Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate

Accepted Manuscript Title: Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate Authors: Junxiao Ye, Xing Pei, Hui Cui, Zhili Yu, Hyukjin Lee, Jianxin Wang, Xu Wang, Lu Sun, Huining He, Victor C. Yang PII: DOI: Reference:

S1226-086X(18)30063-7 https://doi.org/10.1016/j.jiec.2018.02.005 JIEC 3865

To appear in: Received date: Revised date: Accepted date:

2-1-2018 31-1-2018 2-2-2018

Please cite this article as: Junxiao Ye, Xing Pei, Hui Cui, Zhili Yu, Hyukjin Lee, Jianxin Wang, Xu Wang, Lu Sun, Huining He, Victor C.Yang, Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate, Journal of Industrial and Engineering Chemistry https://doi.org/10.1016/j.jiec.2018.02.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA Conjugate Junxiao Yea,b, 1, Xing Peia,1, Hui Cuia, Zhili Yua, Hyukjin Leec, Jianxin Wangd, Xu Wanga, Lu Suna, *, Huining Hea,*, and Victor C. Yanga,e

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a. Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China; b. College of Pharmacy, Tsinghua University, Beijing 100084, China c. College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 13760, Republic of Korea d. Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai , China e. Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA. Corresponding author. E-mail address: [email protected] [email protected]

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Theses authors contributed equally to this work.

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Graphical abstract

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Abstract: The covalent attachment of CPPs to siRNA molecules offers great potential for CPP-mediated siRNA delivery. We recently reported a concise and high-yield synthesis strategy of the cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugate. Herein, cell uptake mechanism and cellular toxicity studies of this

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conjugate were performed to evaluate the potential of LMWP-siRNA conjugate for clinical translation. Cellular uptake mechanism study indicated that the conjugate

could be taken up by cells via multiple pathways, including direct penetration of the

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plasma membrane and clathrin- and caveolae-independent endocytosis. In vitro cytotoxicity study revealed that the conjugation promoted internalization in a

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low-toxic fashion.

Introduction

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Keywords: Cell penetrating peptide, siRNA delivery, Covalent CPP-siRNA chemical conjugate, Cell uptake mechanism, Cytotoxicity

RNA interference (RNAi) is a promising tool for the treatment of human diseases that

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cannot be cured by rational therapies. In principle, RNAi has the potential to provide personalized therapies for various diseases by modulating the expression of virtually

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all disease-relevant mRNAs. In view of the considerable capability in targeting almost all genes and cleaving mRNA efficiently and specifically, siRNA has been investigated as a novel class of therapeutic agents for the treatment of a wide range of

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disorders including infections [1-3], and cancers [4-7]. However, owing to their high molecule size and strong anionic charge of short interfering RNAs (siRNAs), they cannot pass through the highly regulated and restricted plasma membrane [8]. To overcome these problems, multiple delivery methodologies, have been being developed to circumvent this problem. Cell penetrating peptides (CPPs) have been used for the delivery of a wide range of 2

macromolecules including peptides, proteins and antisense oligonucleotides [9-12]. With the recent advancement and understanding of RNAi, CPPs offer an attractive means for the cellular uptake of double-stranded siRNAs to induce an RNAi response. Two major strategies in CPP-mediated siRNA delivery include noncovalent electrostatic complexing and covalent attachment [13], and the former is technically much simpler by condensing siRNAs into aggregates or nanoparticles with the dense

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cationic charge of CPPs to protect the siRNA from the RNase-rich in vivo environment as well as helping siRNA cross cell membranes. However, due to

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insufficient biodistribution, low transfection efficiency, rapid plasma clearance and

cellular toxicity, this strategy has limitations in in vivo application [14]. The other strategy that covalent linkage of CPPs to siRNAs to create small, monomeric

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CPP/siRNA molecules can bypass multiple in vivo complications shown for other

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methodologies utilized for nucleic acid delivery like cationic polymer condensation,

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liposome encapsulation etc. However, the ease that cationic peptides bind to and condense siRNA makes this approach difficult to utilize. Although these two

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approaches each have strengths and weaknesses, it had been demonstrated that even

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though electrostatic condensation of the polyanionic DNA or siRNA with the polycationic CPP was also capable of inducing reasonable intracellular uptake of these drugs via the mechanism of macropinocytosis [15-17], CPP-mediated cell

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translocation of such non-covalently linked complexes was far less effective than the covalent conjugate [2]. Hence, covalent attachment of CPPs to siRNA molecules

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offers the greater potential for CPP-mediated siRNA delivery, and it has been attracting a lot of interest to deal with technical difficulties of the Holy Grail of CPP-mediated siRNA delivery. Recently, our group presented a concise coupling

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strategy that is capable of permitting a high-yield synthesis of the cell-permeable, cytosol-dissociable CPP-siRNA covalent conjugate based on the dynamic motion of PEG. And the CPP used here was the low molecular-weight protamine (LMWP) peptide, a potent CPP that was developed in our own laboratories [18, 19]. Cell culture assessment demonstrates that this chemical conjugate yields by far the most effective intracellular siRNA delivery and its corresponded gene-silencing activities 3

[20]. Although the field of CPP has emerged as a possible future candidate for siRNA delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery [21]. And the potential for cytotoxicity was rarely reported in the references describing siRNA delivery by covalent conjugation to a CPP [13]. Herein, a thorough and crucial analysis of cellular toxicity as well as the

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cell uptake mechanism was performed to further evaluate the potential of LMWP-siRNA covalent conjugate as a platform technology to realize clinical

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translation of virtually all nucleic acid-based drugs including therapeutic genes.

Experimental

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Materials

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LMWP (VSRRRRGGRRRRRR) was prepared by enzymatic degradation according

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the reported protocol developed by our lab [18, 19]. The siRNA used in this study was purchased from Guangzhou Riobobio Co., Ltd. (Guangzhou, China). The sequences

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of siRNA were as follows: sense: 5’-CGUACGCGGAAUACUUCGAdTdT-3’ and antisense: 5’-UCGAAGUAUUCCGCGUACGdTdT-3’. The sense strand was

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modified with sulfhydryl group at 5’ end. Hetero-bifunctional Succinimidyl Carboxymethyl Ester PEG ortho-pyridyl disulfide derivatives (NHS-PEG-OPSS,

DNA

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MW=3500) was synthesized by JenKem Technology Co., Ltd. (Beijing, China). 20 bp Ladder,

Annexin

V-FITC

Apoptosis

Detection

Kit,

3-(4,

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5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and Polyetherimide (PEI, MW=1200) were purchased from Sigma (Saint Louis, Missouri, USA). GoodViewTM Nucleic Acid Stain was obtained from SBS Genetech Co., Ltd. (Beijing,

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China). Agrose B, Low EEO (Biotech Grade) was supplied by BBI Life Sciences Corporation (Shanghai, China). Trypan Blue Staining Cell Viability Assay Kit and Lipo6000TM Transfection Reagent were provided by Beyotime Biotechology (Haimen, China). LDH Cytotoxicity Assay Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reagents for cell culture were all supplied by Invitrogen (Carlsbad, CA, USA). Unless otherwise specified, chemicals used here 4

were purchased as analytical grade from Tianjin Guangfu Fine Chemical Research Institute without further purification.

Synthesis of the LMWP-siRNA chemical conjugate PEG derivatives NHS-PEG-OPSS was dissolved in anhydrous DMSO and added dropwise to LMWP in PBS solution (20 mM sodium phosphate, pH=6.9) gently

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mixed by low-speed vortex, with a final ratio 5:1 (mol/mol, PEG: LMWP). The resulting solution was shaking for 2 h at 37 °C, and further purified by an affinity

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Heparin sepharose chromatography (HiTrapTM 1 mL, GE Healthcare, Sweden) to

obtain the pegylated LMWP conjugate (LMWP-PEG-OPSS). The LMWP-PEG-OPSS was then added to the sulfhydryl modified siRNA drug at the 5’-end of the sense

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strand to yield the LMWP-PEG-S-S-siRNA conjugate (LMWP-siRNA).

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Ultimately, the LMWP-siRNA conjugate was purified by an anion exchange column

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(HiTrapTM DEAE FF 1 mL, GE Healthcare, Sweden) as reported by our group [20].

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Preparation of the other siRNA formulations

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The commonly used siRNA transfection agents PEI and lipid were used as comparative agents to evaluate the in vitro cytotoxicity of the LMWP-siRNA conjugate. The other siRNA formulations except the LMWP-siRNA conjugate, were

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prepared as follows: 2.5 µg siRNA was added to 5 µL Lipo6000TM according to the instructions to form the Lipo/RNA complex. The PEI/siRNA complex was produced

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by slowly mixing PEI with siRNA solution (nitrogen/phosphorous, N/P ratio=10:1). The other two LMWP/siRNA complexes prepared as the process of PEI/RNA

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complex at a ratio of 10:1 (N/P ratio) and 1:1 (mole ratio), respectively.

Cell culture MDA-MB-231 breast cancer cells donated by Prof. Xiaoyue Tan (Nankai University) were cultured in L15 medium with 10% (v/v) fetal calf serum, 100 µg /mL penicillin and 100 µg/mL streptomycin, and maintained in a humidified atmosphere at 37 °C of 5% CO2. 5

Cellular uptake studies MDA-MB-231 cells were plated into twenty four-well plates at a density of 8×104/well and incubated for 18 h, then treated with the 6-TAMRA labeled LMWP-siRNA conjugate at different doses (0.9, 1.8, 4.5, 9 µM) and analyzed by confocal microscopy or incubated with 4.5 µM LMWP-siRNA and analyzed by

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confocal microscopy at different time points.

A comparative cellular uptake study of different siRNA formulations with 4.5 µM

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siRNA was carried out by using six formulations: siRNA alone, physical mixture of LMWP and siRNA with a mole ratio of 1:1 (LMWP/siRNA I), physical mixture of LMWP and siRNA with a mole ratio of 42:1, i.e N/P ratio of 10:1 (LMWP/siRNA II),

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PEI and siRNA complex with a N/P ratio of 10:1 (PEI/siRNA), Lipo6000 and siRNA

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complex (Lipo/siRNA), and the LMWP-siRNA conjugate (Conjugate).

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To investigate the cellular uptake mechanism of LMWP-siRNA conjugate, cells were treated with different membrane entry inhibitors for 1 h. The doses and targets of

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inhibitors were as follows: chlorpromazine (10 µg/mL, inhibitor of clathrin-mediated

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endocytosis), methyl-β-cyclodextrin (M-β-CD, 5 mM, inhibitor of caveolae), dynasore (15 µM, inhibitor of dynamin) and amiloride (50 µM, inhibitor of macropinocytosis). After that, cells were incubated with the FITC-labeled

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LMWP-siRNA conjugate for 2 h. Subsequently, the treated cells were washed with pre-cooled PBS for 3 times. Ultimately, the cells were collected after digested with

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trypsin and the fluorescent intensity of FITC in the cells were analyzed by flow cytometry.

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In vitro cytotoxicity studies: Cell proliferation inhibition evaluated by MTT assay Cell proliferation inhibition was detected by MTT assay based on the cleavage of yellow tetrazolium salt MTT by metabolically active cells to form a purple formazan dye which was quantified by Microplate Reader (Thermo Scientific Multiskan FC, USA). Cells were seeded in 96-well plates with a density of 1×104/well and cultured 6

overnight, and the culture medium was replaced by fresh medium containing different siRNA formulations at the desired concentration (0.34, 0.68, 1.41, 2.81, 5.63, 11.25 µM). After 48 h incubation, the supernatants containing drug were aspirated out, cells were washed gently with PBS. Subsequently, after changing the culture medium, cells were treated with 20 µL fresh MTT solution (0.5 mg/mL) and incubated for 4 h. After discarding the supernatants, 200 µL DMSO was added to the cells, and the

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viability (i.e. IC50 values) was then calculated based on the results.

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absorbance of the samples at 570 nm was recorded by Microplate Reader. The cell

Cell apoptosis tested by Annexin V-FITC/PI double staining assay

The apoptosis rate of MDA-MB-231 cells caused by different transfection reagents

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was examined by Annexin V-FITC apoptosis detection kit. Cells were seeded in

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12-well plates at a density of 1×105/ well and incubated for 24 h. Then drugs were

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introduced to the cells pretreated by different siRNA formulations with different concentrations (0.45, 4.5, 13.5, 22.5 µM) and incubated for 12 h. The cells were

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digested by trypsin and collected. After washed 2 times with PBS, cells were

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resuspended in 1× Binding buffer according to the protocol of Annexin V-FITC apoptosis detection kit. Then cells were incubated for 10 minutes at room temperature after adding 5 µL Annexin V FITC and 10 µL Propidium Iodide Solution. Ultimately,

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the fluorescence of the cells was detected by a flow cytometer.

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Trypan blue staining cell mortality assay The cell mortality was measured by Trypan blue assay. MDA-MB-231 cells were plated in the 24-well plate with a density of 5×104 cells per well and cultured for 24 h.

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The siRNA formulations were added to cells with a concentration of 13.5 µM in culture medium. After 24 h, the supernatant was collected and adherent cells were digested by trypsin. Then the cells and supernatant were mixed and centrifuged for 1 min at 1000 g. After discarding the supernatant, cells were dispersed by resuspension solution, and then treated by the same volume of trypan blue solution. After staining for 3 min, the cells were analyzed by automated cell counter. The cell viability is 7

expressed as the percentage of the death cell number/the total cell number.

LDH release assay LDH release was tested by LDH cytotoxicity assay kit. MDA-MB-231 cells were seeded in 12-well plates at a density of 1×105/ well and incubated for 24 h. After that, cells were exposed to LMWP-siRNA conjugate and other siRNA formulations with

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different siRNA concentrations (0.45, 4.5, 13.5 µM). After 24 h, the supernatants were collected and centrifuged at 1000 rpm for 3 min to test. The LDH activity was

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detected in the 96-well plate according to the procedures of specification as following: First, 20 µL supernatants, 25 µL substrate buffer and 5 µL coenzyme were added to the 96-well plate in turn and mixed. After 15 min incubation at 37 °C, 25 µL 2,

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4-dinitrophenylhydrazine was added to the mixture and incubated continuously for

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another 15 min. Then 250 µL 0.4 mol/L NaOH solution was added, and the mixture

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was incubated at room temperature for 5 min. The absorbance at 450 nm reflecting the LDH activity was ultimately recorded by Microplate Reader. And the final LDH

Statistical analysis

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activity was normalized to total protein.

The statistical analysis was processed with GraphPad prism 5.01. Data was analyzed

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by analysis of variance (ANOVA) followed by Dunnett’s test and expressed as the mean ± SD (standard deviation), asterisks (*) represent statistically significant (*p <

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0.05, **p < 0.01, and ***p < 0.001).

Results and Discussion

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Principles in the design of cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugate The CPP and siRNA conjugate evaluated here is monomeric LMWP-siRNA covalent conjugate synthesized by our group. A cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugate was developed using PEG as a linker. In the conjugate, CPP was conjugated with the siRNA via a PEG linker which can shield 8

LMWP from charge-induced complexation with siRNA during the coupling process; meanwhile, a cytosol-cleavable disulfide (S-S) linkage was introduced between PEG linker and siRNA which resulted in free siRNA duplexes upon exposure to the reducing cytoplasmic environment (Fig. 1). In our previous studies, the conjugation ratios, and structure confirmation of LMWP-siRNA conjugate have been proved by mass spectrum, the exact ratio of LMWP/PEG/siRNA in the hybrid was 1:1:1 [20].

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Intracellular uptake of the LMWP-PEG-S-S-siRNA conjugate includes four separate events: (1) CPP-mediated cell transduction; (2) Cleavage of the disulfide bond

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resulting in the detachment of LMWP from siRNA; (3) Nucleus localization of LMWP; and (4) Specific gene silencing by the cytosol-released siRNA via the RNA-induced silencing complex system. Cell culture assessments have demonstrated

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that this chemical conjugate yields by far the most effective intracellular siRNA

PEG

siRNA

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-S-S-

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LMWP

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delivery and its corresponded gene-silencing activities [20].

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Fig. 1. Schematic illustration of the component of the LMWP-siRNA conjugate. The siRNA duplex with the 5’-end of the sense strand being modified with a

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sulfhydryl group was coupled, via a disulfide linkage, to a PEG-protected cell-penetrating

peptide

(LMWP),

thereby

avoiding

charge-induced

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complexation/aggregation between siRNA and LMWP.

Cell uptake mechanism study of LMWP-siRNA conjugate by MDA-MB-231 cells

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The cell uptake dynamics of the LMWP-siRNA conjugate were presented by the overall cell fluorescence intensity. Results in Fig. 2A and 2B indicated that the uptake displayed time and dose dependent increase in the fluorescence intensity of cells. Generally, CPP-cargo complexes can translocate through the cell membrane via direct translocation or via endocytosis. Therefore the contribution of the energy-independent pathways and endocytosis to CPP-siRNA conjugate internalization by MDA-MB-231 9

cells was evaluated. As known, endocytosis occurs by the action of various pathways and can be classified into caveolae and/or lipid-raft-mediated endocytosis, macropinocytosis, cholesterol-dependent clathrin-mediated endocytosis, or caveolaeand clathrin-independent endocytosis. The cells were incubated with CPP-siRNA conjugate (4.5 μM) under different conditions: (i) at 37 °C (control), (ii) at 4 °C, and (iii) after pretreatment with inhibitors. Incubation of the cells at 4 °C was also known

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to block endocytosis, while treating the cells with different inhibitors provided the information about the distinct pathways of endocytosis that CPP-siRNA conjugate

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might take.

For the analysis of CPP-siRNA conjugate’s uptake under different cellular treatments, the fluorescence intensity of labeled siRNA was quantitated by FACS. As shown in

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Fig. 2C, when endocytosis is hindered by low temperature (Fig. 2C, 4°C), the

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fluorescent signal of CPP-siRNA conjugate displayed an approximate 50% decrease

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compared to the control (Fig. 2C, Control). These results suggested that part of CPP-siRNA conjugate was still internalized by an energy-independent manner when

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endocytosis was blocked. To further document the endocytosis mechanisms involved

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in the cellular uptake of CPP-siRNA conjugate, different receptor-mediated endocytosis pathways were investigated. In the experiments, cells were incubated with CPP-siRNA conjugate under conditions with either the clathrin or the caveolae

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pathway inhibited. It was noted that CPP-siRNA conjugate uptake in MDA-MB-231 cells was affected, neither in the presence of chlorpromazine (a known inhibitor of

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clathrin-mediated endocytotic pathway), nor in the presence of methyl-β-cyclodextrin which perturbs formation of clathrin-coated endocytic vesicles. The cell uptake of CPP-siRNA conjugate was not inhibited with the addition of dynaosre, which blocks

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GTPase activity of dynamin that is essential for clathrin-coated vesicle formation in endocytosis, in transport from the trans Golgi network, as well as for ligand uptake through caveolae. In addition, for the group incubated with the macropinocytosis inhibitor amiloride, CPP-siRNA conjugate internalizes almost the same as control cells without any inhibitor treatment. All these results suggested that part of CPP-siRNA conjugate might internalize cells mainly through clathrin- and 10

caveolae-independent endocytosis. In summary, CPP-siRNA conjugate might be taken up by cells via multiple pathways as reported, including direct penetration of the plasma membrane and clathrin- and caveolae-independent endocytosis in our case.

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Fig. 2. Cell uptake of LMWP-siRNA conjugate by MDA-MB-231 cells.

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LMWP-siRNA conjugate uptake is A. time-dependent and B. dose-dependent. C. LMWP -siRNA conjugate uptake is not impaired by inhibitors targeting different

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endocytic pathways. Scale bar: 20 μm.

Cellular uptake study of different siRNA formulations Cellular uptake of six FITC-labeled siRNA formulations were evaluated by the

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overall cell fluorescence intensity and monitored via confocal microscopy. These six siRNA formulations were siRNA alone, physical mixture of LMWP and siRNA with a

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mole ratio of 1:1 (LMWP/siRNA I), physical mixture of LMWP and siRNA with a mole ratio of 42:1, i.e N/P ratio of 10:1 (LMWP/siRNA II), PEI and siRNA complex with a N/P ratio of 10:1 (PEI/siRNA), Lipo6000 and siRNA complex (Lipo/siRNA),

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and the LMWP-siRNA conjugate (Conjugate). PBS treated cell was used as a control in the study. In order to evaluate the cell uptake efficacy and cytotoxicity of the monomeric LMWP-siRNA conjugate, the commonly used and effective siRNA transfection agent Poly(ethylenimine) (PEI) was applied in our study. The ratio of PEI nitrogens to DNA phosphates is important in terms of transfection efficiency and cell toxicity [22], and the ratio of 10:1(N/P) which has been used successfully for optimal 12

transfection efficiency in the literature [23-25], thereby we applied this optimized ratio in our PEI/siRNA complex. While LMWP/siRNA complex with a N/P ratio of 10:1 exhibited a molar ratio of 42:1, this formulation used here was set for an effective comparative study among PEI/siRNA, LMWP/siRNA I and Conjugate groups. As expected, confocal images (Fig. 3A) showed insignificant cell uptake of the

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FITC-labeled siRNA, as the fluorescence intensity inside the cells was almost

identical to the background intensity shown by the PBS buffer; primarily due to the

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lack of cell internalizing ability by the siRNA alone. On the other hand, both the Lipo/siRNA group as well as the LMWP/siRNA I group displayed slightly improved yet still fairly weak cellular FITC intensities, suggesting the capable yet ineffective

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cell internalization by these two samples. Besides, the confocal images of both

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LMWP/siRNA II and PEI/siRNA groups indicated significant higher cell uptake than

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other groups, which was apparently due to the excessive dose of cationic transfection reagents used. In contrast, the cell uptake decreased when the mole ratio of

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LMWP/siRNA decreased to 1:1(LMWP/siRNA I). Under an equal mole ratio of

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LMWP vs siRNA, the covalent and monomeric (1:1) chemical conjugate could yield a far more effective intracellular siRNA uptake than that by the charge-associated aggregates produced via the conventional method of physically mixing CPP with

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siRNA (LMWP/siRNA I) (Fig. 3B). All these results demonstrated that covalent conjugation of LMWP and siRNA via PEG offered a relatively more effective siRNA

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delivery strategy.

The gene silencing effects of different siRNA formulations including siRNA alone, LMWP/siRNA I, Lipo/siRNA and Conjugate groups have been evaluated in previous

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study [20]. The gene silencing down-regulating effect by anti-EGFP siRNA was assessed using EGFP over-expressed MDA-MB-231-EGFP cells, and characterized by confocal fluorescent images, FACS quantifications and Western blot analysis. According to our reported results, anti-EGFP siRNA alone yielded negligible inhibition on EGFP expression, and on the other hand, while both the Lipo/siRNA and LMWP/siRNA I groups displayed obvious inhibition on EGFP expression in the 13

MDA-MB-231-EGFP cells, the most effective EGFP gene-silencing efficacy was observed in cells treated with the Conjugate group. This implied that the difference of gene silencing effects of these formulations might due to their different cell uptake efficiency.

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Fig. 3. Cell uptake studies carried out on MDA-MB-231 cells with different siRNA formulations. A. Confocal microscopy and B. Fluorescence intensity results of samples containing: (1) PBS, (2) siRNA alone, (3) LMWP/siRNA I, (4) LMWP/siRNA II, (5) PEI/siRNA, (6) Lipo/siRNA, and (7) Conjugate. For 14

comparison, the quantities of siRNA employed in all of these studies were maintained identical. Scale bar: 20 μm.

In vitro cytotoxicity study of different siRNA formulations To systematically evaluate and get a thorough understanding of the in vitro cytotoxicity of the monomeric LMWP-siRNA conjugate, comparative toxicity studies

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were carried out, including MTT assay, Annexin-V FITC/PI double staining assay,

MTT assay

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Trypan blue exclusion test and LDH release assay.

Table 1. IC50 values of different siRNA formulations I

II

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6.43

PEI/siRNA

Lipo/siRNA

10.82

0.94

Conjugate

38.01

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(µmol/L)

LMWP/siRNA

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IC50 value

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Samples

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As shown in Table 1, Lipo/siRNA group indicated significantly higher cytotoxicity (IC50 = 0.94 µmol/L) than the other groups. What’s more, the PEI/ siRNA group and

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LMWP/siRNA II group presented similar cytotoxicity to MDA-MB-231 cell with IC50 values at 10.82 µmol/L and 6.43 µmol/L, respectively. Conjugate (IC50 = 38.01

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µmol/L) and LMWP/siRNA I group at high dose caused slight impact on cell viabilities (IC50 = 169.90 µmol/L), indicating the lower cytotoxicity to the cells.

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Based on the results of comparative cellular uptake study of different siRNA formulations, the lower cytotoxicity of LMWP/siRNA I group compared with Conjugate group might due to its lower cell uptake by MDA-MB-231. All these data suggested that, unlike the commonly used cationic transfection reagents which

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displayed significant cytotoxicity towards cells, LMWP-siRNA conjugate would offer a safer and more effective siRNA delivery strategy.

Annexin-V FITC/PI double staining assay Cell apoptosis induced by the above mentioned five groups (LMWP/siRNA I, 15

LMWP/siRNA II, PEI/siRNA, Lipo/siRNA, Conjugate) was detected by annexin V-FITC/PI double-staining. Our results indicated that all the transfection reagents induced apoptosis in MDA-MB-231 cells in a dose-dependent manner (Fig. 4A). In high dose of PEI/siRNA, LMWP/siRNA II, LMWP/siRNA I, and conjugate exposed group, cells started to enter late stage of apoptosis, while at relatively low dose LMWP/siRNA I, and conjugate groups caused slight impact on cell growth (Fig. 4B).

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The possible explanation might be that the cationic transfection reagents were

cytotoxic through their interactions with membrane and their induction of the

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apoptosis pathway via the production of ROS, which leads to cell death as reported

[26-31]. Compared to other formulations, less cells entered the early and late stage of apoptosis both in the LMWP/siRNA I and conjugate treatment groups. Although

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percentage of late apoptotic cells was slightly higher in the conjugate treatment

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groups than the LMWP/siRNA I treatment groups, considering the higher cell uptake

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of LMWP-siRNA conjugate, LMWP-siRNA conjugate presented a much better

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biocompatibility than the other cationic transfection reagents.

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Fig. 4. Dose-dependent effect of different siRNA formulations on apoptosis in

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MDA-MB-231 cells. MDA-MB-231 cells were treated with various concentrations of five different siRNA formulations for 4 hours followed by staining with Annexin V

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FITC and propidium iodide. A. Representative Annexin V FITC-A vs Propidium

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Iodide-A contour plots from four concentrations of five different siRNA formulations. B. Dose response curve of five different siRNA formulations showing the percentages

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of cells in the population (Annexin V+PI+) gated as shown in Fig. 4A.

Trypan blue exclusion test of cell viability

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Measurements of the indicators of programmed cell death (i.e., apoptosis) and/or necrosis directly reveal the ability of molecule/nanoparticles to induce intracellular suicide mechanisms or destroy cells. Such assays focus largely on measuring

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membrane integrity. Herein, membrane integrity affected by different siRNA formulations was evaluated via trypan blue staining cell viability assay which can provide both the rate of proliferation as well as the percentage of viable cells. As shown in Fig. 5, the mortalities of cells of the five experimental groups were significantly higher than that of the control group (P < 0.001), where Lipo/siRNA, PEI/siRNA and LMWP/siRNA II groups induced about 75% cell mortality at a siRNA 18

concentration of 13.5 µM and the LMWP/siRNA I group induced about 45%. In sharp contrast, the mortality induced by conjugate was only about 17%, which is much lower than other complexes. We may conclude that covalent conjugation of CPP with

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siRNA promoted internalization conjugate in a low-toxic fashion.

Fig. 5. Cytotoxic effect of different siRNA formulations to MDA-MB-231 cells

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examined by Trypan blue exclusion test. MDA-MB-231 cells were cultured with different siRNA formulations for 24 h as indicated in the Materials and Methods. Cell

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viability was then determined based on the trypan blue exclusion test.

LDH release assay In order to obtain undistorted results of the safety evaluation of the siRNA transfection reagents, another cytotoxicity assay - LDH release assay was carried out to quantitatively measure lactate dehydrogenase (LDH) released into the media from damaged cells as a biomarker for cellular cytotoxicity and cytolysis. As shown in Fig. 19

6, the Lipo/siRNA group induced the highest level of LDH leakage among all the siRNA formulations at each test concentration. The LDH leakage of PEI/siRNA, LMWP/siRNA II and LMWP/siRNA I group indicated similar LDH activity as the control group (P > 0.05) at low concentrations (0.45 and 4.5 µM), however, PEI/siRNA and LMWP/siRNA I induced LDH leakage of cells when concentration increased (PEI/RNA at 22.5 µM, LMWP/siRNA I at 13.5 µM). Exposed cells to

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LMWP-siRNA conjugate lead to LDH leakage of cells at all test concentration, and the LDH activity level was stable at about 500 U/gprot. However, the LDH activity

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level in the LMWP-siRNA conjugate treatment group was similar to other three

groups that induced a relatively low LDH leakage of cells. All this suggested that

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covalent conjugation of CPP with siRNA indicated a relatively low cytotoxicity

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Fig. 6. Comparison of LDH release assay results in MDA-MB-231 cells after exposure to different siRNA formulations for 24 h.

Conclusions As a continuation of our previous study [20], the cell uptake mechanism study, as well 20

as a comparative in vitro cytotoxicity analysis of the effective LMWP-siRNA conjugate with a number of commonly used cationic transfection reagents, was carried out. Unlike the involvement of vesicular formation processes in the membrane transduction of CPP like oligoarginine [32-34], LMWP-siRNA conjugate might be taken up by cells via multiple pathways including direct penetration of the plasma membrane and clathrin- and caveolae-independent endocytosis which needs further

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investigation in detail.

Although the number of studies addressing translocation mechanisms of single CPPs

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and different cargo molecules is growing rapidly, only a few toxicity studies are

available. Our previous results have demonstrated that, LMWP-siRNA conjugate displayed outstanding RNAi effect compared to the commonly used lipid based and

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polymer based strategy. The coupling strategy offers the potential to be a safer

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methodology for siRNA delivery. To further assess the possible toxic mechanism of

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the LMWP-siRNA conjugate delivery strategies in the cell structure and viability, cytotoxicity studies have examined by cell viability, cell apoptosis and membrane

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permeability. This thorough and comparative analysis of cytotoxicity study indicated

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that covalent conjugation of CPP with siRNA promoted internalization of conjugate in a low-toxic fashion and offered a very promising platform for delivery of siRNA.

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Acknowledgement

This work was supported in part by the National Key Research and Development Plan

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(2016YFE0119200) and National Natural Science Foundation of China (NSFC, 81361140344). This research was also partially sponsored by Tianjin Municipal Science and Technology Commission (15JCYBJC28700, 15JCQNJC13600 and

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17JCYBJC20700) and the China Postdoctoral Science Foundation (Grant No. 2015M581306).

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