Amphiphilic siRNA Conjugates for Co-Delivery of Nucleic Acids and

Article pubs.acs.org/bc

Amphiphilic siRNA Conjugates for Co-Delivery of Nucleic Acids and Hydrophobic Drugs Soo Hyeon Lee,*,† Jeong Yu Lee,‡ Jee Seon Kim,§ Tae Gwan Park,∥ and Hyejung Mok*,⊥ †

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich (ETHZ), Vladimir-Prelog-Weg, Zurich 8093, Switzerland ‡ Basic Research & Innovation Division, AmorePacific Corporation R&D Unit, Yongin 446-729, Republic of Korea § National Institute of Biomedical Imaging and Bioengineering, 9000 Rockville Pike, Bethesda, Maryland 20892, United States ∥ Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea ⊥ Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea S Supporting Information *

ABSTRACT: Combination therapy of nucleic acids and chemical drugs for cancer treatment is a promising strategy to enhance the therapeutic efficacy by simultaneously regulating multiple troublesome pathways. In this study, we report on polyethylene glycol-siRNA-polycaprolactone (PEG-siRNA-PCL) micelles that encapsulate hydrophobic drugs for efficient co-delivery of siRNA and drugs to cancer cells. Amphiphilic PEG-siRNA-PCL copolymers were synthesized by annealing antisense siRNA-PCL conjugates with sense siRNA-PEG conjugates. After paclitaxel encapsulation, PEG-siRNA-PCL micelles containing antiapoptotic Bcl-2-specific siRNA were stabilized with linear polyethylenimine via electrostatic interactions. Stabilized PEG-siRNA-PCL micelles showed superior anticancer effects, assessed by caspase-3 activity analysis, apoptotic cell staining, and a cytotoxicity test, to those of paclitaxel-free PEG-siRNA-PCL micelles and unmodified siRNAs. The strong anticancer activity of paclitaxelincorporated siRNA micelles can be attributed to the synergistic effect of Bcl-2 siRNA and paclitaxel. This work provides an efficient co-delivery platform for combination anticancer therapy with siRNA and chemotherapy.

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tics.6,7 siRNA can be used to modify abnormal gene expression, treating the cancer itself while also sensitizing tumor cells to chemotherapy and increasing the overall therapeutic effect. For example, siRNA-mediated gene silencing can reduce multidrug resistance or block antiapoptotic signaling, eliciting synergistic therapeutic effects with anticancer chemotherapies.4,8 The advances of conjugation techniques and biomaterials have led to the development of efficient multifunctional drug carriers to increase therapeutic efficacy and prevent adverse effects. Various carriers, e.g., micelles, liposomes, polymeric complexes, and inorganic nanoparticles, have been utilized for

INTRODUCTION

Chemotherapy using small-molecule drugs has long been considered the principle approach to cancer treatments. However, treatment with a single drug often fails to provide the desired clinical outcome, resulting in a poor prognosis and disease relapse, due to cancer complexity and heterogeneity including cases of multidrug resistance and signaling redundancy.1,2 To overcome these challenges, a combination therapy using therapeutic nucleic acids and chemical drugs has been developed as an efficient cancer treatment that makes use of the growing knowledge of the biochemical mechanisms of action underlying cancer development.3−5 Small interfering ribonucleic acid (siRNA) is a widely investigated nucleic acid drug that modulates the expressed levels of disease-causing genes, which are “non-druggable” targets for conventional therapeu© XXXX American Chemical Society

Received: April 19, 2017 Revised: June 29, 2017 Published: July 3, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00222 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Synthetic scheme of (A) antisense siRNA-PCL and (B) sense siRNA-PEG by coupling thiol reactive PCL and PEG, respectively, with thiol functionalized single-stranded siRNA. (C) Micelle preparation with polyethylene glycol-siRNA-polycaprolactone (PEG-siRNA-PCL) conjugates by annealing antisense siRNA-PCL conjugates and sense siRNA-PEG conjugates (upper). Co-delivery of siRNA and a hydrophobic anticancer drug (paclitaxel) using PEG-siRNA-PCL micelles (bottom).

co-delivery of chemical and nucleic acid drugs.3,5,9 Micelles or nanoparticles formed by positively charged amphiphilic polymers can encapsulate hydrophobic drugs, and negatively charged nucleic acid drugs can bind to particle surfaces via electrostatic interaction.10−18 Liposomes or polymersomes have also been widely investigated as dual drug delivery carriers by encapsulating hydrophilic nucleic acid drugs in the aqueous core and entrapping hydrophobic drugs within hydrophobic layers.19,20 Moreover, anthracyclines, such as doxorubicin, can be loaded into the double helix of nucleic acids via intercalation, and nucleic acid drugs can serve as drug delivery scaffolds as well as therapeutics themselves.21,22 Recent studies have shown that conjugation of drugs to carriers could prevent premature drug release and that the timely release of different drugs triggered by internal or external stimuli at their site of action could increase synergistic effects.5,23−25 Therefore, chemical

conjugation of nucleic acid drugs or chemical drugs to carriers with cleavable linkages would be an appropriate strategy to deliver them in a controlled manner. Among various approaches for combination therapy of chemical drugs and nucleic acid drugs, dual treatment with paclitaxel (PTX) and Bcl-2-targeting nucleic acid drugs has been widely investigated for cancer treatments.26−28 PTX is an antineoplastic drug binding to β-tubulin and preventing cell division. It can induce cellular stress and death signals, leading to apoptotic cell death.29 Bcl-2, an antiapoptotic oncogene, plays an important role in regulating cell survival and death. It is overexpressed in many malignant tumors and blocks mitochondrial cytochrome c release, resulting in the survival of tumor cells against death signals.30,31 Down-regulation of Bcl-2 expression using antisense nucleic acid drugs chemosensitizes tumor cells to PTX and enhances anticancer effects in B


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Bioconjugate Chemistry vitro and in vivo.25,32,33 This synergistic effect has been shown in our previous study by calculating combination index after codelivery of PTX and Bcl-2 siRNA to the cancer cells.34 Although recent clinical trials with this combination did not show encouraging results, it was likely due to the absence of an appropriate delivery platform of nucleic acid drugs with favorable biocompatibility and efficacy, resulting in their poor intracellular delivery.28 In this study, we demonstrate an efficient co-delivery platform for siRNA and hydrophobic chemical drugs using a polyethylene glycol-polycaprolactone (PEG-PCL) amphiphilic copolymer containing biologically functional siRNA. Previously, the PEG-PCL copolymer has been shown to have efficient drug delivery capacity by encapsulating hydrophobic drugs in the core of a self-assembled nanostructure.35−37 The PEG-siRNAPCL amphiphilic copolymer used in this study can form micelle structures in aqueous solution, wherein hydrophobic PCL blocks face the core and hydrophilic siRNA-PEG moieties face the external aqueous environment. For simultaneous delivery of chemical drugs and nucleic acid drugs, a hydrophobic drug was encapsulated in the core of the siRNA micelle by the simple and efficient solvent-dialysis method.38 To enhance intracellular delivery efficiencies of micelles, a positively charged polymer, linear polyethylenimine (LPEI), was used to coat the negatively charged siRNA micelle surface via electrostatic interactions. After determination of the optimal PEG density in siRNA micelles that would lead to high in vitro transfection efficiency, the ability of the micelles to silence the target gene was compared with that of intact siRNA. For combinational cancer therapy, PTX and Bcl-2-specific siRNAs were used as a model chemical drug and nucleic acid drug, respectively. Sequencespecific target gene silencing by PTX-encapsulated siRNA micelles was confirmed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Anticancer effects with co-delivery of PTX and Bcl-2 siRNA were determined by measurement of caspase-3 activity, apoptotic cell staining, and a cytotoxicity test, respectively.

Figure 2. (A) Gel electrophoresis of antisense (AS) siRNA-PCL conjugates (left) and PEG-siRNA-PCL conjugates (right) without or with 100 mM of dithiothreitol (DTT) treatment. (B) Atomic force microscopy (AFM) images of PEG-siRNA-PCL micelles (left) and paclitaxel (PTX) encapsulated PEG-siRNA-PCL micelles (right). Scale bar = 200 nm.

RESULTS AND DISCUSSION Synthesis of PEG-siRNA-PCL Conjugates. Hydrophobic PCL was conjugated to siRNA, enabling the new molecule to form self-assembled micelle structures, which could be used as delivery carriers for a hydrophobic drug. PEG was then introduced into siRNA-PCL conjugates to maintain colloidal stability of the micelles in physiological environments. To synthesize PEG-siRNA-PCL conjugates, single-stranded antisense siRNA-PCL conjugates (AS siRNA-PCL) and singlestranded sense siRNA-PEG conjugates (S siRNA-PEG) were synthesized separately, followed by hybridization of two complementary strands in aqueous solution (Figure 1). First, AS siRNA-PCL conjugates containing disulfide bonds were synthesized via thiol−disulfide exchange reactions between AS siRNA containing a sulfhydryl group at the 3′ end and a pyridyl disulfide-functionalized PCL (PCL-pys), as shown in Figure 1A. PCL-pys was produced via a two-step synthesis: PCL functionalization with p-nitrophenyl chloroformate (p-NPC) and subsequent coupling with 2-aminoethyl 2-pyridyl disulfide. NMR spectroscopy showed that 51% and 31% of PCL end groups were functionalized with p-NPC and pyridyl disulfide, respectively (Supporting Information Figure S1). Next, for the preparation of complementary S siRNA-PEG conjugates, amine-functionalized PEG (mPEG-NH2) was activated with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to produce

respectively). The siRNA band shifted to the upper part of the gel after PCL and PEG conjugation due to the increase in molecular size. Since amphiphilic AS siRNA-PCL and PEGsiRNA-PCL conjugates can each form micelles in aqueous solution, some conjugates remained in the wells but most of them migrated in the electric field, which can disrupt micelle structure (Figure 2A). Migration of amphiphilic siRNA molecules on the gel during electrophoresis has also been observed in our previous study.39 Only a negligible amount of free siRNA was observed in the gel, suggesting that most siRNAs were conjugated to polymers. For the intrinsic biological function of siRNA, PCL, and PEG blocks were linked to siRNAs by a cleavable disulfide bond. This bond is susceptible to degradation in a reductive environment, like cytosol, to release the intact form of siRNA at the site of action.40 Treatment of AS siRNA-PCL and PCL-siRNA-PEG conjugates with the reducing agent, dithiothreitol (DTT), caused cleavage of the polymers from the conjugates, resulting in the release of free siRNA (Figure 2A). Paclitaxel Encapsulation in PEG-siRNA-PCL Micelles. PTX was encapsulated into the core of PEG-siRNA-PCL micelles via hydrophobic interactions between PTX and PCL moieties. The loading efficiencies of siRNA and PTX were 83% and 56%, respectively, and the relative molar ratio between siRNA and PTX in micelle solution was 1:4. The morphological

PEG-containing pyridyl disulfide groups, which were then reacted with an S siRNA containing a sulfhydryl group at the 3′ end (Figure 1B). The prepared AS siRNA-PCL was annealed with S siRNA-PEG to obtain PEG-siRNA-PCL conjugates, which readily formed micellar structures in aqueous solution (Figure 1C). Hydrophobic paclitaxel could then be incorporated within the hydrophobic cores of PEG-siRNA-PCL micelles. For facile interaction with cellular surfaces and stable particle structure, the cationic polymer linear polyethylenimine (LPEI) was coated onto the micelles. Successful conjugation of PCL and PEG to siRNA was confirmed by gel electrophoresis (Figures 2A and S2 in the Supporting Information,

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Figure 3. Target GFP silencing efficiency after transfection of polyethylene glycol-siRNA-polycaprolactone (PEG-siRNA-PCL) micelles complexed with linear polyethylenimine (LPEI) in GFP overexpressing MDA-MB-435 cells. Relative GFP expression levels after transfection of (A) PEGsiRNA-PCL micelles containing 0, 1, 10, 50, and 100 mol % PEGylated conjugates with LPEI (siRNA concentration, 75 nM; N/P ratios, 10, 20, and 40), and (B,C) PEG-siRNA-PCL micelles containing 10 mol % PEGylated conjugates with LPEI at various N/P ratios (siRNA concentration 37.5 nM) and various siRNA concentrations (N/P ratio: 20). After transfection of PEG-siRNA-PCL micelles containing 10 mol % PEGylated conjugates with LPEI (siRNA concentration, 75 nM; N/P ratio, 40), (D) intracellular GFP mRNA levels determined by semiquantitative RT-PCR, and (E,F) relative GFP intensities determined by confocal microscopy and flow cytometry, respectively. Scale bar = 50 μm. Values are represented as mean ± SD.

siRNA-PCL micelles containing GFP-specific siRNA were transfected into GFP-expressing breast cancer cells and GFP intensities in the cells were measured. To determine the optimal PEG density for efficient transfection, PEG-siRNAPCL micelles containing various PEG amounts were used to treat the cells. PEG has been widely explored for systemic delivery of nanoparticles because it can enhance particle stability, prolong their circulation time, and prevent undesired immune stimulations. However, PEG can disturb the interaction between particles and cell surfaces due to its large

properties of PEG-siRNA-PCL micelles and PTX-loaded PEGsiRNA-PCL micelles (PTX/PEG-siRNA-PCL) were analyzed by atomic force microscopy (AFM) (Figure 2B). The average diameters of PEG-siRNA-PCL and PTX/PEG-siRNA-PCL were 38.7 ± 17.2 nm and 30.3 ± 11.7 nm, respectively. The size of PEG-siRNA-PCL micelles reduced slightly after PTX loading, presumably due to the strong hydrophobic interaction between PTX and PCL moiety.41 Target GFP Silencing with PEG-siRNA-PCL Micelles. To evaluate siRNA-mediated target gene silencing, PEGD


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Figure 4. Determination of anticancer effects of paclitaxel (PTX) encapsulated polyethylene glycol-siRNA-polycaprolactone (PEG-siRNA-PCL) micelles containing Bcl-2 specific siRNA (siBCL2) or GFP specific control siRNA (siGFP) in HeLa cells. (A) Intracellular Bcl-2 mRNA levels determined by reverse transcription−polymer chain reaction (RT-PCR) after transfection of various siRNA conjugates/linear polyethylenimine (LPEI) complexes (siRNA concentration, 37.5 nM; N/P ratio, 40; PTX concentration, 0.135 μg/mL). LF: lipofectamine. (B) Relative caspase-3 activities of the cells transfected with various siRNA conjugates/LPEI (siRNA concentration, 35.7 nM; N/P ratio, 40; PTX concentration, 0.135 μg/ mL). (C) Distribution and (D) quantitative analysis of early/late apoptotic cells using Annexin V-FITC/propidium iodide staining after transfection of various siRNA conjugates/LPEI (siRNA concentration, 150 nM; N/P ratio, 40; PTX concentration, 0.54 μg/mL). Annexin V-FITC positive cells are considered apoptotic. (E) Relative cell viabilities determined by CCK assay after transfection of various siRNA conjugates/LPEI at an N/P ratio of 40. Values are represented as mean ± SD: * p < 0.05, ** p < 0.05 vs PBS-treated cells, ***p < 0.001 vs PBS-treated cells, and ****p < 0.05 vs all other groups.

hydrodynamic radius, causing poor intracellular uptake. This PEG dilemma can be solved by introducing targeting ligands and stimuli-responsive cleavable linkages in PEG chains.5,42

Besides these, PEG graft density on the particle surface can be systematically adjusted to ensure high physicochemical stability as well as marginal interference of cellular uptake.43 The E


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(PEG-siBCL2-PCL). Intracellular localization of PTX-encapsulated PEG-siBCL2-PCL micelles (PTX/PEG-siBCL2-PCL) was assessed using BODIPY-labeled PTX and fluorescence labeled jetPEI (JetPEI-FluoF). After transfection with fluorescently labeled PTX/PEG-siBCL2-PCL into HeLa cells, intracellular fluorescence was observed by confocal microscopy. Strong fluorescent intensities within cells show successful internalization of PTX and PEG-siRNA-PCL/LPEI micelles (Supporting Information Figure S4). To examine gene silencing, RT-PCR was performed after transfection. PEGsiBCL2-PCL with or without PTX encapsulation reduced the expression level of target Bcl-2 mRNA, while PEG-siRNA-PCL micelles containing GFP-specific siRNA (PEG-siGFP-PCL) did not change its expression level (Figure 4A). As a positive control, Bcl-2-specific unmodified siRNA (siBCL2) was delivered with lipofectamine (LF), a gold standard for in vitro siRNA transfection, resulting in efficient Bcl-2 mRNA silencing. These results suggest that PEG-siRNA-PCL micelles mediating gene silencing were sequence-specific and that PTX encapsulation in micelles did not modify the target gene silencing capacity of siRNA. To demonstrate anticancer effects of PTX/PEG-siBCL2PCL, caspase-3 activity in transfected cells was measured. Caspase-3, an enzyme downstream of the apoptotic pathway, plays an important role in the cleavage of key proteins that cause apoptosis. High caspase-3 activity indicates progression of apoptosis. PTX/PEG-siBCL2-PCL/LPEI elevated caspase-3 activity by 1.5-, 2-, and 4-fold compared with that reported for PTX-free PEG-siBCL2-PCL/LPEI, siBCL2/LPEI, and phosphate buffer solution (PBS) treatment, respectively (Figure 4B). Additionally, the cells were stained with FITClabeled annexin V protein and propidium iodide (PI) to monitor cell population at early and late stages of apoptosis. One day after transfection, the relative number of early/late apoptotic cells after treatment with PTX/PEG-siBCL2-PCL/ LPEI was higher by 7- and 4-fold than that of cells treated with PBS or PTX-free PEG-siBCL2-PCL/LPEI, respectively (Figure 4C,D). PTX-free PEG-siBCL2-PCL/LPEI and siBCL2/LPEI did not show any significant difference. Finally, overall cytotoxicities were determined by measuring mitochondrial activity using tetrazolium salt-containing reagent (Figure 4E). Four days after transfection, the relative viabilities of cells treated with PTX/PEG-siBCL2-PCL/LPEI, PEG-siBCL2PCL/LPEI, siBCL2/LPEI, PEG-siGFP-PCL/LPEI, or LPEI alone were 17.6 ± 1.1%, 59.5 ± 8.1%, 61.2 ± 8.4%, 70.3 ± 14.9%, and 70.5 ± 21.0%, respectively, compared to that of PBS-treated control cells. Considering IC50 (∼700 nM) of PTX in HeLa cells after 24 h treatment, significantly reduced cell viability down to ∼20% by treatment for 5 h might be attributed to synergistic effects of PTX and siBCL2 in PTX/ PEG-siBCL2-PCL/LPEI.46 PTX/PEG-siBCL2-PCL/LPEI induced greater cell death than PTX-free PEG-siBCL2-PCL/ LPEI did. Taking into consideration the confocal microscopy image showing colocalization of PTX and micelles (Supporting Information Figure S4), this synergistic anticancer effect was attributed to the successful co-delivery of PTX and Bcl-2specific siRNA via PEG-siRNA-PCL micelles. It should be noted that simultaneous co-delivery of nucleic acid drugs and chemical drugs loaded in a single delivery carrier, called a “two-in-one system”, has shown a superior therapeutic effect over administration of a physical mixture of these drugs.11,18,47 Combining two drugs into a single formulation can ensure the simultaneous delivery of both

advantage of the current formulation is that PEG density in the micelles can be easily modulated by changing the molar ratios between PEG-siRNA-PCL and siRNA-PCL. For example, PEG-siRNA-PCL micelles containing 10 mol % PEGylated conjugates can be formed by mixing PEG-siRNA-PCL and siRNA-PCL at a molar ratio of 1:9 during micelle preparation. To enhance transfection efficiency, LPEI, a model cationic polymer, was used to coat the negatively charged micelle surface via electrostatic interactions (micelleplex).16,44 PEGsiRNA-PCL micelles with 10 mol % PEGylated conjugates had the highest siRNA transfection efficiency (51.6 ± 0.9% to GFP expression level in control cells) (Figure 3A). Previously we showed that a high PEG grafting density on the siRNA-PEG complexes over 50% caused dramatic decrease of intracellular uptake, while siRNA-PEG complexes with a moderate PEG grafting density (5−10%) exhibited the highest uptake and target gene silencing efficiencies.43 Consistent with that study,43 increase of PEGylated conjugates from 10 to 100 mol % caused a gradual decrease of gene silencing efficacy, presumably due to reduced cellular uptake. However, PEG-free siRNA-PCL micelles (0 mol % PEG micelles) showed lower transfection efficiency (66.2 ± 6.1% to GFP expression level in control cells) than 10 mol % PEG micelles did, suggesting that moderate PEGylation enhanced transfection efficiency due to increased particle stability. Based on these results, further in vitro experiments were performed with PEG-siRNA-PCL micelles containing 10 mol % PEGylated conjugates. Previously, we demonstrated that siRNA micellization using amphiphilic siRNA-PLGA conjugates exhibited increased siRNA delivery efficiency when coated with the cationic polymeric carriers, LPEI.39 We suggested that LPEI could bind to siRNAs facing the external surface of the micelles and deliver them into cells with high efficacy, while unmodified siRNAs were not able to transfect due to their low binding affinity to LPEI.39 To demonstrate whether PEG-siRNA-PCL micelles can be delivered more efficiently than unmodified siRNA, unmodified siRNA was transfected into GFP-overexpressing cells along with PEG-siRNA-PCL micelles coated with LPEI at various N/P ratios (Figures 3B and S3 in the Supporting Information) and various concentrations of siRNA (Figure 3C). Unmodified siRNA/LPEI showed only marginal changes in GFP expression in all conditions while PEG-siRNAPCL/LPEI elicited efficient GFP silencing. GFP expression levels in the cells treated with PEG-siRNA-PCL/LPEI were efficiently reduced by increasing N/P ratios and siRNA concentrations. With 37.5 nM of siRNA in PEG-siRNA-PCL/ LPEI at an N/P ratio of 40 and with 75 nM of siRNA in PEGsiRNA-PCL/LPEI at an N/P ratio of 20, GFP expression levels were 57.2 ± 1.3% and 55.1 ± 10.1%, respectively. Superior silencing of GFP mRNA and protein expression by PEGsiRNA-PCL/LPEI compared to that by unmodified siRNA/ LPEI was also confirmed using RT-PCR (Figure 3D), confocal microscopy (Figure 3E), and flow cytometry (Figure 3F). These data support the previous studies suggesting that structural modification of siRNA, e.g., micellization and multimerization, could be a promising approach to developing siRNA delivery platforms by forming stable siRNA complexes with cationic delivery carriers, thereby increasing their transfection efficiencies.39,40,45 Anticancer Effect of Paclitaxel-Encapsulated PEGsiRNA-PCL Micelles. To apply the delivery platform discussed above to chemotherapy treatment, PTX was incorporated into PEG-siRNA-PCL micelles containing Bcl-2-specific siRNAs F


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thiol-functionalization at their 3′ ends and the primers for RTPCR were obtained from Bioneer Co. (Daejeon, South Korea). The sequences of siRNAs and primers are as follows: GFP specific sense siRNA; 5′-GCA AGC UGA CCC UGA AGU UdTdT-3′, GFP specific antisense siRNA; 5′-AAC UUC AGG GUC AGC UUG CdTdT-3′, Bcl-2 specific sense siRNA; 5′UCA AAC AGA GGC CGC AUG CdTdT-3′, Bcl-2 specific antisense siRNA; 5′-GCA UGC GGC CUC UGU UUG AdTdT-3′, forward GFP primer; 5′-TGG TGA GCA AGG GCG AGG AG-3′, reverse GFP primer; 5′-GGG GGT GTT CTG CTG GTA GT-3′, forward Bcl-2 primer; 5′-ATG TGT GTG GAG AGC GTC AA-3′, reverse Bcl-2 primer; 5′-CAG GAG AAA TCA AAC AGA GGC-3′, forward human β-actin primer; 5′-GTG GGG CGC CCC AGG CAC CAG GGC-3′, reverse human β-actin primer; 5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′. TRI Reagent and Taq DNA polymerase were purchased from Ambion, Inc. (Austin, TX) and Takara (Tokyo, Japan), respectively. Reverse transcriptase and CaspACE colorimetric assay kits were obtained from Promega (Madison, WI). PTX and GFP-overexpressing MDA-MB-435 cells were kindly donated by Samyang Co. (Seoul, South Korea). HeLa cells were obtained from Korean Cell Line Bank (Seoul, South Korea). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), red-orange-fluorescent BODIPY 564/570 Taxol, and Lipofectamine Reagent were obtained from Invitrogen (Carlsbad, CA). Cell counting kit-8 (CCK-8) and ApoScan Annexin V FITC apoptosis detection kit were purchased from Dojindo Laboratories (Kumamoto, Japan) and BioBud (Seoul, South Korea), respectively. Methods. Synthesis of Pyridyl Disulfide Functionalized Polycaprolactone (PCL-pys). To synthesize the thiol-reactive PCL, the end terminals of PCL diol were functionalized with pyridyl disulfide groups (pys). First, 6 g of PCL diol was incubated with 6 g of p-nitrophenyl chloroformate in 10 mL of methylene chloride overnight to produce amine-reactive PCL (1) (molar ratio of hydroxyl groups of PCL:p-nitrophenyl chloroformate = 1:5). The product was purified by washing with methanol, precipitating two times with cold ether, and drying under vacuum. Compound 1 was dissolved in deuterated chloroform (CDCl3) and characterized by 1H NMR analysis using a Bruker AVANCE 400 NMR. The thiol-reactive reagent, 2-aminoethyl 2-pyridyl disulfide (2), was synthesized according to the method described in previous studies.48,49 Briefly, 3.3 g of 2,2′-dithiodipyridine was reacted with cysteamine/HCl in 10 mL of methanol for 5 h (molar ratio of 2,2′-dithiodipyridine:cysteamine:HCl = 3:1:1). After precipitation three times with cold ether, compound 2 was obtained by drying under vacuum. 250 μmol of amine-reactive PCL (compound 1) and 1.5 mmol of compound 2 were reacted in 8 mL of DMSO/methylene chloride cosolvent (volume ratio of DMSO:methylene chloride = 1:1) containing 1.5 mmol of triethlyamine (TEA) overnight. The resulting PCL-pys (compound 3) was purified by precipitating twice with cold methanol and dried in a vacuum chamber. The conjugation efficiency of compound 3 was determined by 1H NMR analysis using a CDCl3 solvent. Synthesis of Antisense siRNA-Polycaprolactone (AS siRNA-PCL) Conjugates. Twenty nanomoles of thiolmodified single-stranded antisense siRNAs (AS siRNA-SH) was incubated in 50 mM of TCEP solution in DEPC-pretreated distilled water (pH 7.4) for 1 h under shaking in order to activate sulfhydryl groups. Then, 150 μL of 9.5 M ammonium acetate and 1.5 mL of cold ethanol were sequentially added to

drugs to the same cell, whereas a physical mixture of multiple formulations leads to heterogeneous drug distribution in tissues and thus fails to induce synergistic effects. Therefore, we believe that the current system of loading both siRNA and PTX into a single micelle can enhance the synergistic therapeutic effect in comparison of the physical mixture of these drugs. Upon intracellular uptake of PTX/PEG-siRNA-PCL micelles, disulfide bonds in PEG-siRNA-PCL conjugates are cleaved due to the reductive cytosolic environment, resulting in a release of free siRNAs, which can participate in target gene silencing mechanism. Encapsulated PTX is then diffused into the cell; micelle destabilization due to disulfide bond cleavage may accelerate PTX release.24

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CONCLUSIONS

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EXPERIMENTAL PROCEDURES

In this work, we synthesized amphiphilic PEG-siRNA-PCL conjugates for co-delivery of siRNAs and chemical drugs for cancer therapy. Amphiphilic PEG-siRNA-PCL conjugates formed self-assembled micelles, hydrophobic PTX was encapsulated into the micelle cores, and hydrophilic siRNA was placed facing the surface. Compared to previous co-delivery systems for chemical drugs and nucleic acid drugs, the current system has several remarkable features. First, it is simple and easy to adjust PEG contents in particles ensuring desirable particle stability and a high transfection efficiency. For in vivo applications to target different tissues via various administration routes, PEG grafting degrees in the system can be finely adjusted. Second, siRNA serves not only as a biologically functional nucleic acid drug but also as a part of the micelle structure to carry the hydrophobic drug, which avoids the excessive use of additives in the delivery system. Third, direct conjugation of siRNAs to the hydrophobic polymer with reducible disulfide bonds prevents premature release of siRNAs from micelles during delivery. After simultaneous delivery of two anticancer reagents, PTX and Bcl-2-targeting siRNA, using PEG-siRNA-PCL conjugates, significant synergistic cancer cell apoptosis was observed. To maximize this synergistic effect, several factors need to be further optimized, such as drug dose, ratio between siRNA and chemical drug, and drug release patterns. In addition, other types of biodegradable and biocompatible cationic polymers could be adopted for surface coating of PEG-siRNA-PCL for in vivo study. With these improvements, our system will provide a promising co-delivery platform for the efficient cancer therapy.

Materials. Polycaprolactone diol (PCL diol, Mw ∼ 2000), methoxypolyethylene glycol amine (mPEG-NH2, Mw 5000), and cysteamine hydrochloride were purchased from Aldrich (Milwaukee, WI), Sunbio Inc. (Anyang, South Korea), and Fluka (Buchs, Switzerland), respectively. Triethylamine and methanol were obtained from Sigma (St. Louis, MO), and pnitrophenyl chloroformate and 2,2′-dithiodipyridine were obtained from TCI (Tokyo, Japan). Tris(2-carboxyethyl)phosphine (TCEP), N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), and Micro-BCA protein assay kit were purchased from Pierce (Rockford, IL), and linear polyethylenimine (LPEI, Mw 25 000) was obtained from Polysciences, Inc. (Warrington, PA). Dimethyl sulfoxide (DMSO) and methylene chloride were obtained from Junsei (Tokyo, Japan), and diethyl ether was obtained from Daejung (Daejeon, South Korea). GFP and Bcl-2-specific siRNAs with or without G


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micelle solution by centrifugation at 15 996 × g for 3 min. SiRNA concentrations in PEG-siRNA-PCL micelles with or without PTX were measured using a spectrophotometer. For AFM observation, salts in micelle solution were removed by washing three times with DEPC-pretreated distilled water using a Vivaspin 500 ultrafiltration device (Sartorius, MWCO 10 000) with centrifugation at 2000 × g for 5 min. PEG-siRNA-PCL micelles with or without PTX (amount of siRNA = 24 pmoles) were loaded on the freshly prepared mica and dried by blowing nitrogen gas. Sample images were scanned from a 1 × 1 μm2 area using psia XE-100 AFM system. To estimate drug encapsulation efficiency, BODIPY-labeled PTX was incorporated into PEG-siRNA-PCL micelles by following the same method for PTX encapsulation described above.20 The mixture of 7.2 μg of PTX and 0.8 μg of BODIPY-PTX (10% w/w BODIPY-labeled PTX) was added to 1.5 nmol of PEG-siRNAPCL mixture, composed of 90 mol % of siRNA-PCL and 10 mol % of PEG-siRNA-PCL conjugates, in 200 μL of DMSO. Micelles containing a mixture of BODIPY-PTX and PTX were produced by dialysis in DEPC-pretreated 1.5 mM PBS solution (pH 7.4). The supernatant was collected after centrifugation at 15 996 × g for 3 min and lyophilized. After dissolving the dried samples in 100 μL of DMSO, the encapsulated PTX amount was estimated by measuring BODIPY amount with NanoDrop (ND-3300) fluorospectrometer (Thermo Fisher. Scientific, Inc. Wilmington, DE) using a white LED excitation source (500− 680 nm) with an emission wavelength of 578 nm. In Vitro GFP Silencing with PEG-siRNA-PCL Micelles. To evaluate siRNA-mediated target GFP silencing, MDA-MB435-GFP cells were seeded in a 12-well plate at a concentration of 1.5 × 105 cells/well 1 day prior to transfection. To determine the optimal PEG density on the PEG-siRNA-PCL micelles for efficient transfection, GFP-specific AS siRNA-PCL was annealed with complementary S siRNA containing 0, 1, 10, 50, and 100 mol % S-siRNA-PEG conjugate, which were prepared by blending S siRNA with S siRNA-PEG. PEGsiRNA-PCL micelles with various PEG densities were complexed with LPEI at N/P ratios of 10, 20, and 40. The complexes were transfected to the cells for 5 h in the absence of serum (siRNA concentration = 75 nM) and the medium was replaced with fresh one. After 2 days of further incubation, the cells were lysed using 1% Triton X-100 solution in PBS, and cellular supernatants containing GFP were collected through centrifugation at 15 996 × g for 5 min. Intensities of GFP in cell supernatants were measured using a spectrofluorophotometer (SLM-AMINCO 8100, SLM Instruments Inc., Urbana, IL) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Assigning the GFP expression level of PBS-treated control cells as 100%, the relative GFP expression levels were calculated. Based on their superior GFP silencing efficiency, PEG-siRNA-PCL micelles composed of 90 mol % of siRNA-PCL and 10 mol % of PEG-siRNA-PCL were used for further experiments. The N/P ratio and siRNA dose-dependent GFP silencing efficiencies were determined by transfecting PEG-siRNA-PCL micelles and unmodified siRNA complexed with LPEI at various N/P ratios (5, 10, 20, and 40) and various siRNA concentrations (9.4, 18.8, 37.5, and 75 nM) into the target cells. For RT-PCR and flow cytometry studies, the cells were seeded in a 6-well plate at a concentration of 3 × 105 cells/well 1 day prior to transfection. Unmodified siRNA or PEG-siRNA-PCL complexed with LPEI were transfected to the cells for 5 h in the absence of serum (siRNA concentration, 75 nM; N/P ratio,

the solution to precipitate AS siRNA-SH. After 20 min incubation at −70 °C, the siRNA pellet was collected by centrifugation at 15 996 × g for 20 min and resuspended in 400 μL DEPC-pretreated distilled water. To remove the residual TCEP completely from the sample, this precipitation procedure was repeated four times. One hundred nanomoles of PCL-pys dissolved in 50 μL of DMSO/methylene chloride cosolvent (volume ratio of DMSO:methylene chloride = 1:1) was added to AS siRNA-SH dissolved in 10 μL DEPC-pretreated PBS solution (2 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, adjusted to pH 8.2 with NaOH) and 40 μL of DMSO. The reaction was performed overnight at room temperature. To purify AS siRNA-PCL conjugates, the reaction solution was dialyzed for 2 days in DMSO, methylene chloride, DMSO, and DEPC-pretreated distilled water sequentially using an MWCO 10 000 membrane. AS siRNA-PCL conjugates (4) in aqueous solution were filtered using a 0.45-μm-sized syringe filter and stored at 4 °C. The amount of siRNA in the solution was quantified using a NanoDrop (ND-1000) spectrophotometer (Thermo Fisher Scientific, Inc. Wilmington, DE). Synthesis of Sense siRNA-Polyethylene Glycol (S siRNA-PEG) Conjugates. Single-stranded sense siRNA-PEG conjugates (S siRNA-PEG) were synthesized according to the procedure described in previous studies.43,50,51 To produce pyridyl disulfide-functionalized PEG (PEG-pys), mPEG-NH2 was activated with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) in PBS (molar ratio of amine:SPDP = 1:10) and the residual SPDP was removed by dialysis for 1 d. Twenty nanomoles of S siRNA-SH, after TCEP treatment as described above, was reacted with 40 nmol of PEG-pys in PBS solution (pH 7.4) overnight. To remove free PEG and S siRNA, the sample was purified by gel permeation chromatography (GPC) using an Agilent HPLC pump system with SuperdexTM 75 10/ 300 GL column (GE Healthcare, Sweden).43,51 S siRNA-PEG was eluted using DEPC-pretreated PBS solution with a flow rate of 0.5 mL/min. S siRNA-PEG conjugates were confirmed by electrophoresis using 12% acrylamide gel, and their concentration was determined using NanoDrop spectrophotometer. Gel Electrophoresis of PEG-siRNA-PCL Conjugates. PEGylated siRNA-PCL conjugates (PEG-siRNA-PCL) were prepared by the annealing of AS siRNA-PCL conjugates with a S siRNA-PEG mixture, containing 90 mol % of S siRNAs and 10 mol % of the S siRNA-PEG conjugates, in PBS (pH 7.4) solution at 37 °C for 1 h. PEG and PCL polymer blocks were linked to siRNA with disulfide bonds. AS siRNA-PCL and PEG-siRNA-PCL conjugates were incubated in 0 and 100 mM of DTT solution for 15 min at room temperature and the band shift of reduced siRNA conjugates was observed by electrophoresis using 1% agarose gel and EtBr staining. Cell Preparation. HeLa cells (human cervical cancer cell lines) and GFP overexpressing MDA-MB-435 cells (MDA-MB435-GFP, human breast cancer cell lines) were maintained in 10% FBS containing DMEM supplemented with 100 units/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in 5% CO2 humidified atmosphere. Paclitaxel Encapsulation in PEG-siRNA-PCL Micelles and Their Morphological Properties. Eight micrograms of PTX and 1.5 nmol of PEG-siRNA-PCL mixture, composed of 90 mol % of siRNA-PCL and 10 mol % of PEG-siRNA-PCL conjugates, were dissolved in 200 μL of DMSO. Organic solvent was removed by dialysis in DEPC-pretreated PBS solution (pH 7.4) and free PTX aggregates were removed from H


Article


12-well plate with a concentration of 1.5 × 105 cells/well 1 day prior to transfection. Cells were treated with various siRNA conjugates/LPEI complexes for 5 h in the absence of serum (siRNA concentration, 35.7 nM; N/P ratio, 40). After 18 h, 100 μL of lysis buffer was added to the cells and 54 μL of cell lysate solution was used for evaluation of relative caspase-3 activities with the CaspACE Assay System according to the manufacturer’s protocol. Values were normalized with total protein amounts in the cell lysates, which were determined by MicroBCA assay according to the manufacturer’s protocol. For analyzing apoptotic cell populations after transfection, HeLa cells were seeded in a 6-well plate with a concentration of 3 × 105 cells/well 1 day prior to transfection. Various siRNA conjugates/LPEI complexes were transfected to the cells for 5 h in the absence of serum (siRNA concentration, 150 nM; N/P ratio, 40) and the cells were replaced with fresh medium. After 24 h of further incubation, apoptotic cells were detected by staining annexin V-FITC and PI according to the manufacturer’s protocol. For the cell viability test, HeLa cells were seeded onto a 96-well plate at a concentration of 1 × 104 cells/ well 1 day prior to transfection. After complexing with LPEI at an N/P ratio of 40, siBCL2, PEG-siBCL2-PCL with or without PTX encapsulation, and PEG-siGFP-PCL were transfected into the cells for 5 h in the absence of serum and the medium was replaced with fresh. After 4 days of further incubation, the relative cell viability was evaluated using CCK-8 solution containing tetrazolium salt according to the manufacturer’s protocol. Assigned the viability of PBS-treated control cells as 100%, the relative cell viabilities were calculated. Statistical Studies. Statistical analysis was assessed using one-way ANOVA combined with Tukey’s post-hoc test. The differences between groups were considered statistically significant at p-values below 0.05.

40), and the cells were further incubated for 24 and 48 h for RT-PCR and flow cytometry, respectively. For RT-PCR, 2 μg of total RNA isolated from the cells using TRI Reagent was reverse transcribed to cDNA by reverse transcriptase at 37 °C for 60 min and 93 °C for 5 min. The amplification was performed with predesigned primer sets and Taq polymerase using the following thermal cycles: 1 cycle at 94 °C for 5 min; 21 cycles at 94 °C for 30 s, at 60 °C for 30 s; and at 72 °C for 40 s; 1 cycle at 72 °C for 10 min. Final products were visualized by 1% agarose gel electrophoresis and EtBr staining. For flow cytometry, the transfected cells were detached by 0.25% (w/v) trypsin treatment, and washed three times with PBS after trypsin inactivation using 10% serum containing medium. The cells were fixed using 3.7% (w/v) formaldehyde solution in PBS and analyzed using a flow cytometer (FACScan, Becton Dickinson, USA) with CELLQUEST software (PharMingen, USA). For confocal microscopy, cells were seeded in a 4-well chamber slide at a concentration of 2 × 105 cells/well 1 day prior to transfection. Unmodified siRNA and PEG-siRNA-PCL micelles were complexed with LPEI and transfected to the cells for 5 h (siRNA concentration, 75 nM; N/P ratio, 40). After 2day incubation, the cells were fixed using 3.7% (w/v) formaldehyde solution in PBS and GFP intensities were observed using a LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). Apoptotic Effect of Paclitaxel-Encapsulated PEGsiRNA-PCL Micelles. To determine Bcl-2 mRNA levels, HeLa cells were seeded onto a 6-well plate at a concentration of 3 × 105 cells/well 1 day prior to transfection. PEG-siBCL2PCL micelles with or without PTX encapsulation were complexed with LPEI at an N/P ratio of 40. PEG-siGFPPCL micelles were used as a negative control with a nonspecific siRNA sequence. These complexes were transfected to the cells for 5 h (siRNA concentration: 37.5 nM). As a control, Bcl-2 siRNA was transfected with LF at a weight ratio of 6. After 19 h of further incubation in fresh medium, Bcl-2 and β-actin mRNA levels in the transfected cells were determined by RT-PCR. Two micrograms of total RNA isolated from the cells using TRI Reagent were reverse transcribed into cDNA by reverse transcriptase at 37 °C for 60 min and 93 °C for 5 min. The amplification was performed with predesigned primer sets and Taq polymerase using the following thermal cycles: 1 cycle at 95 °C for 5 min; 35 and 19 cycles for Bcl-2 and β-actin mRNA, respectively, at 95 °C for 30 s, at 58 °C for 30 s, and at 72 °C for 45 s; 1 cycle at 72 °C for 10 min. Final products were visualized by 1% agarose gel electrophoresis and EtBr staining. For intracellular uptake, Dye-labeled PTX (10% w/w BODIPYlabeled PTX) encapsulated PEG-siRNA-PECL micelles containing 10% PEGylated conjugates were prepared as described in the manuscript. JetPEI-FluoF (jetPEI), a cationic polymer transfection reagent (mean molecular weight of 22 kDa; Polyplus, Illkirsh, France), was mixed with BODIPY-PTX encapsulated PEG-siRNA-PCL micelles at an N/P ratio of 40. These micelleplexes were transfected to HeLa cells for 2 h in the absence of serum (siRNA concentration = 200 nM). After washing three times with PBS, the transfected cells covered with VECTASHIELD Mounting Medium containing DAPI (4′,6-diamidino-2-phenylindole), a nucleus indicator, were observed using a LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). The apoptotic effects were evaluated by the caspase-3 activity test, annexin V-FITC/PI staining, and a cell viability test. For comparison of caspase-3 activity, HeLa cells were seeded in a

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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.bioconjchem.7b00222. NMR data for polycaprolactone functionalized with pnitrophenyl chloroformate (PCL-NPC) and pyridyl sulfide group (PCL-pys) (Figure S1), gel images of electrophoresis with siRNA-PEG (Figure S2), target GFP silencing effect by in vitro transfection with PCL-siRNAPEG before and after LPEI coating at various N/P ratios (Figure S3), and confocal microscopy images of cells after transfection of BODIPY-labeled paclitaxel loaded micelles stabilized with FITC-labeled LPEI (Figure S4) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +82-2-450-0448. ORCID

Jee Seon Kim: 0000-0003-1324-3653 Hyejung Mok: 0000-0002-0665-4449 Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS This study was supported by the Global Innovative Research Center (GiRC) program (NRF-2012K1A1A2A01056094) and a grant (NRF-2014R1A2A1A11049772) through the National Research Foundation funded by the Ministry of Education, Science and Technology, Republic of Korea. We gratefully acknowledge the contribution and inspiration of the late Professor Tae Gwan Park.

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