Tobacco Mosaic Virus-Based 1D Nanorod-Drug Carrier via the

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Tobacco Mosaic Virus Based 1D Nanorod-Drug Carrier via the Integrin-Mediated Endocytosis Pathway Ye Tian, Sijia Gao, Man Wu, Xiangxiang Liu, Jing Qiao, Quan Zhou, Shidong Jiang, and Zhongwei Niu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02801 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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ACS Applied Materials & Interfaces

Tobacco Mosaic Virus Based 1D Nanorod-Drug Carrier via the Integrin-Mediated Endocytosis Pathway Ye Tian, Sijia Gao, Man Wu, Xiangxiang Liu, Jing Qiao, Quan Zhou, Shidong Jiang and Zhongwei Niu* Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China. KEYWORDS: tobacco mosaic virus, cRGD, rod-like nanoparticle, pH sensitivity, integrinmediated endocytosis.

ABSTRACT: For cancer therapy, viruses have been utilized as excellent delivery vehicles due to their facile transfection efficiency in their host cells. However, their inherent immunogenicity has become the major obstacle for their translation into approved pharmaceuticals. Herein, we utilized rod-like plant virus, tobacco mosaic virus (TMV), which is non-toxic to mammals and mainly infects tobacco species, as anti-cancer nanorod-drug vector for cancer therapy study. Doxorubicin (DOX) was installed in the inner cavity of TMV by hydrazone bond, which enabled the pH sensitive drug release property. Conjugation of cyclic Arg-Gly-Asp (cRGD) on the surface of TMV can enhance HeLa cell uptake of the carrier via the integrin-mediated

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endocytosis pathway. Comparing with free DOX, the cRGD-TMV-hydra-DOX vector had similar cell growth inhibition and much higher apoptosis efficiency on HeLa cells. Moreover, the in vivo assay assumed that cRGD-TMV-hydra-DOX behaved similar anti-tumor efficiency but much lower side effect on HeLa bearing Balb/c-nu mice. Our work provides novel insights into potentially cancer therapy based on rod-like plant viral nanocarriers.

1. Introduction Viruses, although extremely simple in structure and composition, provide an ideal basis for tumor therapy due to their naturally evolved high efficiency in transfection of their host cells.1-3 Adenoviruses, retroviruses and lentiviruses, composing of capsid and core proteins as well as viral DNA, are the most commonly used viral vector systems for drug delivery. 4, 5 Oncolytic virotherapy6 such as the newly founded M1 virus7 is also showing promise in clinical trials. Recently human H-ferritin has been developed as a natural nanocarrier that specifically and significantly inhibited tumor growth with a single-dose treatment in murine cancer models.8, 9 While the major concern about the inherent immunogenic nature, the unwanted side effects and the biosafety of the viral vectors has impeded their translation into approved pharmaceuticals.5, 10, 11

Nowadays, the polymeric "artificial virus" that lack harmful genes and equal (or exceed) to

viral vector in terms of target-cell specificity has been proposed for safe and highly efficient nano carrier.10-13 While the polydispersity of the molecular weight is always the critical shortcoming to hamper its clinic translation. To solve the above problems, the bio-synthesized plant virus, which has the absolute biocompatibility, non-infection to mammals, monodispersity


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in morphology and molecular weight, and cost-effective scalable bioproduction, provides ideal substitute for drug delivery. As a plant virus that mainly infects Nicotiana benthamiana, Nicotiana rustica and other tobacco plants, tobacco mosaic virus (TMV) consists of 2130 identical coat protein assembled helically around the single stranded RNA and has a 1D rod-like structure measuring 300 × 18 nm with 4 nm cavity.14-16 The naturally formed three-dimensional structure of the protein cage provides accurate sites for chemical conjugation and better protection of the passenger drug from harsh environments. To provide TMV the tumor-specific performance and do minimal adverse effects to normal tissues, the introducing of a tumor-targeting ligand is necessary. As a tumor-homing peptide, cyclic Arg-Gly-Asp (cRGD) is a candidate ligand with selective affinity for the αvβ3 integrins, which is a protein over expressing on both tumor cells of various origins and endothelial cells associated with growing tumors.17-19 cRGD modified nanoparticles could have greatly improved cellular uptake and tumor accumulation via the integrin-mediated internalization mechanism.20-22 In this contribution, the rod-like TMV was applied as the main body; the tumor-homing peptide cRGD was functionalized to the tyrosine 139 residues on TMV exterior surface to enhance its tumor accumulation; the chemotherapeutic drug doxorubicin (DOX) was chemically conjugated to the glutamic acids 97 and 106 residues in the inner cavity of TMV. To facilitate the release of the anticancer drugs specifically and rapidly in tumor interstitium and cancer cells, hydrazone bond, which is more cleavable at low pH values (tumor microenvironment pH and endosomal pH),23, 24 performs as the drug-protein linker. This plant viral vector will provide a novel strategy in the design of chemotherapeutic drug carrier for successful cancer therapy with the minimal adverse effects.


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2. Materials and Methods 2.1 Materials TMV was extracted from infected Nicotiana benthamiana plants following a literature protocol.25 The anti-cancer drug DOX was purchased from Aladdin Industrial Corporation. ptoluenesulfonic acid (pTSA, Aladdin), 3-aminophenylacetylene (Aladdin), sodium nitrite (NaNO2, Beijing Chemical Works) were used for the alkyne-functionalization of tyrosine residues on the exterior surface of TMV. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxybenzotriazole (HOBt) were all supplied from Aladdin Industrial Corporation and used for the EDC-mediated conjugation onto the interior glutamic acid residues. Cyclic peptide cRGDfK-N3 (SciLight Biotechnology, LLC, Beijing), aminoguanidine (Sigma-Aldrich), ascorbic acid sodium (Sigma-Aldrich), copper (II) sulfate pentahydrate (CuSO4·5H2O, Beijing Chemical Works), ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) were used for the click reaction on alkyne-functionalized TMV. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium hydrate (NaOH), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O), boric acid (H3BO3), sodium carbonate (NaCl) from Beijing Chemical Works were used for preparation of various buffer solutions. Dimethyl sulfoxide (DMSO) was supplied from Aladdin Industrial Corporation. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were all from Life Technologies and used for HeLa cell culture. Cell Counting Kit-8 (CCK-8, DOjinDO Molecular Technologies) and Annexin V-FITC/PI Apoptosis Detection Kit (YeaSen Biotechnology) were used for cell growth inhibition and cell apoptosis measurement, respectively. Hoechst 33342, LysoTracker® Deep Red and


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MitoTracker® Green were all purchased from Life Technologies and applied for the staining of cell nucleus, endo/lysosomes and mitochondria, respectively. 2.2 Alkyne-functionalization of TMV exterior surface The alkyne groups are introduced to the exterior tyrosine residues of TMV following references.25, 26 Mix pre-colded 800 µL of 0.3 M pTSA solution in H2O, 150 µL of 0.68 M 3ethynylaniline solution in acetonitrile and 50 µL of 3.0 M NaNO2 solution in H2O for 1 h at 4 °C, and then add the formed diazonium salt into 15 mg TMV in 4.8 mL of 0.1 M borate buffer pH 8.8 (containing 0.1 M NaCl) at 4 °C. After 1 h of reaction at 4 °C, the modified TMV (alkyTMV) are purified by dialysis in H2O and gel chromatographic separation on a Sephadex G-25 column. The whole reaction is protected from light. 2.3 DOX conjugation on the TMV interior surface Add 92 µL of 10 mg/mL EDC aqueous solution into 7.5 mL of 0.1 M HEPES buffer (pH 7.4) containing 10 mg alky-TMV and mix. After 30 min, add 2 mL of 1.925 mg/mL HOBt in DMSO (50 eq per coat protein) and 250 µL of 10 mg/mL adipic acid dihydrazide in H2O (25 eq per coat protein) into the mixture. Then another two times 92 µL of 10 mg/mL EDC (totally 25 eq per coat protein for the three times) in H2O are added into the mixture at 6 h and 18 h. This reaction is performed at 4 °C for 24 h. After purification by dialysis and gel chromatographic separation, 5 mg of the product, coded as alky-TMV-hydra, is mixed with 0.33 mg of DOX in 5 mL mixture solution of 80% 0.05 M phosphate buffer pH 7.4 and 20% DMSO. After reaction for 48 h at 4 °C and dialysis in 0.01 M phosphate buffer (pH 7.4), the intermediate product, alky-TMV-hydraDOX, is obtained. 2.4 cRGD-functionalization of alky-TMV-hydra-DOX


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Mix 2 mg of alky-TMV-hydra-DOX with 0.36 mg cRGDfK-N3 (5 eq per coat protein), 0.002 M aminoguanidine, 0.002 M ascorbic acid sodium salt and 0.001 M copper sulfate in 1 mL ice colded 0.01 M phosphate buffer (pH 7.4) for 30 min. Then add 20 µL of 0.5 M EDTA/NaOH (pH ≈ 8) and mix for 5 min. After the purification process like the above steps, the final product cRGD-TMV-hydra-DOX is obtained. 2.5 Characterization The morphology of TMV based drug carrier is studied using a JEM-2100 (JEOL, Japan) transmission electron microscope (TEM). Absorption and emission spectra are determined through a U-3900 UV-Vis spectrophotometer and F-4600 fluorescence spectrophotometer (HITACHI, Japan). An AKTA fast protein liquid chromatography (FPLC) with Superdex 200 column (GE) and Bio-Rad SDS-PAGE system are applied to confirm the success of chemical conjugation. 2.6 Cell growth inhibition and cell apoptosis HeLa cells are cultured in cell culture flasks (Corning) in DMEM supplemented with 10% FBS and 1% P/S at 37 °C in 5% CO2. For cell growth inhibition assay, HeLa cells are seeded in a 96well plate at a density of 6000 cells per well and cultured in 5% CO2 at 37 °C overnight. Then the culture medium is replaced by different concentration of samples (cRGD-TMV-hydra-DOX and free DOX) in fresh growth medium. After 24 h or 48 h of incubation, assays are performed by replacing the medium containing different samples with 100 µL fresh growth medium and adding 10 µL CCK-8 reagent solution to each well for 3 h. By recording the absorbance at 450nm using EnSpire Multimode Plate Reader (PerkinElmer), the cell viability is determined as followed: Cell viability (%) = Isample / Icontrol ×100%


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where Isample and Icontrol represent the intensity determined for cells treated with different samples and for control cells (untreated), respectively. For cell apoptosis assay, HeLa cells are seeded in a 6-well plate at a density of 2 × 105 cells per well and cultured in 5% CO2 at 37 °C overnight. Then the culture medium is replaced by fresh growth medium containing different samples (cRGD-TMV-hydra-DOX and free DOX) at the DOX concentration of 0.3 µg/mL. Untreated cells are used as control. At 24 h of incubation, the cells are trypsinized (without EDTA), collected, and resuspended in 500 µL of binding buffer. Thereafter, 5 µL of annexin V-FITC and 5 µL of PI are added and mixed for 30 min in the dark. The stained cells are analyzed using a flow cytometer (BD Calibur). 2.7 Cellular uptake efficiency HeLa cells are seeded in 6-well plates at a density of 2 × 105 cells per well and cultured in 5% CO2 at 37°C overnight. The cells are pretreated with cRGD at 0, 50 and 100 µg/mL for 1 h. Then the cells are incubated with cRGD-TMV-hydra-DOX and TMV-hydra-DOX at a final DOX concentration of 0.4 µg/mL for another 2 h. After being washed with PBS, trypsinized, and resuspended into 10% FBS in PBS, the intracellular uptake efficiency are measured through flow cytometry (BD Calibur). Background fluorescence taken from untreated cells is subtracted from test samples. All conditions were done in triplicates independently. 2.8 Intracellular distribution and co-localization HeLa cells are cultured in glass-bottom cell culture dishes overnight and incubated with cRGD-TMV-hydra-DOX at a final DOX concentration of 0.1 µg/mL for a determined time. After replacing the loading solution with fresh growth medium with 50 nM LysoTracker® Deep Red, 50 nM MitoTracker® Green and 10 µM Hoechst 33342 for 30 min, the cells are imaged directly on Nikon Eclipse Ti confocal laser scanning microscopy (CLSM) with a TDKAI HIT


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live cell imaging system. The cells are imaged by collecting fluorescence channel of 425 nm 475 nm (Hoechst 33342 for nucleus), 510 nm - 550 nm (MitoTracker® Green for mitochondria), 570 nm - 610 nm (DOX) and 650 nm - 690 nm (LysoTracker® Deep Red for lysosomes). 2.9 In vivo therapeutic and side effect evaluation on HeLa bearing Balb/c-nu mice HeLa cells are implanted into female Balb/c-nu mice at 15-17g body weight. When the tumor volume reached 80-100 mm3, the mice are injected intratumor with cRGD-TMV-hydra-DOX (2 mg DOX/kg body weight; n = 6), free DOX (2 mg DOX/kg body weight; n = 6) and saline (100 µL; n = 6) every two days. The body weight of mice, maximum diameter of tumor (A) and minimum diameter of tumor (B) are measured every several days. The tumor volume is calculated as following: Tumor Volume = 1/2 × A × B2 At day 24, all the three group mice are put to death and the ex vivo tumors are weighted. 3. Results and Discussion 3.1 Synthesis For each TMV coat protein (TMVCP), there are three chemical reaction sites in the rod-like TMV protein cage, which are the glutamic acids 97 and 106 residues on TMV interior surface (providing reactive carboxyl groups) and the tyrosine 139 residues on TMV exterior surface (providing reactive phenolic hydroxyl group). Herein, we labeled the tyrosine 139 residues on TMV exterior surface with alkyne bonds following a proven protocol,25 and conjugate diazonium cRGD through click reaction. In the TMV cavity, hydrazine bond was introduced by the EDCmediated conjugation between the glutamic acid residues and adipic acid dihydrazide. Then DOX was chemically loaded into the cavity through the acid cleavable hydrazone bond. This TMV templated DOX carrier decorated with cRGD was referred as cRGD-TMV-hydra-DOX in


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the following. As Figure 1 shows, with the selective affinity between cRGD and the αvβ3 integrins, this cRGD-TMV-hydra-DOX nano carrier should enter into cancer cells through integrin-mediated endocytosis.

Figure 1. Schematic illustration for the design of cRGD-TMV-hydra-DOX nanocarrier and its integrin-mediated endocytosis. After the chemical modifications, the integrity of TMV was characterized by transmission electron microscope (TEM) (Figure 2a, b) and dynamic light scattering (DLS) (Figure S1 in supporting information). From TEM, the rod-like morphology maintains and there is no obvious degradation. DLS data show that some slight broken of the rod-like nanoparticle happened


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during the chemical conjugation, resulting in a smaller hydrodynamic radius with wider polydispersion. Zeta potential data (Figure S2 in supporting information) indicate that TMV still behaves negative charge after the surface modification. In Figure 2c, compared to the UV-Vis spectrum of TMV (black line), the disappearance of the 260 nm - 280 nm peak and the emergence of a new broad band between 300 nm and 400 nm are the proofs for alkyl functionalization (green line).25, 26 And in the spectrum of cRGD-TMV-hydra-DOX, the new absorption peak at 480 nm confirms the conjugation of DOX and the significant decrease in absorbance at 330 nm implies the click reaction between diazonium cRGD and alkyne.26 Moreover, in the fluorescence emission spectrum of cRGD-TMV-hydra-DOX (Figure 2d, red line), the signature emission peak of DOX emerges, also confirming the DOX conjugation. Figure 2e shows the elution time of RNA (260 nm, black line), TMVCP (280 nm, blue line) and DOX (480 nm, red line) for the cRGD-TMV-hydra-DOX. The exactly same elution volume of RNA, TMVCP and DOX illustrated that TMV kept its viral assemble structure after the chemical reaction and DOX has been loaded into the TMV cavity. The chemical modifications of interior and exterior surface of TMV were further confirmed by SDS-PAGE analysis (Figure 2f). The lane 1 and lane 2 show the protein bands of TMV and TMV-hydra, respectively. The emergence of two protein bands in lane 2 indicates that partial glutamic acids residues on TMV interior surface are conjugated with adipic acid dihydrazide, resulting TMVCP band and TMVCP-hydra band, which have different molecular weights. And the existence of red emission band in lane 3 revealed that DOX was chemically conjugated onto the TMVCP. For the exterior surface modification, the lane 4 and lane 5 show the protein bands of TMV and alky-TMV respectively. The alky-TMVCP has a molecular weight increase compared to TMVCP, so its protein band has a slightly up moving in lane 5. In lane 6, the two protein bands mean that partial alky-TMVCP


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was modified with cRGD. The lower band is the non-modified alky-TMVCP, and the upper band is TMVCP-cRGD with a higher molecular weight. By gray scale analysis through ImageJ software, the cRGD modification efficiency was estimated as 48.8%, thus about 1039 cRGD molecules were immobilized onto one single virus. Through UV-Vis spectrophotometer to confirm DOX concentration, together with the Modified Lowry Protein Assay Kit (Solarbio) to determine the exact TMV concentration, the conjugation density of DOX was calculated as 0.22 DOX molecules per TMVCP (469 DOX molecules per virus). Due to the high molecular weight of TMVCP (17540 Da), drug loading content is relative low (0.7 wt%).


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Figure 2. (a, b) TEM images for TMV (a) and cRGD-TMV-hydra-DOX (b), scale bars 100 nm. (c) UV-Vis spectra of TMV (black line), alky-TMV (green line) and cRGD-TMV-hydra-DOX (red line). (d) Fluorescence emission spectra of TMV (black line) and cRGD-TMV-hydra-DOX (red line) excited at 480 nm. (e) FPLC spectra of cRGD-TMV-hydra-DOX. The black line, blue line and red line collected at 260 nm, 280 nm and 480 nm showed the elution curves of RNA, TMVCP and DOX, respectively. (f) SDS-PAGE analysis to prove the interior (the upper lanes) and exterior (the lower lanes) surface modification of TMV. Lane 1 and lane 2 are TMV and TMV-hydra bands, respectively, stained by coomassie brilliant blue; lane 3 is TMV-hydra-DOX fluorescent band excited by UV lamp. Lane 4, lane 5 and lane 6 are TMV, alky-TMV and TMVcRGD bands, respectively, stained by coomassie brilliant blue. 3.2 In vitro drug release An ideal drug carrier should have the ability to retain the drug during the transport in the blood compartments, but releases the drug efficiently in the tumor cells.27 pH triggered release is a frequently used approach to minimize premature drug release from a stable carrier. Considering that the hydrolysis of hydrazone bond is sensitive to low pH value, the conjugated DOX could be released from the carrier in tumor microenvironment. Here, we investigate the release behavior of DOX from the cRGD-TMV-hydra-DOX nanocarrier by using a dialysis membrane (molecular weight cut off 1,000 kDa) in 0.1 M pH 7.4 phosphate buffer solution, pH 6.3 phosphate buffer solution and pH 5.0 acetic acid buffer solution, at 37 ℃. From the releasing kinetics in Figure 3, the pH value remarkably affects the releasing behavior. At pH 7.4, the physiological pH, only less than 20% DOX is leaked within a 120 h period. At pH 6.3, which is the extracellular pH value of tumor site and the intracellular endosome pH value, the DOX release is activated and about 50% of drug was liberated during 120 h. In comparison, at pH 5.0, which is the lysosome


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pH value, the acidic environment significantly accelerates the drug release. Up to 70% of drug was robustly released during the first day and totally 80% of DOX was liberated within 120 h. Such pH-dependent release behavior is entirely in character for the ideal drug release.

Figure 3. In vitro DOX release from the cRGD-TMV-hydra-DOX at different pH values. 3.3 Internalization by HeLa cells and intracellular distribution cRGD is a frequently used tumor homing peptide to target αvβ3 integrins, which are over expressed on both tumor cells and the neovasculature in many forms of cancer.17-19 To demonstrate that the cRGD functionalized TMV enters into HeLa cells through integrinmediated endocytosis pathway, the cells were pretreated with different concentration of cRGD for 1 hour and then co-incubated with cRGD-TMV-hydra-DOX for another 2 hours. From the flow cytometric analysis (Figure 4), at a pretreated cRGD concentration of 50 and 100 µg/mL, the cellular uptake of cRGD-TMV-hydra-DOX was significantly inhibited to 73 % and 49 % of that without cRGD pretreatment. And as a negative control, for the non-cRGD-functionalized TMV-hydra-DOX, the existence of free cRGD did not bring significant difference in cellular uptake behavior. Besides, after a 2-hour incubation, the cRGD functionalized TMV showed approximately 4-fold increase in uptake efficiency compared with the non-functionalized TMV.


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This indicates that cRGD peptide could specifically bind to HeLa cells and considerably improve the cellular internalization.

Figure 4. Internalization efficiency of cRGD-TMV-hydra-DOX and non-cRGD-functionalized TMV-hydra-DOX into HeLa cells pre-treated with different concentration of cRGD. (a) and (b) are the histogram and columnar statistics from flow cytometry, respectively. The destination of the endocytosed substances is different depending on their composition and surface nature. Without subcellular organelle targeting ligand or cellular penetrating ligand, most endocytosed materials end up in endo/lysosomes. Figure S3 (see supporting information) shows that the TMV-hydra-DOX without cRGD decoration ended up in lysosomes. Figure 5 shows the intracellular distribution of cRGD-TMV-hydra-DOX in HeLa cells after 24 hours’ incubation. The nanoparticles, cell nuclei, endo/lysosomes and mitochondria were shown in red (Figure 5a), blue (Figure 5a-e), light blue (Figure 5b) and green (Figure 5c), respectively. It is shown in Figure 5d, the cRGD-TMV-hydra-DOX was mostly accumulated in the endo/lysosomes (the colocalized region showing pink), while no obvious colocalization happened between cRGDTMV-hydra-DOX and mitochondria (the coinciding color of red and green should be yellow).


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This indicated successful internalization of cRGD-TMV-hydra-DOX into endo/lysosomes, rather than mitochondria or cytoplasm. In a normal mammal cell, the pH value in every organelle is highly regulated: the mitochondria pH is basic (~8); cytosolic pH is slightly more acidic (~7.2); the endo/lysosomes show acidic (4.7 - 6.5). The low pH value of endo/lysosomes is helpful to the hydrolysis of hydrazone bond of trapped cRGD-TMV-hydra-DOX, to release DOX free. We further use the Pearson’s correlation coefficient, which is obtained from the CLSM software, to research the endosomal escape of DOX. Pearson’s correlation coefficient is one of the most popular and useful evaluations to characterize the degree of overlap between images, usually two channels in a multidimensional microscopy image recorded at different emission wavelengths. From Figure 5f, at the beginning of co-incubation, cRGD-TMV-hydra-DOX has not highly accumulated in endo/lysosomes, with the Pearson’s correlation of ~0.55. At the 24 hours’ time point, the nanoparticles co-localized with endo/lysosomes perfectly (see Figure 5d), with Pearson’s correlation of ~0.87. In the acidic microenvironment of endo/lysosomes, it is much easier for the hydrazone bond, the connecting chemical bond between DOX and TMV interior surface, to be hydrolyzed. Then the chemically conjugated DOX could be released from the TMV cavity and escape from endo/lysosomes, the evidence for which is the decreasing of Pearson’s correlation between DOX and LysoTracker after 24 hours. Then the released DOX will accumulate in the cell nuclei (the Pearson’s correlation between DOX and Hoechst is continuously increasing from 0.02 to 0.48), and interact with DNA.


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Figure 5. (a-e) Intracellular distribution of cRGD-TMV-hydra-DOX incubated in HeLa cells for 24 hours, getting from confocal laser scanning microscopy (CLSM). Blue color in (a-e) is imaged for cell nuclei marked by Hoechst 33342. (a-c) show the location of DOX (red in a), endo/lysosomes (light blue in b) and mitochondria (green in c), respectively. (d) and (e) are the colocalization of DOX with endo/lysosome and mitochondria, respectively. (f) shows the Pearson’s correlation coefficient getting from the CLSM software. Data missing in line of DOX - Hoechst at 72 hour is due to the serious apoptosis of cell and the damage of cell nuclei. 3.4 In vitro Anti-cancer efficiency for HeLa cells To investigate the in vitro anti-tumor potential of this TMV based DOX carrier, the cell growth inhibition and cell apoptosis were assayed. Upon exposure of the HeLa cell line to different concentrations of cRGD-TMV-hydra-DOX and free DOX for 24 h and 48 h, the cell growth inhibition was studied using Cell Counting Kit-8 (CCK-8) assays. For many nanoparticle


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loaded DOX, the slow intracellular release of DOX may make its inhibition efficiency lower than free DOX. From Figure 6a and 6b, the TMV loaded DOX show similar inhibition efficiency as free DOX on HeLa cells. This may benefit from the high invasion capacity of cRGD-TMVhydra-DOX. Cell apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit. Figure 6c-d showed the percentage of early apoptosis (Q3 quadrant) and late apoptosis (Q2 quadrant) cells treated with different DOX formulations. The cRGD-TMV-hydra-DOX had much higher apoptosis efficiency (totally 29.1% of early and late apoptosis cells) than free DOX (totally 10.22% of early and late apoptosis cells). Overall, the cell growth inhibition and apoptosis studies confirmed that the cRGD-TMV-hydra-DOX vector achieved similar cell growth inhibition and much stronger apoptosis efficiency than free DOX.


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Figure 6. (a, b) Cell growth inhibition of cRGD-TMV-hydra-DOX and free DOX on HeLa cells at 24 h (a) and 48 h (b). (c, d) Cell apoptosis assay of cRGD-TMV-hydra-DOX (c) and free DOX (d) on HeLa cells for 24 h, at a DOX concentration of 0.3 µg/mL. 3.5 In vivo anti-cancer efficiency and side effect for HeLa bearing Balb/c-nu mice To assess the therapeutic effects and DOX-associated toxicity, HeLa-bearing Balb/c-nu mice model was performed. Considering the rapid blood clearance of TMV,16 we chose the intratumor injection on the mice. The tumor volume (Figure 7a) calculated from the tumor maximum and minimum diameters could be used for the evaluation of the anti-tumor effect of different formulations. The results showed that the tumors treated with saline grew rapidly, and the free DOX treatment could remarkably reduce the tumor growth rate at the beginning. The tumors treated with cRGD-TMV-hydra-DOX grew at a similar rate as control group at beginning, and started to show significant suppressing of the tumor growth at day 6. Comparing the three groups, cRGD-TMV-hydra-DOX had similar therapeutic effect as free DOX: The tumor volume of mice treated with cRGD-TMV-hydra-DOX and free DOX was only 16.5% and 23.2%, respectively, of that of the saline group, at the end of the treatment (day 24). The ex vivo tumor tissues from different formulations (see Figure 7a inset) were weighted as 0.28 ± 0.19 g, 0.29 ± 0.08 g and 0.96 ± 0.60 g for cRGD-TMV-hydra-DOX, free DOX and saline, respectively. In addition, body weight of mice was monitored after injection of different formulations to evaluate the in vivo drug toxicity, and the body weight curves were displayed in Figure 7b. As depicted, the body weights of saline control group were higher than those of other groups owing to the remarkable increase in tumor volumes and the growth of mice. The administration of free DOX significantly decreased the body weight, indicating the severe side effects of free DOX. On the contrary, cRGD-TMV-hydra-DOX did not obviously affect the mice body weight, demonstrating


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that the peptide cRGD remarkably increased tumor homing ability, managing much lower adverse effect. Overall, this plant viral vector shows similar therapeutic effects as free DOX but much higher safety.

Figure 7. Antitumor activity and adverse effect of cRGD-TMV-hydra-DOX and free DOX. HeLa cells were implanted into female Balb/c-nu mice at 15-17g body weight. When the tumor volume reached 80-100 mm3, the mice were injected intratumor with cRGD-TMV-hydra-DOX (2 mg DOX/kg body weight; n = 6), free DOX (2 mg DOX/kg body weight; n = 6) and saline (100 µL; n = 6) every two days, and this day was designated as day 0. (a) Tumor growth curves for different mice groups. The inset shows the ex vivo image of tumor tissues from different groups at 24 day post injection. (b) The effect of different formulation treatment on mouse body weight. (bars represent means ± SD, n = 6). 4. Conclusions In conclusion, we have demonstrated a plant virus based vector to deliver anticancer drugs. This vector had little drug leakage at the physiological pH (pH 7.4), activated drug release at the extracellular pH value of tumor site and the intracellular endosome pH value (pH 6.3), and a robust release at the lysosome pH (pH 5.0). Benefitting from the tumor homing peptide cRGD,


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this vector entered into HeLa cells through the integrin-mediated endocytosis pathway, ending up in endo/lysosomes. The acidic microenvironment of endo/lysosomes could trigger DOX releasing into cytoplasm and then into cell nuclei. Comparing with free DOX, the cRGD-TMVhydra-DOX vector had similar cell growth inhibition and much higher apoptosis efficiency on HeLa cells, and behaved similar anti-tumor efficiency but much lower side effect on HeLa bearing Balb/c-nu mice. We predict that plant virus based vectors are very promising in the field of drug/gene delivery. ASSOCIATED CONTENT Supporting Information. DLS, zeta potential data for TMV and cRGD-TMV-hydra-DOX, and CLSM images for HeLa cells treated by TMV-hydra-DOX. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the 973 Program (2013CB933800) and National Natural Science Foundation of China (Grant No. 51303191, 21304103, 21474123 and 51173198). REFERENCES (1) Smith, A. E.; Helenius, A., How Viruses Enter Animal Cells. Science 2004, 304, 237-242.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Liang, K.; Such, G. K.; Johnston, A. P. R.; Zhu, Z.; Ejima, H.; Richardson, J. J.; Cui, J.; Caruso, F., Endocytic pH-Triggered Degradation of Nanoengineered Multilayer Capsules. Adv. Mater. 2014, 26, 1901-1905. (3) Aljabali, A. A.; Shukla, S.; Lomonossoff, G. P.; Steinmetz, N. F.; Evans, D. J., CPMVDOX Delivers. Mol. Pharm. 2013, 10, 3-10. (4) Huh, Y. M.; Lee, E. S.; Lee, J. H.; Jun, Y. w.; Kim, P. H.; Yun, C. O.; Kim, J. H.; Suh, J. S.; Cheon, J., Hybrid Nanoparticles for Magnetic Resonance Imaging of Target-Specific Viral Gene Delivery. Adv. Mater. 2007, 19, 3109-3112. (5) Yoo, J. W.; Irvine, D. J.; Discher, D. E.; Mitragotri, S., Bio-inspired, Bioengineered and Biomimetic Drug Delivery Carriers. Nat. Rev. Drug Discovery 2011, 10, 521-535. (6) Russell, S. J.; Peng, K.-W.; Bell, J. C., Oncolytic Virotherapy. Nat. Biotechnol. 2012, 30, 658-670. (7) Lin, Y.; Zhang, H. P.; Liang, J. K.; Li, K.; Zhu, W. B.; Fu, L. W.; Wang, F.; Zheng, X. K.; Shi, H. J.; Wu, S. H.; Xiao, X.; Chen, L. J.; Tang, L. P.; Yan, M.; Yang, X. X.; Tan, Y. Q.; Qiu, P. X.; Huang, Y. J.; Yin, W.; Su, X. W.; Hu, H. Y.; Hu, J.; Yan, G. M., Identification and Characterization of Alphavirus M1 as a Selective Oncolytic Virus Targeting ZAP-Defective Human Cancers. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E4504-E4512. (8) Liang, M.; Fan, K.; Zhou, M.; Duan, D.; Zheng, J.; Yang, D.; Feng, J.; Yan, X., H-FerritinNanocaged Doxorubicin Nanoparticles Specifically Target and Kill Tumors with a Single-Dose Injection. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 14900-14905. (9) Zhang, L.; Li, L.; Di Penta, A.; Carmona, U.; Yang, F.; Schöps, R.; Brandsch, M.; Zugaza, J. L.; Knez, M., H-Chain Ferritin: A Natural Nuclei Targeting and Bioactive Delivery Nanovector. Adv. Healthcare Mater. 2015, 4, 1305-1310.


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Page 22 of 25

(10) Mastrobattista, E.; van der Aa, M. A. E. M.; Hennink, W. E.; Crommelin, D. J. A., Artificial Viruses: a Nanotechnological Approach to Gene Delivery. Nat. Rev. Drug Discovery 2006, 5, 115-121. (11) Miyata, K.; Nishiyama, N.; Kataoka, K., Rational Design of Smart Supramolecular Assemblies for Gene Delivery: Chemical Challenges in the Creation of Artificial Viruses. Chem. Soc. Rev. 2012, 41, 2562-2574. (12) Unzueta, U.; Cespedes, M. V.; Vazquez, E.; Ferrer-Miralles, N.; Mangues, R.; Villaverde, A., Towards Protein-Based Viral Mimetics for Cancer Therapies. Trends Biotechnol. 2015, 33, 253-258. (13) Karjoo, Z.; McCarthy, H. O.; Patel, P.; Nouri, F. S.; Hatefi, A., Systematic Engineering of Uniform, Highly Efficient, Targeted and Shielded Viral-Mimetic Nanoparticles. Small 2013, 9, 2774-2783. (14) Liu, Z.; Qiao, J.; Niu, Z.; Wang, Q., Natural Supramolecular Building Blocks: from Virus Coat Proteins to Viral Nanoparticles. Chem. Soc. Rev. 2012, 41, 6178-6194. (15) Liu, Z.; Gu, J.; Wu, M.; Jiang, S.; Wu, D.; Wang, Q.; Niu, Z.; Huang, Y., Nonionic Block Copolymers Assemble on the Surface of Protein Bionanoparticle. Langmuir 2012, 28, 1195711961. (16) Wu, M.; Shi, J.; Fan, D.; Zhou, Q.; Wang, F.; Niu, Z.; Huang, Y., Biobehavior in Normal and Tumor-Bearing Mice of Tobacco Mosaic Virus. Biomacromolecules 2013, 14, 4032-4037. (17) Shi, H.; Liu, J.; Geng, J.; Tang, B. Z.; Liu, B., Specific Detection of Integrin αVβ3 by Light-up Bioprobe with Aggregation-induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 9569-9572.


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Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Liang, K.; Richardson, J. J.; Ejima, H.; Such, G. K.; Cui, J.; Caruso, F., Peptide-Tunable Drug Cytotoxicity via One-Step Assembled Polymer Nanoparticles. Adv. Mater. 2014, 26, 23982402. (19) Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.; Arnaout, M. A., Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with an ArgGly-Asp Ligand. Science 2002, 296, 151-155. (20) Yuan, Y.; Kwok, R. T.; Tang, B. Z.; Liu, B., Targeted Theranostic platinum(IV) Prodrug with a Built-in Aggregation-induced Emission Light-up Apoptosis Sensor for Noninvasive Early Evaluation of its Therapeutic Responses in situ. J. Am. Chem. Soc. 2014, 136, 2546-2554. (21) Brogan, A. P.; Sessions, R. B.; Perriman, A. W.; Mann, S., Molecular Dynamics Simulations Reveal a Dielectric-Responsive Coronal Structure in Protein-Polymer Surfactant Hybrid Nanoconstructs. J. Am. Chem. Soc. 2014, 136, 16824-16831. (22) Shukla, S.; Eber, F. J.; Nagarajan, A. S.; DiFranco, N. A.; Schmidt, N.; Wen, A. M.; Eiben, S.; Twyman, R. M.; Wege, C.; Steinmetz, N. F., The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft-Matter Nanorods. Adv. Healthcare Mater. 2015, 4, 874-882. (23) Ding, X.; Liu, Y.; Li, J.; Luo, Z.; Hu, Y.; Zhang, B.; Liu, J.; Zhou, J.; Cai, K., HydrazoneBearing PMMA-Functionalized Magnetic Nanocubes as pH-responsive Drug Carriers for Remotely Targeted Cancer Therapy in vitro and in vivo. ACS Appl. Mater. Interfaces 2014, 6, 7395-7407. (24) Lee, C.-H.; Cheng, S.-H.; Huang, I. P.; Souris, J. S.; Yang, C.-S.; Mou, C.-Y.; Lo, L.-W., Intracellular pH-Responsive Mesoporous Silica Nanoparticles for the Controlled Release of Anticancer Chemotherapeutics. Angew. Chem. Int. Ed. 2010, 49, 8214-8219.


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(25) Bruckman, M. A.; Steinmetz, N. F., Chemical Modification of the Inner and Outer Surfaces of Tobacco Mosaic Virus (TMV). Methods Mol. Biol. 2014, 1108, 173-185. (26) Chen, L.; Zhao, X.; Lin, Y.; Huang, Y.; Wang, Q., A Supramolecular Strategy to Assemble Multifunctional Viral Nanoparticles. Chem. Commun. 2013, 49, 9678-9680. (27) Sun, Q.; Radosz, M.; Shen, Y., Challenges in Design of Translational Nanocarriers. J. Controlled Release 2012, 164, 156-169.


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Tobacco Mosaic Virus-Based 1D Nanorod-Drug Carrier via the

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