Galactose Derivative-Modified Nanoparticles for Efficient siRNA

May 29, 2018 - To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and ... Citing Articles; Related Conten...
0 downloads 0 Views 8MB Size
Download PDF
Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

pubs.acs.org/Biomac

Galactose Derivative-Modified Nanoparticles for Efficient siRNA Delivery to Hepatocellular Carcinoma Kuan-Wei Huang,†,‡ Yu-Tsung Lai,†,§ Guann-Jen Chern,‡ Shao-Feng Huang,§ Chia-Lung Tsai,§ Yun-Chieh Sung,‡,∥ Cheng-Chin Chiang,‡ Pi-Bei Hwang,‡ Ting-Lun Ho,‡ Rui-Lin Huang,‡ Ting-Yun Shiue,‡ Yunching Chen,*,‡,∥ and Sheng-Kai Wang*,§,∥ ‡

Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan ∥ Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan §

S Supporting Information *

ABSTRACT: Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl β-D-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.

■

INTRODUCTION

growth factor (VEGF) from HCC cells in the tumor microenvironment.9,10 Thus, silencing VEGF expression in HCC cells by siRNA may serve as a potent antiangiogenic approach as well as a promising anti-HCC therapy. Despite being a promising therapeutic approach, efficient delivery of siRNAs in vivo remains challenging due to the following reasons: Naked siRNAs are rapidly degraded by nucleases in the blood circulation and are eliminated by renal filtration, and the anionic and hydrophilic properties of siRNAs limit the efficacy of cellular uptake and endosome escape,

Hepatocellular carcinoma (HCC) is a primary liver cancer with high mortality and morbidity rates in East Asia.1,2 The current treatment approaches for HCC, such as surgical resection, chemoembolization, and tyrosine kinase inhibitor (TKI) therapy, show poor prognosis or strong adverse effects due to their complexities.3−5 Thus, developing an effective treatment approach with reduced side effects is an urgent need. Small interfering RNA (siRNA) therapy represents a potential therapeutic strategy6,7 for mediating the downregulation of pro-tumor molecules to suppress cancer progression and exerts beneficial effects against various types of cancer.8 In HCC, tumor progression and metastasis development significantly rely on the formation of neovessels that can be promoted by the secretion of angiogenic factors such as vascular endothelial © XXXX American Chemical Society

Special Issue: Biomacromolecules Asian Special Issue Received: March 1, 2018 Revised: May 15, 2018

A

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules leading to poor bioavailability.11,12 Moreover, selective delivery of siRNAs into cancer cells without inducing cytotoxicity in normal cells is crucial to reducing the side effects.13 Thus, successful siRNA therapy would require a suitable delivery system with selectivity.11,14 In previous studies, we have developed an LCP (lipid/calcium/phosphate) nanoparticle (NP) as a multifunctional siRNA delivery system that overcomes the above obstacles by encapsulating siRNA in a pH stimuli-responsive calcium phosphate core enclosed in a lipid bilayer shell.15 The LCP NPs protect siRNA from degradation by nucleases and thus enhance its stability in the blood circulation. Meanwhile, the CaP (calcium phosphate) core of LCP NPs disassmbles at low pH in the endosome, resulting in the release of nucleic acid cargo to the cytoplasm.16,17 With these advantages, the remaining problem toward an ideal delivery system is the selectivity for cancer cells. More recently, specific delivery to HCC has been widely explored using carbohydrate ligands. The advantages exploiting carbohydrate−receptor interactions include their low toxicity, high avidity, and expression.18,19 One major receptor responsible for this specificity is the asialoglycoprotein receptor (ASGPR), which is found predominately on hepatocytes and HCC cells, but minimally on nonhepatic cells.19 Galactosylated polymers or liposomes targeting ASGPR were developed to serve as drug/gene carriers for the treatment of HCC.20,21 ASGPR is composed of two types of polypeptide subunits (subunits 1 and 2), which form homo- and hetero-oligomers that can coexist on the plasma membrane.22 Upon recognition and binding of ligands, ASGPR is internalized and allows the cargo to be contained in endosomes and then degraded in lysosomes.23 ASGPR selectively binds to galactose (Gal) and N-acetylgalactosamine (GalNAc) and weakly binds to glucose.24,25 To improve the affinity of glycan ligands to ASGPR, recent studies have introduced artificial moieties to galactose. For example, the Ernst group has reduced the anomeric hydroxyl group of GalNAc and extended aromatic moieties on C-2 through a triazole ring.26 The Finn and Mascitti groups have modified galactosides with broad types of moieties on C-1, C-2, C-5, and C-6 to identify the trifluoromethylacetamide derivative that significantly enhanced ASGPR interaction.27 The same groups also developed bicyclic bridged ketal as a GalNAc derivative to target ASGPR.28 In addition to modifying glycan ligands, another approach for targeting ASGPR is through multivalent binding to this oligomeric receptor on the plasma membrane. Despite the unavailability of the ASGPR oligomer crystal structure, the distances between carbohydrate binding sites have been predicted to be 15−25 Å29,30 based on the binding response of trimeric ligands on defined scaffolds.31 Immobilization of ASGPR ligands on nanocarriers may provide multivalent binding to the surface of HCC cells with elevated ASGPR expression. In many cases, including nanoparticle32,33 and micelle34 carriers, galactosides with a C-1 aromatic moiety are employed as ASGPR ligands. To achieve more potent siRNA delivery in HCC, we investigated the effect of the aromatic moiety on ASGPR-based siRNA delivery in HCC. We synthesized various galactoside derivatives and investigated the siRNA delivery capability of LCP NPs modified with those galactoside derivatives in HCC cells. Given the potent ASGPRtargeting property of galactoside derivatives, we encapsulated the therapeutic siRNA (VEGF siRNA) in the ASGPR-targeted LCP NPs and investigated whether VEGF siRNA delivered by

ASGPR-targeted NPs suppressed HCC progression in murine orthotopic HCC models.

■

EXPERIMENTAL SECTION

Materials. High-concentration and phenol red-free Matrigel and phosphate-buffered saline (PBS) were purchased from Corning (U.S.A.). Fetal bovine serum (FBS), trypsin, penicillin-streptomycin, high-glucose Dulbecco’s modified Eagle’s medium (DMEM), MEM α (minimum essential medium α), and F-12K medium (Kaighn’s modification of Ham’s F-12 medium) were obtained from HyClone (U.S.A.). Protein standard, polyacrylamide, and enhanced chemiluminescence (ECL) were purchased from Bio-Rad (U.S.A.). Stellate cell growth supplement was purchased from ScienCell Research Laboratory (U.S.A.). Polyvinylidene fluoride membrane (PVDF) and enhanced chemiluminescence (ECL) were acquired from GE Healthcare Life Sciences (U.K.). HRP IgG conjugate was also purchased from ImmunoReagents, Inc. (U.S.A.). Fluorescein amidite (FAM) was conjugated to the 5′ end of the sense sequence. 5′-FAMlabeled VEGF siRNA, sodium dodecyl sulfate (SDS), and bovine serum albumin (BSA) were obtained from MDBio, Inc. (Taiwan). Asialofeutin, coumarin 6, dimethyl sulfoxide (DMSO), β-actin antibody, methylcellulose, paraformaldehyde, tocopheryl polyethylene glycol succinate (TPGS), methanol, ethanol, acetone, and VEGF siRNA with the sequence 5′-AUGUGAAUGCAGACCAAAGAA-3′ were purchased from Sigma-Aldrich (U.S.A.). RIPA buffer was acquired from Bio Basic Canada, Inc. (Canada). LB broth was purchased from FocusBio (Australia). LB Agar and tris(2-carboxyethyl) phosphine (TCEP) were obtained from ThermoFisher Scientific (U.S.A.). Agarose and sodium chloride were purchased from Avantor (U.S.A.). Optimal cutting temperature (OCT) was purchased from Sakura Finetek (U.S.A.). Peptides were ordered from Kelowna (Taiwan). Cholesterol, l,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (Azido-DSPEPEG) were purchased from Avanti Polar Lipids (U.S.A.). Cell Culture. Murine HCC cells (HCA-1), murine hepatocytes (FL83B), and human HCC cells (Hep3B) were kindly provided by Dr. Dan Duda (MGH, Boston). The HCA-1 and Hep3B cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose and MEM α (minimum essential medium α), respectively. The FL83B cells were cultured in F-12K medium (Kaighn’s modification of Ham’s F-12 medium). All media contained 10% FBS and 1% antibiotics. All cells are maintained at 37 °C in an incubator supplied with 5% CO2. Mice and Orthotopic Tumor Model Establishment. C3H/ HeNCrNarl mice were purchased from National Laboratory Animal Center (Taipei, Taiwan). Mice aged 4−5 weeks were used for pancreatic tumor establishment. HCA-1 cells (106), which were resuspended with a mixture of medium-concentration, high-concentration, and phenol red-free Matrigel, were orthotopically injected into mice for the screening process. The remaining 105 of the tumor cells were used otherwise. All animals received humane care, in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Academy of Sciences. Preparation of Nanoparticles. LCP NPs were prepared using a modified protocol.16,35 Two separate microemulsions (3 mL each) were prepared. To prepare the calcium-loaded microemulsion, siRNA (15 μg) and 40 μL of 500 mM CaCl2 (pH 7) were added to the oil phase of cyclohexane and Igepal-520 (7:3, v/v). To prepare the phosphate buffer-loaded microemulsion, a Na2HPO4 solution (74 μL, 100 mM, pH 9) and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA; 74 μL, 35 mM) were added to the oil phase of cyclohexane and Igepal520 (7:3, v/v). Two separate microemulsions were mixed using a magnetic stir bar and stir plate at the speed of 700−1000 rpm for 20 min at room temperature. The emulsions were then mixed for 20 min to form the condensed CaP cores of CaP/siRNA. Then, 6 mL of 100% EtOH was added to disrupt the emulsion, and the mixture was B

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Scheme 1. Synthesis Scheme of Alkynyl Galactosides Conjugating with Azido-PEG-DSPE for Assembly of NPs

centrifuged at 10000 × g for 20 min to remove free DOPA lipid. After removing the supernatant solution, the precipitated CaP cores were washed twice with 100% EtOH to remove the organic solvents with emulsifying agents. The precipitate (LCP core) was then dried under N2 and suspended in chloroform by sonication. A mixture of free lipids (DOTAP/DSPE-PEG2000/cholesterol = 1:1:2 molar ratio) in chloroform was added to the CaP cores (2.5:1 ratio of total free lipids to DOPA) and then dried under N2. After evaporating the chloroform, 160 μL of water was added to form LCP NPs with the lipid-bilayer core structure followed by sonication. For galactosidedecorated LCP NPs, the mixture of free lipids was changed to (DOTAP/DSPE-PEG2000/cholesterol/glycolipid = 1:0.99:2:0.01 molar ratio). Characterization of Nanoparticles. The NPs were formulated as described above and were recovered in deionized water. The morphology of the LCP NPs was characterized by transmission electron microscopy (TEM; H-7500, Hitachi High-Tech, Tokyo, Japan). The NPs were stained on dried Formvar-coated 100-mesh copper grids at room temperature. All grids were further dried for 2 days before imaging. The particle size and surface charge were examined using a Zetasizer system (Zetasizer nano zs, Malvern Instruments Ltd., Worcestershire, U.K.) at room temperature. Cellular Uptake. Cells and NPs were prepared as mentioned above. The cells were incubated with the NPs for 4 h. The medium was replaced with 4% paraformaldehyde for 10 min, followed by three washes with PBS. The coverslips containing the cells were mounted with 3 μL of DAPI, fixed on slides, and finally imaged using a confocal microscopy (LSM 780, Zeiss, Germany). The images were analyzed using Matlab. Competition Assay. Cells and NPs were prepared as mentioned above. The free form of asialofetuin protein was added to medium 10 min prior to the treatment of the NPs. The cells were incubated with the NPs for another 1 h. The medium was replaced with 4% paraformaldehyde for 10 min, followed by three washes with PBS. The coverslips containing the cells were mounted with 3 μL of DAPI, fixed on slides, and finally imaged using a confocal microscopy (LSM 780, Zeiss, Germany). The images were analyzed using Matlab. qRT-PCR. To evaluate gene silencing by quantitative reverse transcription polymerase chain reaction (qRT-PCR), the cells were seeded in a 12-well plate, grown overnight with 24 or 48 h treatments of various LCP formulations, and then washed with PBS. In total, 1 μg

of each of the isolated RNA samples was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, U.S.A.) in a Piko Thermal Cycler in a 24-well plate (ThermoFisher Scientific) according to the manufacturer’s instructions. Quantitative PCR was carried out using SYBR Green PCR Mastermix with gene-specific PCR primer pairs with a 7500 Real-Time PCR System (Applied Biosystems, U.S.A.). The primer sequences used were as follows: sense, 5′-CTGTGCAGGCTGCTGTAACG-3′ and antisense, 5′-GTTCCCGAAACCCTGAGGAG-3′ for VEGF-A; sense 5′-TGAGAGGGAAATCGTGCGTG-3′ and antisense, 5′TTGCTGATCCACATCTGCTGG-3′ for β-actin; and sense, 5′CTGCCACCCAGAAGACTGTG-3′ and antisense, 5′-GGTCCTCAGTGTAGCCCAAG-3′ for GAPDH. Further validation was performed using RT2 Profiler PCR Arrays Human Fibrosis (Qiagen). cDNA samples along with SYBR Green PCR Mastermix were added into wells preloaded with specific primers in the array. The PCR was carried out according to the manufacturer’s instruction. The data were subsequently analyzed and plotted using online software offered by the manufacturer. Tumor Growth Inhibition Study. Orthotopic HCA-1 tumorbearing mice were intravenously injected with different formulations containing VEGF siRNA (0.7 mg/kg/dose, 3 doses per week). VEGF siRNA in ASGPR-targeted LCP NPs was administered intravenously to mice with orthotopic HCA-1 HCCs. Tumor size in the treated mice was measured using clipper at 2 weeks after the first treatment and estimated by using the following formula: tumor volume = length × width2/2. Statistics. Data analyses were performed using Student’s t test and were presented as mean ± SEM. P-values less than 0.05 were considered statistically significant for all tests.

■

RESULTS AND DISCUSSION Preparation of Galactoside-Decorated LCP Nanoparticles and Synthesis of Alkynyl Galactosides. To optimize the affinity and specificity of the galactoside derivatives to ASGPR on HCC, we first investigated the effect of the aromatic moieties on the galactosides toward the uptake of ASGPR-targeted NPs. We designed two phenyl and naphthyl groups with different linkages and provided a galactoside ligand C

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

In addition, as GalNAc ligands generally initiate stronger ASGPR interactions than their Gal analogs24,25,27 but take more effort or resources to synthesize, we would also like to evaluate the difference between these two families in our siRNA delivery system. These galactosides with alkyne groups were conjugated to commercial azido-PEG-DSPE through the robust Cu(I)catalyzed alkyne−azide cycloaddition (CuAAC) reaction. The resulted glycolipids L1−L6 were employed to assemble the LCP NPs for ASGPR-based delivery (Scheme 1 and Figures S1−S33). The designed alkynyl galactosides can be prepared through straightforward transformations from peracetylated galactose 7 or peracetylated GalNAc 8 (Scheme 2). Glycan 1a and 1b were synthesized in a similar manner, which began from C-1 deacetylated intermediates of 7 and 8, respectively. Then, the intermediates were transformed to trichloroacetimidate and glycosylated with propargyl alcohol to give 1a and 1b after deacetylation with sodium methoxide. Glycans 2a and 3 were prepared with a similar approach by propargylamine coupling and deacetylation to intermediates 9 and 10, which can be carried out by following the reported methods.36 Glycan 2b was obtained through glycosylation of the glycosyl bromide from 8 with 3-hydroxy-N-propargyl-benzamide and followed by deacetylation. Galactosides 4−6 were prepared from the coupling of 11−13 to pentynoic acid and then deacetylation. The key reactant 11 was prepared by known methods,37 and 12 was prepared by phase transfer catalytic (PTC) glycosylation38 of galactosyl bromide of 7 with 5-amino-1-naphthol.39 Likewise, galactoside 13 was prepared through the same PTC glycosylation with 4-iodophenol, followed by reaction with 5nitroindole and by hydrogenation reduction of the nitro group.39 In Vitro Cellular Uptake of ASGPR-Targeted NPs. ASGPR-targeted NPs were prepared according to the methods of previous studies.16,35 First, we utilized a reverse microemulsion to encapsulate the nucleic acid (siRNA) in lipidcoated CaP NPs. Next, the lipid-coated siRNA/CaP NPs were allowed to form a self-assembled lipid-bilayer structure by adding the outer leaflet lipids (DOTAP, cholesterol, DSPEPEG) to the siRNA/CaP cores. To achieve ASGPR targeting, we conjugated galactoside derivatives into the outer leaflet lipids (DSPE-PEG) and assembled the LCP NPs with galactoside derivative-conjugated DSPE-PEG(L1-L6). We further examined the physical properties of siRNA-loaded ASGPR-targeted LCP NPs by transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. The TEM image showed that the ASGPR-targeted LCP NPs were spherical (Figure 1A) with the average diameter of 126.3 ± 0.8 nm and a zeta-potential of −2.72 mV measured by DLS (Figures 1B and S34). The particle size of the ASGPR-targeted LCP NP (L4-LCP) was similar to that of the nontargeted LCP NP and Gal(L1a)-LCP NP. The stability of the LCP NPs was also checked by measuring mean size and PDI after long storage times (up to 14 days), at 4 °C in water. No significant changes in the particle size and PDI were observed (Figure S35). In addition, we also found the percentage of siRNA encapsulation for the L4-LCP NP was 90%. To investigate the cellular uptake of ASGPR-targeted LCP NPs, FAM-siRNA was encapsulated in LCP NPs modified with various galactose analogs, and the uptakes of FAM-siRNA loaded NPs by HCC cells and normal hepatocytes were examined 4 h after treatment (Figure 2). The results (Figure 2A,B) indicate that the simple galactose ligand (L1a) and the

Scheme 2. Scheme of Alkynyl Galactoside Synthesis

Figure 1. Morphology of L4-LCP NPs containing siRNA. (A) Representative TEM images of L4-LCP NPs. (B) Sizes and zeta potentials of nontargeted LCP, Gal-LCP, and L4-LCP NPs. The data are the mean values ± the SD (n = 3). PDI, polydispersity index.

without an aromatic group for comparison as well as extension of an extra aromatic moiety from the phenyl group (Scheme 1). D

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 2. Cellular uptake of FAM-siRNA delivered by ASGPR-targeted LCP NPs. (A) Hep3B cells were treated with FAM-labeled siRNA (75 nM) in different formulations for 4 h and observed with a Zeiss LSM 780 confocal microscope. FAM-siRNA was presented in green, and the nucleus was presented in blue (DAPI). Scale bar = 25 μm. (B) The quantitative data of cellular uptake of FAM-siRNA delivered by ASGPR-targeted LCP NPs. (C, D) Competitive inhibition was performed with excess free asialofetuin. The uptake of FAM-siRNA was imaged and quantified with a confocal microscope. (E, F) L4-LCP NPs exhibited a superior siRNA delivery into murine HCC (HCA-1) cells compared to normal murine hepatocytes (FL83B). Scale bar = 20 μm. The data are the mean values ± the SEM; *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Figure 3. VEGF siRNA delivered by L4-LCP NPs downregulated VEGF expression in HCC cells in vitro. L4-LCP NPs containing VEGF siRNA (75 nM) significantly decreased VEGF expression in both human (Hep3B) and murine (HCA-1) HCC cells in a dose-dependent manner. VEGF expression was detected by qRT-PCR (A, B) and Western blotting (C, D). The data are the mean values ± the SEM; *p < 0.05, ** p < 0.01.

with the nontargeted LCP NPs. However, the meta-benzamide moiety introduced to the galactoside and GalNAc on L2a-LCP

GalNAc analog (L1b) on ASGPR-targeted LCP NPs only slightly improved the cellular uptake of nanocarriers compared E

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 4. VEGF siRNA delivered by L4-LCP NPs suppressed angiogenesis in tumor microenvironment of murine orthotopic HCC models. (A) Treatment schedule of VEGF siRNA (0.7 mg/kg) in different formulations in mice with orthotopic HCA-1 HCCs. (B−E) MVD was measured using anti-CD31 antibodies. Representative immunofluorescence images of VEGF staining (B, C) and CD31 staining (D, E) in the tumor tissues of orthotopic HCA-1 tumor models. L4-LCP NPs significantly decreased the expression of VEGF and inhibited the formation of vessels in HCC tumor tissues. VEGF was presented in green, CD31 was presented in red, and the nucleus was presented in blue (DAPI). Scale bar = 50 μm. The data are the mean value ± the SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

phenyl β-D-galactoside-decorated LCP NPs (L4-LCP NPs; Figures 2 and S36). Furthermore, the uptake of L4-LCP NPs was competitively inhibited by the addition of asialofetuin, a specific ASGPR ligand (Figure 2C,D). These results indicated that the cellular uptake of L4-LCP NPs was via ASGPRmediated endocytosis. However, from current understanding on ASGPR, it is difficult to interpret the observed uptake efficiency based on the receptor binding to different galactoside structures. Recent structure−activity relationship investigation on ASGPR H1 subunit by the Ernst, Finn, and Mascitti groups26,27 have focused on galactosides with modifications at 2, 5, and 6 positions. Although an aromatic modification at the anomeric position of GalNAc was also investigated, those were C-glycosides and developed to increase the rigidity of the molecule. With such purpose, a β-4-methoxyphenyl substituent resulted in 5-fold loss of ASGPR H1 affinity compared to the βmethyl substituent.27 As those C-glycoside ligands significantly limit the motion of their anomeric substituent, they are very different to our present set of galactoside ligands, so that the

and L2b-LCP NPs significantly enhanced the cellular uptake. NPs modified with galactoside with a para-phenylamide moiety (L4-LCP NPs) showed potent cellular uptake in HCC at 9-fold higher than that of nontargeted LCP NPs in HCC cells. Galactoside with naphthyl modification on L3-LCP and L5LCP NPs did not show improvement of HCC uptake when compared with nontargeted LCP NPs. Notably, the indole group extended from the para position of the phenyl moiety on L6-LCP NPs also provided significant uptake improvement over simple galactoside on Gal(L1a)-LCP NPs. Our data indicated that the glycan ligand with phenyl group significantly improved the uptake of siRNA-loaded NPs, and even larger indole group at the para position can be tolerated. We also showed that naphthyl moieties in two different linkages are both not favored for NP delivery. It is also interesting that the NPs modified with GalNAc ligand did not show improved uptake in HCC compared to the NPs modified with their galactose analogs. Among eight derivatives, we demonstrated that the uptake of siRNA was greatest in HCC cells treated with F

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 5. VEGF siRNA delivered by L4-LCP NPs suppressed HCC progression in murine orthotopic HCC models. (A) Treatment with VEGF siRNA (0.7 mg/kg) delivered by L4-LCP NPs significantly reduced tumor sizes in the orthotopic tumor-bearing mice (n = 8). (B, C) Treatment with VEGF siRNA delivered by L4-LCP NPs significantly reduced the number of lung metastatic nodules in the orthotopic tumor-bearing mice. Scale bar = 200 μm. The data are the mean value ± the SEM; ***p < 0.001. ns, not significant.

effect of aromatic moiety cannot be directly compared. Moreover, because the ASGPR is protein oligomer of H1 and H2 subunits reported with varied stoichiometric ratios,40−42 and that recent studies indicated different ASGPR heterooligomers forms affect the ligand specificity,16 the LCP nanoparticle uptake activity arisen from glycan structural modification is difficult to explain solely by the affinity to a single subunit. Despite of the lack of a clear structure−activity relationship explanation for the RNAi delivery efficiency, we continued to investigate the capability of the most potent L4LCP NPs. More interestingly, we demonstrated that L4-LCP NPs exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes (Figure 2E,F). The enhanced uptake of ASGPR-targeted NPs in HCC cells may be due to the upregulation of ASGPR expression in HCC cells compared with normal hepatocytes (Figure S37). With these observations, L4 was chosen as a targeting moiety for HCC in the following studies. VEGF siRNA Loaded in ASGPR-Targeted NPs Silences VEGF Expression in HCC In Vitro and In Vivo. In an attempt to block angiogenesis in HCC effectively and specifically, we encapsulated VEGF siRNA in different formulations and examined the gene silencing effect and capability to suppress angiogenesis in HCC. We first performed qRT-PCR to validate VEGF mRNA expression in HCC cells after treatment of VEGF siRNA loaded in different formulations. As shown in Figure 3A,B, VEGF siRNAs delivered by L4-LCP NPs showed significant inhibition of VEGF mRNA expression. As expected, VEGF expression remained unchanged or slightly changed when cells were treated with VEGF siRNA in the nontargeted LCP NPs or GalLCP NPs, respectively. Furthermore, we also observed a significant decrease in VEGF protein expression in the HCC

cells treated with VEGF siRNA-loaded L4-LCP NPs, as detected by using a Western blot (Figure 3C,D). For the in vivo evaluation, we next established a syngeneic orthotopic murine HCC model with HCA-1 cells intrahepatically implanted into C3H mice. After HCC tumors were established, VEGF siRNAs in different formulations were given at a dose of 0.7 mg/kg, 3 doses per week for 2 weeks of treatment (Figure 4A). The systemic injections of VEGF siRNA in L4-LCP NPs significantly suppressed VEGF expression in HCA-1 tumors, whereas VEGF siRNA delivered by nontargeted LCP only moderately decreased VEGF expression in HCA-1 tumors (Figure 4B,C). Our results indicated that L4-LCP NPs efficiently delivered VEGF siRNAs into HCC and achieved a significant gene silencing effect in vitro and in vivo. The delivery efficacy and the silencing activity of siRNAs loaded in NPs were ligand (L4) dependent. VEGF siRNA Loaded in ASGPR-Targeted NPs Demonstrated Potent Antiangiogenic Activity in HCA-1 Tumors and Suppressed Primary HCC Growth and Distal Metastasis. We next evaluated the antiangiogenic effects of VEGF siRNAs in L4-LCP NPs in orthotopic HCC models. After the HCA-1 tumor was established, VEGF siRNAs loaded in L4-LCP NPs were I.V. (intravenous) administered into HCA-1 tumor-bearing mice. VEGF siRNAs loaded in L4-LCP NPs significantly decreased the mean vessel density (MVD) compared to VEGF siRNA delivered by nontargeted LCP NPs (Figure 4D,E). Given that VEGF siRNAs delivered by ASGPRtargeted LCP NPs demonstrated potent antiangiogenic effects, we further examined whether the treatment could lead to tumor growth inhibition. Indeed, tumor growth inhibition was observed after treatment with VEGF siRNA-loaded L4-LCP NPs in the HCA-1 orthotopic model (Figure 5A), while mice receiving VEGF-siRNAs from Gal-LCP NPs or nontargeted LCP NPs showed moderate or nonsignificant HCC growth G

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 6. Schematic illustration of the anti-HCC mechanisms of VEGF siRNA-loaded ASGPR-targeted LCP NPs, which deliver VEGF siRNA into HCCs cells and suppress angiogenesis in HCC. Therefore, systemic treatment with VEGF siRNA delivered by ASGPR-targeted LCP NPs significantly inhibits both primary and distal HCC progression and serves as a potent therapeutic strategy to treat HCC.

■

inhibition, respectively (Figure 5). Furthermore, VEGF siRNAs delivered by L4-LCP NPs significantly inhibited distal lung metastasis in the orthotopic HCA-1 model (Figure 5B,C). Mice receiving VEGF siRNAs in nontargeted LCP NPs did not show a reduction in metastasis formation compared to the control group. Our results demonstrated that L4-LCP NPs delivering therapeutic VEGF siRNAs significantly suppressed primary tumor progression and metastasis formation in the orthotopic HCC models.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00358. MALDI-TOF mass spectra of commercial N3-PEGDSPE, L1a, L2a, L3, L4, L5, L6, L1b, and L2b; The intensity size distributions from LCP NP, Gal-LCP NP, and L4-LCP NP; The stability of the LCP NPs; Cellular uptake of FAM-siRNA delivered by ASGPR-targeting LCP NPs; The upregulation of ASGPR expression in HCC cells (PDF).

CONCLUSIONS

In summary, we developed a highly efficient siRNA delivery carrier targeting HCC. β-phenyl galactoside modified PEGDSPE (L4) was incorporated into the gene carrier LCP NP to facilitate the delivery of siRNA into HCCs and to achieve an enhanced gene inhibition effect in HCC in vitro and in vivo. Significant suppression of VEGF expression led to MVD reduction in the tumor microenvironment. Finally, systemic treatment with VEGF siRNA delivered by LCP NPs modified with β-phenyl galactoside could serve as a potent anticancer treatment to suppress HCC progression (Figure 6). The ASGPR-targeted NPs may serve as a platform to encapsulate various RNA therapeutic agents for the treatment of HCC or other liver diseases in the future.

■

AUTHOR INFORMATION

Corresponding Authors

*Phone: +886-3-571-5131, ext: 35503. E-mail: yunching@mx. nthu.edu.tw. *Phone: +886-3-571-5131, ext: 33360. E-mail: skwang@mx. nthu.edu.tw. ORCID

Yunching Chen: 0000-0001-6228-5169 Sheng-Kai Wang: 0000-0002-3827-7983 Author Contributions †

These authors contributed equally to this work.

H

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Notes

(8) Lu, P. Y.; Xie, F.; Woodle, M. C. In vivo application of RNA interference: from functional genomics to therapeutics. Adv. Genet. 2005, 54, 117−42. (9) Hanahan, D.; Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86 (3), 353− 64. (10) Folkman, J.; D’Amore, P. A. Blood vessel formation: what is its molecular basis? Cell 1996, 87 (7), 1153−5. (11) Gavrilov, K.; Saltzman, W. M. Therapeutic siRNA: principles, challenges, and strategies. Yale J. Biol. Med. 2012, 85 (2), 187−200. (12) Seth, S.; Johns, R.; Templin, M. V. Delivery and biodistribution of siRNA for cancer therapy: challenges and future prospects. Ther. Delivery 2012, 3 (2), 245−61. (13) Severi, T.; van Malenstein, H.; Verslype, C.; van Pelt, J. F. Tumor initiation and progression in hepatocellular carcinoma: risk factors, classification, and therapeutic targets. Acta Pharmacol. Sin. 2010, 31 (11), 1409−20. (14) Oh, Y. K.; Park, T. G. siRNA delivery systems for cancer treatment. Adv. Drug Delivery Rev. 2009, 61 (10), 850−62. (15) Li, J.; Chen, Y. C.; Tseng, Y. C.; Mozumdar, S.; Huang, L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J. Controlled Release 2010, 142 (3), 416−21. (16) Li, J.; Yang, Y.; Huang, L. Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor. J. Controlled Release 2012, 158 (1), 108−14. (17) Liu, C. H.; Chern, G. J.; Hsu, F. F.; Huang, K. W.; Sung, Y. C.; Huang, H. C.; Qiu, J. T.; Wang, S. K.; Lin, C. C.; Wu, C. H.; Wu, H. C.; Liu, J. Y.; Chen, Y. A multifunctional nanocarrier for efficient TRAIL-based gene therapy against hepatocellular carcinoma with desmoplasia in mice. Hepatology 2018, 67 (3), 899−913. (18) Kawakami, S.; Hashida, M. Glycosylation-mediated targeting of carriers. J. Controlled Release 2014, 190, 542−55. (19) D’Souza, A. A.; Devarajan, P. V. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications. J. Controlled Release 2015, 203, 126−39. (20) Li, M.; Zhang, W.; Wang, B.; Gao, Y.; Song, Z.; Zheng, Q. C. Ligand-based targeted therapy: a novel strategy for hepatocellular carcinoma. Int. J. Nanomed. 2016, 11, 5645−5669. (21) Oh, H. R.; Jo, H. Y.; Park, J. S.; Kim, D. E.; Cho, J. Y.; Kim, P. H.; Kim, K. S. Galactosylated Liposomes for Targeted Co-Delivery of Doxorubicin/Vimentin siRNA to Hepatocellular Carcinoma. Nanomaterials 2016, 6 (8), 141. (22) Renz, M.; Daniels, B. R.; Vamosi, G.; Arias, I. M.; LippincottSchwartz, J. Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (44), E2989−97. (23) Tanabe, T.; Pricer, W. E., Jr.; Ashwell, G. Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J. Biol. Chem. 1979, 254 (4), 1038−43. (24) Kolatkar, A. R.; Leung, A. K.; Isecke, R.; Brossmer, R.; Drickamer, K.; Weis, W. I. Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain. J. Biol. Chem. 1998, 273 (31), 19502−8. (25) Ruiz, N. I.; Drickamer, K. Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor. Glycobiology 1996, 6 (5), 551−9. (26) Stokmaier, D.; Khorev, O.; Cutting, B.; Born, R.; Ricklin, D.; Ernst, T. O.; Boni, F.; Schwingruber, K.; Gentner, M.; Wittwer, M.; Spreafico, M.; Vedani, A.; Rabbani, S.; Schwardt, O.; Ernst, B. Design, synthesis and evaluation of monovalent ligands for the asialoglycoprotein receptor (ASGP-R). Bioorg. Med. Chem. 2009, 17 (20), 7254− 64. (27) Mamidyala, S. K.; Dutta, S.; Chrunyk, B. A.; Preville, C.; Wang, H.; Withka, J. M.; McColl, A.; Subashi, T. A.; Hawrylik, S. J.; Griffor, M. C.; Kim, S.; Pfefferkorn, J. A.; Price, D. A.; Menhaji-Klotz, E.; Mascitti, V.; Finn, M. G. Glycomimetic ligands for the human asialoglycoprotein receptor. J. Am. Chem. Soc. 2012, 134 (4), 1978−81. (28) Sanhueza, C. A.; Baksh, M. M.; Thuma, B.; Roy, M. D.; Dutta, S.; Preville, C.; Chrunyk, B. A.; Beaumont, K.; Dullea, R.; Ammirati,

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the Ministry of Science and Technology (MOST 104-2628-B-007-001-MY3, 105-2628-E007-007-MY3 for Y.C.; MOST 105-2633-M-007-002, 1062633-M-007-004 for S.K.W.) and by the National Institute for Health Research (NHRI-EX107-10609BC). This work was also supported by the Frontier Research Center on Fundamental and Applied Sceinces of Matters from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by Ministry of Education (MOE) in Taiwan.

■

ABBREVIATIONS ASGPR: asialoglycoprotein receptor; BSA: bovine serum albumin; CaP: calcium phosphate; CuAAC: Cu(I)-catalyzed alkyne−azide cycloaddition; DAPI: 4′,6-diamidino-2-phenylindole; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; DLS: dynamic light scattering; DOPA: 1,2dioleoyl-sn-glycero-3-phosphate; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DSPE-PEG: 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; azido-DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000]; ECL: enhanced chemiluminescence; EtOH: ethanol; FAM: fluorescein amidite; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Gal: galactose; GL: glycolipids; HCC: hepatocellular carcinoma; HRP: horseradish peroxidase; I.V.: intravenous; LCP: lipid/calcium/phosphate; MVD: mean vessel density; NP: nanoparticle; GalNAc: N-acetylgalactosamine; PBS: phosphate-buffered saline; PBST: phosphatebuffered saline with Tween 20; PDI: polydispersity index; PEG: polyethylene glycol; PTC: phase transfer catalytic; PVDF: polyvinylidene fluoride; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SEM: standard error of the mean; siRNA: small interfering ribonucleic acid; TKI: tyrosine kinase inhibitor; TEM: transmission electron microscopy; VEGF: vascular endothelial growth factor

■

REFERENCES

(1) Wong, M. C.; Jiang, J. Y.; Goggins, W. B.; Liang, M.; Fang, Y.; Fung, F. D.; Leung, C.; Wang, H. H.; Wong, G. L.; Wong, V. W.; Chan, H. L. International incidence and mortality trends of liver cancer: a global profile. Sci. Rep. 2017, 7, 45846. (2) Yang, J. D.; Roberts, L. R. Hepatocellular carcinoma: A global view. Nat. Rev. Gastroenterol. Hepatol. 2010, 7 (8), 448−58. (3) Kishi, Y.; Hasegawa, K.; Sugawara, Y.; Kokudo, N. Hepatocellular carcinoma: current management and future development-improved outcomes with surgical resection. Int. J. Hepatol. 2011, 2011, 728103. (4) Eun, H. S.; Kim, M. J.; Kim, H. J.; Ko, K. H.; Moon, H. S.; Lee, E. S.; Kim, S. H.; Lee, H. Y.; Lee, B. S. The retrospective cohort study for survival rate in patients with advanced hepatocellular carcinoma receiving radiotherapy or palliative care. Korean J. Hepatol 2011, 17 (3), 189−98. (5) Burak, K. W. Prognosis in the early stages of hepatocellular carcinoma: Predicting outcomes and properly selecting patients for curative options. Can. J. Gastroenterol 2011, 25 (9), 482−4. (6) Zamore, P. D.; Haley, B. Ribo-gnome: the big world of small RNAs. Science 2005, 309 (5740), 1519−24. (7) Sontheimer, E. J.; Carthew, R. W. Silence from within: endogenous siRNAs and miRNAs. Cell 2005, 122 (1), 9−12. I

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules M.; Liu, S.; Gebhard, D.; Finley, J. E.; Salatto, C. T.; King-Ahmad, A.; Stock, I.; Atkinson, K.; Reidich, B.; Lin, W.; Kumar, R.; Tu, M.; Menhaji-Klotz, E.; Price, D. A.; Liras, S.; Finn, M. G.; Mascitti, V. Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor. J. Am. Chem. Soc. 2017, 139 (9), 3528−3536. (29) Rice, K. G.; Weisz, O. A.; Barthel, T.; Lee, R. T.; Lee, Y. C. Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes. J. Biol. Chem. 1990, 265 (30), 18429−34. (30) Lee, Y. C.; Lee, R. T.; Ernst, B.; Hart, G. W.; Sinaý, P. Interactions of Oligosaccharides and Glycopeptides with Hepatic Carbohydrate Receptors. Carbohydrates in Chemistry and Biology; Wiley-VCH Verlag GmbH, 2008; pp 549−561. (31) Huang, X.; Leroux, J. C.; Castagner, B. Well-Defined Multivalent Ligands for Hepatocytes Targeting via Asialoglycoprotein Receptor. Bioconjugate Chem. 2017, 28 (2), 283−295. (32) Zhu, L.; Mahato, R. I. Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation. Bioconjugate Chem. 2010, 21 (11), 2119−27. (33) Hu, Y.; Haynes, M. T.; Wang, Y.; Liu, F.; Huang, L. A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo. ACS Nano 2013, 7 (6), 5376−84. (34) Sakashita, M.; Mochizuki, S.; Sakurai, K. Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry. Bioorg. Med. Chem. 2014, 22 (19), 5212−5219. (35) Goodwin, T. J.; Zhou, Y.; Musetti, S. N.; Liu, R.; Huang, L. Local and transient gene expression primes the liver to resist cancer metastasis. Sci. Transl. Med. 2016, 8 (364), 364ra153. (36) Casoni, F.; Dupin, L.; Vergoten, G.; Meyer, A.; Ligeour, C.; Gehin, T.; Vidal, O.; Souteyrand, E.; Vasseur, J. J.; Chevolot, Y.; Morvan, F. The influence of the aromatic aglycon of galactoclusters on the binding of LecA: a case study with O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-biphenyl and O-naphthyl aglycons. Org. Biomol. Chem. 2014, 12 (45), 9166−79. (37) Cao, S. D.; Meunier, S. J.; Andersson, F. O.; Letellier, M.; Roy, R. Mild Stereoselective Syntheses of Thioglycosides under Ptc Conditions and Their Use as Active and Latent Glycosyl Donors. Tetrahedron: Asymmetry 1994, 5 (11), 2303−2312. (38) Cecioni, S.; Praly, J. P.; Matthews, S. E.; Wimmerova, M.; Imberty, A.; Vidal, S. Rational design and synthesis of optimized glycoclusters for multivalent lectin-carbohydrate interactions: influence of the linker arm. Chem. - Eur. J. 2012, 18 (20), 6250−63. (39) Huang, S. F.; Lin, C. H.; Lai, Y. T.; Tsai, C. L.; Cheng, T. R.; Wang, S. K. Development of Pseudomonas aeruginosa Lectin LecA Inhibitors using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds. Chem. - Asian J. 2018, 13, 686. (40) Henis, Y. I.; Katzir, Z.; Shia, M. A.; Lodish, H. F. Oligomeric structure of the human asialoglycoprotein receptor: nature and stoichiometry of mutual complexes containing H1 and H2 polypeptides assessed by fluorescence photobleaching recovery. J. Cell Biol. 1990, 111 (4), 1409−18. (41) Bider, M. D.; Wahlberg, J. M.; Kammerer, R. A.; Spiess, M. The oligomerization domain of the asialoglycoprotein receptor preferentially forms 2:2 heterotetramers in vitro. J. Biol. Chem. 1996, 271 (50), 31996−2001. (42) Ramadugu, S. K.; Chung, Y. H.; Fuentes, E. J.; Rice, K. G.; Margulis, C. J. In silico prediction of the 3D structure of trimeric asialoglycoprotein receptor bound to triantennary oligosaccharide. J. Am. Chem. Soc. 2010, 132 (26), 9087−95.

J

DOI: 10.1021/acs.biomac.8b00358 Biomacromolecules XXXX, XXX, XXX−XXX