PEGylated Fab (fragment antigen binding) into CD.siRNA complexes using a “postinsertion-like approach”. (b) Targeted formulation will specifically bind IL-3Rα, a well-known antigen overexpressing on the cell surface of LSCs, achieving cellular uptake via receptor-medicated endocytosis. Following endolysosomal escape, targeted formulation can activate efficient RNAi and downregulate the corresponding gene (BRD4, an epigenetic reader), leading to myeloid differentiation, leukemia apoptosis, and synergistic therapeutic efficacy in combination with the chemotherapeutic cytarabine (Ara-C).

The Fab was prepared using human IL-3Rα monoclonal antibody (Clone #32703, R&D Systems, U.K.) with the Pierce Fab Preparation Kit (Thermo Scientific). Thiolated Fab (FabSH) was prepared using Traut’s reagent (Thermo Scientific) in accordance with the manufacturer’s instructions. In order to achieve DSPE-PEG-Fab (Figure 1), the micelle solution of 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG-Maleimide, Nanocs, USA) was prepared as previously described;24 this was followed by the addition of the Fab-SH, and incubation at 16 °C overnight with slight shaking.21 For the targeted CD.siRNA.DSPE-PEG-Fab formulation (Figure 1), DSPE-PEG-Fab was incubated with the CD.siRNA at 60 °C for 1 h with slight shaking.24 The nontargeted CD.siRNA.DSPE-PEG formulation was prepared in the same way as the targeted formulation except that DSPE-PEG-Maleimide was replaced by 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG, Nanocs, USA). The CD.siRNA.DSPE-PEG-Fab was purified using a Sepharose CL4B size-exclusion column to remove free antibody, as previously described.22,25 Physicochemical Characterization of the Nanoparticles. siRNA binding with CD, CD.DSPE-PEG, and CD.DSPE-PEG-Fab was examined using gel electrophoresis. Briefly, complexes containing 0.5 μg of Negative Control siRNA (siNeg; sense strand, sense sequence 5′-UUC UCC

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MATERIALS AND METHODS Nanoparticle Preparation. The amphiphilic cationic βcyclodextrin “SC12-click-propylamine-CD” (CD) was synthesized and prepared as previously described.13 Briefly to prepare 941


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Molecular Pharmaceutics

were first treated with CellScrub (Genlatins) to remove complexes associated with the cell surfaces (uninternalized complexes) according to manufacturer’s instructions. Cells were then washed with cold PBS and analyzed using flow cytometry. The intracellular trafficking of complexes containing FAM siRNA was examined using confocal microscopy. KG1 cells (60,000 per well) were seeded in 24-well culture plates 1 day before transfection. Cells were transfected with 100 nM FAM siRNA complexed with CD.DSPE-PEG-Fab and incubated under the normal growth conditions for 10 min, 30 min, and 2 h. In order to label late endosomes and lysosomes, 75 nM LysoTracker Deep Red (Molecular Probes, Invitrogen) was added to the cells for 30 min at 37 °C prior to imaging. The cells were washed with fresh growth medium, transferred into 12-well plates with glass bottoms (MatTek), and centrifuged briefly. Images were acquired on a FluoView FV1000 confocal microscope and analyzed using Olympus FluoView ver 2.1b software. Nanoparticle-Mediated Gene Silencing in KG1 Cells. KG1 cells (60,000 per well) were seeded in 24-well culture plates. Cyclodextrin NPs containing BRD4 siRNA (siBRD4; siRNA ID number s23902, Life Technologies) were applied to cells with a final siRNA concentration of 100 nM and incubated under the normal growth conditions for 72 h. Following incubation, total RNA was isolated from cells using the GenElute Mammalian Total RNA Miniprep Kit (SigmaAldrich), according to manufacturer’s instructions. First-strand cDNA was generated from total RNA samples using HighCapacity cDNA Reverse Transcription Kits (Applied Biosystems). Gene expression was assessed by real-time quantitative PCR (qPCR) using the Applied Biosystems Real Time PCR System (model 7300). Assays were performed using appropriate primers for BRD4 and GAPDH (TaqMan, Applied Biosystems). Amplification was carried out by 40 cycles of denaturation at 95 °C (15 s) and annealing at 60 °C (1 min). The quantitative level of each BRD4 mRNA was measured as a fluorescent signal corrected according to the signal for GAPDH mRNA.26 Following 72 h post-transfection, KG1 cells were lysed using RIPA buffer [Tris-HCl (50 mM; pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, NaCl (150 mM), EGTA (1 mM), sodium orthovanadate (1 mM), sodium fluoride (1 mM), cocktail protease inhibitors (Roche, Welwyn, Hertforshire, U.K.) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (200 mM)]. Protein concentrations were quantified using the bicinchoninic acid (BCA) assay (Thermo Scientific). 30−50 μg of protein per sample was loaded onto an SDS− polyacrylamide gel and electrophoresed at 100 V for ∼2.5 h. Protein was then transferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) for 1 h at 90 V. Membranes were incubated overnight with appropriate antibodies (Anti-Brd4 antibody, ab128874, abcam; c-Myc Antibody, #9402, Cell Signaling Technology; GAPDH antibody, #D16H11, Cell Signaling Technology) at 4 °C. Antibody reactive bands were detected using a LI-COR Odyssey infrared imaging system (IRDye 680RD, G926-68071, LI-COR Biosciences UK Ltd.). Densitometry analysis of bands was performed using ImageJ, and all results were normalized to GAPDH control. Nanoparticle-Mediated Gene Silencing in AML Primary Patient Samples. Patient samples were collected from 6 newly presenting patients following informed consent and

GAA CGU GUC ACG U-3′, Sigma-Aldrich) were loaded onto a 1% (w/v) agarose gel in Tris/Borate/EDTA (TBE, SigmaAldrich) buffer containing Safeview (NBS Biologicals Ltd., U.K.). Electrophoresis was performed at 120 V for 30 min, and the resulting gels were photographed under UV. The incorporation of IL-3Rα Fab was assessed by SDS− PAGE. The gels were run under nonreducing conditions at a constant voltage of 100 V in a Tris/glycine/SDS buffer (Trizma base, glycine, and sodium dodecyl sulfate, Sigma-Aldrich) and stained with Coomassie Brilliant Blue G (Sigma-Aldrich) to detect the protein. The antibody incorporation efficiency was determined by scanning densitometry using ImageJ. The morphology of complexes was studied using transmission electron microscopy (TEM). Complexes containing 0.5 μg of siNeg were left on 400-mesh carbon-filmed copper grids (Agar Scientific) for several minutes. Subsequently, grids were blotted with filter paper. The grids were then stained with 2% (w/w) uranyl acetate, blotted again, and left at rt overnight. Samples were analyzed using a JEOL 2000 FXII transmission electron microscope (Jeol Ltd., Tokyo, Japan). Particle sizes and zeta potentials were measured by dynamic light scattering (DLS) with a Malvern Nano-ZS (Malvern Instruments, U.K.). DIW (0.2 μm membrane-filtered) was added to the complexes and made up to 1 mL. The concentration of CD was fixed at 0.2 mg/mL. The effects of salt- and serum-containing media on the aggregation of the complexes were investigated by incubating complexes in either 50% fetal bovine serum (FBS, SigmaAldrich) for 24 h at 37 °C or 90% OptiMEM transfection medium (Life Technologies) for 24, 48, and 72 h at 37 °C. Size measurements were then carried out by DLS. In addition, siRNAs complexed with or without cyclodextrins were incubated with 50% FBS for 4 and 24 h. The stability of siRNAs was assessed using 1.5% (w/v) agarose gels, and the resulting gels were photographed under UV. In Vitro Characterization of the Nanoparticles. KG1 cells (Sigma-Aldrich) were maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Sigma-Aldrich) supplemented with 20% FBS and 2 mM L-glutamine (Sigma-Aldrich). K562 cells (kindly donated from Cork Cancer Research Centre, Ireland) were maintained in RPMI-1640 medium (SigmaAldrich) supplemented with 10% FBS. For the competitive binding study, 60,000 KG1 cells/well were seeded in 24-well plates and incubated for 24 h under normal growth conditions. On the following day, free IL-3Rα Fab (10 μg/mL) was added to KG1 cells for 1 h at 4 °C prior to the incubation of formulations containing 50 nM FAM siRNA (sense sequence 5′-UUC UCC GAA CGU GUC ACG U-3′, modified by 6-FAM on 5′ of sense sequence, SigmaAldrich) (the final concentration of siRNA per well, unless otherwise mentioned). Following 24 h incubation at 4 °C cells were washed with cold phosphate buffered saline solution (PBS, Sigma-Aldrich) and were resuspended in 1000 μL of cold PBS in polystyrene round-bottom tubes (Becton Dickinson). Ten thousands cells were measured for each sample following the Becton Dickinson FACScalibur manual. The level of cellular uptake mediated by complexes containing FAM siRNA was assessed using flow cytometry. 60,000 KG1 and 60,000 K562 cells/well were seeded in 24-well plates and incubated for 24 h under normal growth conditions. Cells were then transfected by 50 nM FAM siRNA complexed CD.DSPE-PEG and CD.DSPE-PEG-Fab and incubated for 24 h under normal growth conditions. Following incubation, cells 942


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weight ratio (WR) 10 have achieved efficient in vitro and in vivo gene knockdown17,18 and were therefore used in this study to prepare the antibody-targeted formulation. A “postinsertion” technique has been described to prepare antibody-targeted liposomes which are generally termed “immunoliposomes”.21,22,25 The whole antibodies or antibody fragments can be coupled to DSPE-PEG and then transferred in a simple incubation step into the outer layer of preformed, drug-loaded liposomes. This versatile method facilitates a combinatorial approach to the design of targeted liposomes that minimizes manufacturing complexities, allowing PEG and antibodies to be inserted into a variety of preformed liposomes.21,22,25 In this study, a similar postinsertion-like approach was used to incorporate DSPE-PEG or DSPE-PEGFab into the preformed amphiphilic CD.siRNA complexes (Figure 1). As indicated by dynamic light scattering (DLS), the particle size of CD.siRNA (WR10) complex was significantly (p < 0.05) increased following incorporation of DSPE-mPEG, and the surface charge was significantly (p < 0.05) reduced (Table S1). These results suggest that successful incorporation of DSPEPEG into the CD.siRNA complexes occurred. At a CD to DSPE-mPEG molar ratio (MR) of 0.35 the resulting CD.siRNA.DSPE-PEG formulation demonstrated nanoscale particle size (∼200 nm) and nearly neutral surface charge (∼6 mV) (Table S1). Due to favorable particle size and zeta potential, the MR of 0.35 between CD and DSPE-mPEG (also DSPE-PEG-Maleimide) was chosen for preparation of the antibody-targeted formulation. It has been reported that positively charged NPs can nonspecifically bind serum proteins causing aggregation when administrated systemically. These large aggregates maybe entrapped inside the lung endothelial capillary bed or taken up by the reticuloendothelial system [RES, also known as mononuclear phagocyte system (MPS)].29 Coating the NPs with PEG can provide charge shielding and steric effects, therefore stabilizing the NPs against salt-, protein-, and complement-induced inactivation.30 Incorporation of an Antibody Targeting Ligand. AML LSCs exhibit unique biological properties, including infrequent entrance into the cell cycle, self-renewal capacity, and resistance to conventional chemotherapeutics.7 The identification of LSCs has important clinical implications, as their elimination should reduce the potential for relapse.31 Recently, a number of AML LSC antigens have been discovered, including CD33, CD44, CD47, C-type lectin-like molecule-1 (CLL-1), and IL-3Rα (also known as CD123).32 When the human IL-3 receptor, a cell surface heterodimer composed of α and β subunits, binds IL-3 (Interleukin-3, a cytokine) it stimulates cell cycle progression and differentiation and inhibits apoptosis of hematopoietic cells.33 It has been reported that IL-3Rα is expressed on the myeloid progenitors and a subpopulation of B lymphocytes but in contrast is not expressed on lymphoid progenitors, peripheral T cells, natural killer cells (NK-cells), or mature myeloid cells (i.e., platelets and red blood cells).33,34 It has also been demonstrated that LSCs with high expression of IL-3Rα are biologically distinct from their normal stem cell counterparts where a lower density of this antigen is found.35,36 Recently, therapeutic agents conjugated onto fusion proteins or antibodies targeting IL3Rα have demonstrated in vitro and in vivo antileukemia effects,37−39 suggesting a promising role for IL-3Rα in targeted delivery of therapeutic agents in the treatment of AML.

ethical approval [ref: ECM 4(gg) 07/01/14)]. All newly diagnosed adult patients presenting to Cork University Hospital were eligible for the study. Sequential patients consented to donate the BM and/or PB so that an EDTA sample with a minimum blast count of 5 × 109/L was obtained. Samples were collected within 4 h and processed fresh on the day of harvest. Mononuclear cells (MNCs) were prepared using Histopaque-1077 (Sigma-Aldrich) and used freshly. Primary AML cells (500,000 per well) were seeded in 24-well culture plates with RPMI-1640 medium supplemented with 10% FBS 1 day before transfection. Cyclodextrins containing BRD4 siRNA were applied to the cells with a final siRNA concentration of 200 nM and incubated under the normal growth conditions for 72 h. The BRD4 mRNA and protein levels were assessed by real-time qPCR and Western blotting as described above. In Vitro and ex Vivo Therapeutic Studies using the Nanoparticles. Cell proliferation was determined in KG1 cells using Cell Counting Kit-8 (CCK-8) (Dojindo, Japan), which is reduced by dehydrogenases in living cells to give orange colored formazan products. KG1 cells (10,000 per well) were seeded in 96-well plates under the normal growth conditions. Following 1 day incubation, CD.DSPE-PEG-Fab containing 25, 50, 100, and 200 nM siNeg and siBRD4 was added to the cells and incubated for 24 h. 50 nM cytarabine (Ara-C) was then added and incubated for 48 h. Subsequently, CCK-8 solution was added to the cells and incubated for 4 h before measuring the absorbance at 450 nm using a microplate reader. The viability of the cells treated with CD.siRNA.DSPE-PEG-Fab, Ara-C, and the combination was normalized relative to the untreated control. The combined therapeutic effects were determined as previously described.27 In brief, the value of coefficient of drug interaction (CDI) was determined using the following formula: CDI = SAB/(SA × SB), where SA and SB are the viability of CD.siRNA.DSPE-PEG-Fab and Ara-C relative to untreated control; the SAB is the viability of the combination treatment relative to the control. CDI < 1, CDI = 1, and CDI > 1 suggest synergistic, additive, and antagonistic effects, respectively. Antiproliferative effects of the cyclodextrin.siRNA NPs were also determined in primary leukemic cells using the colony formation assay.28 AML primary cells were prepared as described above and seeded in 24-well plates (300,000 cells per well) 1 day before transfection. Following incubation, cells were treated with 200 nM siBRD4 complexed with CD.DSPEPEG and CD.DSPE-PEG-Fab, and 200 nM siNeg complexed with CD.DSPE-PEG-Fab for 24 h. Subsequently 200 nM Ara-C was added and incubated with the cells (80,000 per 35 mm dish) in MethoCult H4034 Optimum (Stemcell Technologies), according to the instructions supplied. After 1 week incubation cell counts were obtained using the trypan blue assay (SigmaAldrich) from Bio-Rad TC10 Automated Cell Counter. The combined therapeutic effects were determined as described above. Following 1 week incubation, cells were spun onto glass slides and analyzed for morphological signs of myeloid differentiation by Wright−Giemsa staining in accordance with the manufacturer’s instructions (Sigma-Aldrich).

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RESULTS AND DISCUSSION Formulation of Neutral Nanosized CD-Based siRNA Nanoparticles. The aim of this work was to formulate a nanosized, neutral, antibody-targeted cyclodextrin.siRNA delivery system (CD.siRNA.DSPE-PEG-Fab) using the approach outlined in Figure 1. Previously, CD.siRNA complexes at 943


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the free Fab is visible in each case (Figure 2a). In contrast, with DSPE-PEG-Maleimide to Fab at the higher MR20, ∼ 95% incorporation of antibody was achieved (measured using ImageJ software) where free Fab is now negligible (Figure 2b). The cellular uptake of the CD.siRNA.DSPE-PEG-Fab was assessed in KG1 cells using flow cytometry (Figure S2), and results indicated that the internalization of siRNA was slightly higher (∼28%, [C] = siRNA) (p > 0.05) at MR20 (DSPEPEG-Maleimide and Fab) when compared to MR10 (∼26%) and MR40 (∼24%). Physicochemical Characterization of the AntibodyTargeted CD-Based Prototype Formulation. Based on the results outlined above, a prototype targeted CD.siRNA.DSPEPEG-Fab formulation (CD to siRNA WR10; CD to DSPEPEG-Maleimide MR0.35; DSPE-PEG-Maleimide to Fab MR20) was selected for all future studies (Table S2). Following purification, CD.siRNA.DSPE-PEG-Fab was treated with heparin (1000 IU/mL) for 1 h at rt to release the siRNA.16 As controls, CD.siRNA and CD.siRNA.DSPE-PEG were treated under the same conditions. The yield of CD.siRNA.DSPE-PEG-Fab was approximately 100% in terms of siRNA concentration relative to CD.siRNA and CD.siRNA.DSPE-PEG (Figure S3). Gel electrophoresis confirmed that the incorporation of DSPE-PEG or DSPE-PEG-Fab did not impair the complexation of siRNA with CD (Figure 3a). The particle size and zeta

Several disadvantages of using whole antibodies as targeting moieties have been reported, including the following: (1) the complex process involved in production and the subsequent high cost; (2) whole antibodies are normally immunogenic and are rapidly cleared from the circulation through Fc (fragment, crystallizable)-mediated uptake by macrophages;40 and (3) whole antibodies may increase the overall particle size, thus negatively affecting biodistribution and cellular uptake of NPs.41 In addition, it is important to note that the Fc of human IL-3Rα antibodies has antibody-related therapeutic effects, inducing the innate immune system and inhibiting LSC homing and proliferation in an AML mouse model.42 As the purpose of this study is to develop a targeted cyclodextrin NP for delivery of siRNA, a Fab fragment of human IL-3Rα antibodies (Clone #32703, R&D Systems, U.K.), but not whole antibodies, was used in this study for targeted delivery of siRNA to AML LSCs. The incorporation of IL-3Rα Fab was verified using SDS− PAGE (Figure 2). The use of SDS−PAGE is the routine state

Figure 2. Incorporation of IL-3Rα Fab was confirmed using SDS− PAGE. (a) The CD.siRNA.DSPE-PEG-Fab was prepared without purification. Lane 1: protein marker (Life Technologies). Lane 2: 2 μg of free Fab. Lane 3: CD.siRNA.DSPE-PEG-Fab (WR10 of CD to siRNA; MR0.35 of CD to DSPE-PEG-Maleimide; MR2.5 of DSPEPEG-Maleimide to Fab). Lane 4: CD.siRNA.DSPE-PEG-Fab (MR5 of DSPE-PEG-Maleimide to Fab). Lane 5: CD.siRNA.DSPE-PEG-Fab (MR 10 of DSPE-PEG-Maleimide to Fab). The incorporation efficiency of Fab was 20%, 30%, and 80% at MR2.5, MR5, and MR10 of DSEP-PEG-Maleimide to Fab respectively, which were measured using ImageJ software. (b) The incorporation of IL-3Rα Fab was confirmed using SDS−PAGE. The CD.siRNA.DSPE-PEG-Fab was prepared with purification. Lane 1: protein marker (Life Technologies). Lane 2: 2 μg of free Fab. Lane 3: DSPE-PEG-Fab (containing 2 μg of Fab). Lane 4: CD.siRNA.DSPE-PEG-Fab (WR10 of CD to siRNA; MR0.35 of CD to DSPE-PEG-Maleimide; MR20 of DSPE-PEG-Maleimide to Fab) (containing 2 μg of Fab). The incorporation efficiency of Fab was 95% at MR20 of DSEP-PEGMaleimide to Fab, which was measured using ImageJ software.

Figure 3. Physicochemical characterization of cyclodextrin.siRNA formulations (Table S2). (a) Complexation of siRNA (0.5 μg) with cyclodextrins was confirmed using 1% agarose gel electrophoresis, and the resulting gels were analyzed under UV. (b) Particle sizes and zeta potentials of CD.siRNA, CD.siRNA.DSPE-PEG, and CD.siRNA.DSPE-PEG-Fab (mean ± SD, n = 6) were analyzed using DLS (*p < 0.05 compared to nontargeted formulation). (c) Morphology of CD.siRNA, CD.siRNA.DSPE-PEG, and CD.siRNA.DSPE-PEG-Fab was analyzed using TEM (scale bar = 200 nm).

of the art analysis used for such antibody conjugates as described in recent references.21,43 Here, in comparison with the protein marker, Fab was seen to have a molecular weight range in the region of ∼40 kDa (Figure 2). The DSPE-PEGFab can be seen to be largely overlapping (versus Fab) but with a very slightly higher molecular weight range (Figure 2b). Because of polydispersity in molecular weights of both DSPEPEG (2941 Da average) and Fab, any further differentiation between the species is unlikely by this gel method. When the MR of DSPE-PEG-Maleimide to Fab was increased (i.e., MR of 2.5, 5, and 10) the antibody incorporation was improved (Figure 2a). As the DSPE-PEG-Maleimide to Fab molar ratio range (MR 2.5−10) was not enough to complete the coupling,

potential of cyclodextrin.siRNA complexes were measured using DLS (Figure 3b; it is worth noting that the particle size and zeta potential of CD.siRNA.DSPE-PEG-Fab were not significantly changed before and after purification). Results demonstrated that the incorporation of the antibody significantly (p < 0.05) increased the overall size of CD.siRNA.DSPEPEG-Fab (∼210 nm) in comparison with CD.siRNA.DSPEPEG (∼200 nm) (Figure 3b); in contrast, the surface charge remained unaffected [CD.siRNA.DSPE-PEG-Fab (∼5 mV) and 944


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Figure 4. In vitro characterization of cyclodextrin.siRNA formulations. (a) For the competitive binding study, free antibody was added to KG1 cells (antigen-positive cells) to block IL-3Rα prior to the addition of formulations. Cyclodextrin complexes were then incubated with cells for 24 h at 4 °C. The competition with free IL-3Rα Fab dramatically reduced the binding of targeted formulation (50 nM siRNA) to KG1 cells. (b) Fluoresceinpositive KG1 and K562 (antigen-negative) cells (%, mean ± SD) 24 h post-transfection at 37 °C with cyclodextrin formulations (50 nM siRNA). Targeted formulation demonstrated significantly higher cellular uptake in KG1 cells (∼50% fluorescein-positive cells) relative to K562 cells (∼8%) (n = 3, **p < 0.01). (c) Intracellular distribution of targeted formulation (100 nM siRNA) in KG1 cells at time intervals of 10 min, 2 and 24 h posttransfection using confocal microscopy (40×). In the images for each time point, left panels and right panels represented the merged images of FAM siRNA (green) and LysoTracker Deep Red (red) and the merged images of FAM siRNA and cells under the transmission light.

group.46 In contrast, CD.siRNA.DSPE-PEG was significantly changed, showing a similar pattern to that previously published,20 suggesting a “shielding” layer around the particles. In addition, an irregular looped morphology was observed for CD.siRNA.DSPE-PEG-Fab, which was not seen for CD.siRNA and CD.siRNA.DSPE-PEG. Clearly, these differences in morphology support changes to particle structure following PEGylation and incorporation of the PEGylated antibody. It has been reported that NPs can protect siRNA from nuclease degradation when compared to unmodified or uncomplexed siRNA.47 The targeted formulation protected siRNA from degradation when incubated within 50% FBScontaining medium for up to 24 h when compared to naked siRNA (Figure S5). In addition, stability studies in serum- and salt-containing media indicated that the PEGylated formulations significantly inhibited aggregation in comparison with the non-PEGylated counterpart (Figure S6). These favorable physicochemical characteristics, in terms of size, charge, and stability, suggest that the PEGylated formulations may avoid the nonspecific recognition of RES,29 thus prolonging circulation in vivo and resulting in enhanced interaction of the targeted formulation with leukemia cells in the blood.9 In Vitro Characterization of the Antibody-Targeted CD-Based Prototype Formulation. To investigate the selective targeting of CD.siRNA.DSPE-PEG-Fab, two leukemic cell lines were utilized, namely, KG1 (an AML leukemia stem and progenitor cell line48) and K562 (a chronic myeloid leukemia cell line). The antigen expression study showed that up to 70% of KG1 cells expressed IL-3Rα whereas K562 cells were IL-3Rα negative (Figure S7). A competitive binding study was performed using pretreatment with excess IL-3Rα Fab. The binding of nontargeted formulation to KG1 cells was not

CD.siRNA.DSPE-PEG (∼6 mV)]. These results further support the formation of antibody-targeted cyclodextrin.siRNA NPs. In addition, the average polydispersity index (PDI) of CD.siRNA, CD.siRNA.DSPE-PEG, and CD.siRNA.DSPEPEG-Fab were 0.208, 0.255, and 0.257 respectively, suggesting the formation of mainly monodisperse particles. Modified amphiphilic CDs, such as the example in this study, incorporate lipid groups, and these mesomolecular amphiphiles have been shown by our group to self-assemble into micelles and vesicles.44 In addition to the characterization shown in this manuscript, we have previously investigated complexes formed between cationic amphiphilic CDs and plasmid DNA (pDNA) by using small-angle X-ray scattering.44 Results show that these modified CDs form bilayer vesicles where the DNA lies between the polar layers of the CD protonated amino groups in alternation with the lipid bilayers. In addition we have also shown that amphiphilic PEGylated CDs can be coformulated with their amphiphilic cationic CD counterparts to form “liposomal-like” structures with morphology similar to that in this current work. 45 We have also shown that this coformulation approach can be extended to incorporate DSPE-PEG into the lipidic bilayer structure of the amphiphilic CDs.20 These previously published studies support the concept that lipid-like DSPE-PEG (DSPE-PEG2000 and DSPE-PEG2000antibody) can be formulated with amphiphilic CDs using a “postinsertion-like approach” to form “liposomal-like” structures. Indeed, when DSPE-PEG and DSPE-PEG-Fab were added into CD.siRNA, the PEGylated formulations showed distinct structures (Figure 3c and Figure S4). As indicated by TEM in this study, CD.siRNA demonstrated a solid structure, which was similar to what has been previously reported by our 945


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landscapes where oncogenic gene expressions are enforced by certain chromatin regulatory machinery.57 Recently, it has been demonstrated by Zuber et al. that the epigenetic reader BRD4 is critically required to maintain LSCs and prevents terminal myeloid differentiation.23 In addition, the expression of BRD4 mRNA and protein was found in various AML cell lines (i.e., KG1, HL60, U937, MV4-11, and MOLM-13) and primary samples from all AML patients examined (n = 37),58 suggesting a role for BRD4 as a therapeutic target in AML. Therefore, the in vitro therapeutic effects were assessed in KG1 cells using targeted formulation containing BRD4 siRNA. The knockdown efficiency of BRD4 mRNA and protein in KG1 cells was assessed by real time qPCR (Figure 5a) and

significantly affected with or without pretreatment of free IL3Rα Fab; in contrast, with the targeted formulation a dramatic reduction in binding to KG1 cells occurred (Figure 4a). This data suggest selective binding of CD.siRNA.DSPE-PEG-Fab to IL-3Rα positive cells. In addition to cell surface binding specificity, successful delivery of siRNA using targeted formulation is also dependent on efficient internalization. The internalization can vary depending on the particle size, surface charge, shape and chemistry of NPs, and the interaction between targeting moieties and receptors.49,50 The cellular uptake of targeted formulation was assessed in KG1 and K562 cells using flow cytometry. Results in Figure 4b show that CD.siRNA.DSPEPEG-Fab achieved significantly higher cellular uptake in KG1 cells (∼50% fluorescein-positive cells) when compared to the untargeted counterpart (∼10% fluorescein-positive cells). In addition, as shown in Figure 4b, targeted formulation also demonstrated significantly higher cellular uptake in KG1 cells relative to K562 cells (∼8% fluorescein-positive cells). In contrast, the nontargeted counterpart did not achieve a significant level of internalization into either KG1 or K562 cells. Therefore, these results further confirmed cell surface binding specificity due to the antibody incorporation. Although PEGylation offers enhanced NP stability, it has been reported to impair cell binding and uptake.51,52 However, in this study the conjugation of Fab to the PEG chain has been shown to facilitate selective targeting and enhance cellular uptake of the PEGylated formulation into IL-3α positive cells via a receptor-mediated pathway, thereby minimizing nonspecific internalization in antigen negative cells. Following receptor-mediated endocytosis, siRNA escape from the endosomal/lysosomal compartment into the cytoplasm remains a significant challenge.53−55 Therefore, intracellular trafficking of the targeted formulation using 5carboxyfluorescein-labeled (FAM) siRNA was studied in KG1 cells using confocal microscopy (Figure 4c). Ten minutes after transfection, FAM siRNA was attached to the cell membrane, suggesting efficient binding of the targeted formulation. Endosomal escape of siRNA was evident at 2 h posttransfection, as free siRNA was found inside the cytoplasm of the cells, although some siRNA remains trapped in the endosomes, where the yellow staining observed was colocalized with LysoTracker Deep Red (as shown by the arrow). At 24 h post transfection, the green fluorescence (free siRNA) had become predominately released (Figure 4c). The efficient trafficking of siRNA into the cytoplasm of AML cells is most likely due to the presence of protonated amine groups on the CD triggering the proton sponge effect (Figure S1, CD has previously demonstrated successful intracellular trafficking of siRNA in various cell lines14,17). In Vitro Therapeutic Effects of the Antibody-Targeted CD-Based Prototype Formulation. Gene expression can be regulated by epigenetic pathways through controlling and interpreting chromatins (a macromolecular complex of DNA and histone proteins). Modifications to DNA and histones remodel chromatin structure by altering noncovalent interactions within and between nucleosomes (the fundamental subunit of chromatin), which can serve as docking sites for specialized proteins (i.e., epigenetic readers) that recognize these modifications.56 The epigenetic readers, playing a role as the effectors of the modification, will recruit additional chromatin modifiers and remodeling enzymes. It is now known that cancer cells are characterized by altered epigenetic

Figure 5. In vitro therapeutic effects of antibody-targeted formulation. (a) mRNA and (b) protein downregulation of BRD4 in KG1 cells 72 h post-transfection by naked BRD4 siRNA (100 nM), Negative Control siRNA with targeted formulation, and BRD4 siRNA with targeted and nontargeted formulation. (NS = no significance, *p < 0.05 compared to targeted formulation with Negative Control siRNA) (mean ± SD, n = 3). (c) KG1 cells were transfected with targeted formulation containing BRD4 or Negative Control siRNA at different concentrations (25, 50, 100, and 200 nM) for 24 h. Following 1 day transfection, 50 nM cytarabine (Ara-C) was added to cells and incubated for 48 h. After incubation, viability was measured using CCK-8. (# represents synergistic effect as per the coefficient of drug interaction) (mean ± SD, n = 3).

Western blot (Figure 5b) respectively. Results in Figure 5a indicate that CD.siBRD4.DSPE-PEG-Fab (100 nM siRNA) achieved up to 40% BRD4 mRNA inactivation relative to Negative Control siRNA counterpart (p < 0.05). In addition, CD.siBRD4.DSPE-PEG-Fab significantly reduced BRD4 protein level by ∼50% relative to Negative Control siRNA counterpart (p < 0.05) (Figure 5b) (Figure S8). c-MYC plays an important function in growth control, differentiation and apoptosis, and its abnormal expression is associated with many tumors, i.e., the c-MYC transcriptional network has an vital role in AML proliferation and LSC self-renewal.59 As a downstream target of the BET family, c-MYC is positively regulated by BRD4 and therefore, inhibition of BRD4 has been shown to be a therapeutic strategy to inhibit c-MYC.60 In this study, the expression of c-MYC protein was also reduced by ∼55% using CD.siBRD4.DSPE-PEG-Fab when compared to Negative Control siRNA counterpart (Figure 5b) (Figure S8), suggesting that the targeted formulation is able to impair the regulatory function of BRD4 on c-MYC. 946


newly diagnosed PB newly diagnosed PB newly diagnosed PB newly diagnosed PB newly diagnosed BM relapsed PB to

to

to

WHO AML AML AML MDS AML AML/MDS AML/MDS

BRD4 and c-MYC protein levels

6.1 26.3 2.6 21 6.3 27.6

23 85 61 88 4 46

PB

karyotype

therapeutic effects

del 9q 11q23 t(11:19)(q23:p13.3) trisomy 8 normal monosomy 7 del 5q

ND MLL(KMT2A) ND ND ND ND

molecular defects

significant inhibition of AML growth relative to Negative Control siRNA formulation (p < 0.05); synergistic therapeutic effect in combination with Ara-C slight inhibition of AML growth relative to Negative Control siRNA formulation; synergistic therapeutic effect in combination with Ara-C significant inhibition of AML growth relative to Negative Control siRNA formulation (p < 0.05); synergistic therapeutic effect in combination with Ara-C significant inhibition of AML growth relative to Negative Control siRNA formulation (p < 0.05); synergistic therapeutic effect in combination with Ara-C significant inhibition of AML growth relative to Negative Control siRNA formulation (p < 0.05). synergistic therapeutic effect in combination with Ara-C significant inhibition of AML growth relative to Negative Control siRNA formulation (p < 0.01); synergistic therapeutic effect in combination with Ara-C

50 65 54 90 20 40

BM

% blast cells

∼45% reduction in BRD4 protein expression and ∼35% reduction in c-MYC protein expression, relative to Negative Control siRNA formulation ∼65% reduction in c-MYC protein expression, relative to Negative Control siRNA formulation

NT

NT

NT

∼30% BRD4 mRNA inactivation relative Negative Control siRNA formulation ∼35% BRD4 mRNA inactivation relative Negative Control siRNA formulation ∼40% BRD4 mRNA inactivation relative Negative Control siRNA formulation ∼40% BRD4 mRNA inactivation relative Negative Control siRNA formulation ∼60% BRD4 mRNA inactivation relative Negative Control siRNA formulation to

NT to

M6 M2 M2 M1 M2 M2

FAB

NT

BRD4 mRNA level

68 46 67 72 34 66

age

WBC (1 × 10̂9 cells/L)

FAB = French−American−British classification; WHO = World Health Organization classification; WBC = white blood cell; PB = peripheral blood; BM = the bone marrow; Ara-C = cytarabine; UC = unclassifiable on FAB; ND = none detectedFlt3, NPM1, MLL wild type; AML/MDS is WHO AML with MDS related change.

a

6

5

4

3

2

1

source

female female female male male female

1 2 3 4 5 6

AML patient no.

gender

AML patient no.

diagnosis

Table 1. Therapeutic Effects of Targeted Formulation Containing BRD4 siRNA on Primary Leukemia Cellsa

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combined therapy with Ara-C), results obtained from patient 5 (Figure 6) and patient 6 (Figure 7) will be discussed in detail in this section.

Following successful BRD4 knockdown, the antileukemia mechanisms of the targeted formulation were investigated using KG1 cells. AML is characterized by an expanded self-renewal capacity linked with an inability to complete terminal myeloid differentiation. When the differentiation of KG1 cells was investigated using Wright−Giemsa staining, results showed that treatment with the targeted formulation containing BRD4 siRNA (100 nM) altered the morphology (i.e., large round nuclei) of KG1 cells from myelomonocytic blasts to cells with a monocyte-like structure (i.e., bean-shaped or kidney-shaped nuclei) in comparison with other controls (Figure S9), suggesting that inhibition of BRD4 and c-MYC led to myeloid differentiation.23 In addition, CD.siBRD4.DSPE-PEG-Fab (100 nM siRNA) significantly induced apoptosis in KG1 cells (p < 0.001) relative to Negative Control siRNA formulation (Figure S10). To examine whether these antileukemia mechanisms produce a therapeutic effect, the growth inhibition of KG1 cells was assessed using CCK-8 (WST-8), the detection sensitivity of which is higher than that of other tetrazolium salts such as MTT, XTT, MTS, or WST-1 (Dojindo, Japan). As shown in Figure 5c, CD.siBRD4.DSPE-PEG-Fab achieved significantly higher cell death (∼40% at 200 nM siRNA) relative to Negative Control siRNA counterpart (∼15% at 200 nM siRNA) (p < 0.05). The role of c-MYC in the development of drug resistance has been described in a variety of cancers, and inhibition of c-MYC sensitizes various cancers to chemotherapeutics.61,62 When administered alone the targeted formulation resulted in ∼40% cell death (200 nM siRNA), and treatment with Ara-C alone produced ∼20% cell death. In contrast, when the treatments were used in combination, a synergistic therapeutic effect, with ∼80% cell death, was detected as the knockdown of BRD4 and c-MYC enhanced the sensitivity of the KG1 cells to the chemotherapeutic (Figure 5c). In addition, it has been shown by antiproliferation CCK-8 assay (Figure 5c) that the amounts of NPs used are consistent with very high cell viability, as NPs containing Negative Control siRNA did not cause significant cell death (>85% cell viability at 200 nM siRNA); in contrast, NPs containing BRD4 siRNA achieved significant antiproliferative effect (∼60% cell viability at 200 nM siRNA). In addition, when combined with cytarabine, NPs containing BRD4 siRNA resulted in a synergistic antiproliferative effect (∼20% cell viability at 200 nM siRNA) in comparison to NPs with Negative Control siRNA (∼80% cell viability at 200 nM siRNA) (Figure 5c). It has been shown that the differentiation (Figure S9) and apoptosis (Figure S10) were induced in leukemia cells only following the downregulation of BRD4; these phenotypes have also published by others when they silenced BRD4.23,58 Therefore, the antiproliferation (Figure 5C) is not due to cell death or cytotoxicity from the NPs. Ex Vivo Therapeutic Effect of the Antibody-Targeted CD-Based Prototype Formulation. The ability of targeted formulation to deliver therapeutic siRNA (BRD4) was further confirmed using primary leukemic cells of freshly diagnosed and relapsed AML patients (n = 6, Table 1). Due to the fact, under the experimental conditions used, that the amounts of fresh BM or PB samples from patients 5 (a newly diagnosed patient) and 6 (a relapsed patient) (but not from patients 1 to 4) were sufficient for a full range of investigations including gene knockdown (mRNA and protein levels) and therapeutic efficacies (antibody-targeted CD as a monotherapy and as a

Figure 6. Ex vivo therapeutic effects of antibody-targeted formulation in a newly diagnosed AML (patient 5 newly diagnosed, Table 1). (a) mRNA and (b) protein downregulation of BRD4 72 h posttransfection in MNCs of a newly diagnosed AML by naked BRD4 siRNA, Negative Control siRNA with targeted formulation, BRD4 siRNA with targeted and nontargeted formulations, and BRD4 formulated with Lipofectamine 2000 (*p < 0.05 compared to targeted formulation with Negative Control siRNA) (mean ± SD, n = 3). (c) AML blasts were transfected with various formulations containing Negative Control or BRD4 siRNA (200 nM) for 24 h. Following 1 day transfection, 200 nM cytarabine (Ara-C) was added to cells and incubated within MethoCult H4034 Optimum for 1 week. After incubation, morphologic signs of myeloid differentiation were analyzed by Wright−Giemsa staining using a bright-field microscope (40×). (d) In addition, viability was measured using trypan blue assay (*p < 0.05 compared to formulated Negative Control siRNA; # represents synergistic effect as per the coefficient of drug interaction) (mean ± SD, n = 3).

The mononuclear cells (MNCs) purified from the PB and BM of AML patients are mainly composed of leukemic cells and LSCs, both of which are known to highly express CD123.32,33 The expression of IL-3Rα on AML MNCs was confirmed using flow cytometry (Figure S11). In this study 200 nM siBRD4 was used, due to the effective synergistic efficacy achieved by 200 nM siBRD4, in Figure 5c. As shown in Figures 6a and 6b, BRD4 mRNA and protein levels in blasts of a newly diagnosed AML patient (Table 1, patient 5) were significantly reduced by the targeted formulation (∼40% and ∼45%, respectively; the protein level of c-MYC was reduced by ∼35%) relative to the negative control siRNA formulation (p < 0.05). Following treatment morphological signs of myeloid maturation were detected (Figure 6c). The targeted formulation also demonstrated significantly higher gene knockdown relative to a “gold standard” transfection reagent Lipofectamine 2000 (Figures 6a, 6b, and 7a), indicating the potential of the cyclodextrin construct as a nanoparticulate siRNA delivery system for leukemia. Since LSCs reside in a mostly quiescent state, they are thought to be resistant to current chemotherapeutic regimens, which mainly function by targeting cycling cells.7 Therefore, therapeutic strategies that can selectively eradiate LSCs independent of cell cycle status are urgently required. As 948


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blast death (Figure 7d), which is significantly higher than Ara-C alone (∼50% at 200 nM) (p < 0.05). This is most likely due to the fact that IL-3Rα was highly expressed on blasts from the relapsed AML patient relative to the same population from the newly diagnosed patients (Figure S11). Further research focusing on therapeutic effects using more newly diagnosed and relapsed samples will be required to confirm this hypothesis. Suspension cells, such as primary blood cells and leukemia cells, are known to be difficult to transfect by conventional lipid- and polymer-based delivery strategies; although successfully used in other studies with leukemia cells,64,65 in this work Lipofectamine 2000 did not downregulate the targeted genes, Figures 6a, 6b, and 7a. In this study, results shown in Figures 6 and 7 indicate that the targeted CD-based formulation achieved marked therapeutic efficacies by specifically and efficiently downregulating the targeted genes (BRD4 and C-MYC). More importantly, a combination of the targeted formulation and Ara-C achieved a synergistic therapeutic effect, which can dramatically destroy AML blasts (Figure 7d). These results indicate significant therapeutic potential for the targeted formulation to block the growth of leukemic cells and LSCs through BRD4 inhibition, thus providing a novel treatment concept combining RNAi therapeutics and chemotherapeutics in patients with relapsed or refractory AML.

Figure 7. Ex vivo therapeutic effects of antibody-targeted formulation in a relapsed AML (patient 6 relapsed patient, Table 1). (a) mRNA and (b) protein downregulation of BRD4 72 h post-transfection in blasts of a relapsed AML by various formulations containing BRD4 siRNA or Negative Control siRNA (*p < 0.05 compared to formulated Negative Control siRNA) (mean ± SD, n = 3). (c) AML blasts were transfected with various formulations containing Negative Control or BRD4 siRNA (200 nM) for 24 h. Following 1 day transfection, 200 nM cytarabine (Ara-C) was added to cells and incubated within MethoCult H4034 Optimum for 1 week. After incubation, morphologic signs of myeloid differentiation were analyzed by Wright−Giemsa staining using a bright-field microscope (40×). (d) In addition, viability was measured using trypan blue assay after transfection in combination with exposure to Ara-C (*p < 0.05 compared to 200 nM Ara-C; # represents synergistic effect) (mean ± SD, n = 3).

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CONCLUSION A range of nonviral materials, including lipid/liposomes25,66−68 and polymers,48,69 have been used to formulate gene delivery systems for AML treatment. However, to date the clinical application of siRNA therapeutics for AML therapy is still hindered due to disappointing safety and efficacy data. In this study, an antibody-targeted cyclodextrin to specifically target AML LSCs and efficiently deliver siRNA to silence BRD4 was developed. Efficient knockdown of an epigenetic reader, BRD4, mediated by the targeted formulation, was shown at both the mRNA and protein levels in IL-3Rα positive KG1 cells and primary samples, which led to myeloid differentiation, induced leukemia apoptosis, and reduced blast proliferation. In addition, the targeted formulation achieved synergistic therapeutic effects when combined with the clinically available chemotherapeutic Ara-C, indicating a novel combination therapy for AML treatment. Interestingly, the targeted formulation achieved superior therapeutic effects in relapsed AML samples where IL3Rα is highly expressed, in comparison with newly diagnosed AML where IL-3Rα is partially expressed, thus further strengthening the potential of the strategy targeting LSCs for AML therapy. It is now known that fresh surgical patient materials or human tumor engraftments implanted in immunodeficient mice have been widely used as disease models.70 A patientderived xenograft (PDX) model for leukemia may be achieved by iv administration of primary patient samples, resulting in significant engraftment in mouse spleen and BM.71 As a consequence, the disease progression and leukemia biology in PDX models have been shown to be similar to those in human. Therefore, establishment of an AML PDX model imitating LSC development and the assessment of the CD-based targeted formulation in this model will be the focus of future work. AML is a heterogeneous disease at both the phenotypic and molecular levels with a variety of distinct genetic alterations, and recent studies have demonstrated that such heterogeneity also extends to AML LSCs. Using a postinsertion-like

shown in Figure 6d, inhibition of BRD4 and c-MYC mediated by the targeted formulation (200 nM siBRD4) significantly decreased the proliferation of blasts when compared to Negative Control siRNA formulation (p < 0.05); more importantly, a synergistic therapeutic effect was also achieved in combination with Ara-C (200 nM, a clinical achievable concentration63) (Figure 6d). When leukemic cells treated with CD.siBRD4.DSPE-PEG-Fab were replated into in MethoCult H4034 Optimum, they failed to self-renew or proliferate in culture (Figure S12), suggesting that the targeted formulation is able to impair the leukemic clonogenicity, a hallmark of LSC self-renewal. In addition, similar therapeutic efficacies were also evident with patients 1 to 4 (newly diagnosed AML patients, see therapeutic efficacies in Table 1). Taken together, these results in Figure 6 indicate that the targeted formulation led to BRD4 inhibition in newly diagnosed AML patients via CD123mediated pathway accompanied by terminal myeloid differentiation and elimination of leukemic cells and LSCs, achieving synergistic therapeutic effects in combination with Ara-C. In comparison, in a relapsed AML patient (Table 1, patient 6) CD.siBRD4.DSPE-PEG-Fab demonstrated a more significant therapeutic efficacy (Figure 7). As shown in Figure 7a and Figure 7b, the targeted formulation (200 nM siBRD4) achieved significantly higher downregulation of the corresponding genes (∼60% BRD4 mRNA reduction and ∼65% c-MYC protein reduction) (p < 0.01) when compared to the Negative Control siRNA formulation (neither BRD4 mRNA nor c-MYC protein was downregulated by the Negative Control siRNA formulation relative to naked siRNA), leading to also blast differentiation (Figure 7c). Importantly, inhibition of BRD4 and c-MYC by targeted formulation (200 nM siBRD4) achieved up to 80% 949


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Corbett, R.; Dhalla, N.; Guin, R.; He, A.; Hirst, C.; Hirst, M.; Holt, R. A.; Jones, S.; Karsan, A.; Lee, D.; Li, H. I.; Marra, M. A.; Mayo, M.; Moore, R. A.; Mungall, K.; Parker, J.; Pleasance, E.; Plettner, P.; Schein, J.; Stoll, D.; Swanson, L.; Tam, A.; Thiessen, N.; Varhol, R.; Wye, N.; Zhao, Y. J.; Gabriel, S.; Getz, G.; Sougnez, C.; Zou, L. H.; Leiserson, M. D. M.; Vandin, F.; Wu, H. T.; Applebaum, F.; Baylin, S. B.; Akbani, R.; Broom, B. M.; Chen, K.; Motter, T. C.; Nguyen, K.; Weinstein, J. N.; Zhang, N. Z.; Ferguson, M. L.; Adams, C.; Black, A.; Bowen, J.; Gastier-Foster, J.; Grossman, T.; Lichten-Berg, T.; Wise, L.; Davidsen, T.; Demchok, J. A.; Shaw, K. R. M.; Sheth, M.; Sofia, H. J.; Yang, L. M.; Downing, J. R.; Eley, G.; Alonso, S.; Ayala, B.; Baboud, J.; Backus, M.; Barletta, S. P.; Berton, D. L.; Chu, A. L.; Girshik, S.; Jensen, M. A.; Kahn, A.; Kothiyal, P.; Nicholls, M. C.; Pihl, T. D.; Pot, D. A.; Raman, R.; Sanbhadti, R. N.; Snyder, E. E.; Srinivasan, D.; Walton, J. S.; Wan, Y. H.; Wang, Z. N.; Issa, J. P. J.; Le Beau, M.; Carroll, M.; Kantarjian, H.; Kornblau, S.; Bootwalla, M. S.; Lai, P. H.; Shen, H.; Van den Berg, D. J.; Weisenberger, D. J.; Link, D. C.; Walter, M. J.; Ozenberger, B. A.; Mardis, E. R.; Westervelt, P.; Graubert, T. A.; DiPersio, J. F.; Wilson, R. K. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. N. Engl. J. Med. 2013, 368 (22), 2059−2074. (6) Horton, S. J.; Huntly, B. J. Recent advances in acute myeloid leukemia stem cell biology. Haematologica 2012, 97 (7), 966−74. (7) Konopleva, M. Y.; Jordan, C. T. Leukemia Stem Cells and Microenvironment: Biology and Therapeutic Targeting. J. Clin. Oncol. 2011, 29 (5), 591−599. (8) Borkhardt, A.; Heidenreich, O. RNA interference as a potential tool in the treatment of leukaemia. Expert Opin. Biol. Ther. 2004, 4 (12), 1921−1929. (9) Guo, J.; Cahill, M. R.; McKenna, S. L.; O’Driscoll, C. M. Biomimetic nanoparticles for siRNA delivery in the treatment of leukaemia. Biotechnol. Adv. 2014, 32 (8), 1396−1409. (10) Guo, J.; McKenna, S. L.; O’Dwyer, M. E.; Cahill, M. R.; O’Driscoll, C. M. RNA interference for multiple myeloma therapy: targeting signal transduction pathways. Expert Opin. Ther. Targets 2016, 20 (1), 107−21. (11) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 2013, 12 (11), 967−77. (12) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 2014, 15 (8), 541−55. (13) O’Mahony, A. M.; Godinho, B. M. D. C.; Ogier, J.; Devocelle, M.; Darcy, R.; Cryan, J. F.; O’Driscoll, C. M. Click-Modified Cyclodextrins as Nonviral Vectors for Neuronal siRNA Delivery. ACS Chem. Neurosci. 2012, 3 (10), 744−752. (14) Fitzgerald, K. A.; Guo, J.; Tierney, E. G.; Curtin, C. M.; Malhotra, M.; Darcy, R.; O’Brien, F. J.; O’Driscoll, C. M. The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics. Biomaterials 2015, 66, 53−66. (15) McMahon, A.; O’Neill, M. J.; Gomez, E.; Donohue, R.; Forde, D.; Darcy, R.; O’Driscoll, C. M. Targeted gene delivery to hepatocytes with galactosylated amphiphilic cyclodextrins. J. Pharm. Pharmacol. 2012, 64 (8), 1063−73. (16) Guo, J. F.; Ogier, J. R.; Desgranges, S.; Darcy, R.; O’Driscoll, C. Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery to prostate tumours in mice. Biomaterials 2012, 33 (31), 7775−7784. (17) Godinho, B. M.; Ogier, J. R.; Darcy, R.; O’Driscoll, C. M.; Cryan, J. F. Self-assembling modified beta-cyclodextrin nanoparticles as neuronal siRNA delivery vectors: focus on Huntington’s disease. Mol. Pharmaceutics 2013, 10 (2), 640−9. (18) McCarthy, J.; O’Neill, M. J.; Bourre, L.; Walsh, D.; Quinlan, A.; Hurley, G.; Ogier, J.; Shanahan, F.; Melgar, S.; Darcy, R.; O’Driscoll, C. M. Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system. J. Controlled Release 2013, 168 (1), 28−34. (19) Godinho, B. M.; McCarthy, D. J.; Torres-Fuentes, C.; Beltran, C. J.; McCarthy, J.; Quinlan, A.; Ogier, J. R.; Darcy, R.; O’Driscoll, C. M.; Cryan, J. F. Differential nanotoxicological and neuroinflammatory

formulation technique, the antibody-targeted cyclodextrin can be flexibly tuned to target other LSC antigens (i.e., CD33, CD44, CD47, and CLL-1) for cell-specific delivery. In addition, as more genes are identified as causative factors for AML and relapse, this delivery system can be easily and readily modified to incorporate the appropriate siRNA, thereby facilitating the production of personalized medicines. At a time when the evidence for the therapeutic efficacy of chimeric antigen recognition (CAR) T cells is mounting for the treatment of leukemia,72,73 effective nonviral delivery technology could be evaluated in other settings, and the concept of targeted therapy using cyclodextrin technology to transfect plasmid DNA into the T cells may be explored in the future.15

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01150. Particle size, surface charge, CD and siRNA formulations, flow cytometry, gel electrophoresis, TEM, stability, antigen and protein expression, and differentiation studies (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-431-85619716. Fax: +86-431-85619252. E-mail: [email protected]. *Tel: +353-21-4901396. Fax: +353-21-4901656. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge funding from the Government of Ireland Postdoctoral Fellowship from the Irish Research Council (GOIPD/2013/150), and the Irish Cancer Society via a project grant (PCI11ODR). The authors would like to thank Dr. Michael F. Cronin for his helpful comments on the manuscript.

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REFERENCES

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