Efficient Self-Assembled MicroRNA Delivery System Consisting of

May 2, 2019 - Department of Chemical and Biomolecular Engineering, College of Engineering, Yonsei University, .... Okubo, Miyazaki, Yuba, and Harada...
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Efficient Self-Assembled MicroRNA Delivery System Consisting of Cholesterol-Conjugated MicroRNA and PEGylated Polycationic Polymer for Tumor Treatment Byeonggeol Mun,† Eunji Jang,‡ Seungmin Han,† Hye Young Son,§,∥ Yuna Choi,§ Yong-Min Huh,*,§,∥,⊥,# and Seungjoo Haam*,†

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†

Department of Chemical and Biomolecular Engineering, College of Engineering, Yonsei University, Seoul 120-749, Republic of Korea ‡ MediBio-Informatics Research Center, Novomics Co. Ltd., Seoul 07217, Korea § Department of Radiology, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea ∥ YUHS-KRIBB Medical Convergence Research Institute, Seoul 120-752, Republic of Korea ⊥ Severance Biomedical Science Institute(SBSI), Seoul 120-752, Republic of Korea # Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea

ABSTRACT: MicroRNA (miR), a key molecule involved in endogenous RNA interference, is a promising therapeutic agent. In vivo delivery of miR, however, is a major factor limiting its application because its polyanionic nature and vulnerability to breakdown make delivery of miR to targeted lesions difficult. To overcome these challenges, we developed a self-assembled miR delivery system consisting of cholesterol-conjugated miR and polyethylene glycol-grafted polyethylene imine. Nanosized complexes of miR with polyethylene imine, which protected miR and its delivery into targeted lesions in vivo, were successfully synthesized by polyethylene glycol grafting. The hydrophobicity of cholesterol improved the structural stability of the complex, preventing the loss of miR. Here, we report the preparation of this self-assembled complex. We examined the delivery efficiency and validated the therapeutic efficacy of the complex. In conclusion, our miR delivery system shows considerable potential for effective in vivo delivery of miR. KEYWORDS: self-assembled miRNA delivery, RNA interference, cholesterol conjugation, polycationic polymer, cancer therapy

1. INTRODUCTION

of these molecules, making lesion targeting difficult. The polyanionic nature of miR reduces its cellular uptake and causes poor intracellular trafficking; sequestration in endosomes is also a major limitation.7−9 Intravenous administration of naked miRNA leads to poor tissue distribution because miRNA is degraded by serum RNases.10−14 To overcome this limitation, chemical modification can alter the properties of nucleotides by conferring nuclease resistance, increasing binding affinity, and aiding in cellular uptake.15,16 Chemical

Since the discovery that RNA interference (RNAi) can be used for gene silencing technology, treatments have been developed for many diseases, including cancer. MicroRNAs (miRs), small noncoding RNA molecules containing approximately 22 nucleotides, are key molecules in endogenous RNAi and show potential as therapeutic agents for treating diseases. miRs play important roles in regulating basic cell functions, including proliferation, differentiation, and apoptosis as tumor suppressors or oncomirs.1−6 In the past few years, several groups have examined the miR delivery system. In vivo delivery of miR is difficult because of the polyanionic nature and vulnerability to serum breakdown © XXXX American Chemical Society

Received: March 5, 2019 Accepted: April 24, 2019

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DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials Scheme 1. Schematic Illustration of the Self-Assembled miR Delivery System with cmiR and PEG-g-PEIa

a

Annealing of cmiR (A), synthesis of PEG-g-PEI (B), and delivery of self-assembled PEG-g-PEI/cmiR in tumor cells (C).

Figure 1. Gel retardation assay to confirm annealing of miR and cmiR (A). FT-IR (B) and 1H NMR (C) spectra of synthesized PEG-g-PEI.

modification of siRNAs, including cholesterol conjugation, has been found to markedly improve pharmacological activities in vitro and in vivo.17−21 Additionally, new formulation strategies have been reported for forming miRNA complexes with nanomaterials. Delivery of DNA via nanomaterials vectors, including inorganic, lipid, and polymer vectors, shows potential for avoiding the immune response and may be able to manage larger payloads.22,23 Of these nonviral carriers, cationic polymers such as polyethylenimine (PEI) are well-characterized gene delivery carriers, and many groups have demonstrated effective intracellular gene delivery using these carriers. PEI induces a relatively low immune response and causes rapid transfection efficiency in the endosome based on its proton sponging capability.23−26 However, a major concern of using the PEI polymer as a nanocarrier is its dose-dependent cytotoxicity because of its strong polycationic characteristics inside cells.27−29

In this study, we developed an efficient self-assembled miR delivery system by using cholesterol-conjugated miR and pegylated polycationic polymer (Scheme 1). We predicted that cholesterol conjugated miRNAs (cmiRs) would markedly improve pharmacological properties in vivo. The hydrophobicity of cholesterol protected conjugated miR from outside inhibitors, avoiding the loss of miR and facilitating cell fusion or endosomal internalization of the carrier.30 We also used a polyethylene glycol (PEG)ylation strategy as a polycationic polymer to enhance the in vivo circulation times to reach the tumor and reduce cytotoxicity inside cells.31−36 Nanosized complexation of cmiR with PEG-grafted-PEI (PEGg-PEI), which effectively protected miR and its delivery into targeted lesions in vivo, was successfully self-assembled based on charge interactions between the cmiRs and PEG-g-PEI which reduced cytotoxicity by decreasing the cationic properties of PEI. Here, we report the preparation of PEG-g-PEI and B

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials the self-assembled PEG-g-PEI/cmiR complex. Synthesis of PEG-g-PEI/cmiR was confirmed by evaluating the changes in the chemical structure and properties of the complex. We also analyzed the characteristics of PEG-g-PEI/cmiR. Complexation of PEG-g-PEI/cmiR and its stability against polyanionic materials and RNase were examined. To investigate the biocompatibility of PEG-g-PEI/cmiR, we conducted in vitro tests. To evaluate transfection efficiency and validate therapeutic efficacy of PEG-g-PEI/cmiR, in vitro tests were carried out using a model cell line. In in vivo studies, we examined the delivery efficiency and circulation time of PEG-gPEI/cmiR in tumors of nude mice. Our self-assembled miR delivery system with PEG-g-PEI/cmiR shows considerable potential for effective in vivo delivery of miR.

2. RESULTS AND DISCUSSION Annealing of the two single-stranded RNAs with the microRNA-34a sequence (5p: 5′-UGG CAG UGU CUU AGC UGG UUG U UG UG-3′, 3p: 5′-[CHOLESTERTL] GAA GCA AUC AGC AAG UAU ACU GCC CU-3′, 5′chol3p: 5′-[CHOLESTERTL] GAA GCA AUC AGC AAG UAU ACU GCC CU-3′), of which one strand was conjugated to cholesterol, was conducted and verified by agarose gel electrophoresis (Figure 1 (A)). Cholesterol-conjugated 3p single-stranded RNA (lane 2) was observed at a higher position than that without cholesterol (lane 3), which has the same charge because of the relatively heavy weight of cholesterol. miR (lane 4) and cmiR (lane 5) appeared as one line, demonstrating the annealing of miR and cmiR. PEG-g-PEI was synthesized by PEGylation on branched PEI, which is useful as a gene carrier, and its chemical structure was analyzed by FT-IR and 1H NMR. The CO stretching bands (1660 cm−1) and the N−H bending bands (1540 cm−1) of the amide bond37 resulting from branched PEI and PEG conjugation were confirmed by FT-IR (Figure 1 (B)). The peaks of PEG-g-PEI from 3.7 ppm (−CH2CH2O−) and 2.9− 2.6 ppm (−NH− and −NH2 of PEI) were confirmed by 1H NMR38 (Figure 1 (C)). The degree of PEG grafting in branched PEI was determined by calculating the ratio of proton to the amount of −NH− or −NH2− peak of PEI in the −CH2CH2− peak of PEG,38 and the value was 14.67. These results demonstrate that PEG-g-PEI was successfully synthesized. The miR condensation ability of the synthesized PEG-g-PEI was investigated by agarose gel electrophoresis. The results revealed the influence of the charge ratio on cNC (Figure 2 (A)) and NC (Figure 2 (B)) complexation by PEG-g-PEI. NC is the DNA of the same nucleotide sequence as the miRNA used in Figure 1 (A), and cNC is the NC that has cholesterol conjugation. To precisely determine the molar ratio of the complexes, we used those as alternatives to prevent the loss or damage of miR during the gel retardation assay. As the amount of PEG-g-PEI was increased in forming the complex, the gene migration showed a greater delay than naked gene. The migration of cNC or NC was completely retarded when the complexes were formed at a molar ratio of 0.6 for PEG-g-PEI. That is, cholesterol conjugation had no effect on miR condensation. Accordingly, PEG-g-PEI may be suitable as a gene delivery nanocarrier because of its capacity to condense miR. Additionally, cholesterol conjugation to the miR did not significantly impact the condensation ability. Complexes with a molar ratio of 0.6 were used in subsequent experiments.

Figure 2. Determination of the molar ratio used to prepare PEG-gPEI/cmiR complexes. Gel retardation assay of PEG-g-PEI/cNC (A) and PEG-g-PEI/NC (B) at various molar ratios (cNC or NC: PEG-gPEI = 0.01, 0.05, 0.1, 0.4, 0.5, 0.6, 0.7, and 1.0). TEM image (C) and SEM image (D) of the 0.6 molar ratio PEG-g-PEI/cmiR.

After determining the conjugation ratio of PEG-g-PEI, the complex size and surface charge of PEG-g-PEI/cmiR and PEGg-PEI/miR were measured by dynamic light scattering and zeta potential analysis. PEG-g-PEI/cmiR was approximately 216.9 (±24.1) nm in size and had a surface charge of +3.64 (±0.4) mV. A strong positive charge can improve transfection efficiency, but it can induce cytotoxicity by damaging the cell membrane.39,40 Decreasing the surface charge to +3.6 from +13.04 mV (at moral ratio = 0.01) indicated that the negative charge of the genes was completely conjugated with the positive charge of PEG-g-PEI via strong charge interactions. The size and shape of the complexes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The TEM images of negatively stained complexes showed a spherical shape approximately 200 nm in size (Figure 2 (C)). The SEM images showed similar results (Figure 2 (D)). Heparin is an anionic polysaccharide that can compete for binding with miR to disrupt stability. To determine whether modification of miR conjugated with cholesterol increases the resistance of miR complexes to disassembly, a heparin assay was carried out using various concentrations (0, 0.1, 0.3, 0.5, and 1.0 UI/mL) of heparin. This range was included the heparin concentration (0.26−0.30 UI/mL) that is normally present in human plasma.41,42 Also, we used the alternatives of miR to pay attention to the stability of the complexes against the inhibitor, heparin, without any other effect. As shown in Figure 3, PEG-g-PEI/cNC resisted degradation in the 0.1 UI/ mL heparin environment. In contrast, PEG-g-PEI/NC was degraded. Remaining genes in the complex were determined by measuring the intensity of the unmigrated gene line. The results showed that PEG-g-PEI/cNC was more durable than PEG-g-PEI/NC at all heparin volumes. Thus, modification of cmiR in PEG-g-PEI protected against polyanions such as heparin, enabling PEG-g-PEI/cmiR to remain relatively intact C

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 3. Heparin dissociation assay of PEG-g-PEI/cNC complex (A) and PEG-g-PEI/NC complex (B) at various heparin volumes. Band intensity of remaining gene showing resistance to heparin (C) (n = 3) (*P < 0.01).

Figure 4. Serum dissociation assay of PEG-g-PEI/cmiR (A) and PEG-g-PEI/miR (B) at various serum volumes. Band intensity of remaining gene in the complex revealed resistance to degradation by the serum (C) (n = 3).

and increase transfection efficiency. Notably, cholesterol conjugation improved the stability of complexes against competitive binding and indicated that the complexes will be stable in the human body. Degradation induced by RNase in the serum is a major limitation to the application of miRs. Serum dissociation assay was performed at various concentrations of FBS (1, 3, 5, 10, and 20%) to investigate the difference in tolerance of the complexes to serum due to cholesterol deformation. Naked cmiR34a and miR34a were completely disassembled at 20% FBS. In contrast, PEG-g-PEI/cmiR34a and PEG-g-PEI/ miR34a were protected against serum-induced degradation. To compare the two complexes, the remaining genes in the complex were measured in a dissociation assay to determine the intensity of the line in the same location as the standard. As shown in Figure 4 (C), PEG-g-PEI/cmiR34a was more stable than PEG-g-PEI/miR34a at 5 and 10% FBS. These results demonstrate that PEG-g-PEI and modification of miRconjugated cholesterol protected from serum degradation compared to the naked miR group and unmodified miR group. Additionally, we performed characterization on the complexes that underwent serum dissociation assay using 10% FBS. As a result, the complexes were thought to be intact

because there was no significant difference in size and zeta potential, and was confirmed that properties of those did not change significantly in the presence of serum. Notably, the ability of PEG-g-PEI/cmiR34a complexes to resist serum degradation is essential for successful miR delivery in vivo. Although PEI has been extensively characterized as a gene delivery carrier in many studies, it exhibits dose-dependent cytotoxicity because of its polycationic properties. PEGylation can be used to overcome this limitation. PEGylated PEI not only has a lower charge than PEI alone but also has several advantages such as prolonged residence, decreased degradation by metabolic enzymes, and reduced or eliminated protein immunogenicity.43 We carried out an MTT assay at various concentrations to confirm the nontoxicity of the complexes. We used the alternatives to precisely identify the toxicity and prevent the increase of cell viability due to the therapeutic effects of miR. We also used the HS746T gastric cancer cell line, where miR34a is known to inhibit cell growth and induce apoptosis.44 As shown in Figure 5, no polyplexes showed toxicity when they contained 100 nM of the gene. As the gene concentration increased, the toxicity of polyplexes was observed when PEI was used. Thus, PEGylation lowered the D

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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and (B), Cy5.5-miR (orange) was delivered slightly more to the cells than Cy5.5 cmiR (blue). PEG-g-PEI markedly enhanced cell uptake in comparison of Cy5.5 cmiR (blue) and PEG-g-PEI/Cy5.5 cmiR (red). Additionally, PEG-g-PEI/ Cy5.5 cmiR (red) was delivered into the cells as well as with Lipofectamine 2000 (purple). Confocal microscopy was used to confirm that the measured fluorescence was within the cells using DAPI (blue) (Figure 6 (C)). PEG-g-PEI/cy5.5 cmiR was found to be scattered in the absence of cells. However, when cells were present, it was located around and inside the DAPIstained cell. The fifth slice of the 11 Z-stack images clearly confirmed that it was present in the cells. Thus, cholesterol and PEG-g-PEI not only played a protective role but also increased cellular uptake. To validate the therapeutic efficacy of PEG-g-PEI/cmiR, qRT-PCR was conducted to measure target gene expression of Nanog,45 Oct3/4,46 Snail,45 and Bcl247 by treating HS746T cells with the complexes. The therapeutic efficacy of cmiR delivered by Lipofectamine 2000 (Lipo.2000), a common transfection agent, was validated. For PEG-g-PEI/cmiR treatment, Nanog and Oct3/4, the transcription factor of cancer stemness, Snail, which regulates epithelial to mesen-

Figure 5. Cytotoxicity of complexes with cNC or NC at various concentrations (100−1000 nM) using PEI-g-PEI, Lipofectamine 2000 (Lipo2000), or PEI against Hs746T cells after 24 h incubation (n = 4).

toxicity of PEI, and the complexes had no toxic effects at the concentrations evaluated. To evaluate the transfection efficiency of PEG-g-PEI/cmiR, HS746T cells were treated with complexes consisting of Cy5.5 cmiR or Cy5.5-miR. Cell uptake was measured by fluorescence-activated cell sorting. As shown in Figure 6 (A)

Figure 6. Flow cytometry histograms illustrating cell uptake of complexes with Cy 5.5 cmiR or Cy5.5-miR using none, PEG-g-PEI, or Lipofectamine 2000 against Hs746T cells (n = 3) (A) and bar diagrams showing the cell uptake (%) of those (B). Confocal images of cell uptake of PEG-g-PEI/Cy5.5 cmiR (red) using DAPI (blue) (C). E

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 7. Validation of therapeutic efficacy of PEG-g-PEI/cmiR. Real-time PCR (qRT-PCR) analysis based on comparisons with nontreatment. RQ value was determined using the 2−△△Ct method (A) (n = 3). Confocal images of apoptosis assay using FITC (green) and PI (red) solution (B) and number of apoptosis cells (C). Wound healing assay for 96 h (D).

Figure 8. In vivo fluorescence images of tumor-bearing nude mice to confirm delivery efficiency and circulation time at various times (preinjection, 0, 1, 2, 4, 24, and 48 h) after injection of PEG-g-PEI/cNC and PEG-g-PEI/NC (A). Ex vivo fluorescence images of the tumor and normal organs (liver, kidney, and muscle) extracted from mice at 48 h after administration (B).

F

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials chymal transition, and Bcl2, an apoptosis regulator, were remarkably decreased (Figure 7A). Additionally, an apoptosis assay and cell-growth inhibition analysis were performed to examine the therapeutic effectiveness of PEG-g-PEI/cmiR. The apoptosis assay was confirmed by confocal image using the FITC Annexin V apoptosis detection kit. FITC fluorescence (green) was stained in cells that underwent apoptosis, and PI fluorescence was stained in dead cells. That is, early apoptotic cells are stained only with FITC fluorescence, and cells in the end stage of apoptosis are stained with both FITC and PI fluorescence. As with nontreatment image in Figure 7 (B), cells stained only with PI are dead cells through necrotic processes. The images of miR and cmiR treatments showed that the selected miRNAs caused apoptosis in HS746T cells. Apoptosis was observed in both images of PEG-g-PEI/miR and PEG-g-PEI/cmiR treatments. The same results were undoubtedly obtained when cmiR was treated with Lipo.2000, which was commercialized (Figure 7 (B)). However, it was difficult to compare the extent of apoptosis with confocal images. The number of apoptotic cells was measured by fluorescence-activated cell sorting (FACS). We reaffirmed that miR and cmiR were positive for apoptosis via FACS and confirmed that cholesterol conjugation of cmiR and polyplex formation using PEG-g-PEI increased apoptosis. This number was superior to that of Lipo.2000, although the size of the error bars was large. Thus, we verified that our system effectively increased apoptosis in vitro (Figure 8 (C)). The wound healing assay showed that cell migration was considerably disturbed by PEG-g-PEI/miR at 48 h. Additionally, cholesterol conjugation enhanced the therapeutic effects in both tests at 72 h, showing similar results as PEG-g-PEI. Thus, the therapeutic efficacy of cmiR was valid and can be effectively delivered to gastric cancer cells by using PEG-g-PEI and cholesterol conjugation in vitro. The delivery efficiency and circulation time of the complexes were examined in tumor-bearing mice. We used the alternatives tagged fluorescence to determine if the stability achieved by cholesterol conjugation and complex formation enhances delivery deficiency in vivo. As shown in Figure 8, PEG-g-PEI/cNC was present at remarkably higher fluorescence levels than PEG-g-PEI/NC in tumors at 4 h after injection. Additionally, PEG-g-PEI/cNC was found at higher fluorescence levels in tumor tissues than in nontumor tissues, while PEG-g-PEI/NC was mostly detected in the liver and kidney rather than in the tumor. Therefore, cholesterol conjugation prolonged the in vivo circulation time of miR and enhanced miR delivery to tumors.

Therefore, the self-assembled miR delivery system using cmiR and PEG-g-PEI demonstrated the clinical applications of RNAi gene therapy by stably transferring miR into tumors.

4. EXPERIMENTAL SECTION 4.1. Annealing the Cholesterol-Conjugated MicroRNA (cmiR). Two single-stranded RNAs with the sequence of microRNA-34a were purchased from Bioneer, Inc. (Daejeon, Korea); one of these RNAs was conjugated to cholesterol (5′-UGG CAG UGU CUU AGC UGG UUG U UG UG-3′ and 5′-[CHOLESTERTL] GAA GCA AUC AGC AAG UAU ACU GCC CU-3′). The mixture of the RNAs was annealed by denaturation for 5 min at 95 °C and cooled gradually to room temperature using a PCR cycler (Eppendorf MasterCycler Pro S, Eppendorf, Hamburg, Germany). Production of the annealed cmiR was verified by 2% agarose gel electrophoresis in 0.5× TBE and stored at −20 °C until use. 4.2. Synthesis of PEG-g-PEI. PEG-g-PEI was synthesized by conjugating branched PEI (25k, Sigma-Aldrich, St. Louis, MO, United States) and thiolated PEG (SH-PEG, MW 5000, Laysan Bio, Arab, AL, United States). Branched PEI (0.8 μmol) was dissolved in 10 mL of ultrapure deionized water and preactivated with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Thermo Fisher, Waltham, MA, United States) (16 μmol) for 30 min under vigorous stirring, followed by addition of SH-PEG (8 μmol). After 2 h, the solution was centrifuged at 5000g for 30 min through an Amicon Ultra-15 centrifugal filter (50k nominal molecular weight limit) to remove unreacted materials (SH-PEG and sulfoSMCC). The purified PEI-g-PEG was lyophilized and stored at −20 °C until use. Conjugation of PEG-g-PEI was confirmed by Fouriertransform infrared spectroscopy (FT-IR, Excalibur, Varian, Palo Alto, CA, United States) and 1H NMR spectroscopy (JUM-ECP300, JEOL, Ltd., Tokyo, Japan) with deuterium oxide (Sigma-Aldrich). 4.3. Formation and Characterization of PEG-g-PEI/cmiR. The complex of PEG-g-PEI and cmiR (PEG-g-PEI/cmiR) was prepared by mixing each solution (Tris-EDTA (TE) buffer) in various molar ratios (0.01, 0.05, 0.1, 0.4, 0.5, 0.6, 0.7, and 1.0:1 cmiR) for 30 min by gentle vortex mixing. Optimal condition for complexation of PEG-g-PEI/cmiR was examined by 2% agarose gel electrophoresis in 0.5× TBE. The hydrodynamic size and zeta potential of PEG-g-PEI/cmiR were measured by laser scattering (ELS-Z, Otsuka Electronics, Osaka, Japan). The morphology of PEGg-PEI/cmiR, negatively stained with 0.3% phosphotungstic acid, was observed by TEM (JEM-F200, JEOL) and SEM (JEOL-7800F, JEOL). 4.4. Cell Culture and Transfection. The HS746T human gastric cancer cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained with Dulbecco’s Modified Eagle’s Media (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic in a humidified incubator with 5% CO2 and 95% air at 37 °C (General TF condition). 4.5. Heparin Assay. To determine the stability of the complex against polyanion, a competition assay was carried out using heparin sulfate (170 U/mg). PEG-g-PEI/cNC (5′-[CHOLESTEROL]-CAG TAC TTT TGT GTA GTA CAA-3′ and 5′-CAG TAC TTT TGT GTA GTA CAA-3′, Bioneer, Inc.) and PEG-g-PEI/NC (NC: 5′-TTG TAC TAC ACA AAA GTA CTG-3′ and 5′-CAG TAC TTT TGT GTA GTA CAA-3′, Bioneer, Inc.) were prepared at a molar ratio of 0.6. The complexes were incubated with heparin for 30 min at 37 °C using different concentration of heparin (0, 0.1, 0.3, 0.5, and 1.0 IU/ mL). The solutions were then separated on 2% agarose gels electrophoresis in Tris-borate-DETA (TBE) buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, and pH 8.0) and visualized with a gel image analysis system (i-max, CoreBio System Co., Ltd., Seoul, Korea). 4.6. Serum Assay. The serum stability assay was performed using FBS to determine the effect of PEG-g-PEI on protecting cmiR and miR from serum degradation. Complexes were prepared at a molar ratio of 0.6 and incubated with serum for 30 min at 37 °C and different concentration of FBS (0, 1, 3, 5, 10, and 20%). Additionally,

3. CONCLUSION In this study, we successfully developed an efficient selfassembled miR delivery system consisting of cholesterolconjugated miR and pegylated polycationic polymer. The selfassembled complex with well-annealed cmiR and synthesized PEG-g-PEI showed the desirable size, zeta potential charge, spherical shape, and biocompatibility. This complex had improved resistance to competitive reactions and RNA degradation. In addition, the complex showed efficient transfection into the HS746T gastric cancer cell line in vitro and in vivo. Therapeutic efficacy was validated by RNAi, apoptosis, and cell-growth inhibition in vitro. G

DOI: 10.1021/acsabm.9b00186 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials free miR and cmiR were separately mixed with 2 μL FBS. The free miR, cmiR, and PEG-g-PEI/cmiR (0.5 mg) complexes were separately mixed with FBS. The solutions were analyzed by 2.0% agarose gel electrophoresis in TBE buffer and visualized with a gel image analysis system (i-max). 4.7. Toxicity. The toxicity of PEG-g-PEI/cmiR toward HS746T cells was evaluated by measuring the inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HS746T cells were seeded into a 96-well cell culture plate (1 × 104 per well) and incubated overnight in a humidified atmosphere at 37 °C with 5% CO2. Next, PEG-g-PEI/NC and PEG-gPEI/cNC were used various concentrations of NC and cNC to treat the cells for 24 h. Likewise, Lipofectamine 2000 was used as the manual. After treatment, the cells were rinsed with 100 μL Dulbecco’s phosphate-buffered saline (DPBS, WelGENE, Inc.). After further incubation 24 h, the MTT assay was performed using the principle that tetrazolium salt (yellow) was reduced to formazan crystals (purple) in metabolically active cells. The relative percentage of cell viability was determined as the ratio of formazan intensity in cells treated with PEG-g-PEI/NC to in nontreated cells (control). 4.8. Cellular Uptake. HS746T cells were seeded in 6-well plates at a density of 3 × 105 cells in 2 mL of DMEM and incubated for 24 h in a humidified atmosphere at 37 °C with 5% CO2. Complexes were prepared at a molar ratio of 0.6 in DMEM using miR and cmiRconjugated Cy5.5 respectively. After washing with PBS, complex solutions were used to treat the cells for 30 min. The cells were harvested after washing three times with PBS and centrifuged at 210g for 3 min. The supernatants were discarded, and the cells were suspended in 3.5% paraformaldehyde for fixation. Flow cytometry analysis was conducted using a flow cytometer (LSR Fortessa, BD Biosciences, Franklin Lakes, NJ, United States). The fluorescence of the cells was also observed by confocal laser scanning microscopy (LSM 880, Carl Zeiss, Oberkochen, Germany). 4.9. Real-Time PCR. To determine the target mRNA expression levels in HS746T cells after PEG-g-PEI/cmiR treatment, real-time quantitative reverse transcriptase polymerase chain reaction (qRTPCR) assay was performed with internal standards. Total RNA was isolated from HS746T cells using the MasterPure Complete DNA and RNA Purification Kit (Epicenter, Madison, WI, United States) according to the manufacturer’s instructions. RNA concentration was determined using aNanodrop 2000 (Thermo Fisher Scientific). The High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, United States) was used to synthesize cDNA from 2 μg of total RNA and diluted in a total of 100 μL. qRT-PCR analysis and quantification were performed using the QuantiMix SYBR Kit (PhileKoreaTechnology, Daejoen, Korea) on a real-time PCR System (LightCycler 480 System, HNS Bio, Seoul, Korea). The reactions were carried out in a 10-μL reaction mixture containing 1 μL of each forward and reverse primers, 3 μL of cDNA, and 5 μL of SYBR Green mixture. Primer sequences were as follows: Nanog (forward: 5′-GAT TTG TGG GCC TGA ACA AA-3′), (reverse: 5′-CTT TGG GAC TGG TGG AAG AA-3′); OCT3/4 (forward: 5′-CTG AAG CAG AAG AGG ATC AC-3′), (reverse: 5′-GAC CAC ATC CTT CTC GAG CG-3′); SNAIL (forward: 5′-AAG ATG CAC ATC CGA AGC CA-3′), (reverse: 5′-CTC TTG GTG CTT GTG GAG CA-3′); Bcl-2 (forward: 5′-CAT GTG TGT GGA GAG CGT CA-3′), (reverse: 5′GCC GGT TCA GGT ACT CAG TC-3′). PCR was initiated for 2 min at 95 °C, followed by 45 cycles of amplification (95 °C for 5 s, 60 °C for 10 s, and 72 °C for 10 s). Melting curves for each reaction were generated to confirm the purity of the amplification products. PCR was performed in triplicate for each sample, and the data were analyzed by the comparative Ct method, also referred to as the 2−ΔΔCt method. UBB, ACTB, and GAPDH were used as internal standards for each sample. 4.10. Apoptosis Assay. To confirm whether the complexes caused apoptosis of HS746T cells, an apoptosis assay was carried out using an FITC Annexin V apoptosis Detection kit (BD Biosciences) according to the manufacturer’s instructions. After resuspending the HS746T cells in 100 μL of 1× annexin-binding buffer, 5 μL of annexin V-FITC solution and 5 μL of PI solutions were injected and

incubated with the cells for 15 min at room temperature in the dark. The fluorescence-stained HS746T cells were analyzed by flow cytometry (LSR Fortessa) by monitoring the fluorescence-activated cells after adding 400 μL of 1× annexin-binding buffer. The fluorescence of the stained cells was also observed by confocal laser scanning microscopy (LSM 880, Carl Zeiss, Oberkochen, Germany). 4.11. Wound Healing Assay. To validate the therapeutic efficacy of PEG-g-PEI/cmiR, cell-growth inhibition analysis was performed. HS746T cells were seeded into 6-well plates at a density of 2.5 × 105 cells in 2 mL of DMEM and incubated for 24 h in a humidified atmosphere at 37 °C with 5% CO2. Complexes were prepared at a molar ratio of 0.6 in DMEM using miR and cmiR, respectively. After washing with PBS, the complex solutions were treated for 24 h. Using a pipet tip, a straight scratch was made in each well, simulating a wound. After washing with PBS, wound closure was observed at various times (0, 48, 72, and 96 h) using an inverted light microscope (Olympus CKX41, Olympus Co. Ltd., Tokyo, Japan). 4.12. Delivery of miR in vivo. All animal experiments were conducted with the approval of the Association for Assessment and Accreditation of Laboratory Animal Care International. Nude mice anesthetized by intraperitoneal injection of a Zoletil-Rompun mixture were injected with 200 μL of saline suspension containing 5.0 × 106 HS746T cells into the femoral region. One hundred microliters of saline and complexes prepared in a molar ratio of 0.6 with Cy5.5 conjugated NC and cNC were administered, followed by observation with an IVIS Spectrum (Caliper Life Sciences, Waltham, MA, United States) (at various times (preinjection, 0, 1, 2, 4, 24, and 48 h). Next, the tumor and organs were extracted and examined using an IVIS Spectrum. 4.13. Statistical Analysis. All experiments were performed in triplicate and tested with Student’s t-test.

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AUTHOR INFORMATION

Corresponding Authors

*Seungjoo Haam: E-mail: [email protected]; Tel.: +82-21232751. *Yong-Min Huh: E-mail: [email protected]; Tel.: +82-22280886. ORCID

Seungjoo Haam: 0000-0003-1533-8357 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (NRF2018M3A9E2022819), by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2017M3A9G5083322 and NRF2015M3A9D7029878), and by the Development of Platform Technology for Innovative Medical Measurements Program (KRISS-2018-GP2018-0018) from the Korea Research Institute of Standards and Science.

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