Delivery of Liver-Specific miRNA-122 Using a Targeted

Subscriber access provided by WEBSTER UNIV

Biological and Medical Applications of Materials and Interfaces

Delivery of Liver Specific miRNA-122 Using a Targeted Macromolecular Prodrug Toward Synergistic Therapy For Hepatocellular Carcinoma Qian Ning, Yufeng Liu, Pengju Ye, Pei Gao, Zhiping Li, Siyue Tang, Dong-xiu He, Shengsong Tang, Hua Wei, and Cui-Yun Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00634 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38 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

ACS Applied Materials & Interfaces

Delivery of Liver-Specific miRNA-122 Using a Targeted Macromolecular Prodrug Toward Synergistic Therapy For Hepatocellular Carcinoma Qian Ning 1,2,3,#, Yu-Feng Liu 1,3,#, Peng-Ju Ye 1,3, Pei Gao 4, Zhi-Ping Li 1,3, Si-Yue Tang1,3, Dong-Xiu He 1,3, Sheng-Song Tang 1,2,3, Hua Wei 1,2,3,*, Cui-Yun Yu 1,2,3,* 1Hunan

Province Cooperative Innovation Center for Molecular Target New Drug Study,

University of South China, Hengyang, 421001, China 2Hunan

Province Key Laboratory for Antibody-based Drug and Intelligent Delivery System,

Hunan University of Medicine, Huaihua, 418000, China 3

Institute of Pharmacy & Pharmacology, Learning Key Laboratory for Pharmacoproteomics

of Hunan Province, University of South China, Hengyang, 421001, China 4Chemistry

Department, Eastern Kentucky University, Richmond, Kentucky, 40475, USA

KEYWORDS: miRNA-122, GC-FU, co-delivery, macromolecular prodrug, hepatocellular carcinoma, synergistic therapy

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

ABSTRACT: Hepatocellular carcinoma (HCC) has consistuted a great threat to human health. The elegant combination of gene therapy and chemotherapy by nanocarriers has been repeatedly highlighted to realize enhanced therapeutic efficacy relative to mono-treatment. However, the leading strategy to achieve the efficient co-delivery of gene and drug remains the electrostatic condensation with the nucleic acid and the hydrophobic encapsulation of drug molecules by the nano-carriers, which suffers substantially from premature drug leakage during circulation and severe off-target-associated side effects. To address these issues, we reported in this study the co-delivery of liver-specific miRNA-122 and anti-cancer drug 5-fluorouracil (5-Fu) using a macromolecular prodrug approach, i.e., electrostatic condensation with miRNA-122 using N-galactosylated-chitosan-5-fluorouracil (GC-FU). The delivery efficacy was evaluated comprehensively in vitro and in vivo. Specifically, the biocompatibility of GC-FU/miR-122 nanoparticles (NPs) was assessed by hemolysis activity analysis, BSA adsorption test, and cell viability assay in both normal liver cells (L02 cells) and endothelial cells (ECs cells). The resulting co-delivery systems showed enhanced blood and salt stability, efficient proliferation inhibition of HCC cells, and further induction apoptosis of HCC cells, as well as down-regulated expression of ADAM17 and Bcl-2. This strategy developed herein is thus a highly promising platform for an effective co-delivery of miRNA-122 and 5-Fu with facile fabrication and great potential for the clinical translation toward HCC synergistic therapy.


Page 2 of 38

Page 3 of 38 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


INTRODUCTION Hepatocellular carcinoma (HCC) accounts for 75-85% of primary liver cancers, thus represents a major health burden and is predicted to be the fourth leading cause of cancer deaths worldwide in 2018.1-3 Approximately 383,000 people die from liver cancer every year in China. HCC has been revealed to be an extremely complex disease.4 In the early stages, surgical are suitable curative treatments. In the intermediate or advanced stages, the current curative treatments couldn’t work effectively anymore.5-6 Chemotherapy is the most effective treatment in HCC therapy but strictly limited for the poor targeting efficency and serious side effects.7 The elegant combination of gene therapy and chemotherapy by nanocarriers has been repeatedly highlighted to realize enhanced therapeutic efficacy relative to mono-treatment. However, to our knowledge, the leading strategy to achieve the efficient co-delivery of gene and drug remains the electrostatic condensation with the nucleic acid and the hydrophobic encapsulation of drug molecules by the nano-carriers, which suffers substantially from premature drug leakage during circulation and severe off-target-associated side effects.8 To address these issues, we reported in this study the co-delivery of liver-specific miRNA-122(miR-122) and anti-cancer drug fluorouracil (5-Fu) using a macromolecular prodrug approach. MiR-122 is a liver-specific miRNA that accounts for 70% of the total miRNA population and is also the most abundant one in the liver tissue.9 It is normally expressed greatly in the human liver cell, but has a very low expression in most



Page 4 of 38

tissues.10-11 Nevertheless, miR-122 plays a critical role in regulating hepatocyte development, differentiation, lipid metabolism, and stress response.12 miR-122 could inhibit

hepatocarcinogenesis,

epithelial-mesenchymal

transition

(EMT)

and

angiogenesis by targeting Bcl-w, Bcl-2, IGF-1R, Wnt1, CUTL1, AKT3, ADAM10, ADAM17 and so on.13-17 Both viral and non-viral vectors have been extensively used for gene transfer.18-19 While high transfection efficiency, the clinical translation of viral gene vectors has been seriously hampered by their serious immunotoxicity, adverse effects, scaling-up difficulties, high production cost and risk of mutagenesis.20-22 Cationic polymers and lipids as the most investigated non-viral vectors for plasmid delivery can efficiently condense nucleic acid via electrostatic interactions to form a cationic polyplex, which contributes to the charge-enhanced cellular uptake and higher miRNA-mediated gene-overexpression effect.23-27 The development of cationic polymer-based miRNA delivery systems is highly dependent on the understanding and adoption of the structure-property relationship of cationic polymers for advanced materials design including cytotoxicity, miRNA binding efficiency, and polyplex stability.23 In this work, to realize optimal synergistic anti-tumor efficacy, we developed the co-delivery systems of 5-Fu and miR-122 by GC-FU-based nanovector capable of releasing the gene and drug at the desired target in a precisely controlled manner. A novel approach was reported herein for the synthesis of miR-122 and 5-Fu co-delivery system via a combination of an electrostatically driven physical assembly and an


Page 5 of 38 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


amide condensation reaction. We utilized galactosylated chitosan (GC) and fluorouracil acetic acid (FUA) to prepare macromolecular prodrugs of galactosylated chitosan-5-fluorouracil acetic acid (GC-FU), and introduced liver specific expression gene, miR-122, to compose a macromolecule prodrug/gene co-delivery system. Finally, the anti-tumor effects of the co-delivery systems were evaluated comprehensively on HCC cells in vitro and in vivo.

RESULTS AND DISCUSSION We have synthesized GC-FU-NPs for HCC therapy in our previous study.28-29 The resulting GC-FU-NPs indicated excellent biocompatibility in the hemolysis activity examinations and BSA adsorption. In vitro assessment also proved a lower cytotoxicity of GC-FU-NPs relative to free 5-Fu. This system was therefore used in the current study for the co-delivery of 5-Fu and miR-122 with synergistic effects for cancer treatment.

Physicochemical characteristics of GC-FU/miR-122-NPs The detailed characteriation of the chemical structure of GC-FU copolymer was preiously reported.29 The typical FT-IR and 1H NMR spectra were included in the supporting information as well (Figure S2 and S3). It is worth pointing out that the repeated attempts to characterize the molecular weight of GC-Fu by GPC measurements in various aqueous (acetic buffer with different pH values) and organic (DMF and THF) phases



Page 6 of 38

were unsuccessful. We couldn’t acquire any elution peaks for GC-Fu due to certain unclear reasons. Neither GC nor GC-FU can form NPs due to their hydrophilicity. However, the addition of sodium tripolyphosphate can promote the formation of GC-NPs and GC-FU-NPs by electrostatic interactions. The mean size and zeta potential (ζ) of GC-FU-NPs and GC-NPs are determined by dynamic light scattering (DLS) to be 178.5±2.8 nm, 27.0±0.45 mV and 89±1.2 nm, 31.0±0.45 mV, respectively (Figure 1). The formation of GC-FU/miR-122-NPs was illustrated in Scheme 1. The drug loading content

(LC%)

that

was

defined

as

the

weight

percentage

of

FUA

in

GC-FU/miR-122-NPs was determined to be 22.25% by HPLC. The agarose gel retardation experiment was carried out to investigate the binding ability of miR-122 by GC-FU. The condensation ability of polymer increased with the mass ratio of polymer and miRNA, and the full condensation was observed at the mass ratio higher than 256:1 (Figure 2A). Such low loading content of miR-122 is most likely attributed to the remaining low positive charge and weak condensation ability of GC-FU. The successful loading of miR-122 by GC-FU nanovector was also confirmed by the size and zeta-potential measurements (Figure 2 and 3), which reveals a mean size range from 95±5.2 nm and a slightly positive surface charge of 16.3±1.9 mV at a mass ratio of 1024:1 or higher values. An increase of the mass ratio leads to a decrease of the particle size and an increase of the surface charge. This is expected since the use of more cationic polymers for polyplex formation results in more compact structure and higher positive charge of polyplexes. Therefore, in addition to the nanosize-enhanced


Page 7 of 38 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


passive tumor targeting via the enhanced permeability and retention (EPR) effect, the positive surface potential of a polycation further contribute to the endocytosis through charge-mediated interactions with the cell membrane.

30-32

To acquire the optimal

mass ratio of GC-FU and miR-122 for efficient plasmid transfection, miR-122 plasmid was labeled with a green fluorescent protein, GFP, and the percentage of transfected live cells in all the cell population was quantified by flow cytometry (Figure 2B). The cells detected with the green fluorescence of GFP were regard as the transfected live cells. The results show clearly an increased trend of the transfected live cells with an increase of the mass ratio of GC-FU and miR-122. Notably, the transfected live cells using a high ratio of 1024:1 reached a great value of approximately 95%, which confirms the optimal transfection mediated by GC-Fu at this ratio. Furthermore, TEM images (Figure 3A), SEM images (Figure 3B) and distribution of particle size (Figure 3C) revealed that the GC-FU/miR-122-NPs polyplexes prepared at a mass ratio of 1024:1 show uniform and regular spherical shape with an average diameter of 100 nm. The invisibility of drug crystal indirectly reflects the successfully simultaneous loading of miR-122 and 5-Fu by GC-FU based nanovector. The release profiles of 5-Fu from GC-FU/miR-122-NPs were investigated at three different pHs of 7.4, 6.8 and 5.0 and 37 oC. The results showed clearly pH-dependent cumulative release of 5-Fu with the greatest percentage of 85.4% at pH 5.0 after 48 hrs (Figure 3E). The recorded sustained release profiles are believed to exert greater therapeutic efficacy for cancer.



Biocompatibilities of GC-FU/miR-122-NPs Excellent biocompatibilities is the most important requirement for nanodrugs because the ultimately clinical applications. Therefore, the abilities of nanomedicine to resist protein adsorption and eliminate hemolysis were highly desirable. Firstly, the protein adsorption assay was performed with BSA. As shown in Figure 4A, under all the tested concentrations (0.125, 0.25, 0.50, 1.0 g·L-1) and incubation periods (2, 4 and 6 hrs), the GC-FU/miR-122-NPs only showed a slight adsorption of BSA, while free 5-Fu interacted strongly with BSA. In addition, the hemolysis radio (HR) of GC-FU/miR-122-NPs was evaluated by a hemolysis assay. As presented in Figure 4B, when the RBCs were exposed to the GC-FU/miR-122-NPs at different concentrations for 2 hrs. The GC-FU/miR-122-NPs showed only less than 5% hemolysis to RBCs even at a high concentration of 1.0 g·L-1, suggesting that GC-FU/miR-122-NPs have desirable properties for practical applications. To investigate the cell biocompatibility of GC-FU/miR-122, the proliferation inhibitions experiment of normal cells(ECs and L02 cells) were evaluated by a CCK-8 assay. As displayed in Figure 4C and Figure 4D, the cell survival rate was detected after dealing with GC-NPs at different concentrations for 48 hrs. A high survival ratio of 90% for both ECs and L02 cell lines incubated with GC-NPs even at a high concentration of 0.10 g·L-1 confirms apparently the excellent cell biocompatibility of the GC-FU/miR-122-NPs.


Page 8 of 38

Page 9 of 38 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


Cellular internalization To provide a direct evidence that miR-122 entered the cells, miR-122 plasmid was labled with GFP and examined under a confocal microscope.33-34 The results demonstrated substantial localization of miR-122 plasmid in the cytoplasm and nucleus (Figure 5B). The asialoglycoprotein receptor (ASGP-R) could bind to galactose and N-acetylgalactosamine-terminaled glycoproteins. The receptor-mediated cellular uptake of 5-Fu and miR-122 from GC-FU/miR-122-NPs were evaluated by competition studies on HPLC and confocal microscopy, respectively (Figure 5). The significantly decrease of the recorded amount of 5-Fu and the observed green fluorescence of miR-122 with the pretreatment of glucose and galactose compared to those

of

non-treated

samples

strongly

supported

the

glucose/galactose

receptor-mediated endocytosis.

Cellular proliferation inhibitions The proliferation inhibitions of HCC cells (SMMC7721 and HepG2 cells) were evaluated by CCK-8 assay (Figure 6A and S1). All the formulations including free 5-Fu, GC-FU-NPs, GC/miR-122-NPs and GC-FU/miR-122-NPs show a both time and dose-dependent proliferation inhibition effects for HCC cells. Compared to 5-Fu, the GC-FU/miR-122 nanomedicine exhibited significantly greater activity in inhibiting proliferation of HepG2 and SMMC7721 cells, which is likely attributed to the



co-delivery of miR-122 sequence by GC-FU/miR-122-NPs nanomedicine toward enhanced therapeutic efficacy of 5-Fu.

Cell apoptosis and cell cycle arrest To further clarify the apoptosis mechanism of HepG2 cells cultured with the nanomedicines, cells were double stained for viability (with PI) and apoptosis (with Annexin V-FITC) investigations. GC-FU/miR-122-NPs induced higher apoptosis than GC-FU-NPs and GC/miR-122-NPs, most likely due to the synergistic effect of drug and gene (Figure 6B). We also compared the effects of this formulation on cell cycle progression. As displayed in Figure 6C, after 24 hrs incubation, GC-FU/miR-122-NPs and GC-FU-NPs at 0.25 g·L-1 could arrest cell growth at the same G2/M phase, with the increased rates being 27.46% for GC-FU-NPs and 28.99% for GC-FU/miR-122-NPs, respectively, compared to 10.99% for control and 4.15% for free 5-Fu. This suggested that GC-FU/miR-122-NPs might play different roles in treatments.

In vitro anti-metastatic effects Firstly, the wound healing assay was carried out to evaluate the inhibition ability of GC-FU-NPs, GC/miR-122-NPs, GC-FU/miR-122-NPs and free 5-Fu on cell motility and interactions. As demonstrated in Figure 7A, control group displayed strong migration healing ability on the scratch in 24 hrs, which suggested the high metastasis


Page 10 of 38

Page 11 of 38 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


of HepG2 cells. All the agents indicated obviously inhibitory effect, compared to control. The healing rate of free 5-Fu, GC/miR-122-NPs, GC-FU-NPs and GC-FU/miR-122-NPs groups, is 55.42%, 21.7%, 31.36% and 11.30%, respectively. Similar trends were observed in SMMC7721 cells as well with the healing rate of 41.79%, 63.57%, 23.21%, and 5.36% for GC-FU-NPs, free 5-Fu, GC/miR-122-NPs and GC-FU/miR-122-NPs groups, respectively (Figure S1A and C). The results confirmed the strongest inhibitory effect of GC-FU/miR-122-NPs among all the formulations. The invasion experiment was carried out to further estimate the effects of nanomedicine on cellular motility ability of HCC cells. The control group showed the high invasion ability, which is consistent with the manifestation of wound healing assay (Figure 7B). All agents demonstrated inhibitory effects on cellular motility of HepG2

cells.

The

formulations

of

GC-FU/miR-122-NPs

GC/miR-122-NPs,

GC-FU-NPs and 5-Fu displayed somewhat inhibition on invasion of HepG2 cells with invasion rates at 32.67%, 34.72%, 43.8% and 53.6%, respectively. But the influences of GC-FU/miR-122-NPs on cell invasion were more remarkable than that of 5-Fu and GC-FU-NPs. Similar trends were also observed in SMMC7721 cells with the invasion rates of 80.24%, 91.04%, 40.40%, and 36.42% for GC-FU-NPs, free 5-Fu, GC/miR-122-NPs and GC-FU/miR-122-NPs groups, respectively(Figure S1B and D). The results showed better inhibitory effect of GC-FU/miR-122-NPs.



The expression of Bcl-2, ADAM17 The anti-apoptotic Bcl-2 family members are viable targets for HCC given the fact that they are up-regulated in these tumors. This is also supported by many preclinical studies that show that Bcl-2 family members are implicated in apoptosis regulation in model systems of these tumors.

35

ADAM17 modulated vital cellular functions

through cleavage of transmembrane substrates including TGF-α, amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). Furthermore, ADAM17 weaken the adhesion of cells by splicing cell adhesion molecules, which provides a prerequisite for the metastasis of tumor cells.

36, 37

As exhibited in Figure 7E, GC-FU/miR-122-NPs

mediated slightly decreased expression of Bcl-2 and Adam17 proteins with statistical significances relative to the other groups. Note that the differences between the expression levels of Bcl-2 for each formulation is slight based on the image results, however, the decreased trend observed from the column plot is statistical significant between each group. The results were consistent with the flow cytometer detection of apoptosis and the data of migration and invasion, which suggests the significant metastasis of HCC and constitutes the foundation for the following in vivo evaluations.

In vivo antitumor efficacies To estimate the hepatoma targeting ability and antitumor efficacy of nanomedicine in vivo ， HepG2 cells were first culcured for subcutaneous xenografts in the armpit of BALB/c nude mice. When tumors volume reached to more than 200 mm3, in about


Page 12 of 38

Page 13 of 38 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


two weeks after cell implantation, the mice were randomly divided into 7 groups (8 mice in each group), and then were given treatments on day 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. The 7 groups were given as follows, saline, 5-Fu, free 5-Fu + miR-122 and

nanomedicines

from

GC-NPs,

GC-FU-NPs,

GC/miR-122-NPs,

GC-FU/miR-122-NPs. As shown in Figure 8, the tumors volume and body weight were measured in the therapeutic duration of 20 days. The results revealed that the GC-FU/miR-122-NPs-based co-delivery nanomedicine were the most effective construct to suppress the tumor growth, relative to free 5-Fu, GC-FU prodrug and GC/miR-122-NPs formulation. To further explain the anti-tumor effect in a molecular level, the tumor tissue was sliced up, further stained with H&E, whereafter under observation by microscopy. Spontaneous necrosis would occur on account of intraplastic hypoxia in tumor, hence H&E were photographed near the edge of the tumor. For a clear observation, in the H&E staining, all of tissues were stained with dark blue and showed a full state. As demonstrated in Figure 9A, the mice treated with saline, GC-NPs, free 5-Fu or GC/miR-122-NPs displayed insignificant necrosis or apoptosis. However, most of the tumors treated with GC-FU-NPs or GC-FU/miR-122-NPs were necrosis or apoptosis, and the results were in line with the anti-tumor effect. For immunohistochemistry analysis, as shown in Figure 9, ADAM17(B) or Bcl-2(C) could be down-regulated by GC-FU/miR-122-NPs, GC-FU-NPs, GC/miR-122-NPs and free 5-Fu in vivo.



As for safety assessment, as demonstrated in Figure 8B, during the whole treatment, there were insignificant weight loss in the tumor-bearing mice after the treatment of nanomedicines, supporting the excellent biocompatibility of the used nanomedicines. Nevertheless, the tumor-bearing mice treated with 5-Fu using equivalent dose showed slight weight loss, possibly attributed to the minor acute-phase plasma concentration of 5-Fu. The biodistribution of agents directly reflects their possible toxicity on organs and antitumor effects in vivo, therefore is deemed as a highly crucial parameters for drug delivery systems. For this purpose, the tissue distribution of GC-FU/miR-122-NPs was investigated using the HPLC. The results confirm a substantial accumulation of most free 5-Fu in the liver with the increase of time (Figure 10A). For clinical translations, the following index parameters reflect the function of liver (ALT, AST), heart (CK, CK-MB and LDH) and kidney (BUN and Cr). Chemotherapeutics generally cause grievous toxic and side effects. To evaluate the systematic toxicities of GC-FU/miR-122 nanomedicines and free 5-Fu, variation of clinical index parameters in SD rats after treatment were detected. As demonstrated in Figure 10B, the most parameters of all treatment groups maintained within normal limits, manifesting that the simultaneous use of GC-FU/miR-122 nanomedicines and free 5-Fu unaltered the functionality of liver, heart and kidney of SD rats during the treatments.

CONCLUSIONS


Page 14 of 38

Page 15 of 38 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


In summary, a innovative assembly strategy based on a liver-specific miR-122 and prodrug GC-FU delivery system was developed to realize the co-delivery of 5-Fu and miR-122 for HCC therapy. This strategy resulted in markedly induced HepG2 cells apoptosis, cell cycle arrest, inhibited HCC cellls proliferation, migration and invasion, and down-regulated expression of Bcl-2 and ADAM17 in HepG2 cells as well as greater antitumor efficacy in vivo, thus addressed the tradeoff amongst miRNA delivery efficiency and toxicities of system. This strategy promoted the clinical translation of miRNA treatment by developing a facile route to realize effective dlelivery of miR-122. The GC-FU/miR-122-NPs developed herein represent a promising platform for HCC synergistic therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental section, additional in vitro anti-metastatic effects, cellular proliferation inhibitions of SMMC7721 cellls, FT-IR and 1H-NMR spectra are available in Figure S1-S3.

AUTHOR INFORMATION Corresponding Authors *E-mail addresses: [email protected] (C.Y. Yu) or [email protected] (H. Wei)

Present Addresses



Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, 421001, China Author Contributions All authors have given approval to the final version of the manuscript. #These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We deeply acknowledge the sponsorship from National Natural Science Foundation of China (81471777), the Outstanding Youth Fund of Natural Science Foundation of Hunan province (2017JJ1024), Hunan Provincial Science and Technology Plan Key Projects of China (2017SK2183), Hunan Provincial Natural Science Foundation of China (2019JJ50416), and the Chuanshan Talent Program of the University of South China.

REFERENCES (1) Laursen, L. A Preventable Cancer. Nature 2014, 516, S2-S3. (2) Schmidt, S.; Follmann, M.; Malek, N.; Manns, M. P.; Greten, T. F. Critical Appraisal of Clinical Practice Guidelines for Diagnosis and Treatment of Hepatocellular Carcinoma. J. Gastroenterol. Hepatol. 2011, 26, 1779-1786. (3) Yu, C. Y.; Wang, Y. M.; Li, N. M.; Liu, G. S.; Yang, S.; Tang, G. T.; He, D. X.; Tan, X. W.; Wei, H. In Vitro and In Vivo Evaluation of Pectin-Based Nanoparticles for Hepatocellular Carcinoma Drug Chemotherapy. Mol. Pharmaceutics 2014, 11, 638-644.


Page 16 of 38

Page 17 of 38 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


(4) Wang, F. S.; Fan, J. G.; Zhang, Z.; Gao, B.; Wang, H. Y. The Global Burden of Liver Disease: the Major Impact of China. Hepatology 2014, 60, 2099-2108. (5) Koberle, V.; Kronenberger, B.; Pleli, T.; Trojan, J.; Imelmann, E.; Peveling-Oberhag, J.; Welker, M. W.; Elhendawy, M.; Zeuzem, S.; Piiper, A.; Waidmann, O. Serum microRNA-1 and microRNA-122 Are Prognostic Markers in Patients with Hepatocellular Carcinoma. Eur. J. Cancer 2013, 49, 3442-3449. (6) Utsunomiya, T.; Shimada, M.; Morine, Y.; Tajima, A.; Imoto, I. Specific Molecular Signatures of Non-Tumor Liver Tissue May Predict A Risk of Hepatocarcinogenesis. Cancer Sci. 2014, 105, 749-754. (7) Khaliq, N. U.; Sandra, F. C.; Park, D. Y.; Lee, J. Y.; Oh, K. S.; Kim, D.; Byun, Y.; Kim, I. S.; Kwon, I. C.; Kim, S. Y.; Yuk, S. H. Doxorubicin/Heparin Composite Nanoparticles for Caspase-Activated Prodrug Chemotherapy. Biomaterials 2016, 101, 131-142. (8) Teo, P. Y.; Cheng, W.; Hedrick, J. L.; Yang, Y. Y. Co-Delivery of Drugs and Plasmid DNA for Cancer Therapy. Adv. Drug Delivery Rev. 2016, 98, 41-63. (9) Zhou, X.; Cao, P.; Zhu, Y.; Lu, W.; Gu, N.; Mao, C. Phage-Mediated Counting by the Naked Eye of miRNA Molecules at Attomolar Concentrations in A Petri Dish. Nat. Mater. 2015, 14, 1058-1064. (10)Song, K.; Han, C.; Zhang, J.; Lu, D.; Dash, S.; Feitelson, M.; Lim, K.; Wu, T. Epigenetic Regulation of MicroRNA-122 by Peroxisome Proliferator Activated Receptor-gamma and Hepatitis B Virus X Protein in Hepatocellular Carcinoma Cells. Hepatology 2013, 58, 1681-1692. (11)Tsai, W. C.; Hsu, S. D.; Hsu, C. S.; Lai, T. C.; Chen, S. J.; Shen, R.; Huang, Y.; Chen, H. C.; Lee, C. H.; Tsai, T. F.; Hsu, M. T.; Wu, J. C.; Huang, H. D.; Shiao, M. S.; Hsiao, M.; Tsou, A. P. MicroRNA-122 Plays A Critical Role in Liver Homeostasis and Hepatocarcinogenesis. J. Clin. Invest. 2012, 122, 2884-2897. (12)Nakao, K.; Miyaaki, H.; Ichikawa, T. Antitumor Function of microRNA-122 Against Hepatocellular Carcinoma. J. Gastroenterol. 2014, 49, 589-593.



(13) Tsai, W. C.; Hsu, P. W.; Lai, T. C.; Chau, G. Y.; Lin, C. W.; Chen, C. M.; Lin, C. D.; Liao, Y. L.; Wang, J. L.; Chau, Y. P.; Hsu, M. T.; Hsiao, M.; Huang, H. D.; Tsou, A. P. MicroRNA-122, A Tumor Suppressor microRNA that Regulates Intrahepatic Metastasis of Hepatocellular Carcinoma. Hepatology 2009, 49, 1571-1582. (14) Fornari, F.; Gramantieri, L.; Giovannini, C.; Veronese, A.; Ferracin, M.; Sabbioni, S.; Calin, G. A.; Grazi, G. L.; Croce, C. M.; Tavolari, S.; Chieco, P.; Negrini, M.; Bolondi, L. MiR-122/cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells. Cancer Res. 2009, 69, 5761-5767. (15) Xu, H.; He, J. H.; Xiao, Z. D.; Zhang, Q. Q.; Chen, Y. Q.; Zhou, H.; Qu, L. H. Liver-Enriched Transcription Factors Regulate microRNA-122 that Targets CUTL1 during Liver Development. Hepatology 2010, 52, 1431-1442. (16) Xu, J.; Zhu, X.; Wu, L.; Yang, R.; Yang, Z.; Wang, Q.; Wu, F. MicroRNA-122 Suppresses Cell Proliferation and Induces Cell Apoptosis in Hepatocellular Carcinoma by Directly Targeting Wnt/beta-catenin Pathway. Liver Int. 2012, 32, 752-760. (17) Zeng, C.; Wang, R.; Li, D.; Lin, X. J.; Wei, Q. K.; Yuan, Y.; Wang, Q.; Chen, W.; Zhuang, S. M. A Novel GSK-3 Beta-C/EBP Alpha-miR-122-Insulin-Like Growth Factor 1 Receptor Regulatory Circuitry in Human Hepatocellular Carcinoma. Hepatology 2010, 52, 1702-1712. (18) Sun, Q.; Kang, Z.; Xue, L.; Shang, Y.; Su, Z.; Sun, H.; Ping, Q.; Mo, R.; Zhang, C. A Collaborative Assembly Strategy for Tumor-Targeted siRNA Delivery. J. Am. Chem. Soc. 2015, 137, 6000-6010. (19) Wei, H.; Volpatti, L. R.; Sellers, D. L.; Maris, D. O.; Andrews, I. W.; Hemphill, A. S.; Chan, L. W.; Chu, D. S.; Horner, P. J.; Pun, S. H. Dual Responsive, Stabilized Nanoparticles for Efficient In Vivo Plasmid Delivery. Angew. Chem., Int. Ed. 2013, 52, 5377-5381. (20) Wei, H.; Pahang, J. A.; Pun, S. H. Optimization of Brush-Like Cationic Copolymers for Nonviral Gene Delivery. Biomacromolecules 2013, 14, 275-284.


Page 18 of 38

Page 19 of 38 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


(21) Duerner, L. J.; Schwantes, A.; Schneider, I. C.; Cichutek, K.; Buchholz, C. J. Cell Entry Targeting Restricts Biodistribution of Replication-Competent Retroviruses to Tumour Tissue. Gene Ther. 2008, 15, 1500-1510. (22) Mastorakos, P.; Kambhampati, S. P.; Mishra, M. K.; Wu, T.; Song, E.; Hanes, J.; Kannan, R. M. Hydroxyl PAMAM Dendrimer-Based Gene Vectors for Transgene Delivery to Human Retinal Pigment Epithelial Cells. Nanoscale 2015, 7, 3845-3856. (23) Schroeder, A.; Levins, C. G.; Cortez, C.; Langer, R.; Anderson, D. G. Lipid-Based Nanotherapeutics for siRNA Delivery. J. Intern. Med. 2010, 267, 9-21. (24) Zhu, G.; Mei, L.; Vishwasrao, H. D.; Jacobson, O.; Wang, Z.; Liu, Y.; Yung, B. C.; Fu, X.; Jin, A.; Niu, G.; Wang, Q.; Zhang, F.; Shroff, H.; Chen, X. Intertwining DNA-RNA Nanocapsules Loaded with Tumor Neoantigens as Synergistic Nanovaccines for Cancer Immunotherapy. Nat. Commun. 2017, 8, 1482. (25) Yang, X. Z.; Dou, S.; Sun, T. M.; Mao, C. Q.; Wang, H. X.; Wang, J. Systemic Delivery of siRNA with Cationic Lipid Assisted PEG-PLA Nanoparticles for Cancer Therapy. J. Controlled Release 2011, 156, 203-211. (26) Ornelas-Megiatto, C.; Wich, P. R.; Fréchet, J. M. Polyphosphonium Polymers for siRNA Delivery: An Efficient and Nontoxic Alternative to Polyammonium Carriers. J. Am. Chem. Soc. 2012, 134, 1902-1905. (27) Yu, T.; Liu, X.; Bolcato-Bellemin, A. L.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.; Rocchi, P.; Behr, J. P.; Peng, L. An Amphiphilic Dendrimer for Effective Delivery of Small Interfering RNA and Gene Silencing In Vitro and In Vivo. Angew. Chem., Int. Ed. 2012, 51, 8478-8484. (28) Yu, C. Y.; Li, N. M.; Yang, S.; Ning, Q.; Huang, C.; Huang,W.; He, Z. N.; He, D. X.; Tan, X.W.; Sun, L. C. Fabrication of Galactosylated Chitosan-5-fluorouracil Acetic Acid Based Nanoparticles for Controlled Drug Delivery. J. Appl. Polym. Sci. 2015, 132, 42625-42631. (29) Huang, C.; Li, N. M.; Gao, P.; Yang, S.; Ning, Q.; Huang, W.; Li, Z. P.; Ye, P. J.; Xiang, L.; He, D. X.; Tan, X. W.; Yu, C. Y. In Vitro and In Vivo Evaluation of



Page 20 of 38

Macromolecular Prodrug GC-FUA Based Nanoparticle for Hepatocellular Carcinoma Chemotherapy. Drug Delivery 2017, 24, 459-466. (30) Tang, S.; Yin, Q.; Su, J. H.; Sun, H. P.; Meng, Q. S.; Chen, Y.; Chen, L. L.; Huang,Y. Z.; Gu, W. W.; Xu, M. H.; Yu, H. J.; Zhang, Z. W.; Li, Y. P. Inhibition of Metastasis and Growth of Breast Cancer by pH-Sensitive Poly(β-Amino Ester) Nanoparticles Co-Delivering Two siRNA and Paclitaxel. Biomaterials 2015, 48, 1-15. (31) Ozpolat, B.; Sood, A. K.; Lopez-Berestein, G. Liposomal siRNA Nanocarriers for Cancer Therapy. Adv. Drug Delivery Rev. 2014, 66, 110-116. (32) Ballarin-Gonzalez, B.; Howard, K. A. Polycation-Based Nanoparticle Delivery of RNAi Therapeutics: Adverse Effects and Solutions. Adv. Drug Delivery Rev. 2012, 64, 1717-1729. (33)

Jiang,

H.

L.;

Kwon,

J.

T.;

Kim,

E.

M.

Galactosylated

Poly(Ethylene

Glycol)-Chitosan-Graft-Polyethylenimine as A Gene Carrier for Hepatocyte-Targeting. J. Controlled Release 2008, 131, 150-157. (34) Alex, S. M.; Rekha, M. R. Spermine Grafted Galactosylated Chitosan for Improved Nanoparticle Mediated Gene Delivery. Int. J. Pharm. 2011, 410, 125-137. (35) Adams, J. M. The Bcl-2 Protein Family: Arbiters of Cell Survival. Science 1998, 281, 1322-1326. (36) Cavadas, M.; Oikonomidi, I.; Gaspar, C. J. Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE. Cell Rep. 2017, 21, 745-757. (37) Veit, M.; Koyro, K. I.; Ahrens, B.; Bleibaum, F.; Munz, M.; Rövekamp, H.; Andrä, J.; Schreiber, R.; Kunzelmann, K.; Sommer, A.; Bhakdi, S.; Reiss, K. Anoctamin-6 Regulates ADAM Sheddase Function. Biochim. Biophys. Acta, Mol. Cell Res. 2018, 1865, 1598-1610.


Page 21 of 38 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


Scheme 1. Preparation of GC-FU/miR-122 and hepatoma-targeted co-delivery of miR-122 and 5-Fu.



Figure 1. (A) Particle size of GC-FUA-NPs and GC-NPs (C). (B) ζ potential of GC-FUA-NPs and GC-NPs (D) formed by the addition of sodium tripolyphosphate to


Page 22 of 38

Page 23 of 38 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


GC-FU and GC. Note that the neither GC nor GC-FU can form NPs due to their hydrophilicity. However, the addition of sodium tripolyphosphate can promote the formation of GC-NPs and GC-FU-NPs by electrostatic interactions.



Figure 2. Physicochemical characteristics of GC-FU/miR-122-NPs. (A) Agarose gel electrophoresis assay of GC-FU/miR-122 at different ω:ω ratios. (B) Flow cytometry analysis of transfected live cells after treatment with GC-Fu/miR-122-NPs for 24 hrs in HepG2 cells at various ω:ω ratios. (C) Particle sizeand at different ω:ω ratios. (D) ζ potential at different ω:ω ratios.


Page 24 of 38

Page 25 of 38


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



Page 26 of 38

Figure 3. (A) TEM image of GC-FU/miR-122-NPs (ω:ω = 1024:1), scale bar: 50 nm. (B) SEM image of GC-FU/miR-122-NPs (ω:ω = 1024:1), scale bar: 2.0 µm. (C) Size distribution of GC-FU/miR-122-NPs (ω:ω = 1024:1) using the DLS measurement. (D)Zeta potential distribution

of

GC-FU/miR-122-NPs

(ω:ω

=

1024:1).

GC-FU/miR-122-NPs in PBS at pH 5.5 (a), 6.8 (b) and 7.4 (c).


(E)5-Fu

release

from

Page 27 of 38 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


Figure

4.

Biocompatibilities

of

GC-FU/miR-122-NPs.

(A)

BSA

adsorption

on

GC-FU/miR-122-NPs and equivalent 5-Fu after incubation at 37 °C, different concentrations and periods of time: 2, 4 and 6 hrs. (B) Percentage of RBCs hemolysis incubated with GC-FU/miR-122-NPs. (C) Cell viabilities of L02 and ECs cells (D) incubated with GC-NPs.



Figure 5. Cell uptake of the 5-Fu(A) and miR-122(B) incubated with nanomedicines from GC-FU/miR-122-NPs (a, b, c) and equivalent free 5-Fu (d) for 24 hrs: with the incubation of 1.0 g·L-1 glucose (a) or 1.0 g·L-1 galactose(b) or without the incubation of glucose or galactose(c), Scale bar: 50 μm.


Page 28 of 38

Page 29 of 38


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



Figure 6. Cellular proliferation inhibitions, cell apoptosis and cell cycle arrest. (A) Viability of HepG2 cells after incubation with various constructs including GC-FU-NPs, GC/miR-122-NPs, GC-FU/miR-122-NPs, a physical mixture of 5-Fu and miR-122, and free 5-Fu for 48 hrs. The concentration of 5-Fu was used for all the constructs containing the drug. For GC/miR-122 formulation without 5-Fu, the concentration of GC was used instead. The used concentrations from 1 to 5 represent 0.01, 0.05, 0.1, 0.5, 1.0 mg/ml, respectively. (B) Flow cytometry analysis of apoptosis of HepG2 cells induced by PBS, free 5-Fu, GC-NPs, GC/miR-122-NPs, GC-FU-NPs, and GC-FU/miR-122-NPs for 24 hrs. (C) Flow cytometry analysis of cycle of HepG2 cells induced by PBS (control), free 5-Fu, GC/miR-122-NPs, GC-NPs, GC-FU-NPs and GC-FU/miR-122-NPs for 24 hrs.


Page 30 of 38

Page 31 of 38


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



Figure 7. In vitro anti-metastatic effects and the expression of Bcl-2, ADAM17. The wound healing images (A) and quantitative analysis (C, **p < 0.05, vs GC/miR-122-NPs group; ##p < 0.01, vs 5-Fu group; &p < 0.05 vs GC-FU-NPs group) after scratch for 24 hrs. Microscopy images of invasion (B) and quantitative analysis (D, #p < 0.05 vs GC-FU-NPs, **p < 0.01 vs 5-Fu) of HepG2 cells that passed through the membrane. Cells were pre-incubated with GC-FU-NPs, GC/miR-122-NPs, GC-FU/miR-122-NPs and free 5-Fu for 48 hrs. Cells without treatments were used as control. (E) The expression of Bcl-2, ADAM17 in HepG2 cells and quantitative analysis

(n = 3, ***p < 0.001, **p < 0.01).


Page 32 of 38

Page 33 of 38 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


Figure 8. In vivo antitumor efficacies. Tumor volume (A) and body weight changes (B) of tumor-burdened BALB/c nude mice treated with saline, free 5-Fu + miR-122, GC-NPs, GC-FU-NPs, GC/miR-122-NPs, GC-FU/miR-122-NPs and free 5-Fu. Each formulation was administered on day 0, 2, 4 ,6, 8, 10, 12, 14, 16, 18 and 20 by injection with a dosage of 2.0 mg 5-Fu per kg body weight for injection of free 5-Fu and nanomedicines. (C) Representative digital pictures of tumors. Data were presented as mean ± standard deviation (n ≥ 6) (*p < 0.001).




Page 34 of 38

Page 35 of 38


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



Figure 9. (A) Histopathologic examination of the tumors on day 20 after the first administration. (B) Representative microphotos of tumor sections stained for anti-ADAM17. (C) Representative microphotos of tumor sections stained for anti-Bcl-2.


Page 36 of 38

Page 37 of 38 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


Figure 10. (A) Biodistribution of 5-Fu in SD rats. (B) Evaluation of ALT, AST, BUN, Cr, CK, CK-MB and LDH levels after all the treatments of GC-FU/miR-122-NPs (a), Saline (b), and free 5-Fu (c) (n ≥ 6).



Graphical Table of Contents (TOC)


Page 38 of 38

Delivery of Liver-Specific miRNA-122 Using a Targeted

Recommend Documents