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Integration of Cell-Penetrating Peptides with Rod-like Bionanoparticles: Virus-Inspired Gene Silencing Technology Ye Tian, Mengxue Zhou, Haigang Shi, Sijia Gao, Guocheng Xie, Meng Zhu, Man Wu, Jun Chen, and Zhongwei Niu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b01805 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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Integration of Cell-Penetrating Peptides with Rodlike Bionanoparticles: Virus-Inspired Gene Silencing Technology Ye Tian,† Mengxue Zhou,‡ Haigang Shi,† Sijia Gao,† Guocheng Xie,† Meng Zhu,† Man Wu,† Jun Chen‡ and Zhongwei Niu*,†,§ †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute

of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun East Road, Beijing 100190 (P.R. China) ‡

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of

High Energy Physics, Chinese Academy of Sciences, No. 19(B) Yuquan Road, Beijing 100049 (P.R. China) §

School of Future Technology, University of Chinese Academy of Sciences, No.19(A) Yuquan

Road, Beijing 100049 (P.R. China)

ACS Paragon Plus Environment

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ABSTRACT. Inspired by the high gene transfer efficiency of viral vectors and to avoid side effects, we present here a 1D rod-like gene silencing vector based on a plant virus. By decorating the transacting activator of transduction (TAT) peptide on the exterior surface, the TATmodified tobacco mosaic virus (TMV) achieves a tunable isoelectric point (from ~3.5 to ~9.6), depending on the TAT dose. In addition to enhanced cell internalization, this plant virus-based vector (TMV-TAT) acquired endo/lysosomal escape capacity without inducing lysosomal damage, resulting in both high efficiency and low cytotoxicity. By loading silencer GFP siRNA onto the TMV-TAT vector (siRNA@TMV-TAT) and interfering with GFP-expressing mouse epidermal stem cells (ESCs/GFP) in vitro, the proportion of GFP-positive cells could be knocked down to levels even lower than 15% at a concentration of ~100% cell viability. Moreover, by interfering with GFP-expressing highly metastatic hepatocellular carcinoma (MHCC97-H/GFP) tumors in vivo, treatment with siRNA@TMV-TAT complexes for 10 days achieved a GFPnegative rate as high as 80.8%. This work combines the high efficiency of viral vectors and the safety of non-viral vectors and may provide a promising strategy for gene silencing technology.

KEYWORDS. tobacco mosaic virus, cell-penetrating peptide, gene silence, endosomal escape, viral vector.

Profiting from the development of nanotechnology, genetics and molecular biology, RNA interference (RNAi) provides an attractive strategy for silencing gene expression and has proven to be promising for treating cancer, HIV-1, hepatitis and so on.1-4 Due to the negative charge of naked nucleic acids, a safe and efficient vector becomes the primary challenge in gene silencing.


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Viral vectors such as adeno- and retroviruses have covered most gene vectors in clinical trials, showing high gene transfection efficacy,5-6 but their inherent immunogenicity, pathogenicity and probable chromosomal integration become major barriers for their clinical trials.7 Especially with the reported death and occurrence of leukemia of patients treated with adenoviral or retroviral vectors, the clinical restrictions on viral vectors have become ever more serious.8-9 In the past decades, non-viral gene vectors such as polyetherimide (PEI), polyamidoamine and chitosan have been greatly exploited.10-11 They show low immunogenicity and can achieve improved efficiency and specificity through intelligent design. Because gene transfection efficiency is still not comparable to that of viral vectors, and due to the polydispersity of their size and molecular weight, it is a great challenge for non-viral vectors to obtain FDA approval. Inspired by the high efficiency of viruses and to avoid side effects, various virus-derived nanoparticles, such as adeno-associated virus (AAV) and virus-like nanoparticles, have been utilized for gene therapy, performing well in both safety and therapeutic efficacy.12-13 Recently, more research studies have demonstrated that rod-like nanoparticles with high aspect ratio may be propitious to circulation, biodistribution and internalization as a drug or gene carrier.14-15 As a rod-like plant virus with a high aspect ratio, tobacco mosaic virus (TMV), which measures 300 nm × 18 nm and shows no infection in mammals, may provide an alternative for gene delivery.1620

Benefiting from its well-defined structure, size and shape, site-selective functionalization and

genetic programmability, TMV and its virus-like nanoparticles have been widely exploited in biomedical fields such as cell behavior regulation,21-22 imaging,23 drug delivery24-26 and even gene delivery in plants or phytopathogenic fungi.27-29 Previous studies show that TMV lacks endosomal escape activity in mammalian cells,24 which is critical for a gene carrier to bring cargos into the cell cytoplasm.30 The tyrosine 139 residues on the TMV exterior surface provide


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chemically reactive sites for decorating cell-penetrating peptides (CPPs), which are capable of translocating through biological membranes. As one commercially available CPP, transacting activator of transduction (TAT) peptide has a +8 charged sequence of amino acids,31-32 and has already been widely employed to facilitate not only cytosolic or intranuclear delivery but also crossing of the blood−brain barrier (BBB).33 Herein, we present the design and construction of a 1D rod-like gene silencing vector based on TMV (Scheme 1). Specifically, TAT peptide was decorated to the exterior surface of TMV to provide endosomal escape property, and silencer green fluorescent protein (GFP) siRNA was loaded onto the TMV-TAT vector through electrostatic interactions. The siRNA released in the cell cytoplasm will bind to RNA-induced silencing complex (RISC) with the target mRNA and knock down GFP expression in GFP-expressing cells. This work exploits the uniform shape, size, molecular weight, non-immunogenicity and modifiable surface to realize a plant virusbased gene vector with high efficiency and safety, providing a potentially safer approach for gene silencing.


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Scheme 1. (a) Schematic illustration for the design of TAT-decorated TMV and siRNA loading. (b) Schematic illustration of the gene silencing process. The TAT decoration onto the TMV exterior surface was achieved through a click reaction on alkyne-modified TMV (alky-TMV). For each TMV coat protein (TMVCP), there is only one reaction-active phenolic hydroxyl group exposed on the TMV exterior surface, which is from the


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tyrosine 139 residue. By following a proven protocol,34 the phenolic hydroxyl group of tyrosine 139 can be modified with alkyne bonds and then conjugated with 5-azidopentanoic acidYGRKKRRQRRR (TAT-N3) through a click reaction (Scheme 1). To assess the dose-dependent efficiency, different equivalents of TAT-N3 (0.5, 5 and 50 eq of TAT-N3 per coat protein) were used in the click reaction. Transmission electron microscope (TEM) images (Figure 1a) showed that the chemical modification would not disrupt the rod-like structure of TMV. SDS-PAGE analysis (Figure 1b) confirmed the success and efficiency of the TAT decoration. After alkyne modification, TMVCP has a molecular weight increase of 128, leading to a slight upward movement of the protein band. Due to steric hindrance resulting from high molecular weight (1685 Da) and the high cation density of TAT, the efficiency for the click reaction could not reach up to 100%. Therefore, in the TMV-TAT lanes in Figure 1b, there are two protein bands in each lane: the lower bands are from the non-modified alky-TMVCP with lower molecular weight, and the upper bands are from TMVCP-TAT with higher molecular weight. By gray scale analysis through ImageJ software, the grafting efficiency was estimated as 5% (TMV-TAT5), 45% (TMV-TAT45) and 60% (TMV-TAT60) at TAT equivalents of 0.5, 5 and 50, respectively. From MALDI-TOF MS analysis (Figure S1), based on the theoretical m/z value of TMVCP (17,534) and TMVCP-TAT (19,347), the emergence of m/z value of 19,198 confirms the success of the TAT modification. To assess the internalization efficiency and intracellular distribution, rhodamine B (RB) was labeled in the TMV cavity through chemical conjugation to the glutamic acids 97 and 106 residues.35 The red emission in the protein bands (Figure S2) revealed that RB was labeled onto the TMV-TAT. For each sample, the four emission bands from the bottom to the top represent single RB-labeled TMVCP, dual RB-labeled TMVCP, single RB-labeled TMVCP-TAT and dual RB-labeled TMVCP-TAT, respectively.


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Figure 1. (a) TEM image of TAT-decorated TMV; scale bar: 100 nm. (b) SDS-PAGE analysis stained by Coomassie brilliant blue for TMV, alky-TMV and TMV-TAT with different TAT equivalents (0.5, 5 and 50) in the exterior surface modification; TAT conjugation efficiency obtained from ImageJ analysis of the SDS-PAGE image. (c) Zeta potentials of TMV, TMVTAT5, TMV-TAT45 and TMV-TAT60 at different pH values in 0.01 M buffer. (d) Isoelectric points of TMV, TMV-TAT5, TMV-TAT45 and TMV-TAT60 obtained from zeta potential analysis of image (c). Similar to most viral nanoparticles, TMV behaves as a negative surface charge under neutral conditions. The TAT peptide is well known for its positively charged property. Through TAT decoration, the zeta potential of TMV-TAT becomes more positive in a dose-dependent manner


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(Figure 1c). At neutral pH (pH = 7.4), the surface property of TMV gradually reversed from negatively charged (TMV) to positively charged (TMV-TAT60), which is advantageous for cellular internalization. From the previous reports, it was found that chemical modifications can only change the isoelectric point of a virus or peptide over a small range. For example, acid modification of the M13 bacteriophage decreases the isoelectric point from 4.3 to 3.7,36 conjugation of fluorous ponytails on the carboxyl groups of TMV slightly increase the isoelectric point from 3-4 to 4-5,37 and conjugation of cationic CPPs on HE oligopeptide sequences increase the isoelectric point from 6.20 to 6.77.38 In this work, TAT decoration increased the isoelectric point of TMV from ~3.5 to ~9.6 (Figure 1d). We then investigated the effect of the TAT dose on the internalization efficiency of different cell lines. HeLa cells and smooth muscle cells (SMCs) were incubated with the TMVRB and TMVRB-TAT samples for 0.5, 5, and 20 h. The internalization efficiency was quantified through flow cytometry. As shown in Figure 2a-b and Figure S3-4, compared to TMVRB, the TAT decoration on the TMV exterior surface leads to significantly enhanced cellular uptake. Even the negatively charged TMVRB-TAT5 (zeta potential -17 mV at pH 7.4) and nearly neutral TMVRBTAT45 (zeta potential -0.5 mV at pH 7.4) show enhanced cell penetration properties. All three TMVRB-TAT samples showed significant differences in cell internalization compared to that of TMVRB. TMVRB-TAT5 showed 2-2.5-fold uptake efficiency compared with that of TMVRB at each time point for both HeLa cells and SMCs. There is no significant difference between the TMVRB-TAT45 and TMVRB-TAT60 groups. In HeLa cells, TMVRB-TAT45 and TMVRB-TAT60 displayed 3-4-fold uptake efficiency compared with that of TMVRB at 5 and 20 h, and 6-8-fold at 0.5 h; in SMCs, they penetrated the cells with an efficiency of 3-4-times that of TMVRB at each


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time point. This enhanced cellular uptake may benefit from both the α-helical secondary structure and the positive charge of TAT.


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Figure 2. (a, b) Columnar statistics from flow cytometry of the internalization efficiency of HeLa cells (a) and SMCs (b) incubated with TMVRB, TMVRB-TAT5, TMVRB-TAT45 and TMVRB-TAT60 for 0.5, 5 and 20 h. (c-e) Intracellular distribution of TMVRB-TAT45 in HeLa cells at 1 h of incubation as obtained from CLSM. Blue and red colors in all images are for cell nuclei and TMVRB-TAT45, respectively. Green color shows the intracellular location of endo/lysosomes (c), mitochondria (d) and the Golgi apparatus (e). Scale bar: 20 µm. (f) Cell growth inhibition of TMV, TMV-TAT5, TMV-TAT45 and TMV-TAT60 on HeLa cells for 24 h. (g) Immunofluorescence localization of cathepsin B (green color) in HeLa cells incubated with TMVRB-TAT45 (red color) for 24 h. Blue color is for cell nuclei. Scale bar: 20 µm. (h) Hemolysis ratios and photograph of the hemolysis assay after incubation with TMV-TAT45 for 2 h. (i) CLSM image of the erythrocytes treated with 100 µg/mL of TMV-TAT45 for 2 h. All data represent mean value ± SD (n = 3). * p ≤ 0.05 between the two groups; ** p ≤ 0.01 between the two groups. It is commonly accepted that some CPPs can directly penetrate the plasma membrane and achieve cytosolic cargo delivery.39 To evaluate the intracellular distribution of the TMV-TAT vectors, HeLa cells were incubated with TMVRB, TMVRB-TAT5, TMVRB-TAT45 and TMVRBTAT60 for 1 h, and the endo/lysosomes, mitochondria and Golgi apparatus were separately labeled. From confocal laser scanning microscopy (CLSM) images (Figure 2c-e and Figure S5), without subcellular organelle targeting ligand or membrane interaction ligand, endo/lysosomes were the main target organelles for TMVRB (the colocalized region is shown in yellow in Figure S5a). No obvious colocalization occurred between TMVRB and mitochondria or the Golgi apparatus (Figure S5d, S5g). As shown in Figure 2c-e and Figure S5, none of the three TMVRBTAT

samples

(TMVRB-TAT5,

TMVRB-TAT45

and

TMVRB-TAT60) localized

to

the


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endo/lysosomes, mitochondria or Golgi apparatus. The samples may achieve a cytoplasmic distribution from either endosomal escape or through direct membrane penetration. This cytosolic localization provides TMV-TAT the opportunity to function as gene delivery vector. Cationic polymer vectors such as PEI normally escape from the endo/lysosomes through a “proton sponge” effect, leading to membrane leakage and significant cytotoxicity. Here, from the cell growth inhibition assay (Figure 2f), TMV-TAT5, TMV-TAT45 and even the highly positive TMV-TAT60 show low cytotoxicity. Cations causing lysosomal damage is one of the main reasons for toxicity. To assess lysosomal damage caused by TMV-TAT vectors, we evaluated the intracellular localization of the lysosomal enzyme cathepsin B through fluorescence immunostaining.40-41 Cathepsin B in normal cells is usually distributed in a punctate manner (as shown in Figure S6a), indicating the integrity of the lysosomal membrane. It will show a diffuse cytosolic release in cells when lysosomes are ruptured.40-41 From Figure S6 and Figure 2g, it can be seen that neither TMVRB nor TMVRB-TAT samples caused lysosomal disruption. The cathepsin B was still safely enveloped in intact lysosomes, showing a punctate distribution. These data indicate that the TMV-TAT vectors will not induce lysosomal damage, resulting in low toxicity. We further assessed the erythrocyte toxicity of the TMV-TAT45 (Figure 2h,i and Figure S7). The photographs show that saline and the TMV-TAT45 samples induced little hemolysis compared to that of dd water (positive control). Through the quantitative assessment and statistical analysis presented in Figure 2h, TMV-TAT45 is shown to have a low hemolysis ratio (< 1%) even at a high concentration of 500 µg/mL. There is no significant difference between TMV-TAT45 samples and saline in hemolysis ratio at a TMV-TAT45 concentration of ≤300 µg/mL. CLSM images showed that, unlike the ruptured structure in dd water, the erythrocytes retained the same morphology with TMV-TAT45 as in saline. These results indicate


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that

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for

both

cytocompatibility

and

hemocompatibility. Then, silencer GFP siRNA was loaded on the TMV-TAT vectors through electrostatic interactions to form the siRNA@TMV-TAT complex. To assess the binding capacity of TMVTAT vectors to siRNA, agarose gel electrophoresis was performed. Figure S8a reveals that TMV-TAT5 lacked the ability to form a stable complex with siRNA even at a TMVTAT5/siRNA molar ratio as high as 0.05:1. The vectors with higher isoelectric point, TMVTAT45 and TMV-TAT60, could form stable complexes with siRNA at a TMV-TAT/siRNA molar ratio ≥ 0.01 (Figure 3a and Figure S8b). The formed complexes remained intact during the electrophoresis process, as evidenced by the disappearance of free siRNA bands.


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Figure 3. (a) Agarose gel electrophoresis of the siRNA@TMV-TAT45 complex at a variety of TMV-TAT45/siRNA molar ratios. (b-d) GFP knockdown efficiency in ESCs/GFP cells treated with siRNA@TMV-TAT45 complexes at a final TMV-TAT45 concentration of 100 µg/mL. The TMV-TAT45/siRNA molar ratios of 1.34, 0.268, 0.0536 and 0.0107 resulted in corresponding final siRNA concentrations of 0.002, 0.01, 0.05 and 0.25 µM, respectively. (b) GFP mRNA level determined by RT-PCR. (c) Histograms from flow cytometry. (d) Columnar statistics from flow cytometry. (e) GFP silencing efficiency (column bar charts) and cell viability (line charts) of TMV-TAT45, Lipo 2000 and PEI25k at different vector concentrations at a final siRNA


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concentration of 0.05 µM. (f) Ratios of cell viability to gene silencing efficiency of TMV-TAT45, Lipo 2000 and PEI25k at different vector concentrations obtained from (e). * p ≤ 0.05 compared with control group; ** p ≤ 0.01 compared with control group. Since TMV-TAT45 and TMV-TAT60 showed similar binding capacities to siRNA, we took siRNA@TMV-TAT45 to evaluate the gene silencing efficiency in GFP-expressing mouse epidermal stem cells (ESCs/GFP), which were incubated with siRNA@TMV-TAT45 complexes at a final TMV-TAT45 concentration of 100 µg/mL, at which point the ESCs/GFP cell viability is ~100% (Figure S9). The siRNA@TMV-TAT45 complexes were prepared at TMV-TAT45/siRNA molar ratios of 1.34, 0.268, 0.0536 and 0.0107, at which point the final siRNA concentrations were 0.002, 0.01, 0.05 and 0.25 µM, respectively. Real-time PCR (RT-PCR) results revealed that GFP mRNA expression was significantly knocked down to

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