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A biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo Zongguang Tai, Xiaoyu Wang, Jing Tian, Yuan Gao, Lijuan Zhang, Chong Yao, Xin Wu, Wei Zhang, Quangang Zhu, and Shen Gao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501777a • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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A biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo
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Zongguang Taia‡, Xiaoyu Wanga‡, Jing Tiana‡, Yuan Gaoa, Lijuan Zhanga, Chong Yaoa, Xin
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Wub , Wei Zhanga, Quangang Zhu a, c,**, Shen Gaoa,*
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a
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Shanghai 200433, China.
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b
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University School of Medicine, Shanghai 200080, China
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Department of Pharmaceutics, Changhai Hospital, Second Military Medical University,
Department of Pharmaceutics, Shanghai First People’s Hospital, Shanghai Jiaotong
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c
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Medicine，Shanghai University of Traditional Chinese Medicine，Shanghai 200437, China
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KEWORDS: arginine; histidine; stearyl; disulfide bond; peptide; siRNA delivery
Department of Pharmacy, Yueyang Hospital of Integrated Traditional Chinese and Western
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ABSTRACT
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RNA-based delivery system for cancer therapy remains a challenge. In this study, a
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stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine and stearyl moiety.
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Further, the stearyl-peptides were cross linked by disulfide bonds to obtain cross-linked
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polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4).
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The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a
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hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and
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confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation
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behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in
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cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with
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SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on
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Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay
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rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker
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expression of red fluorescence protein was also observed on SHRss2/simCh-treated
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mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce
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cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies
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demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies
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indicated that the SHRss are promising and efficient non-viral vectors for siRNA delivery.
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INTRODUCTION
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Small interfering RNA (siRNA) can specifically inhibit gene expression and are being
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researched in many disease areas, especially in cancer, inflammation, diabetes, and
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neurodegenerative therapy
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potential to inhibit gene expression and evaluation in human therapeutic application of RNAi
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5-7
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siRNA during the therapeutic procedure 9, 10. Therefore, developing a vector which has siRNA
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binding capacity, improved serum stability, high cell uptake, endosomal escape, as well as
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siRNA dissociation in the cytoplasm and tissue penetration for systemic delivery is prime
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1-4
. Various studies show that RNA interference (RNAi) has a
. However, there are still some challenges
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such as instability and poor cellular uptake of
importance 11.
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It was reported that cell-penetrating peptides (CPPs, also called peptide transduction
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domain) have the ability to enhance intracellular uptake of macromolecules including nucleic
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acids in a low-toxic manner
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which are considered as critical residues for intracellular uptake 14. Arginine is cationic amino
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acid, anionic siRNA can form stable complexes with arginine through strong electrostatic
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interactions, leading to efficient cellular uptake of siRNA complexes 15. In addition, due to the
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formation of bidentate ionic interactions with cell surface proteoglycans, arginine residues can
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result in a close association with the cell membrane
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arginine-rich peptides or polymers resulting in cell penetration and gene delivery
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used arginine to package siRNA into electropositive nanoparticles and mediate cellular uptake
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of siRNA.
12, 13
. Some of the CPPs are rich in cationic arginine residues,
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. Several studies have focused on 17-20
. So we
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Intracellular uptake is just one of the obstacles for RNAi. In cells, siRNA complexes should
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be able to escape from the endosomal pathway and siRNA should dissociate from the 3 ACS Paragon Plus Environment
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carrier/siRNA complexes. Histidines have a potent proton-accepting moiety, so the carriers
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containing histidines are possible to allow carrier/siRNA complexes to escape from endosomal
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compartments by raising the pH of endosomes, leading to osmotic swelling and bursting
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Thus, the imidazole groups in histidine provide a “proton sponge effect” to enhance
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endosomal/lysosomal escape.
21-23
.
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Furthermore, it was reported that stearylation improved intracellular uptake and endosomal
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escape of siRNA carriers 24-27. Due to the lipophilic property, a stearyl moiety can increase the
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interaction of the complexes with the membrane of the cell and endosome. Therefore a stearyl
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moiety was introduced as a hydrophobic moiety to enhance cellular uptake and endosomal
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escape of siRNA complexes.
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Disulfide cross-linked polymers can achieve efficient siRNA delivery with a low
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cytotoxicity 28, 29. They can rapidly degrade in the reductive intracellular cytoplasm 30, leading
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to a rapid siRNA release into the cytoplasm 31. Moreover, the disulfide cross-linked polymers
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have higher charge density than the non cross-linked polymer, which can enhance the siRNA
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package capacity as well as intracellular uptake.
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Due to the special properties of arginine, histidine and stearyl, we combined the strategies
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and developed a new disulfide cross-linked stearyl-peptide containing arginine and histidine
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(SHRss) (Scheme 1 and Figure 1). The SHRss can effectively condense siRNA into nano-scale
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complexes. The chemical structure of SHRss and characteristics of SHRss/siRNA complexes
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were determined. Furthermore, the cell viability as well as the gene interference efficiency of
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SHRss/siRNA on Luciferase-expressing Hela cells and mCherry-expressing HEK293 cells was
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examined. In addition, the gene silencing efficiency in vivo was evaluated.
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Scheme. 1. Disulfide cross-linked SHRss tends to self-assemble into cationic complexes and
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further formed nanoparticles with negatively charged siRNA based on electrostatic interactions.
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After cellular uptake by endocytosis pathway, the SHRss/siRNA complexes realize endosomal
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escape dependent on “proton sponge effect” mediated by histidine and membrane penetrating
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capability mediated by stearyl moiety, then disulfide bonds are cleaved in the reductive
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cytoplasm, and siRNA are released into the cytoplasm after dissociation of SHRss/siRNA
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complexes. Subsequently, siRNA develops into RNA-induced silencing complex (RISC) and
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guides endonucleolytic cleavage of the target mRNA.
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EXPERIMENTAL SECTION
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Materials. L-histidine, L-arginine, L-cysteine, stearic acid, and 30% H2O2 solution were
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purchased from Sangon Biotech (Shanghai, China). Ethidium bromide (EtBr), dithiothreitol
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(DTT), 4',6-diamidino-2-phenylindole (DAPI), chlorpromazine hydrochloride and amiloride
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hydrochloride were purchased from Sigma Aldrich (St. Louis, MO, USA). Filipin Ⅲ was
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purchased from Cayman Chemical (Michigan, USA). Passive Lysis 5× Buffer and luciferase
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assay kit were obtained from Promega (Wisconsin, USA). Lipofectamine 2000 (LF2000),
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ZeocinTM and puromycin were purchased from Invitrogen (Carlsbad, CA, USA); Dulbecco’s
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modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin
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solution (5 kU/mL) were from Life Technologies (Grand Island, USA). GelredTM was obtained
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from biotium (CA, USA). Enhanced BCA Protein Assay Kit was purchased from Beyotime
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(Nanjing, China). Negative Control siRNA (NC-siRNA, sense: 5’-UUC UCC GAA CGU GUC
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ACG UTT-3’, antisense: 5’-ACG UGA CAC GUU CGG AGA ATT-3’), luciferase targeting
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siRNA (siLuc, sense: 5’-CUUACGCUGAGUACUUCGATT-3’, antisense: 5’-UCG AAG UAC
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UCA GCG UAA GTT-3’), mCherry targeting siRNA (simCh, sense: 5’-CAU GGC CAU CAU
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CAA GGA GTT-3’, antisense:5’-CUC CUU GAU GAU GGC CAU GTT-3’) and Cy-3 labeled
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NC-siRNA (Cy3-siRNA) were synthesized by GenePharma Co. Ltd. (Shanghai, China). Cy-5
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labeled NC-siRNA (Cy5-siRNA) was from RiboBio Co. Ltd. (Guangzhou, China).
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Cell lines and Cell culture. Luciferase-expressing (GL3) Hela cells (Luc-Hela cells) were
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obtained from SBO Medical Biotechnology (Shanghai, China). mCherry-expressing HEK293
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cells (mCherry-HEK293 cells), which express red fluorescence protein, were kindly donated
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from Dr. Fang (Department of Urology of Changhai Hospital in Shanghai, China). All cells 6 ACS Paragon Plus Environment
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were grown in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin and
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streptomycin). To maintain luciferase expression, 200 µg/mL ZeocinTM was added in Luc-Hela
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cells. 5 µg/mL puromycin was added in mCherry-HEK293 cells to maintain mCherry
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expression.
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Synthesis and Characterization of polymers. Peptide H3CR5C (HR) was synthesized using
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the F-moc-solid-phase peptide synthesis method. To increase membrane affinity, we introduced
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stearyl moiety to the N-terminal of HR using the same synthesis method of HR, and the
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product was purified by reverse-phase HPLC. The molecular weight of stearyl-HR (SHR) was
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determined by MALDI-TOF MS (Bruker Daltonics, Germany). The single sulfydryl of
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L-Cysteine can block the formation of disulfide bond and control the the degree of
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polymerization through disulfide bond. To obtain disulfide cross-linked polypeptides (SHRss)
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with different molecular weight, 50 mg SHR was mixed with different mole ratios of
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L-Cysteine hydrochloride monohydrate (Table 1) and dissolved in 9.5 mL distilled water (pH
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7.0), then 0.5 mL 10% H2O2 aqueous solution was added into the mixed solutions under
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stirring. After 12 h reaction, the products were dialyzed in distilled water for 12 h using a
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dialysis membrane (MWCO 1000) and lyophilized. The synthesized polymers were
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characterized by 1H-NMR at 600 MHz (Varian, CA, USA) and gel permeation chromatography
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(GPC) (HLC-8220, TOSOH Corporation, Tokyo, Japan). To obtain controls vector, the
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polypeptides omitting stearyl chain or histidine residues (named HRss2 and SRss2 respectively)
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were synthesized by crossing link H3CR5C and stearyl-CR5C according to the method above.
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Preparation and characterization of complexes. SHRss was complexed with siRNA in
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PBS (50 µL per 0.5 µg of siRNA, pH 6.0). The N/P ratio of SHRss to siRNA was calculated as
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follow. 1.06 µg SHRss per 1µg siRNA was equal to N/P=1.The samples were vortexed and 7 ACS Paragon Plus Environment
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incubated for 30 min at room temperature before use. LF2000 was complexed with siRNA in
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OptiMEM according to the operation manual. The particle size and zeta potential of the
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SHRss/siRNA complexes were determined using a Zetasizer Nano ZS (Malvern, Westborough,
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MA, USA). The morphology of the SHRss2/siRNA complexes at N/P=10 was examined by
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transmission electron microscopy (TEM). Briefly, one drop of a solution containing
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SHRss2/siRNA complexes was placed on a 200-mesh copper grid coated with carbon film. The
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excess solution was removed by a filter paper. Images were recorded under 75 kV acceleration
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voltage.
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Agarose gel electrophoresis. The ability of polymers to condense siRNA was determined
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by agarose gel electrophoresis. The carrier/siRNA complexes were prepared at N/P ratios of 1–
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10. After incubation for 30 min at room temperature, samples containing 1 µg siRNA were
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electrophoresed by a 1.0% agarose gel containing GelredTM at 100 V for 15 min in
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Tris-acetate-EDTA (TAE) buffer. To investigate the influence of polymerization on siRNA
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binding ability as well as the polymer behaviors under reductive environments, 100-fold DTT
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(mole ratio to SHR monomer) was added to solutions of carrier/siRNA complexes. After
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treatment for 1 h, samples containing 1 µg siRNA were electrophoresed as mentioned. The
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location of nucleic acid bands was visualized under UV light.
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Ethidium bromide exclusion assay. Ethidium bromide (EtBr) exclusion assay was
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performed to examine the siRNA condensation efficiency of polymers. Briefly, 4 µg siRNA
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was complexed with polymers with different N/P ratio. The complexes were diluted to a final
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volume of 180 µL using PBS. After a 30-min incubation at room temperature, the diluted
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complexes were mixed with 20 µL of EtBr (50 µg/mL) solution thoroughly. Fluorescence
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intensity of each sample was measured using a fluorescence spectrophotometer (Promega, 8 ACS Paragon Plus Environment
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USA) with excitation at 525 nm and emission at 610 nm.
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Serum stability. The SHRss2/siRNA and SHR/siRNA complexes were incubated in 50%
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FBS solution at a siRNA concentration of 14 µg/mL (1 µM). Naked siRNA was used as control.
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The mixtures were incubated at 37°C for different time intervals. To release the loaded siRNA,
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the mixtures were treated with 10 µL heparin solution (500 U/mL) and subsequently
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electrophoresed by a 1.0% Agarose gel containing GelredTM at 100 V for 15 min in TAE buffer.
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The undegraded siRNA were visualized under UV light.
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Cellular uptake assay. The siRNA uptake by Luc-Hela cells was analyzed using flow
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cytometry. Luc-Hela cells were seeded onto 12-well plates at 2 × 105 cells per well and
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incubated for 24 h. After replacing the culture medium, SHRss/Cy3-siRNA complexes,
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LF2000/Cy3-siRNA, SHR/Cy3-siRNA were added into Luc-Hela cells with a final Cy3-siRNA
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concentration of 75 nM, respectively. After incubation for 3 h, the cells were washed,
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trypsinized, centrifuged, and resuspended in PBS. The cells were analyzed on a FACScan flow
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cytometer (Becton Dickinson, San Jose, CA, USA).
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Transmembrane mechanism study. Luc-Hela cells were seeded onto 12-well plates at
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2×105 cells per well and incubated for 24 h. To probe the internalization mechanism of the
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complexes, Luc-Hela cells were pretreated with various endocytic inhibitors including
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chlorpromazine (30µM), filipin Ⅲ (1µg/ml) and amiloride (30 µM) for 1 h. Then the
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SHRss2/Cy3-siRNA complexes were added into culture medium for an additional 2 h
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incubation. After incubation, cells were washed three times with PBS followed by incubation
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with 400 ml lysis buffer for 0.5 h. The lysates were centrifuged at 5000 rpm for 3 min and the
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supernatant was analyzed by fluorescence spectrophotometer with excitation at 525 nm and
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emission at 610 nm. The cellular internalization of SHRss2/Cy3-siRNA was also visualized 9 ACS Paragon Plus Environment
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under a confocal laser scanning microscope (CLSM) (Olympus, Japan).
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Gene silencing assay. In vitro gene silencing efficacy of SHRss/siRNA complexes was
3
evaluated with Luc-Hela cells and mCherry-HEK293 cells, respectively. For luciferase
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expression silencing assay, Luc-Hela cells were seeded onto 24-well plates at a density of 6 ×
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104 cells per well and incubated for 24 h. After replacement of the culture medium by
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serum-free DMEM, cells were added with SHRss/siLuc complexes and incubated at 37°C.
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LF2000/siLuc, SHR/siLuc, HRss2/siLuc and SRss2/siLuc complexes were used as controls.
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After 3 h incubation, the medium was replaced by fresh culture medium and incubated for
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another 24 h. Before luciferase assay, cells were washed with PBS and lysated in 200 µL of
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lysis buffer for 0.5 h. Then the lysates were centrifuged at 5000 rpm for 3 min and the
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supernatant was collected. The substrate (100 µl) was added to 20 µl of cell lysates and the
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samples were analyzed by luminometer (Promega, USA).
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For mCherry expression silencing assay, mCherry-HEK293 cells were seeded onto 24-well
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plates at a density of 1× 105 cells per well and incubated for 24 h. After replacement of the
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culture medium by serum-free DMEM, cells were added with SHRss2/simCh, SHR/ simCh
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and LF2000/ simCh complexes for 3 h respectively, then medium was replaced and cells were
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incubated with culture medium for another 24 h before analysis. The cells were then analyzed
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using a flow cytometer after trypsinization or imaged with a CLSM to obtain RFP signals of
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each sample after paraformaldehyde fixation. The untreated mCherry-HEK293 cells were used
20
as the control.
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CLSM observation. Luc-Hela cells were seeded onto glass-bottom 24-well plates at a
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density of 5.0 × 104 cells per well and incubated for 24 h. The culture medium was replaced
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with fresh medium containing SHRss/Cy3-siRNA, LF2000/Cy3-siRNA and SHR/Cy3-siRNA 10 ACS Paragon Plus Environment
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complexes, respectively. After incubation for 3 h, the medium was replaced and the culture was
2
expanded. At different times after transfection, the cells were fixed using 4% paraformaldehyde
3
and treated with DAPI for nucleus staining. Further, the cells were washed, sealed with
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mounting medium, and imaged using a CLSM.
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Cytotoxicity assay. The cytotoxicity of SHRss on Luc-Hela and mCherry-HEK293 cells
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under transfection were evaluated by Cell Counting Kit-8 (CCK-8) assay. Cells (1.5×104 of
7
Luc-Hela cells and 4×104 of mCherry-HEK293 per well) in 100 µL of DMEM-containing 10%
8
FBS were seeded into 96-well plates and incubated for 24 h. Then cells were transfected as
9
described above with SHRss/siLuc complexes at N/P=10, which was the optimum N/P ratio for
10
transfection. After transfection for 3 h, the cells were washed with PBS and cultured in 100 µL
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DMEM-containing 10% FBS. Then after 24 h incubation, 10 µL of CCK-8 solution was added
12
to the culture medium and incubated for another 1.5 h. The absorbance of each well was
13
measured by a microplate reader (Thermo, IL, USA) at 450 nm. Cell viability was expressed as
14
a percentage relative to the absorbance of the untreated cells.
15
Cytotoxicity of blank polymers was taken as follows. Luc-Hela cells were seeded and
16
incubated as mentioned above. Then cells were incubated with culture medium containing
17
different concentrations of polymers (1-200 µg/mL) for 24 h, and CCK-8 assay was used to
18
analyze dose-dependent cytotoxicity of polymers. Here SHR and BPEI (25 kDa) were used as
19
controls.
20
In vivo distribution and gene silencing efficiency. All of the animal studies were
21
investigated using the xenograft tumor model. Female Balb/c nude mice 4-weeks old (~ 17 g)
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(Shanghai Experimental Animal Center of Chinese Academic of Sciences, Shanghai, China)
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were subcutaneously injected with 5.0×106 Luc-Hela cells. Two weeks after inoculation, the 11 ACS Paragon Plus Environment
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mice were randomly divided into 2 groups. For tissue distribution study, naked Cy5-siRNA and
2
SHRss2/Cy5-siRNA complexes (N/P=10) were injected via vein tail respectively at a single
3
siRNA dose of 1.5 mg/kg (300 µL solution). After 4 h injection, the mice were anaesthetized
4
and then the images were recorded by Xenogen IVIS-200 imaging system equipped with a
5
CCD camera (Perkin Elmer Inc., MA, USA) at excitation of 640 nm. The mean fluorescence
6
intensity of tumor site was calculated by the software that ships with Xenogen IVIS-200
7
system.
8
For in vivo gene silencing assay, SHRss2/siLuc and SHRss2/NC-siRNA complexes were
9
injected via vein tail respectively at a siRNA dose of 1.5 mg/kg everyday for 3 days. Before the
10
injection and 24 h after the final injection, 150 mg/kg potassium D-luciferin was injected
11
intraperitoneally and the luminescent images were recorded using IVIS imaging system.
12
Further, the mice were sacrificed and the tumors were homogenized in lysis buffer (10 ml lysis
13
buffer per gram) followed by centrifugation at 10,000 g for 5 min. The luciferase activity was
14
determined as described previously. All procedures were performed in accordance with the
15
guideline of the Committee on Animals of the Second Military Medical University (Shanghai,
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China).
17
Statistical analysis. Data were shown as the mean ± SD. One-way analyses of variance
18
(ANOVA) were conducted. P-value < 0.05 was considered to be statistically significant.
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RESULTS AND DISCUSSION
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Polymer synthesis and characterization. The peptide H3CR5C (HR) was synthesized with
21
high purity ( > 95%) and precise molecular weight (1416.65). Then the stearyl moiety was
22
grafted onto the N-terminal of HR with a precise molecular weight of SHR of 1683.14 using
23
the solid-phase peptide synthesis method (Figure 1A). Figure 1B shows 1H-NMR results of 12 ACS Paragon Plus Environment
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SHRss2, which indicates that peaks at δ0.72-1.34 and δ2.08 were from the proton of stearyl
2
moiety (signal a, b, c, and f, respectively). The peak at δ1.54 and the double peak at δ1.66 and
3
δ1.72 were attributed to -CH2- close to the tertiary carbon in arginine and histidine (signal e).
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The peaks at δ2.80 and δ2.85 were from -CH2- of cysteine (signal g). Signal d (δ1.54) and h
5
(δ3.10) were attributed to the rest of -CH2- in arginine. The peaks at δ4.23-4.62 (signal i) were
6
due to protons of tertiary carbon in the polymer. The peak at δ7.17 (signal j) and δ8.54 (signal
7
k) were from protons of imidazole in histidine.
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The mass-average molecular weight (MW) of polymers were measured by GPC analysis.
9
As shown in Table 1, as mole ratio of SHR to cysteine increased, the MWs of SHRss also
10
increased. The 1H-NMR and GPC data demonstrated that the SHRss were synthesized
11
successfully. The proposed synthetic procedure could produce low- or high-MW polymers
12
depending on the mole ratio of SHR and cysteine. Low-concentration H2O2 was used to as a
13
oxidizing agent to form disulfide bonds in a near-neutral pH solution and introduced cysteine,
14
which contains single sulfhydryl group to control the degree of polymerization. Compared with
15
DMSO, iodine and other oxidizing agents, low-concentration H2O2 makes the processes of
16
cross link and after-treatment more simple.
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Table 1 Synthetic conditions and molecular weight of SHRss
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Mole ratios of Polymers
SHR(mg)
Cysteine(mg)
Mw(kDa)a
SHR per Cys
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a
SHRss1
50
1.44
2.5
8.9
SHRss2
50
0.72
5.0
15
SHRss3
50
0.36
10.0
29
SHRss4
50
0.24
15.0
39
obtained by gel permeation chromatography (GPC) analysis 13 ACS Paragon Plus Environment
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Figure 1. Synthesis and 1H-NMR determination of SHRss. (A) Synthesis of the disulfide
3
cross-linked stearyl-peptide (SHRss), Arg: Arginine, His: Histidine, Cys: Cysteine. (B)
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1
5
cysteine (SHRss2).
H-NMR spectra of disulfide bonds linked SHR with blocking oxidative polymerization by 20%
6
Nanoparticle characterization and siRNA condensation. Zeta potential and particle size
7
of SHRss/siRNA are closely related to the N/P ratio. When the N/P ratio was less than 2.5,
8
most of complexes carried a negative surface charge (Figure 2A). As N/P ratio increases, the
9
surface charge of complexes reached a plateau (25–40 mV, Figure 2A), and the smallest
10
particle size was obtained at N/P=10 (Figure 2B), indicating that SHRss appeared on the
11
surface of complexes at this N/P ratio. The degree of polymerization of SHRss influenced the
12
zeta potential and particle size. The complexes compose of high molecular weight polymers
13
showed a higher zeta potential than monomer at the same N/P ratio except SHRss2 (Figure 2A).
14
Compared with the SHR/siRNA complexes, smaller vesicles (150–300 nm) were formed by
15
most SHRss/siRNA complexes when the N/P ratio was >5 (Figure 2B). Of all the polymers, the 14 ACS Paragon Plus Environment
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particle size and zeta potential of SHRss2/siRNA were more appropriate (Figure 2). The
2
average mean diameter and zeta potential of SHRss2/siRNA complexes were 163 ± 9.0 nm and
3
24.6 ± 3.7 mV, respectively. TEM analyses also showed that the SHRss2/siRNA complexes
4
formed a compact nanostructure, which was typically < 200 nm at an N/P ratio of 10 (Figure
5
2C). The compact structure with the appropriate size and zeta potential of the SHRss/siRNA
6
complexes was expected to exhibit efficient cellular uptake 32.
7 8
Figure 2. Characterization of
SHRss/siRNA complexes. (A) Zeta potential of various
9
SHRss/siRNA complexes at different N/P ratios. (B) Particle size of various SHRss/siRNA
10
complexes at different N/P ratios. (C) A typical DLS size profile on the distribution and a
11
representative transmission electron microscope (TEM) image of SHRss2/siRNA complexes
12
after formulation in water at N/P = 10. (D) Surface zeta potential distribution of
13
SHRss2/siRNA at N/P=10 measured by DLS. Data are expressed as mean ± SD (n= 3).
14
**p < 0.01, significant difference between these two groups. 15 ACS Paragon Plus Environment
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We determined the condensation of siRNA with SHRss using agarose gel electrophoresis
3
and EtBr Green exclusion assay. After SHR was cross linked by disulfide bonds, the binding
4
ability to siRNA of SHRss improved. The complete retardation of siRNA was achieved at an
5
N/P ratio of 5 for SHRss2/siRNA complexes, while siRNA was totally retarded at an N/P ratio
6
of 7.5 for SHR/siRNA complexes (Figure 3A). The same trend was detected through EtBr
7
Green exclusion assay, and the siRNA binding ability of SHRss2 was much stronger than that
8
of SHR (Figure 3B). To verify if polymerization could influence the binding ability to siRNA,
9
DTT was added to break the disulfide bonds 33. The depolymerized SHRss2 by DTT showed a
10
weaker siRNA binding ability (Figure 3A). Based on the electrophoresis result at presence of
11
DTT, We hypothesized that the disulfide bond reduction in reducing environments of
12
cytoplasm could weaken the binding affinity between siRNA and SHRss. Although disulfide
13
bonds formed by oxidation of sulfhydryl groups are relatively stable in the extracellular space,
14
the stability is easily reversed under reductive intracellular homeostasis 4. In particular,
15
disulfide cross-linked peptides can provide great advances in terms of enhanced complexes
16
formation and cytoplasm-sensitive dissociation.
17
Stability of siRNA in serum is important for therapeutic RNAi application. The effect of
18
siRNA protection ability of SHRss2 were investigated in 50% FBS. It is found that the naked
19
siRNA was completely degraded in 50% FBS between 8-12 h, whereas SHRss2 could protect
20
the siRNA from complete degradation for up to 48 h (Figure 3C). The protection time of
21
SHRss2 for siRNA degradation doubled as compared with SHR (Figure 3C). Intracellular
22
transfection of siRNA may come up several hours after systemic administration, hence it seems
23
to be sufficient for the increased stability of SHRss2/siRNA complexes. 16 ACS Paragon Plus Environment
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Figure 3. Agarose gel electrophoresis and EtBr exclusion assay. (A) siRNA condensation
3
ability with SHRss2 and DTT-triggering siRNA release from SHRss2/siRNA complexes. (B)
4
EtBr exclusion assay of SHRss2/siRNA complexes at different N/P ratios. (C) Stability test of
5
SHRss2/siRNA complexes in 50% FBS conditions at 37°C. Data are expressed as mean ± SD
6
(n = 3).
7 8
Cellular uptake and transmembrane mechanism. Cy3-labeled siRNA was used as an
9
indicator to determine the cellular uptake of complexes in Luc-Hela cells. Flow cytometry data
10
shows that the cellular uptake efficiency of SHR or SHRss was much higher than LF2000 in
11
Luc-Hela cells (Figure 4). Further, it is shown that an increase of disulfide bonds formation
12
enhanced cellular uptake of siRNA in Luc-Hela cells. Importantly, the ratio of Cy3-siRNA
13
positive cells was nearly 100% of the cell population mediated by SHRss1-4, while LF2000
14
was only 49.3% and SHR was 89.7%. According to the previous reports
15
introduction of lipophilic stearyl moiety greatly improved the cellular uptake of
16
polymer/siRNA complexes. The polymerization through disulfide bonds also contributed to the
17
abundant uptake of siRNA. The cellular uptake assay helps to clarify the connection between 17 ACS Paragon Plus Environment
34, 35
, we infered that
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the amount of the internalized siRNA and their gene silencing activity. The significantly
2
promoted uptake of SHRss/siRNA complexes implies potentially high RNA interference
3
efficiency. Interestingly, the cellular uptake of siRNA mediated by SHRss1- SHRss4 showed
4
no difference, although the gene knockdown by SHRss/siRNA was not equal to some extent.
5 6
Figure 4. Flow cytometry data of SHRss/Cy3-siRNA uptake. Cellular uptake analysis of
7
various Cy3-siRNA complexes (N/P=10) (A) and the ratio of Cy3-siRNA positive cells (B)
8
after 3 h transfection in Luc-Hela cells (final Cy3-siRNA concentration, 75 nM). The untreated
9
cells were used as the control. Data are expressed as mean ± SD (n= 3) . *p
Article
A biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo Zongguang Tai, Xiaoyu Wang, Jing Tian, Yuan Gao, Lijuan Zhang, Chong Yao, Xin Wu, Wei Zhang, Quangang Zhu, and Shen Gao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501777a • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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A biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo
4
Zongguang Taia‡, Xiaoyu Wanga‡, Jing Tiana‡, Yuan Gaoa, Lijuan Zhanga, Chong Yaoa, Xin
5
Wub , Wei Zhanga, Quangang Zhu a, c,**, Shen Gaoa,*
6
a
7
Shanghai 200433, China.
8
b
9
University School of Medicine, Shanghai 200080, China
1 2
Department of Pharmaceutics, Changhai Hospital, Second Military Medical University,
Department of Pharmaceutics, Shanghai First People’s Hospital, Shanghai Jiaotong
10
c
11
Medicine，Shanghai University of Traditional Chinese Medicine，Shanghai 200437, China
12
KEWORDS: arginine; histidine; stearyl; disulfide bond; peptide; siRNA delivery
Department of Pharmacy, Yueyang Hospital of Integrated Traditional Chinese and Western
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ABSTRACT
2
RNA-based delivery system for cancer therapy remains a challenge. In this study, a
3
stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine and stearyl moiety.
4
Further, the stearyl-peptides were cross linked by disulfide bonds to obtain cross-linked
5
polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4).
6
The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a
7
hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and
8
confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation
9
behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in
10
cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with
11
SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on
12
Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay
13
rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker
14
expression of red fluorescence protein was also observed on SHRss2/simCh-treated
15
mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce
16
cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies
17
demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies
18
indicated that the SHRss are promising and efficient non-viral vectors for siRNA delivery.
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INTRODUCTION
2
Small interfering RNA (siRNA) can specifically inhibit gene expression and are being
3
researched in many disease areas, especially in cancer, inflammation, diabetes, and
4
neurodegenerative therapy
5
potential to inhibit gene expression and evaluation in human therapeutic application of RNAi
6
5-7
7
siRNA during the therapeutic procedure 9, 10. Therefore, developing a vector which has siRNA
8
binding capacity, improved serum stability, high cell uptake, endosomal escape, as well as
9
siRNA dissociation in the cytoplasm and tissue penetration for systemic delivery is prime
10
1-4
. Various studies show that RNA interference (RNAi) has a
. However, there are still some challenges
8
such as instability and poor cellular uptake of
importance 11.
11
It was reported that cell-penetrating peptides (CPPs, also called peptide transduction
12
domain) have the ability to enhance intracellular uptake of macromolecules including nucleic
13
acids in a low-toxic manner
14
which are considered as critical residues for intracellular uptake 14. Arginine is cationic amino
15
acid, anionic siRNA can form stable complexes with arginine through strong electrostatic
16
interactions, leading to efficient cellular uptake of siRNA complexes 15. In addition, due to the
17
formation of bidentate ionic interactions with cell surface proteoglycans, arginine residues can
18
result in a close association with the cell membrane
19
arginine-rich peptides or polymers resulting in cell penetration and gene delivery
20
used arginine to package siRNA into electropositive nanoparticles and mediate cellular uptake
21
of siRNA.
12, 13
. Some of the CPPs are rich in cationic arginine residues,
16
. Several studies have focused on 17-20
. So we
22
Intracellular uptake is just one of the obstacles for RNAi. In cells, siRNA complexes should
23
be able to escape from the endosomal pathway and siRNA should dissociate from the 3 ACS Paragon Plus Environment
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carrier/siRNA complexes. Histidines have a potent proton-accepting moiety, so the carriers
2
containing histidines are possible to allow carrier/siRNA complexes to escape from endosomal
3
compartments by raising the pH of endosomes, leading to osmotic swelling and bursting
4
Thus, the imidazole groups in histidine provide a “proton sponge effect” to enhance
5
endosomal/lysosomal escape.
21-23
.
6
Furthermore, it was reported that stearylation improved intracellular uptake and endosomal
7
escape of siRNA carriers 24-27. Due to the lipophilic property, a stearyl moiety can increase the
8
interaction of the complexes with the membrane of the cell and endosome. Therefore a stearyl
9
moiety was introduced as a hydrophobic moiety to enhance cellular uptake and endosomal
10
escape of siRNA complexes.
11
Disulfide cross-linked polymers can achieve efficient siRNA delivery with a low
12
cytotoxicity 28, 29. They can rapidly degrade in the reductive intracellular cytoplasm 30, leading
13
to a rapid siRNA release into the cytoplasm 31. Moreover, the disulfide cross-linked polymers
14
have higher charge density than the non cross-linked polymer, which can enhance the siRNA
15
package capacity as well as intracellular uptake.
16
Due to the special properties of arginine, histidine and stearyl, we combined the strategies
17
and developed a new disulfide cross-linked stearyl-peptide containing arginine and histidine
18
(SHRss) (Scheme 1 and Figure 1). The SHRss can effectively condense siRNA into nano-scale
19
complexes. The chemical structure of SHRss and characteristics of SHRss/siRNA complexes
20
were determined. Furthermore, the cell viability as well as the gene interference efficiency of
21
SHRss/siRNA on Luciferase-expressing Hela cells and mCherry-expressing HEK293 cells was
22
examined. In addition, the gene silencing efficiency in vivo was evaluated.
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1 2
Scheme. 1. Disulfide cross-linked SHRss tends to self-assemble into cationic complexes and
3
further formed nanoparticles with negatively charged siRNA based on electrostatic interactions.
4
After cellular uptake by endocytosis pathway, the SHRss/siRNA complexes realize endosomal
5
escape dependent on “proton sponge effect” mediated by histidine and membrane penetrating
6
capability mediated by stearyl moiety, then disulfide bonds are cleaved in the reductive
7
cytoplasm, and siRNA are released into the cytoplasm after dissociation of SHRss/siRNA
8
complexes. Subsequently, siRNA develops into RNA-induced silencing complex (RISC) and
9
guides endonucleolytic cleavage of the target mRNA.
10
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EXPERIMENTAL SECTION
2
Materials. L-histidine, L-arginine, L-cysteine, stearic acid, and 30% H2O2 solution were
3
purchased from Sangon Biotech (Shanghai, China). Ethidium bromide (EtBr), dithiothreitol
4
(DTT), 4',6-diamidino-2-phenylindole (DAPI), chlorpromazine hydrochloride and amiloride
5
hydrochloride were purchased from Sigma Aldrich (St. Louis, MO, USA). Filipin Ⅲ was
6
purchased from Cayman Chemical (Michigan, USA). Passive Lysis 5× Buffer and luciferase
7
assay kit were obtained from Promega (Wisconsin, USA). Lipofectamine 2000 (LF2000),
8
ZeocinTM and puromycin were purchased from Invitrogen (Carlsbad, CA, USA); Dulbecco’s
9
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin
10
solution (5 kU/mL) were from Life Technologies (Grand Island, USA). GelredTM was obtained
11
from biotium (CA, USA). Enhanced BCA Protein Assay Kit was purchased from Beyotime
12
(Nanjing, China). Negative Control siRNA (NC-siRNA, sense: 5’-UUC UCC GAA CGU GUC
13
ACG UTT-3’, antisense: 5’-ACG UGA CAC GUU CGG AGA ATT-3’), luciferase targeting
14
siRNA (siLuc, sense: 5’-CUUACGCUGAGUACUUCGATT-3’, antisense: 5’-UCG AAG UAC
15
UCA GCG UAA GTT-3’), mCherry targeting siRNA (simCh, sense: 5’-CAU GGC CAU CAU
16
CAA GGA GTT-3’, antisense:5’-CUC CUU GAU GAU GGC CAU GTT-3’) and Cy-3 labeled
17
NC-siRNA (Cy3-siRNA) were synthesized by GenePharma Co. Ltd. (Shanghai, China). Cy-5
18
labeled NC-siRNA (Cy5-siRNA) was from RiboBio Co. Ltd. (Guangzhou, China).
19
Cell lines and Cell culture. Luciferase-expressing (GL3) Hela cells (Luc-Hela cells) were
20
obtained from SBO Medical Biotechnology (Shanghai, China). mCherry-expressing HEK293
21
cells (mCherry-HEK293 cells), which express red fluorescence protein, were kindly donated
22
from Dr. Fang (Department of Urology of Changhai Hospital in Shanghai, China). All cells 6 ACS Paragon Plus Environment
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were grown in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin and
2
streptomycin). To maintain luciferase expression, 200 µg/mL ZeocinTM was added in Luc-Hela
3
cells. 5 µg/mL puromycin was added in mCherry-HEK293 cells to maintain mCherry
4
expression.
5
Synthesis and Characterization of polymers. Peptide H3CR5C (HR) was synthesized using
6
the F-moc-solid-phase peptide synthesis method. To increase membrane affinity, we introduced
7
stearyl moiety to the N-terminal of HR using the same synthesis method of HR, and the
8
product was purified by reverse-phase HPLC. The molecular weight of stearyl-HR (SHR) was
9
determined by MALDI-TOF MS (Bruker Daltonics, Germany). The single sulfydryl of
10
L-Cysteine can block the formation of disulfide bond and control the the degree of
11
polymerization through disulfide bond. To obtain disulfide cross-linked polypeptides (SHRss)
12
with different molecular weight, 50 mg SHR was mixed with different mole ratios of
13
L-Cysteine hydrochloride monohydrate (Table 1) and dissolved in 9.5 mL distilled water (pH
14
7.0), then 0.5 mL 10% H2O2 aqueous solution was added into the mixed solutions under
15
stirring. After 12 h reaction, the products were dialyzed in distilled water for 12 h using a
16
dialysis membrane (MWCO 1000) and lyophilized. The synthesized polymers were
17
characterized by 1H-NMR at 600 MHz (Varian, CA, USA) and gel permeation chromatography
18
(GPC) (HLC-8220, TOSOH Corporation, Tokyo, Japan). To obtain controls vector, the
19
polypeptides omitting stearyl chain or histidine residues (named HRss2 and SRss2 respectively)
20
were synthesized by crossing link H3CR5C and stearyl-CR5C according to the method above.
21
Preparation and characterization of complexes. SHRss was complexed with siRNA in
22
PBS (50 µL per 0.5 µg of siRNA, pH 6.0). The N/P ratio of SHRss to siRNA was calculated as
23
follow. 1.06 µg SHRss per 1µg siRNA was equal to N/P=1.The samples were vortexed and 7 ACS Paragon Plus Environment
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1
incubated for 30 min at room temperature before use. LF2000 was complexed with siRNA in
2
OptiMEM according to the operation manual. The particle size and zeta potential of the
3
SHRss/siRNA complexes were determined using a Zetasizer Nano ZS (Malvern, Westborough,
4
MA, USA). The morphology of the SHRss2/siRNA complexes at N/P=10 was examined by
5
transmission electron microscopy (TEM). Briefly, one drop of a solution containing
6
SHRss2/siRNA complexes was placed on a 200-mesh copper grid coated with carbon film. The
7
excess solution was removed by a filter paper. Images were recorded under 75 kV acceleration
8
voltage.
9
Agarose gel electrophoresis. The ability of polymers to condense siRNA was determined
10
by agarose gel electrophoresis. The carrier/siRNA complexes were prepared at N/P ratios of 1–
11
10. After incubation for 30 min at room temperature, samples containing 1 µg siRNA were
12
electrophoresed by a 1.0% agarose gel containing GelredTM at 100 V for 15 min in
13
Tris-acetate-EDTA (TAE) buffer. To investigate the influence of polymerization on siRNA
14
binding ability as well as the polymer behaviors under reductive environments, 100-fold DTT
15
(mole ratio to SHR monomer) was added to solutions of carrier/siRNA complexes. After
16
treatment for 1 h, samples containing 1 µg siRNA were electrophoresed as mentioned. The
17
location of nucleic acid bands was visualized under UV light.
18
Ethidium bromide exclusion assay. Ethidium bromide (EtBr) exclusion assay was
19
performed to examine the siRNA condensation efficiency of polymers. Briefly, 4 µg siRNA
20
was complexed with polymers with different N/P ratio. The complexes were diluted to a final
21
volume of 180 µL using PBS. After a 30-min incubation at room temperature, the diluted
22
complexes were mixed with 20 µL of EtBr (50 µg/mL) solution thoroughly. Fluorescence
23
intensity of each sample was measured using a fluorescence spectrophotometer (Promega, 8 ACS Paragon Plus Environment
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1
USA) with excitation at 525 nm and emission at 610 nm.
2
Serum stability. The SHRss2/siRNA and SHR/siRNA complexes were incubated in 50%
3
FBS solution at a siRNA concentration of 14 µg/mL (1 µM). Naked siRNA was used as control.
4
The mixtures were incubated at 37°C for different time intervals. To release the loaded siRNA,
5
the mixtures were treated with 10 µL heparin solution (500 U/mL) and subsequently
6
electrophoresed by a 1.0% Agarose gel containing GelredTM at 100 V for 15 min in TAE buffer.
7
The undegraded siRNA were visualized under UV light.
8
Cellular uptake assay. The siRNA uptake by Luc-Hela cells was analyzed using flow
9
cytometry. Luc-Hela cells were seeded onto 12-well plates at 2 × 105 cells per well and
10
incubated for 24 h. After replacing the culture medium, SHRss/Cy3-siRNA complexes,
11
LF2000/Cy3-siRNA, SHR/Cy3-siRNA were added into Luc-Hela cells with a final Cy3-siRNA
12
concentration of 75 nM, respectively. After incubation for 3 h, the cells were washed,
13
trypsinized, centrifuged, and resuspended in PBS. The cells were analyzed on a FACScan flow
14
cytometer (Becton Dickinson, San Jose, CA, USA).
15
Transmembrane mechanism study. Luc-Hela cells were seeded onto 12-well plates at
16
2×105 cells per well and incubated for 24 h. To probe the internalization mechanism of the
17
complexes, Luc-Hela cells were pretreated with various endocytic inhibitors including
18
chlorpromazine (30µM), filipin Ⅲ (1µg/ml) and amiloride (30 µM) for 1 h. Then the
19
SHRss2/Cy3-siRNA complexes were added into culture medium for an additional 2 h
20
incubation. After incubation, cells were washed three times with PBS followed by incubation
21
with 400 ml lysis buffer for 0.5 h. The lysates were centrifuged at 5000 rpm for 3 min and the
22
supernatant was analyzed by fluorescence spectrophotometer with excitation at 525 nm and
23
emission at 610 nm. The cellular internalization of SHRss2/Cy3-siRNA was also visualized 9 ACS Paragon Plus Environment
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under a confocal laser scanning microscope (CLSM) (Olympus, Japan).
2
Gene silencing assay. In vitro gene silencing efficacy of SHRss/siRNA complexes was
3
evaluated with Luc-Hela cells and mCherry-HEK293 cells, respectively. For luciferase
4
expression silencing assay, Luc-Hela cells were seeded onto 24-well plates at a density of 6 ×
5
104 cells per well and incubated for 24 h. After replacement of the culture medium by
6
serum-free DMEM, cells were added with SHRss/siLuc complexes and incubated at 37°C.
7
LF2000/siLuc, SHR/siLuc, HRss2/siLuc and SRss2/siLuc complexes were used as controls.
8
After 3 h incubation, the medium was replaced by fresh culture medium and incubated for
9
another 24 h. Before luciferase assay, cells were washed with PBS and lysated in 200 µL of
10
lysis buffer for 0.5 h. Then the lysates were centrifuged at 5000 rpm for 3 min and the
11
supernatant was collected. The substrate (100 µl) was added to 20 µl of cell lysates and the
12
samples were analyzed by luminometer (Promega, USA).
13
For mCherry expression silencing assay, mCherry-HEK293 cells were seeded onto 24-well
14
plates at a density of 1× 105 cells per well and incubated for 24 h. After replacement of the
15
culture medium by serum-free DMEM, cells were added with SHRss2/simCh, SHR/ simCh
16
and LF2000/ simCh complexes for 3 h respectively, then medium was replaced and cells were
17
incubated with culture medium for another 24 h before analysis. The cells were then analyzed
18
using a flow cytometer after trypsinization or imaged with a CLSM to obtain RFP signals of
19
each sample after paraformaldehyde fixation. The untreated mCherry-HEK293 cells were used
20
as the control.
21
CLSM observation. Luc-Hela cells were seeded onto glass-bottom 24-well plates at a
22
density of 5.0 × 104 cells per well and incubated for 24 h. The culture medium was replaced
23
with fresh medium containing SHRss/Cy3-siRNA, LF2000/Cy3-siRNA and SHR/Cy3-siRNA 10 ACS Paragon Plus Environment
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complexes, respectively. After incubation for 3 h, the medium was replaced and the culture was
2
expanded. At different times after transfection, the cells were fixed using 4% paraformaldehyde
3
and treated with DAPI for nucleus staining. Further, the cells were washed, sealed with
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mounting medium, and imaged using a CLSM.
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Cytotoxicity assay. The cytotoxicity of SHRss on Luc-Hela and mCherry-HEK293 cells
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under transfection were evaluated by Cell Counting Kit-8 (CCK-8) assay. Cells (1.5×104 of
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Luc-Hela cells and 4×104 of mCherry-HEK293 per well) in 100 µL of DMEM-containing 10%
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FBS were seeded into 96-well plates and incubated for 24 h. Then cells were transfected as
9
described above with SHRss/siLuc complexes at N/P=10, which was the optimum N/P ratio for
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transfection. After transfection for 3 h, the cells were washed with PBS and cultured in 100 µL
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DMEM-containing 10% FBS. Then after 24 h incubation, 10 µL of CCK-8 solution was added
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to the culture medium and incubated for another 1.5 h. The absorbance of each well was
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measured by a microplate reader (Thermo, IL, USA) at 450 nm. Cell viability was expressed as
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a percentage relative to the absorbance of the untreated cells.
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Cytotoxicity of blank polymers was taken as follows. Luc-Hela cells were seeded and
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incubated as mentioned above. Then cells were incubated with culture medium containing
17
different concentrations of polymers (1-200 µg/mL) for 24 h, and CCK-8 assay was used to
18
analyze dose-dependent cytotoxicity of polymers. Here SHR and BPEI (25 kDa) were used as
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controls.
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In vivo distribution and gene silencing efficiency. All of the animal studies were
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investigated using the xenograft tumor model. Female Balb/c nude mice 4-weeks old (~ 17 g)
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(Shanghai Experimental Animal Center of Chinese Academic of Sciences, Shanghai, China)
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were subcutaneously injected with 5.0×106 Luc-Hela cells. Two weeks after inoculation, the 11 ACS Paragon Plus Environment
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mice were randomly divided into 2 groups. For tissue distribution study, naked Cy5-siRNA and
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SHRss2/Cy5-siRNA complexes (N/P=10) were injected via vein tail respectively at a single
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siRNA dose of 1.5 mg/kg (300 µL solution). After 4 h injection, the mice were anaesthetized
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and then the images were recorded by Xenogen IVIS-200 imaging system equipped with a
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CCD camera (Perkin Elmer Inc., MA, USA) at excitation of 640 nm. The mean fluorescence
6
intensity of tumor site was calculated by the software that ships with Xenogen IVIS-200
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system.
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For in vivo gene silencing assay, SHRss2/siLuc and SHRss2/NC-siRNA complexes were
9
injected via vein tail respectively at a siRNA dose of 1.5 mg/kg everyday for 3 days. Before the
10
injection and 24 h after the final injection, 150 mg/kg potassium D-luciferin was injected
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intraperitoneally and the luminescent images were recorded using IVIS imaging system.
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Further, the mice were sacrificed and the tumors were homogenized in lysis buffer (10 ml lysis
13
buffer per gram) followed by centrifugation at 10,000 g for 5 min. The luciferase activity was
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determined as described previously. All procedures were performed in accordance with the
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guideline of the Committee on Animals of the Second Military Medical University (Shanghai,
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China).
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Statistical analysis. Data were shown as the mean ± SD. One-way analyses of variance
18
(ANOVA) were conducted. P-value < 0.05 was considered to be statistically significant.
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RESULTS AND DISCUSSION
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Polymer synthesis and characterization. The peptide H3CR5C (HR) was synthesized with
21
high purity ( > 95%) and precise molecular weight (1416.65). Then the stearyl moiety was
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grafted onto the N-terminal of HR with a precise molecular weight of SHR of 1683.14 using
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the solid-phase peptide synthesis method (Figure 1A). Figure 1B shows 1H-NMR results of 12 ACS Paragon Plus Environment
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SHRss2, which indicates that peaks at δ0.72-1.34 and δ2.08 were from the proton of stearyl
2
moiety (signal a, b, c, and f, respectively). The peak at δ1.54 and the double peak at δ1.66 and
3
δ1.72 were attributed to -CH2- close to the tertiary carbon in arginine and histidine (signal e).
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The peaks at δ2.80 and δ2.85 were from -CH2- of cysteine (signal g). Signal d (δ1.54) and h
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(δ3.10) were attributed to the rest of -CH2- in arginine. The peaks at δ4.23-4.62 (signal i) were
6
due to protons of tertiary carbon in the polymer. The peak at δ7.17 (signal j) and δ8.54 (signal
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k) were from protons of imidazole in histidine.
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The mass-average molecular weight (MW) of polymers were measured by GPC analysis.
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As shown in Table 1, as mole ratio of SHR to cysteine increased, the MWs of SHRss also
10
increased. The 1H-NMR and GPC data demonstrated that the SHRss were synthesized
11
successfully. The proposed synthetic procedure could produce low- or high-MW polymers
12
depending on the mole ratio of SHR and cysteine. Low-concentration H2O2 was used to as a
13
oxidizing agent to form disulfide bonds in a near-neutral pH solution and introduced cysteine,
14
which contains single sulfhydryl group to control the degree of polymerization. Compared with
15
DMSO, iodine and other oxidizing agents, low-concentration H2O2 makes the processes of
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cross link and after-treatment more simple.
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Table 1 Synthetic conditions and molecular weight of SHRss
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Mole ratios of Polymers
SHR(mg)
Cysteine(mg)
Mw(kDa)a
SHR per Cys
19
a
SHRss1
50
1.44
2.5
8.9
SHRss2
50
0.72
5.0
15
SHRss3
50
0.36
10.0
29
SHRss4
50
0.24
15.0
39
obtained by gel permeation chromatography (GPC) analysis 13 ACS Paragon Plus Environment
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Figure 1. Synthesis and 1H-NMR determination of SHRss. (A) Synthesis of the disulfide
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cross-linked stearyl-peptide (SHRss), Arg: Arginine, His: Histidine, Cys: Cysteine. (B)
4
1
5
cysteine (SHRss2).
H-NMR spectra of disulfide bonds linked SHR with blocking oxidative polymerization by 20%
6
Nanoparticle characterization and siRNA condensation. Zeta potential and particle size
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of SHRss/siRNA are closely related to the N/P ratio. When the N/P ratio was less than 2.5,
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most of complexes carried a negative surface charge (Figure 2A). As N/P ratio increases, the
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surface charge of complexes reached a plateau (25–40 mV, Figure 2A), and the smallest
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particle size was obtained at N/P=10 (Figure 2B), indicating that SHRss appeared on the
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surface of complexes at this N/P ratio. The degree of polymerization of SHRss influenced the
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zeta potential and particle size. The complexes compose of high molecular weight polymers
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showed a higher zeta potential than monomer at the same N/P ratio except SHRss2 (Figure 2A).
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Compared with the SHR/siRNA complexes, smaller vesicles (150–300 nm) were formed by
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most SHRss/siRNA complexes when the N/P ratio was >5 (Figure 2B). Of all the polymers, the 14 ACS Paragon Plus Environment
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particle size and zeta potential of SHRss2/siRNA were more appropriate (Figure 2). The
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average mean diameter and zeta potential of SHRss2/siRNA complexes were 163 ± 9.0 nm and
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24.6 ± 3.7 mV, respectively. TEM analyses also showed that the SHRss2/siRNA complexes
4
formed a compact nanostructure, which was typically < 200 nm at an N/P ratio of 10 (Figure
5
2C). The compact structure with the appropriate size and zeta potential of the SHRss/siRNA
6
complexes was expected to exhibit efficient cellular uptake 32.
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Figure 2. Characterization of
SHRss/siRNA complexes. (A) Zeta potential of various
9
SHRss/siRNA complexes at different N/P ratios. (B) Particle size of various SHRss/siRNA
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complexes at different N/P ratios. (C) A typical DLS size profile on the distribution and a
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representative transmission electron microscope (TEM) image of SHRss2/siRNA complexes
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after formulation in water at N/P = 10. (D) Surface zeta potential distribution of
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SHRss2/siRNA at N/P=10 measured by DLS. Data are expressed as mean ± SD (n= 3).
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**p < 0.01, significant difference between these two groups. 15 ACS Paragon Plus Environment
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We determined the condensation of siRNA with SHRss using agarose gel electrophoresis
3
and EtBr Green exclusion assay. After SHR was cross linked by disulfide bonds, the binding
4
ability to siRNA of SHRss improved. The complete retardation of siRNA was achieved at an
5
N/P ratio of 5 for SHRss2/siRNA complexes, while siRNA was totally retarded at an N/P ratio
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of 7.5 for SHR/siRNA complexes (Figure 3A). The same trend was detected through EtBr
7
Green exclusion assay, and the siRNA binding ability of SHRss2 was much stronger than that
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of SHR (Figure 3B). To verify if polymerization could influence the binding ability to siRNA,
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DTT was added to break the disulfide bonds 33. The depolymerized SHRss2 by DTT showed a
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weaker siRNA binding ability (Figure 3A). Based on the electrophoresis result at presence of
11
DTT, We hypothesized that the disulfide bond reduction in reducing environments of
12
cytoplasm could weaken the binding affinity between siRNA and SHRss. Although disulfide
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bonds formed by oxidation of sulfhydryl groups are relatively stable in the extracellular space,
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the stability is easily reversed under reductive intracellular homeostasis 4. In particular,
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disulfide cross-linked peptides can provide great advances in terms of enhanced complexes
16
formation and cytoplasm-sensitive dissociation.
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Stability of siRNA in serum is important for therapeutic RNAi application. The effect of
18
siRNA protection ability of SHRss2 were investigated in 50% FBS. It is found that the naked
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siRNA was completely degraded in 50% FBS between 8-12 h, whereas SHRss2 could protect
20
the siRNA from complete degradation for up to 48 h (Figure 3C). The protection time of
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SHRss2 for siRNA degradation doubled as compared with SHR (Figure 3C). Intracellular
22
transfection of siRNA may come up several hours after systemic administration, hence it seems
23
to be sufficient for the increased stability of SHRss2/siRNA complexes. 16 ACS Paragon Plus Environment
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Figure 3. Agarose gel electrophoresis and EtBr exclusion assay. (A) siRNA condensation
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ability with SHRss2 and DTT-triggering siRNA release from SHRss2/siRNA complexes. (B)
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EtBr exclusion assay of SHRss2/siRNA complexes at different N/P ratios. (C) Stability test of
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SHRss2/siRNA complexes in 50% FBS conditions at 37°C. Data are expressed as mean ± SD
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(n = 3).
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Cellular uptake and transmembrane mechanism. Cy3-labeled siRNA was used as an
9
indicator to determine the cellular uptake of complexes in Luc-Hela cells. Flow cytometry data
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shows that the cellular uptake efficiency of SHR or SHRss was much higher than LF2000 in
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Luc-Hela cells (Figure 4). Further, it is shown that an increase of disulfide bonds formation
12
enhanced cellular uptake of siRNA in Luc-Hela cells. Importantly, the ratio of Cy3-siRNA
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positive cells was nearly 100% of the cell population mediated by SHRss1-4, while LF2000
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was only 49.3% and SHR was 89.7%. According to the previous reports
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introduction of lipophilic stearyl moiety greatly improved the cellular uptake of
16
polymer/siRNA complexes. The polymerization through disulfide bonds also contributed to the
17
abundant uptake of siRNA. The cellular uptake assay helps to clarify the connection between 17 ACS Paragon Plus Environment
34, 35
, we infered that
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the amount of the internalized siRNA and their gene silencing activity. The significantly
2
promoted uptake of SHRss/siRNA complexes implies potentially high RNA interference
3
efficiency. Interestingly, the cellular uptake of siRNA mediated by SHRss1- SHRss4 showed
4
no difference, although the gene knockdown by SHRss/siRNA was not equal to some extent.
5 6
Figure 4. Flow cytometry data of SHRss/Cy3-siRNA uptake. Cellular uptake analysis of
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various Cy3-siRNA complexes (N/P=10) (A) and the ratio of Cy3-siRNA positive cells (B)
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after 3 h transfection in Luc-Hela cells (final Cy3-siRNA concentration, 75 nM). The untreated
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cells were used as the control. Data are expressed as mean ± SD (n= 3) . *p
