Biomimetic Dextran–Peptide Vectors for Efficient and Safe siRNA

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Biomimetic Dextran−Peptide Vectors for Efficient and Safe siRNA Delivery Xinjian Qu,†,‡ Yang Hu,‡,§ Huifeng Wang,‡ Haiqing Song,‡ Megan Young,‡ Fujian Xu,§ Ying Liu,‡ and Gang Cheng*,‡ †

School of Life Science and Medicine, Dalian University of Technology, Panjin 124221, China Department of Chemical Engineering, University of Illinois at Chicago, Chicago, Illinois 60607, United States § State Key Laboratory of Chemical Resource Engineering, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China Downloaded via OCCIDENTAL COLG on April 3, 2019 at 09:21:27 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Small interfering RNA (siRNA)-based therapeutics have the potential to treat a series of hereditary and acquired diseases. However, one serious obstacle for siRNA therapy is the lack of an efficient strategy to transport the siRNA to the targeted organ/cell with minimal toxicity. To take advantage of the good biocompatibility and degradability of natural polymers, and to understand how the peptide sequence affects the properties of the vector, four biomimetic vectors (D10-K3H7, D10R3H7, D20-K3H7, and D20-R3H7) were designed and synthesized by conjugating the peptide (K3H7 or R3H7) and dextran with a molecular weight of 10 or 20 kDa. Taking the commercial cellular transfection reagent Lipofectime RNAiMAX as a control, dextran−peptide/siRNA complexes exhibited smaller particle sizes, lower ζ potentials, and lower toxicity with the same value of N/P ratio. To evaluate the potential of this system for therapeutics, siRNA targeting the mRNA of the PCSK9 gene was chosen as a gene drug model to knock down the PCSK9 expression in the HepG2 cell line. Dextran−peptide/siRNA complexes exhibit a more consistent and higher knockdown efficiency than Lipofectamine RNAiMAX/PCSK9 siRNA complexes in a medium with 20% fetal bovine serum (FBS). D20-R3H7/PCSK9 siRNA complexes could knock down the level of PCSK9 mRNA by 85.2%, and they demonstrated a higher efficiency than Lipofectamine RNAiMAX, having 70.5% knockdown in the medium with 20% FBS at the PCSK9 siRNA concentration of 100 nM. These results suggest that the dextran−peptide-based vector has more efficient therapeutic agent properties for a siRNA-based drug transporter. KEYWORDS: siRNA delivery, dextran, peptide, histidine, PCSK9

1. INTRODUCTION

effective mechanisms to uptake naked nucleic acids proactively.6,7 Negatively charged siRNAs have a poor affinity to anionic cell surfaces, and they are susceptible to enzymatic degradation, which limits their passive transport through the plasma membrane of eukaryotic cells.8 Taken together, the small size and natural chemistry of siRNAs lead to their short half-life in the biological milieu.9 Protection and condensation of siRNAs by delivery vectors are essential to prevent their

RNA interference (RNAi), as a high-profile gene therapeutic approach in the personalized and precision medical era, has made significant advances especially in the treatments of both genetic and acquired diseases.1,2 The small interfering RNA (siRNA) is an effective RNAi tool for silencing specific protein coding genes.3 Accordingly, siRNAs are widely used to evaluate the personal contributions of genes to specific cellular phenotypes in vitro,4 for target gene studies in animal hereditary disease models, and for the purpose of treatment of disease-causing viruses in vivo.5 However, one serious obstacle that must be addressed is that normal cells lack © XXXX American Chemical Society

Received: November 14, 2018 Accepted: March 17, 2019

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DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 1. Schematic diagram of the synthetic and preparation route of dextran−peptide/siRNA complexes.

miRNA) and facilitate the endosomal escape of vector/DNA (or miRNA) complexes to achieve a high transfection.24,25 It was observed that specific sequences of peptides are significantly correlated to delivery efficiency.25 We hypothesize that peptides with a higher histidine to arginine ratio can further improve the delivery efficiency, reduce toxicity, and increase the blood stability of the vector owing to its hydrophobic nature and low pKa (6) of the imidazole side chain of the histidine. To verify our hypothesis, we synthesized two new peptides and conjugated the peptides to dextran for siRNA delivery. The success of siRNA therapy requires the selective and efficient transport of the specific siRNA to the target cells.29 According to experimental studies, the PCSK9 gene plays a critical role in regulating the cholesterol and fatty acid metabolism because the protease encoded by this gene binds to the low-density lipoprotein receptor (LDLR) to promote its degradation.30 Mutations in this gene have been associated with autosomal dominant familial hypercholesterolemia. In this study, we aimed to investigate the physiochemical characteristics, cytotoxicity, and in vitro gene transfection efficiency of several dextran−peptides conjugates to deliver PCSK9 siRNA to HepG2 cell lines.

interactions with enzymes and renal clearance, to improve their stability in a biological fluid environment.10 Therefore, the clinical success of siRNA therapeutics depends on the development of siRNA delivery vectors.11 The siRNA vectors for therapeutic applications need a good immune compatibility, low cost, and effective delivery properties.12,13 Cationic lipids are the most widely used biomaterials, especially in nonviral strategies for RNA delivery;14 however, the serum stability of lipid-based vectors, their immune response, and cytotoxicity are serious barriers in nucleic acid transport.15 Recently, polycations have attracted increasing attention owing to their simple synthesis and flexible design strategies.16 Several polycations, such as poly(L-lysine), polyethylenimine (PEI), and polyamidoamine, have exhibited favorable properties for in vitro gene delivery applications.17−19 However, cationic polymers also face several intrinsic drawbacks, including their low transfection efficiency, high toxicity, and nonbiodegradability.20 Such obstacles must be overcome for a safer and more efficient transport vector. Dextran is an indispensable biopolymer in pharmaceutics owing to its biocompatible, antithrombotic, nonantigenic, low toxicity, and biodegradable properties.21 Dextran can be used as an expander of plasma during surgical processes.22,23 As reported earlier by our group, dextran conjugated with cationic peptides demonstrated great potential as a nonviral and hemocompatible plasmid DNA and microRNA delivery system.24,25 In this system, cationic peptides are composed of lysine (K) or arginine (R) as the nucleic acid binding functional domain, histidine (H) as the endosomal binding moiety, and cysteine as a linker to dextran that serves as a macromolecular binder for short peptides.26,27 Peptides have similar characteristics to histones, which can condense DNA and protect it from degrading into target cells.28 A previous work by our group has demonstrated that dextran conjugated with the R5H5 peptide can efficiently condense DNA (or

2. MATERIALS AND METHODS 2.1. Materials. Dextran (Dex) (10 and 20 kDa MW), 4dimethylamino-pyridine (DMAP), glycidyl methacrylate (GMA), and dimethyl sulfoxide (DMSO) were purchased from Sigma− Aldrich (St Louis, MO, USA). The peptides (>95% purity) RRRHHHHHHHC (R3H7) and KKKHHHHHHHC (K3H7) were purchased from GenScript (Piscataway, NJ, USA). The reagents trypsin−EDTA, agarose, streptomycin−penicillin, sodium pyruvate, GlutaMAX, Lipofectamine 2000 (Lipo2000), Lipofectamine RNAiMAX (LipoRNAiMAX), nuclease-free water, ProLong Gold Antifade Reagent, SuperScript IV First-Strand Synthesis System, and PowerUp SYBR Green Master Mix were purchased from Thermo Fisher B

DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials Table 1. Physicochemical Characteristics of Dextran−Peptide/NC siRNA Complexes complexes D10-K3H7/siRNA-NC

D10-R3H7/siRNA-NC

D20-K3H7/siRNA-NC

D20-R3H7/siRNA-NC

Lipo-RNAiMAX/siRNA-NC

N/P

size (d, nm)

SD (±d, nm)

ζ (mV)

SD (±mV)

0.5 1 2 3 4 5 10 20 0.5 1 2 3 4 5 10 20 0.5 1 2 3 4 5 10 20 0.5 1 2 3 4 5 10 20 0.5 1 2 3 4 5 10 20

158.4 144.6 137.8 126.9 113.4 105.9 101.7 95.8 165.6 149.6 128.3 121.4 110.5 104.3 94.6 88.5 146.5 131.8 122.2 115.6 104.9 97.6 87.4 80.2 151.8 128.9 114.5 98.5 86.6 81.4 71.7 68.5 232.6 217.8 204.4 187.2 183.8 181.6 176.4 167.8

5.2 4.6 3.2 3.1 3.4 2.8 2.5 3.2 6.3 5.2 4.4 4.1 3.5 3.2 2.6 2.2 2.6 2.3 3.6 3.1 2.3 2.4 2.2 1.6 4.6 2.9 2.3 1.8 2.1 1.8 1.3 0.9 5.1 4.3 4.2 3.7 3.3 2.8 2.5 2.6

−7.96 −4.33 0.21 2.28 4.54 5.79 7.26 8.66 −6.78 −3.25 1.23 3.37 5.21 7.84 10.21 14.02 −7.29 −3.44 0.91 4.83 6.45 6.89 7.24 8.41 −6.51 −3.16 1.84 3.41 5.34 7.96 10.43 15.23 0.2 16.3 28.6 35.3 50.2 52.4 54.2 58.6

0.87 0.36 0.14 0.13 0.13 0.23 0.32 0.51 0.62 0.21 0.13 0.68 0.34 0.4 1.46 1.81 0.46 0.21 0.15 0.26 0.56 0.32 0.22 0.24 0.28 0.23 0.22 0.32 0.24 0.36 1.35 1.76 0.03 1.23 1.62 1.56 1.71 2.22 2.31 3.42

atmosphere at 25 °C with stirring for 4 days. To neutralize the DMAP and to stop the reaction, an equimolar amount of concentrated HCl was added to the flask. The mixture was dialyzed using a dialysis membrane with 7K MWCO (Spectrum Laboratories, Inc., Massachusetts, USA) against deionized water at 25 °C for 4 days and lyophilized to produce Dex-MA powder. NMR results show that 66% of dextran glucosides (10 kDa) were functionalized with the MA group, while 75% of dextran glucosides (20 kDa) were functionalized with MA. Next, synthetic Dex-peptides were synthesized. First, an aqueous solution (in which 40 mg of the peptide was dissolved) was added to 22 μL of Tris(carboxyethyl)phosphine hydrochloride (TCEP) at a concentration of 200 mM in a 1.5 mL centrifuge tube and mixed at room temperature for 5 min. To neutralize the TCEP and trifluoroacetic acid, 80 μL of NaOH (1 M) was added to the solution. Four 1.5 mL centrifuge tubes contained, respectively, 5.84 mg of Dex10K-MA for producing Dex10-R3H7, 5.21 mg of Dex10KMA for producing Dex10-K3H7, 4.90 mg of Dex20K-MA for producing Dex20-R3H7, and 4.38 mg of Dex20K-MA for producing Dex20-K3H7. Then, 70 μL of deionized water was added to the centrifuge tube to dissolve each calculated Dex-MA at 25 °C for 10 min and then was mixed with the peptide solution. Subsequently, the

Scientific (Rockford, IL, USA). The siRNAs were designed by our group and synthesized by IDT, Inc. (Coralville, IA, USA). The sense sequence of NC siRNA is rUrUrC rUrCrC rGrArA rCrGrUrGrUrC rArCrG rUTT. The antisense of NC siRNA is rArCrG rUrGrA rCrArC rGrUrUrCrGrG rArGrA rATT. The targeted sense sequence in PCSK9 for the RNA interference is 5′-GCCAATCCTTTAG CAGATCA-3′, and the antisense in PCSK9 for the RNA interference is 5′-GCAGCACCTGAAATCAACA-3′. 2.2. Cell Culture. HepG2 cells were cultured as described in Tang et al. (2015).25 Briefly, HepG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) with concentrations of 10% or 20% fetal bovine serum (FBS), supplemented with nonessential amino acids, GlutaMAX, sodium pyruvate, and penicillin−streptomycin, and maintained in 5% CO2 at 37 °C. 2.3. Synthesis of Dextran−Peptide Conjugates. The synthetic process of dextran−peptide conjugates is shown in Figure 1. The cationic dextran−peptide conjugates were synthesized in two steps.24 Briefly, 1.5 g of dextran and 1.35 g of DMAP were weighed separately, added to a 100 mL round-bottom flask, and dissolved by adding 20 mL of DMSO under a nitrogen atmosphere for 20 min. Then, 2 mL of GMA was added. The mixture was kept under a nitrogen C

DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials mixture was incubated at 25 °C for 2 days after the solution pH value was adjusted to 7.5. After the reaction, the mixture was purified using the Zebra Spin Desalting Column (Thermo Fisher Scientific Inc., Rockford, IL, USA) and lyophilized. The powder product was analyzed by 400 MHz 1H nuclear magnetic resonance (NMR). 2.4. Preparation of Dextran−Peptide/siRNA Complexes. An siRNA stock solution (2 μg/μL) was prepared using nuclease-free sterile water and then diluted to 1 μM in PBS. Each dextran−peptide was dissolved in sterile DI water to prepare a 1 mg/mL stock solution and further diluted to PBS to various concentrations, which depend on the dextran−peptide that will be needed to form complexes at specific N/P ratio values. Here, the value of the N/P ratio, as an important indicator, was defined as the molar ratio of an arginine residue (N) in the dextran−peptide vector to the phosphate group (P) in siRNA.25 The dextran−peptides/siRNA complexes were formed by mixing equal volumes of dextran−peptides/siRNA corresponding to different N/P ratio values, whereas the siRNA solutions were kept at the concentration of 1 μM. The solutions were incubated at 25 °C for 20 min, ensuring the peptide and RNA molecules interacted completely. 2.5. RNA Interference Assay. HepG2 cells were grown in 24well plates and subcultured at a density of 5 × 104 cells/well with DMEM media. The prepared dextran−peptide/siRNA complexes were dropped into wells according to the different N/P ratio values when the cell confluency reached 40−50%, approximately. After 48 h, each well was washed twice with PBS, and then 0.3 mL of the Trizol reagent was added to each well to extract the mRNA, which was reverse transcribed into cDNA by reverse transcriptase. GAPDH and PCSK9 were detected using qRT-PCR with the corresponding PCR primers. The products were stained by using SYBR Green I fluorescent dye and amplified according to the protocol provided. As per the Bio-Rad CFX96 operating guide, the real-time quantitative PCR operation was repeated three times in the experiment and PCR data was analyzed using the 2−ΔΔCT method. 2.6. Gel Retardation Electrophoresis Assay. Dextran− peptide/siRNA complex solutions were performed with N/P ratio values of 0, 0.5, 1, 2, 3, 4, 5, 10, and 20. An aliquot of each solution (up to 1 μg siRNA) was pipetted onto a 10% agarose gel, and gel electrophoresis was carried out in 1× TAE buffer at 120 V for 30 min. The gel was then stained and photographed using the Azure C150 Gel Imaging System (Dublin, CA, USA). 2.7. Cytotoxicity Assay. HepG2 cells were counted and seeded in 96-well plates at a density of 2 × 104 cells/well for 24 h. The culture medium was replaced with 10 μL of dextran−peptide/siRNANC and 90 μL of fresh medium. After 48 h of culture at 37 °C, the medium was replaced with 10 μL of a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/mL in PBS) and 100 μL of fresh DMEM medium, and the cells were continuously incubated for 4 h. Then, DMSO was added to each sample (150 μL per well) to dissolve the crystals after the removal of the medium. The absorbance of each sample was detected six times though a Tecan Infinite 200 microplate reader (Männedorf, Switzerland) at 570 nm. Taking the untreated cells as a control, the data of cell viability was shown as a percentage of the control (100%). 2.8. Statistical Analysis of Experimental Data. Experimental data were compared to positive controls (cells treated with Lipo2000 and LipoRNAiMAX) and negative controls (cells treated with siRNANC). All data were presented as mean ± standard deviation and are normally distributed. The statistical analysis of experimental data was performed using Student’s t test. When P < 0.05, the data were presented as significantly different.

and D20-K3H7 (Figure 1). In our design, the ester bond between the dextran and peptide can be cleaved by either hydrolysis or the esterase in the cells. Over two-thirds of the glucosides of dextran (10 or 20 kDa) were functionalized with MA groups according to the 1H NMR (Figure S1 and Figure S2). The average degree of substitution (DS) of peptide side chains for D10-K3H7, D10-R3H7, D20-K3H7, and D20-R3H7 was 18.4% (Figure S3), 18.0% (Figure S4), 18.3% (Figure S5), and 18.1% (Figure S6), respectively. For an efficient siRNA vector, a suitable capacity to condense siRNA is a prerequisite. In this work, the condensability of dextran−peptide was evaluated by dynamic light scattering, ζ potential, and gel retardation assay. Taking different values of N/P ratios as indicators, dextran−peptide conjugates were used to form complexes with synthesized siRNA in a predetermined ratio. As presented in Table 1, the particle size together with the ζ potential of dextran−peptide/ siRNA-NC complexes in a predetermined value of N/P ratio were measured. As the N/P ratio value increased, the hydrodynamic sizes of all complexes decreased, while the ζ potentials of complexes changed from negative to positive, ranging from approximately −8 to +15 mV. All dextran− peptide/siRNA-NC complexes follow a similar trend as the N/ P ratio value changes. At a low value of N/P ratio (0.5 and 1), complexes are detected to be negatively charged, while the ζ potential gradually increases and exhibits a positively charged trend with the increasing value of N/P ratio. The ζ potential of LipoRNAiMAX/siRNA was 0.2 ± 0.03 mV at an N/P ratio value of 0.5 and increased to 16.3 ± 1.23 mV, 28.6 ± 1.62 mV, and 35.3 ± 1.56 mV with N/P ratio values of 1, 2, and 3, respectively. When the value of N/P ratio increased over 4, the ζ potential of the complexes was over 50 mV. It should be noted that a high ζ potential (>15 mV) of the complexes often leads to a high cytotoxicity.29 As derivatives of dextran with the same molecular weight (10 or 20 kDa), Dex-R3H7/siRNA-NC complexes demonstrated a smaller size than Dex-K3H7/siRNA-NC complexes at the same value of N/P ratio. However, the ζ potential of DexR3H7/siRNA-NC is higher than that of Dex-K3H7/siRNANC complexes. This result is consistent with previous findings, indicating that the guanidine group of arginine shows a stronger bind affinity to nucleic acids owing to the charge and hydrogen-bond-forming capability.31 The imidazole group of histidine provides a strong pH-buffering capacity during endosomal escape.32 Because dextran scaffolds have the same peptide grafting density and each peptide has the same number of H residues, Dex-R3H7 carries stronger nucleic acid binding groups; hence, it condenses the anionic siRNA-NC into smaller particles. Dextran is composed of complex branched glucans of various lengths and has unique biological activities (such as excellent blood solubility) and a variety of biologically functional groups.21 Because they have the same grafting density, dextran (10 kDa)−peptide/siRNA-NC complexes have smaller particle sizes than dextran (20 kDa)−peptides/ siRNA-NC with the same peptide sequence. This study revealed that both the dextran molecular weight and architecture are important parameters that determine the properties of the complexes. Taking the LipoRNAiMAX/ siRNA-NC complexes as the control, dextran−peptide/siRNANC complexes, especially D20-R3H7/siRNA-NC complexes, exhibited smaller particle sizes and lower ζ potentials at the same value of N/P ratio.

3. RESULTS AND DISCUSSION Dextrans (10 and 20 kDa) were first reacted with GMA, producing Dex10K-MA and Dex20K-MA, respectively, and then dextran−peptide conjugates were synthesized via the reaction of the cysteine linker of R3H7C or K3H7C with the MA of Dex-MA. The corresponding dextran−peptide conjugates were denoted as D10-R3H7, D10-K3H7, D20-K3H7, D

DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 2. Electrophoretic mobility of siRNA in the complexes of (a) D10-K3H7, (b) D10-R3H7, (c) D20-K3H7, (d) D20-R3H7, and (e) LipoRNAiMAX at various N/P ratio values. The red box indicates the threshold value of the N/P ratio that prevents siRNA from leaking out of the dextran−peptide/siRNA complexes.

respectively. With an N/P ratio of 10, the cytotoxicity depended on the type of dextran−peptide: cell viability was 82.1% and 81.5% for D10-R3H7 and D20-R3H7 and 79.2% and 75.7% for D10-K3H7 and D20-K3H7, respectively. However, for Lipo2000 and LipoRNAiMAX, the cell viability decreased to 56.2% and 54.3%, respectively. With an N/P ratio value of 20, the cell viability of dextran−peptide/siRNA-NC complexes was above 75%, except for D20-K3H7 (74.4% of cell viability), while the cell viability was 51.3% and 47.9% for the Lipo2000 and LipoRNAiMAX complexes, respectively. The results suggest that dextran−peptide vectors possess a much lower cytotoxicity than Lipo2000 and LipoRNAiMAX. Considering the toxicity results, complexes with an N/P ratio value of 2 were selected for the transfection study. The PCSK9 gene, together with the low-density lipoprotein receptor (LDLR) and apolipoprotein B (ApoB), plays a key role in familial hypercholesterolemia. PCSK9 downregulates the LDLR and leads to the elevated levels of low-density lipoprotein cholesterol in the plasma.34,35 It has been shown that the down-regulation of PCSK9 was an effective method for the treatment of hypercholesterolemia because the liver predominantly controls the LDL metabolism and the HepG2 cells express the PCSK9 gene in higher amounts than other normal cells.36 Therefore, the siRNA transfection efficiency of dextran−peptide conjugates against PCSK9 mRNA was evaluated by measuring the mRNA level of the PCSK9 gene via qRT-PCR in HepG2 cells. It has been reported by many studies that blood stability is the major barrier for a cationic polymeric vector-mediated RNA delivery system.37 Many transfection studies were performed in a medium with no serum or low serum, thus resulting in a significant inconsistency between the in vitro and in vivo transfection studies.38,39 To avoid unnecessary usage of animals, the in vitro transfection study should be conducted under high serum conditions (>10%). To evaluate how the serum content in culture medium affects the delivery of dextran−peptides, 10% and 20% FBS were added in the culture medium and cells were transfected at concentrations of 30 and 100 nM siPCSK9. As Figure 4a shows, under the condition of a concentration of 30 nM siPCSK9, the PCSK9 mRNA expression levels in HepG2 with 10% serum were 54.2%, 41.2%, 45.3%, and 22.8% for the D10-K3H7/siPCSK9,

A gel retardation assay was used to evaluate the binding affinity between anionic siRNA and cationic dextran−peptides. As shown in Figure 2, the N/P ratio value of 0 represents the free siRNA. For the siRNA to escape from the complexes under an electrical field, the threshold value of N/P ratio for D10-K3H7, D10-R3H7, and D20-K3H7 was of 2, and it was higher than that of D20-R3H7 and LipoRNAiMAX, which had a threshold value of an N/P ratio of 1. For all dextran− peptides, the mobility of siRNA gradually decreased with the value of the N/P ratio gradually increasing for all dextran− peptides, indicating the compact formation of dextran− peptide/siRNA complexes. Low cytotoxicity is critical for all nucleic acid delivery systems. Herein, an MTT assay was adopted to detect the cytotoxicity of dextran−peptide/siRNA-NC complexes in comparison with LipoRNAiMAX/siRNA-NC complexes in the HepG2 cell line because HepG2 is the commonly used human tumor model in drug metabolism and cytotoxicity studies.33 The quantity of dextran−peptides was adapted corresponding to the value of N/P ratio. Two different concentrations of siRNA were tested: 30 and 100 nM. In general, cell viability reduced as the value of N/P ratio increased because of the toxicity caused by excess polycations. Compared to Lipofectamine/siRNA-NC, a lower cytotoxicity was observed for all dextran−peptide/siRNA-NC complexes at the same siRNA concentration. As shown in Figure 3a, dextran−peptide/siRNA complexes demonstrated a similar cytotoxicity. Cells treated with D10-R3H7 or D20-R3H7/ siRNA complexes were more viable than those treated with D10-K3H7 or D20-K3H7/siRNA complexes at a siRNA concentration of 30 nM and at a value of N/P ratio of 2. The cytotoxicity increased at the value of N/P ratio of 4, but the cell viabilities remained approximately 90% for all dextran− peptide/siRNA-NC complexes. However, the cell viability of Lipo2000 and Lipo2000 complexes was under 80% at an N/P ratio value of 4. More importantly, the cytotoxicity of dextran− peptide complexes with the same N/P ratio value was lower than that of Lipofectamine/siRNA-NC complexes. As shown in Figure 3b, at the siRNA concentration of 100 nM and with an N/P ratio of 3, the viability of all dextran−peptide/siRNANC complexes was around 90%, whereas the viabilities of Lipo2000 and LipoRNAiMAX were 81.4% and 81.2%, E

DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 3. Cell viability of dextran−peptides/siRNA-NC complexes in comparison with Lipofectamine/siRNA-NC complexes in different siRNA concentrations. (a) HepG2 cells were treated with a 30 nM siRNA concentration at different N/P ratio values for 24 h with each vector (mean ± SD, n = 6). (b) HepG2 cells were treated with a 100 nM siRNA concentration at different N/P ratio values for 24 h with each vector (mean ± SD, n = 6).

Figure 4. Relative expression of PCSK9 mRNA levels treated with dextran−peptide/siRNA-NC complexes in comparison with Lipofectamine/siRNA-NC complexes in different siRNA concentrations. (a) HepG2 cells treated at a 30 nM siPCSK9 concentration with different vectors after 48 h at an N/P ratio of 2 (mean ± SD, n = 3). (b) HepG2 cells treated at a 100 nM siPCSK9 concentration with different vectors after 48 h at an N/P ratio of 2 (mean ± SD, n = 3).

D10-R3H7/siPCSK9, D20-K3H7/siPCSK9, and D20-R3H7/ siPCSK9 complexes, respectively. This expression changed slightly to 57.6%, 43.5%, 47.6%, and 23.4% for the respective complexes in a culture medium with 20% serum. However, there was no significant difference between the vectors. On the contrary, with the increase in serum from 10% to 20%, the mRNA knockdown was reduced significantly for Lipo2000 complexes (from 44.1% to 57.1% of the PCSK9 mRNA level) and LipoRNAiMAX complexes (from 23.6% to 32.5% of the PCSK9 mRNA level). As Figure 4b shows, the PCSK9 mRNA levels treated with D10-K3H7/siPCSK9, D10-R3H7/siPCSK9, D20-K3H7/siPCSK9, and D20-R3H7/siPCSK9 complexes at a concentration of 100 nM siPCSK9 in 10% serum medium were 44.2%, 23.2%, 38.3%, and 14.8%, respectively. In the 20% serum culture condition, the PCSK9 mRNA levels were 47.6%, 24.3%, 39.6%, and 15.7%. There was no significant difference for the same complex under the different serum conditions. However, when the serum concentration changed from 10% to 20%, the expression of the PCSK9 mRNA level changes from

36.1% to 48.4% for Lipo2000/siPCSK9 and from 14.6% to 29.5% for LipoRNAiMAX/siPCSK9. The results show that the siRNA knockdown efficiency of the dextran−peptide-based carrier was more stable and efficient than that of the LipoRNAiMAX-based carrier in the more complex medium with higher concentrations of serum proteins. Throughout comparison, the order of the siRNA interference efficacy of dextran−peptide conjugates is D20-R3H7 > D10-R3H7 > D20-K3H7 > D10K3H7. Dextran−peptide conjugates with arginine-rich peptides (R3H7C) have a higher affinity with cell membranes, which can improve the siRNA interference efficacy more than that of conjugates with lysine-rich peptides (K3H7C). F

DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

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4. CONCLUSIONS In this study, we designed and synthesized a dextran−peptide conjugate platform for siRNA delivery. Our data demonstrated the importance of various factors that affect the cytotoxicity and intracellular siRNA effectiveness when dextran−peptide conjugates are used as a carrier. The vector/siRNA ratio and the siRNA concentration are crucial parameters for efficient siRNA delivery. All dextran−peptide conjugates showed a strong RNA binding ability with the increase in N/P ratio values. With a higher molecular weight, the dextran-derived dextran−peptide conjugates demonstrated a higher siRNA transfection efficiency than that composed of the lower molecular weight dextran. The results of the PCSK9 gene knockdown assay demonstrated that Dex-R3H7 has a better siRNA interference performance than Dex-K3H7 and LipoRNAiMAX. Particularly, D20-R3H7 conjugates demonstrated a higher siRNA knockdown efficiency and lower cytotoxicity than the commercially available transfection reagent LipoRNAiMAX at high serum conditions. All of the results suggest that dextran−peptide conjugates could be promising for the transport of siRNA.

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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/acsabm.8b00714. 1 H NMR spectra of dextran−MA and dextran−peptide (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Fujian Xu: 0000-0002-1838-8811 Gang Cheng: 0000-0002-7170-8968 Author Contributions

X.Q. performed the work, analyzed the data, and prepared the manuscript. Y.H. synthesized the dextran−peptides, and H.W. and M.Y. contributed to the NMR data analysis. F.X. and Y.L. provided additional information and supervised the work, and G.C. supervised the work, analyzed the data, and revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the U.S. National Science Foundation (DMR-1206923, DMR-1454837), the Natural Science Foundation of Liaoning Province (201601048), the Fundamental Research Funds for the Central of China (DUT16QY28), and the National Natural Science Foundation of China (51829301).

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REFERENCES

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DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

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DOI: 10.1021/acsabm.8b00714 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX