Sialic Acid-Targeted Nanovectors with Phenylboronic Acid-Grafted

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Sialic acid-targeted nanovectors with phenylboronic acid-grafted polyethylenimine robustly enhance siRNA-based cancer therapy Manyi Ji, Ping Li, Nan Sheng, Lanlan Liu, Hong Pan, Ce Wang, Lintao Cai, and Yifan Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11866 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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ACS Applied Materials & Interfaces

Sialic

acid-targeted

nanovectors

with

phenylboronic

acid-grafted

polyethylenimine robustly enhance siRNA-based cancer therapy

Manyi Jia, b†, Ping Li a†, Nan Shenga, b†, Lanlan Liua, Hong Pana, Ce Wanga, Lintao Caia, Yifan Maa*

a

Key Lab of Health Informatics of Chinese Academy of Sciences, Guangdong Key Laboratory of

Nanomedicine, Shenzhen Laboratory of Fully Human Antibody Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, Shenzhen 518055, P. R. China. b

Nano Science and Technology Institute, University of Science and Technology of China,

Suzhou, 215123, China

† These authors equally contributed to this paper.

*Correspondence to: Dr. Yifan Ma. Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen 518055, P. R. China. Tel: +86-755-86395216. Fax: +86-755-86392299. Email: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Small interference RNA (siRNA)-based therapy holds a great potential for cancer treatment. However, its clinical application remains unsatisfied due to the lack of safe and effective RNA delivery system. Aberrantly elevated sialyation on cell membrane has been reported as an attractive target for cancer diagnosis and therapy. In this study, phenylboronic acid (PBA) was conjugated onto low molecular weight polyethylenimine (PEI1.8k) to generate amphiphilic PBA-grafted PEI1.8k (PEI-PBA) nanovector, which was designed to facilitate cancer-targeted RNA delivery through the recognition of sialic structures on cancer cell membrane. PEI-PBA simultaneously encapsulated siRNA to form PEI-PBA/siRNA nanocomplexes with great biocompatibility, serum stability and RNase resistance. The cell culture study showed that PEI-PBA/siRNA dramatically increased siRNA uptake up to 70-90% in several cancer cell lines, which relied on the interaction between PBA and sialic acid on cell membrane. Moreover, PEI-PBA nanovector effectively promoted the lysosome escape of siRNA, decreasing the expression of target gene Polo-like kinase 1 (PLK-1) in cancer cells. The systemic administration of

PEI-PBA/PLK-1

siRNA

(PEI-PBA/siPLK1)

nanocomplexes

not

only

facilitated

tumor-targeted siRNA delivery, but also significantly decreased PLK-1 expression in tumors, thereby robustly inducing tumor apoptosis and cell cycle arrest. Additionally, the administration of PEI-PBA/siPLK1 didn’t cause significant systemic toxicity or immunotoxicity. Hence, sialic acid-targeted PEI-PBA could be a highly efficient and safe nanovector to improve the efficacy of cancer siRNA therapy.

Keywords: sialic acid; phenylboronic acid; low molecular weight polyethylenimine; siRNA delivery; cancer-targeting 2


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1. INTRODUCTION Cancers are leading cause of mortality worldwide with approximately eight million cancer related death each year.1-2 Inhibition of tumor-specific pathways or gene expressions by siRNA-based gene therapy has emerged as a promising therapeutic strategy for cancer treatment.3-4 However, its therapeutic efficacy remains unsatisfied due to rapid siRNA degradation, quick renal excretion, poor tissue penetration and cellular uptake, as well as potential off-target effect.5-6 In the past decade, cationic nanoparticle-based nonviral vectors, such as liposomes, cationic polymers, and micelles, have been developed for siRNA delivery in order to improve its gene silencing efficacy.7-8 Among them, polyethylenimine (PEI), a polycation with high ionic charge density, is one of the most promising siRNA vectors, which is able to condense siRNA as well as facilitate its cellular uptake and intracellular endosomal escape, thereby improving RNA interference efficacy.9-11 The transfection efficiency and cytotoxicity of PEIs strongly depend on their molecular weight. High-molecular-weight (HMW) PEIs, especially PEI with molecular weight at 25 kDa (PEI25k), demonstrate potent transfection efficacy and have been applied as a golden standard for DNA/RNA transfection. However, their clinical application remains limited due to the non-degradability and significant cytotoxicity. In contrast, although the low-molecular-weight (LMW) PEIs are less toxic, their transfection efficacy is unsatisfied.12 Therefore, it is highly desirable to develop novel PEI derivatives with high transfection efficacy and low cytoxicity in order to achieve successful siRNA therapy. Chemical modifications, such as incorporating hydrophobic moieties or biodegradable linkages, have been reported as an effective strategy to improve transfection efficacy of LMW PEIs.12-13 Moreover, active tumor targeting appears to be another plausible strategy to increase siRNA uptake and transfection through the interaction of ligands on nanoparticles and specific receptors on tumor cells. Till dates, a variety of molecules, including antibodies, aptamers, 3



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peptides, and small molecules, have been incorporated onto nanoparticles in order to enhance the recognition by receptors on cancer cells.14-17 More recently, aberrant glycosylation on cell surface has been reported as another signature marker for many cancers.18 In particular sialylation, which adds sialic acids, an anionic monosaccharide, onto the termini of glycoproteins or glycolipids, are significantly elevated on the majority of cancer cells.19-20 The hypersialylation of cell membrane during malignant transformation not only contributes to tumor growth and metastasis, but also is strongly associated with the poor prognosis in cancer patients.21 Hence, targeting aberrant sialylation has been considered as a novel strategy for cancer diagnosis and treatment. Phenylboronic acid (PBA) derivatives are synthetic mimics of lectin, which has been reported to selectively recognize sialic acid residues at pH 7.4 with a high K value23-24, thereby allowing targeting sialic acid moieties on cancer cells. Deshayes et al. reported that PBA-installed polymeric micelles (PBA-PEG-PLGA) robustly enhanced the cellular uptake of drug by cancer cells through targeting sialylated epitopes on cell surface.24 Peng et al. showed that incorporating PBA moieties onto LMW PEI significantly enhanced their gene transfection efficacy in vitro.25 More recent study reported that PBA moieties enhanced tumor-targeted DNA delivery of crosslinked LMW PEI both in vitro and in vivo.26 Hence, the functionalization within PBA moieties appears to be a promising strategy for tumor-targeted drug and gene delivery. Herein, we synthesized amphiphilic PBA-functionalized PEI1.8k (PEI-PBA) nanoparticles as a simple and versatile siRNA deliver system, and investigated their anti-cancer RNA interference efficacy both in vitro and in vivo. Our results showed that PEI-PBA nanoparticles simultaneously encapsulated siRNA to form stable PEI-PBA/siRNA nanocomplexes, which not only robustly enhanced the cellular uptake of siRNA through the interaction between PBA and sialic acid on cancer cell surface, but also facilitated the lysosomal escape of siRNA, thereby effectively inhibiting the expression of target gene. The gene silencing efficacy and anti-tumor effect of 4


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PEI-PBA/siRNA were further evaluated in nude mice, and the results showed that sialic acid-targeted PEI-PBA significantly improved the efficacy of siRNA-based cancer therapy. These data suggested that PBA moieties could enhance LMW PEI-mediated cancer siRNA therapy through at least two mechanisms: (1) to promote nanoparticle assembly and RNA condensation through the hydrophobic interaction; (2) to react with cis-diol of sialic acid residues on cancer cell surface to form reversible boronate ester, thereby achieving cancer-targeted RNA delivery (Scheme 1). 2. MATERIALS AND METHODS 2.1. Materials, cell culture and animals Branched PEI25k and PEI1.8k, 4-carboxyphenylboronic acid, 3-aminophenylboronic acid hydrochloride and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (Waltham, USA). Opti–MEM and trypsin-EDTA solution were purchased from Gibco Life Technologies. LysoTracker™ Red, Hochest 33358 were purchased from Invitrogen (CA, USA). All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (MO, USA). All siRNAs, including negative control siRNA, FAM-siRNA and siRNA targeting PLK1 (siPLK1) were supplied by Shanghai GenePharma Co. Ltd. (Shanghai, China). The primers for Real-Time PCR were purchased from Invitrogen (CA, USA). The sequences of siRNA and primers were listed in Supplementary Information Table S1. human breast cancer cell lines (MCF-7 & MDA-MB-231) and Hela cervical carcinoma cell line were obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM medium supplemented with 10% FBS. Female nude mice (4-6 weeks old) were supplied by Guangdong Province Laboratory Animal 5



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Center (Guangzhou, China), and maintained in the institutional animal facility. All animal protocols were approved by IACUC of Shenzhen Institutes of Advanced Technology. 2.2. Synthesis and characterization of PEI-PBA 4-carboxyphenylboronic acid was conjugated to branched PEI1.8k via amide bonds using carbodiimide chemistry. Briefly, 100 mg of 4-carboxyphenylboronic acid was dissolved in 10 mL CH3OH. 280 mg of EDC and 100 mg of NHS were then added into the above solution and reacted at room temperature for 4 h. The resulted solution was dropped into 300 mg of PEI1.8K solution (carboxyphenylboronic acid: PEI = 1: 3, w/w) and stirred at room temperature for 24 h, followed by precipitation in diethyl ether thrice. The crude product was then dissolved in double distilled water and dialyzed with a dialysis bag (MWCO= 600 Da) to obtain purified PEI-PBA. Proton nuclear magnetic resonance (1H NMR) spectra and Fourier transform infrared (FTIR) spectra of PEI-PBA were recorded on a Bruker 400 MHz nuclear magnetic resonance instrument and Fourier transform infrared spectrometer (Bruker vertex 70), respectively. 2.3. Preparation and characterization of PEI-PBA/siRNA nanocomplexes The PEI-PBA/siRNA nanocomplexes with N/P ratios from 10 to 50 were prepared simply by mixing siRNA solution with desired amount of PEI-PBA solution at room temperature for 15 min. The size and zeta potential of PEI-PBA/siRNA were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS90 (Malvern, UK). Morphologies of PEI-PBA/siRNA nanocomplexes were observed by transmission electron microscopy (TEM) using a FEI-F20 microscope (FEI, USA) operating at an acceleration voltage of 100 kV. To evaluate the stability of PEI-PBA/siRNA, the nanocomplexes were suspended in double distilled water (ddH2O), DMEM medium with 10% fetal bovine serum, or FBS at 37 °C. The sizes of the nanocomplexes were monitored at different time intervals. 2.4 Agarose gel electrophoresis assay 6


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To determine siRNA loading capacity of PEI-PBA and PEI1.8k, siRNA nanocomplexes at varied N/P ratios were prepared by gently mixing siRNA with appropriate amount of polymers as described previously. The obtained nanocomplexes were loaded into 1% (w/v) agarose gel and subjected to electrophoresis at 120 V for 15 min in TAE buffer (40 mM Tris-HCl, 1% v/v acetic acid, 1 mM EDTA). For RNA protection assay, PEI-PBA/siRNA nanocomplexes were pretreated with 0.2 mg/mL of RNase A (Takara, Japan) at 37 °C for 0.5-8 h, follow by incubation with heparin sodium (1 mg/mL) for another 10 minutes before electrophoresis. The siRNA was then visualized by Gel Red staining using a Dolphin-Doc Molecular Imaging System (Wealtec, USA). 2.5. Cell viability assessment To determine the in vitro cytotoxicity of PEI-PBA and PEI-PBA/siRNA nanocomplexes, MCF-7 cells were seeded into 96-well plates (4×103 cells/well) and treated with 3-aminophenylboronic acid, PEI1.8k, PEI25k, PEI-PBA with or without PLK1 siRNA at 37 °C for 24 h-48 h. MTT stock solution (5 mg/mL) was then added to each well and incubated for additional 4 h. At the end of the experiment, the medium was replaced with 150 µL of DMSO to dissolve the formazan crystals, and the absorbance was measured at 490 nm using a microplate reader (Synergy 4, BioTec, USA). The cell viability was calculated using following formula: Cell viability (%) = (ODexp-ODblank)/ (ODcontrol-ODblank) ×100%. 2.6. Cellular uptake, binding and intracellular trafficking of siRNA To assess the cellular uptake and binding of siRNA, cancer cells were incubated with 100 nM of free FAM-siRNA or PEI-PBA/FAM-siRNA nanocomplexes at 37 °C or 4 °C for 2 h. In some experiments, cancer cells were pretreated with 20 µM of 3-aminophenylboronic acid hydrochloride to block sialic acid on the cell surface. The percentage of siRNA uptake and binding was determined by measuring FAM-siRNA positive cells using Quanta SC Flow cytometer (Beckman Coulter, CA, USA). 7



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To determine the intracellular localization of siRNA, MCF-7 cells were seeded into an 8-well chambered cover glass and incubated with free FAM-siRNA or PEI-PBA/FAM-siRNA nanocomplexes for 2 h. The cells were then labeled with Lyso-Tracker and Hoechst 33258 to identify lysosomes and nuclei, respectively. The intracellular localization of FAM-siRNA was recorded by confocal microscopy (Leica TCS SP5, Germany). 2.7. Real-time PCR analysis Cancer cells were seeded into 24-well plates at 2×104 cells per well in 0.5 mL of DMEM with 10% FBS and cultured at 37 °C overnight. On next day, the medium was replaced with Opti-MEM medium containing 100 nM of free siRNA or siRNA formulated by PEI1.8k, PEI25k, PEI-PBA with the N/P ratio at 40. After 48 h, the cells were harvested and total RNA was isolated using total RNA Miniprep Kit (Axygen, USA) according to manufacturer's instruction. The mRNA levels of PLK1 were then determined by the quantitative real-time PCR analysis followed by normalization to the housekeeping gene β-actin. To determine the mRNA levels of PLK1 in tumor tissues, total RNA in tumor tissue was isolated by Trizol Reagent (Invitrogen, Life Technologies, Paisley, UK) according to the manufacturer’s instruction, followed by chloroform extraction and isopropyl alcohol precipitation. The mRNA expression of PLK1 in tumors was analyzed by qRT-PCR. 2.8. Cell apoptosis and cell cycle assessment MCF-7 cells were with treated 100 nM of free PLK1 siRNA or siRNA formulated by PEI1.8k, PEI25k, PEI-PBA as previously described. After 36 h, the apoptotic cells were labeled by AnnexinV/PI apoptosis detection kit and detected using flow cytometry. To determine the cell cycle of MCF-7 cells, the cells were harvested at the end of the experiment and fixed with 70% ethanol at 4 °C overnight. The fixed cells were washed with PBS and incubated with propidium iodide (PI, 20 µg/mL, Sigma, MI, USA) and RNase A (0.2 mg/ml, Takara, Japan) for 30 min. The 8


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cell cycle of MCF-7 cells was detected by flow cytometry and analyzed using Flowjo software (Emerald Biotech, China) 2.9. In vivo biodistribution of PEI-PBA/siRNA nanocomplexes MCF-7 cells (3 × 106) were injected into the flank region of female nude mice (about 20 g) to establish breast cancer xenograft model. When the tumor size reached around 200-300 mm3, the mice were i.v. injected with the mixture of free Cy5.5 + siRNA or Cy5.5-PEI-PBA formulated siRNA nanocomplexes. The dose of siRNA was 1250 pmol/mouse/dose with the N/P ratio at 40. The in vivo fluorescence images were recorded at 0, 24 h, and 48 h using the CRi Maestro in vivo imaging system (CRi maestro, Woburn, MA). At the end of the experiment, tumor and major organs (heart, liver, spleen, lung and kidney) were removed for fluorescent imaging. The average fluorescence intensity of tumor and different oragnse was calculated by the following formula: average signals (scaled counts/s/pixel) = total counts/exposure time (s)/area (pixel). To determine the localization of siRNA in tumors, tumor-bearing mice were i.v. injected with 2000 pmol of Cy3-siRNA with or without PEI-PBA encapsulation. After 24 h, tumor sections were labeled with Hoechst 33342 to identify nuclei, and siRNA in tumor tissues were observed using confocal microscopy. 2.10. Animal experiments The MCF-7 breast cancer xenograft model was established as described previously. When tumor volumes reached to 100 mm3, the mice were randomly divided into three groups (6/group) and i.v. injected with PBS, PEI-PBA/siNC nanocomplexes, or PEI-PBA/siPLK1 nanocomplexes every other day for 6 dosages. The dosages of siRNA and PEI-PBA were 2000 pmol /mouse/injection with the N/P ratio at 40. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. The estimated volume was calculated according to the following formula: tumor volume (mm3) =0.5 × length ×width2. Mice were 9



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sacrificed 48 h after the last injection and all tumor tissues were collected, weighted, and then for further experiment. At the end of the experiment, tumors were snap-frozen and cut into 5 um sections. The apoptosis in tumor sections was determined by TUNEL assay according to manufacturer's instruction (Promega, MI, USA). The fluorescent images were recorded using a fluorescent microscopy, and TUNEL+ cells in each field were quantified using the image pro software. The percentage of apoptotic cells was calculated using following formula: % of apoptosis= TUNEL+ cells / total cells ×100%. The proliferation in tumor tissues was determined by Ki67 staining. Briefly, tumor cryosections were blocked with 1% BSA for 30 min, and then incubated with anti-Ki67 antibody (Cell signaling, Beverly, MA) for 2 h, followed by Alexa488-conjugated secondary antibody (Molecular probes, Eugene, OR) for another 1 h. The expression of Ki67 in tumor tissues was recorded using confocal microscopy. 2.11. Safety evaluation Tumor-bearing nude mice were i.v. injected with PBS, PEI-PBA/siNC or PEI-PBA/siPLK1 as described previously. Blood samples were collected at 24 h after first injection, and the serum levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) were quantified using ELISA according to manufacturer's instruction (Biolegend, USA). At 24 h after last treatment, the liver function was evaluated by measuring the plasma level of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) using AST and ALT activity Assay Kit (JianCheng Biotech, China). The renal function was evaluated by measuring the plasma levels of urea nitrogen (BUN) and creatinine (CRE) using colorimetry according to manufacture’s instruction (JianCheng Biotech, China). Major organs (heart, liver, spleen, lung, and kidney) were fixed in 4% paraformaldehyde overnight and cut into 5 um sections for hematoxylin and eosin (H&E) staining, and the histology of different organs was assessed using Olympus microscope. 10


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2.12. Statistical analysis Data are reported as mean ± SE. The differences among groups were determined using one-way ANOVA analysis using GraphPad Prism software (GraphPad Software, CA, USA). 3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of PEI-PBA The PEI-PBA conjugates were synthesized as shown in Scheme 1. Briefly, the carboxy group of 4-carboxyphenylboronic acid was activated by EDC/NHS, and then reacted with amino groups of PEI1.8k to form amide linkages. The resulted PEI-PBA was precipitated in cold diethyl ether and purified by lyophilization. The final products were analyzed by using 1H NMR using D2O as the solvent. The modification degree, which was defined by the number of PBA groups on each PEI molecule, was calculated by comparing the integrals between the phenyl proton signals (7.6–7.2 ppm) of 4-carboxyphenylboronic acid groups and the ethylene proton signals (3.0–2.0 ppm) of PEI in the 1H NMR spectra (Fig. 1A). The results showed that the modification degree of PEI-PBA was about 3. The structure of polymer was further determined by FTIR spectra (Fig. 1B). The characteristic absorption bands at 1344 cm−1 were attributable to B−O stretching

27-28

, whereas the bands at 1641 cm−1 (C═O stretching) and 1564 cm−1 (−NH−CO−

stretching) were the characteristic absorbance of the amide group of PEI-PBA. Hence, the conjugation of PBA to PEI was successful. The cytotoxicity of PEI-PBA was evaluated in MCF-7 cells using MTT assay. Although PEI25k dramatically decreased the viability of MCF-7 cells to less than 25% even at a low dosage (20 µg/mL), 20-100 µg/mL of PEI1.8K did not affect the viability of MCF-7 cells at 24 h (Fig. 1C), indicating great biocompatibility of LMW PEIs.29-30 Moreover, neither free PBA nor PEI-PBA significantly decreased the viability of MCF-7 cells (Fig. 1C, S1). These data suggested that the 11



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functionalization with PBA moieties did not affect the biocompatibility of PEI1.8k. 3.2. Preparation and characterization of PEI-PBA/siRNA nanocomplexes Upon dissolved in ddH2O, amphiphilic PEI-PBA spontaneously self-assembled into nanoparticles with hydrodynamic size around 190 nm. These data suggested that PBA moieties could promote nanoparticle assembly through the hydrophobic interaction. The TEM image showed that PEI-PBA nanoparticles had a homogeneous distribution of spherical nanoparticles with particle size around 20-30 nm (Fig. 2A). The inconsistency of TEM measured particle size and hydrodynamic size should be due to changed secondary structure of nanoparticles in dry state and in H2O. The siRNA solution was then mixed with PEI-PBA conjugates at room temperature and spontaneously formed PEI-PBA/siRNA nanocomplexes (scheme 1). The electrostatic attraction between positively charged amine groups on PEI-PBA and negatively charged phosphates on siRNA resulted in simultaneous formation of PEI-PBA/siRNA nanocomplexes with hydrodynamic size at 156.9 ± 3.8 nm. The TEM image of PEI-PBA/siRNA nanocomplexes (N/P = 40) showed a homogeneous distribution of spherical nanoparticles with particle size around 10-30 nm (Fig. 2B). Notably, PEI-PBA/siRNA showed similar zeta potential but smaller hydrodynamic size as compared with PEI1.8k/siRNA (Fig. 2C), suggesting the involvement of both electrostatic attraction and hydrophobic interaction in siRNA encapsulation, which allowed the formation of PEI-PBA/siRNA nanocomplexes with more compact size 29. The RNA condensation capability of PEI-PBA was evaluated by gel electrophoretic assay. As shown in Fig. 2D, both PEI-PBA and PEI1.8k completely prevented siRNA migration at the N/P ratio of 15 or above. However at the N/P ratio of 10, the siRNA migration of PEI-PBA/siRNA nanocomplexes was more significant than that of PEI1.8k/siRNA. These data suggested slightly decreased RNA binding capability of PEI-PBA, which could be due to reduced amine groups caused by PBA conjugation and increased steric hindrance pertinent to phenyl 12


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group29. When the N/P ratio increased from 10 to 50, the zeta potential of PEI-PBA/siRNA gradually increased from 30 mV to 50 mV, whereas their hydrodynamic sizes were maintained at 150 - 200 nm (Fig. S2). In addition to the capability of RNA condensation, an ideal RNA delivery system must be able to maintain RNA stability in order to achieve effective gene silencing. Herein, we first evaluated the RNA protection capability of PEI-PBA/siRNA nanocomplexes. The result showed that naked siRNA was susceptible to RNase A and quickly degraded within 0.5 h of incubation. In contrast, PEI-PBA encapsulated siRNA did not degrade even after incubation with RNAase A for 8 h. These results demonstrated the strong potency of PEI-PBA/siRNA nanocomplexes to prevent RNA degradation (Fig. 2E). Cationic nanovectors often tend to aggregate or disassemble in serum due to non-specific protein absorption, which usually leads to therapeutic failure. Herein, the serum stability of PEI-PBA/siRNA was determined by measuring their hydrodynamic sizes in FBS. Surprisingly, the hydrodynamic size of PEI-PBA/siRNA nanocomplexes did not significantly change during 48-h storage in FBS, suggesting their great serum stability (Fig. 2F). We next evaluated whether PEI-PBA/siRNA nanocomplexes could protect siRNA from degradation in the presence of serum. The result showed that PEI-PBA/siRNA nanocomplexes successfully protected siRNA from RNase degradation in FBS/PBS (1:1, v/v) solution (Fig. S3). Overall, PEI-PBA could effectively bind siRNA and simultaneously generate stable PEI-PBA/siRNA nanocomplexes, thereby enhancing RNA stability. 3.3. The cellular uptake of siRNA by MCF-7 breast cancer cells Previous studies showed that PBA derivatives were able to selectively react with sialic acid residues at physiological pH, facilitating tumor-targeted drug delivery.24 However, whether it could enhance tumor-targeted RNA delivery remains unknown. In the present study, the effect of PBA functionalization on cellular binding and uptake of siRNA was evaluated by MCF-7 cells, 13



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which overexpress sialic acid on the cell surface.31 MCF-7 cells were incubated with free FAM-siRNA or different siRNA nanocomplexes at 37 °C or 4 °C (Fig. 3A-B). After 2 h incubation, the cellular uptake of free FAM-siRNA by MCF-7 cells was not detectable, consistent with previous studies. The uptake and binding of PEI1.8k/siRNA by MCF-7 cells was less than 10%, indicating poor effect of PEI1.8k on RNA uptake. However, PEI-PBA/siRNA dramatically increased RNA binding and uptake by MCF-7 cells up to 75% and 95%, respectively. Hence, the functionalization with PBA moieties dramatically promoted tumor-targeted siRNA delivery by PEI1.8k. To further determine the role of PBA-sialic acid interaction in RNA uptake, MCF-7 cells were pretreated with 3-aminophenylboronic acid for 1 h to block sialic acid residues on cell surface. The result showed that 3-aminophenylboronic acid significantly diminished both binding and cellular uptake of PEI-PBA/siRNA in MCF-7 cells (Fig. 3A-B). We also pretreated MCF-7 cells with sialidase to hydrolyze terminal sialic acid residues on cell surface. Similarly, the pretreatment of sialidase dramatically attenuated cellular uptake of PEI-PBA/siRNA (supplementary Fig. S4). These data demonstrated that PEI-PBA effectively facilitated the uptake of siRNA through the interaction between PBA and sialic acid residues on cancer cell surface. The capability of tumor-targeted siRNA delivery by PEI-PBA was also assessed in Hela and MDA-MB-231 cells. Similar to the results in MCF-7 cells, the cellular uptake of siRNA by Hela and MDA-MB-231 cells was remarkably increased to 60-70% by PEI-PBA/siRNA nanocomplexes, whereas the pretreatment of 3-aminophenylboronic acid significantly decreased siRNA uptake to 10-20% (Fig. 3C-D). Overall, PEI-PBA robustly promoted siRNA uptake by interacting with sialic acids on cancer cell surface. The intracellular localization of siRNA was further investigated by confocal imaging. As shown in Fig. 3E, the fluorescent signal of PEI-PBA/siRNA was significantly higher than that of free siRNA and PEI1.8k/siRNA, confirming increased siRNA uptake. Blocking sialic acid with 14


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3-aminophenylboronic acid dramatically attenuated intracellular siRNA, confirming the essential role of the PBA-sialic acid interaction. Moreover, a significant amount of PEI-PBA formulated siRNA was located in the cytoplasm rather than in lysosomes. These data indicated effective endosome/lysosome escape of siRNA, which could be caused by the protonation of amine groups of PEI-PBA similar to “proton sponge effect” of PEI.12 Hence, PEI-PBA/siRNA nanocomplexes not only remarkably enhanced siRNA uptake by cancer cells through the PBA-sialic acid interaction, but also promoted siRNA release in the cytosol, both which are critical for effective RNA interference. 3.4. In vitro RNA interference by PEI-PBA in cancer cells Polo-like kinase 1 (PLK1) is a key regulator of cell division, playing a crucial role in mammalian cell mitosis and in the maintenance of genomic stability. Previous studies have shown that PLK1 is overexpressed in the majority of human cancers and participates in the oncogenic transformation.32-33 Therefore, we selected PLK1 as a target gene for cancer treatment. MCF-7 cells were incubated with free PLK1 siRNA (siPLK1) or siPLK1 formulated with PEI1.8k, PEI25k, or PEI-PBA with different N/P ratios for 48 h. The results showed that neither free siPLK1 nor PEI1.8k/siPLK1 significantly affected PLK1 mRNA, which should be due to the poor uptake of siRNA. However, PEI25k/siPLK1 significantly reduced PLK1 mRNA by 80%. PEI-PBA formulated siPLK1 with N/P ratios from 20 to 50 effectively decreased PLK1 mRNA in MCF-7 cells by 50%-70%, whereas blocking sialic acid with 3-aminophenylboronic acid effectively recovered PLK1 mRNA level (Fig.4A-B). These data indicated the robust gene silencing of PEI-PBA that could be attributable to its strong potency of tumor-targeted RNA delivery through the recognition of sialic acids on cancer cells. The gene silencing efficacy of PEI-PBA/siRNA nanocomplexes was also investigated in Hela and MDA-MB-231 cells. The results showed that PEI-PBA/siPLK1 nanocomplexes significantly decreased PLK1 mRNA in Hela and 15



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MDA-MB-231 cells to 50-60%, whereas blocking sialic acid with 3-aminophenylboronic acid effectively restored the mRNA level of PLK1 (Fig.4C-D). These data suggested that PEI-PBA robustly enhanced gene silencing efficacy of siRNA in through targeting sialic acids on cancer cell surface. 3.5. The anti-cancer effect of PEI-PBA/siPLK1 nanocomplexes in MCF-7 cells PLK1 inhibition has been reported to suppress tumor growth by inducing mitotic defects, causing significant G2/M cell cycle arrest, and triggering cell apoptosis.34 Herein, the anti-cancer effect of PEI-PBA/siPLK1 nanocomplexes was evaluated in vitro. MCF-7 cells were incubated with 100 nM siPLK1 or PEI-PBA/siPLK1 in or without the presence of serum, and the cell viability was determined by MTT assays. As shown in Fig. 5A-B, free siPLK1 or PEI1.8K/siPLK1 did not affect the viability of MCF-7, which should be attributed to poor siRNA uptake and ineffectively RNA interference. Although PEI25K/siPLK1 significantly reduced cell viability of MCF-7 cells by 45% in serum free medium, the presence of serum significantly abolished its anti-cancer effect. PEI-PBA/siPLK1 significantly reduced the viability of MCF-7 cells by 40% in serum free medium, which was similar to that of PEI25K/siPLK1. More interestingly, PEI-PBA/siPLK1 significantly reduced the viability of MCF-7 cells about 30% in the presence of serum (Fig. 5A-B). These results suggested serum-tolerant transfection activity of PEI-PBA/siPLK1 nanocomplexes, which would be critical for successful RNA therapy in vivo. Previous study reported that the surface hydroxylation could improve serum stability and serum-tolerant transfection efficacy of PEI25K.35 In the present study, since each PBA group contains two hydroxyl groups, PBA functionalization could increase surface hydroxylation of PEI-PBA, thereby contributing to serum-tolerant transfection activity. The decreased viability of MCF-7 cells by PLK1 gene deletion could be attributable to 16


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apoptosis and G2/M cell cycle arrest.36 Herein, the effect of different treatments on MCF-7 cell apoptosis was investigated using Annexin V/PI staining. The results showed that neither free siPLK1 nor PEI1.8k/siPLK1 significantly induced apoptosis in MCF-7 cells, consistent with the result of MTT assay. In contrast, PEI25k/siPLK1 (N/P ratio=10) resulted in 27.8% of Annexin V+ apoptotic cells in MCF-7 cell culture. The administration of PEI-PBA/siPLK1 led to 19.6% of cell apoptosis, whereas the replacement of siPLK1 with control siRNA (siNC) significantly diminished apoptosis (Fig. 5C and 5E). These data suggested that PEI-PBA/siPLK1 triggered cancer cell apoptosis by interfering PLK1 gene expression. In addition to cell apoptosis, the effect of PEI-PBA/siPLK1 on the cell cycle of MCF-7 was also evaluated. The results showed that free siPLK1 and PEI11.8k/siPLK1 did not affect the cell cycle of MCF-7 cells. In contrast, PEI25k/siPLK1 and PEI-PBA/siPLK1 both significantly increased the percentage of G2/M over 50%, which was typical G2/M cell cycle arrest. However, the replacement of siPLK1 with siNC recovered the cell cycle in MCF-7 cells (Fig. 5D and 5F). Overall, PEI-PBA/siPLK1 successfully decreased PLK1 gene, thereby inhibiting MCF-7 cell growth by inducing the apoptosis and G2/M cell cycle arrest. 3.6. In vivo biodistribution of PEI-PBA/siRNA nanocomplexes in tumor-bearing mice. The in vivo biodistribution of PEI-PBA/siRNA nanocomplexes was investigated in tumor-bearing nude mice. Mice were i.v. injected with Cy5.5-PEI-PBA formulated siRNA (Cy5.5-PEI-PBA/siRNA) or the mixted solution of free Cy5.5 + siRNA from tail veins. The fluorescent signals of free dye or nanocomplexes in tumors were recorded at 0, 24, 48 h post-injection using an in vivo imaging system. The results showed that the fluorescent signal was barely detectable in tumors of mice injected with Cy5.5+siRNA. However, the injection of Cy5.5-PEI-PBA/siRNA gradually increased fluorescent signals in tumors from 24-48 h after injection (Fig. 6A-B). The fluorescent intensity of Cy5.5-PEI-PBA/siRNA of tumor tissues was 17



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about 3 fold higher than that of Cy5.5+siRNA, which suggested tumor-targeting ability of PEI-PBA/siRNA nanocomplexes (Fig. 6C, S5). To further determine the accumulation of siRNA in tumors, tumor-bearing mice were i.v. injected with Cy3-siRNA with or without PEI-PBA encapsulation. Similarly, free Cy3-siRNA was not detectable in tumor tissues at 24 h, consistent with the result of in vivo imaging. Although mice injected with PEI1.8k/siRNA didn’t show significant siRNA signal in tumors, PEI-PBA/siRNA nanocomplexes effectively increased siRNA accumulation in tumors (Fig. 6D). Hence, sialic acid-targeted PEI-PBA is an effective nanovector facilitating tumor-targeted siRNA delivery. The biodistribution of PEI-PBA/siRNA nanocomplexes in major organs were assessed at 48 h after injection. Compared with Cy5.5-PEI-PBA/siRNA groups, the injection of Cy5.5+siRNA led to weaker fluorescent signals in major organs except kidney, suggesting short blood circulation of free dye due to the renal excretion.37 In contrast, mice treated with Cy5.5-PEI-PBA/siRNA showed significantly elevated fluorescent signals in the liver (Fig. 6C, S5). The undesirable liver accumulation might be due to increased entrapment in liver reticuloendothelial system (RES) caused by the positive charge of the nanocomplexes.38 Nonetheless, PEI-PBA/siRNA nanocomplexes effectively accumulated in tumors, which would consequently promote tumor-specific siRNA delivery and gene silencing. 3.7. The antitumor effect of PEI-PBA/siPLK1 nanocomplexes in vivo The antitumor effect of PEI-PBA/siPLK1 nanocomplexes was investigated in a MCF-7 tumor xenografted mice model. Nude mice bearing MCF-7 tumors (size ≥ 100 mm3) were i.v. injected with PBS, PEI-PBA/siPLK1 or PEI-PBA/siNC every other day for 6 dosages. During the experiment, three groups didn’t show significant body weight (Fig. 7B), suggesting the treatment of nanocomplexes was tolerable. Although PBS or PEI-PBA/siNC did not affect the tumor 18


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growth, PEI-PBA/siPLK1 nanocomplexes significantly suppressed tumor growth. At the end of the experiment (day 18), the tumor volume and tumor weight were both reduced over 50% by PEI-PBA/siPLK1 nanocomplexes (Fig. 7A and 7C). Moreover, the administration of PEI-PBA/siPLK1 rather than PEI-PBA/siNC effectively decreased PLK1 mRNA in tumors about 30% as compared with PBS group (Fig. S6). These data indicated that PEI-PBA/siPLK1 inhibited tumor growth through down-regulating PLK1 gene expression in tumors. Since PLK1 gene deletion led to cell apoptosis and cell cycle arrest of MCF-7 cells in vitro, we next evaluated the effect of PEI-PBA/siPLK1 on tumor cell apoptosis and proliferation in vivo. The result of TUNEL assay showed that the treatment of PEI-PBA/siPLK1 but not PBS or PEI-PBA/siNC significantly elevated fluorescent signals in tumor sections (Fig. 7D-E), suggesting increased tumor cell apoptosis. Ki-67 is a nuclear antigen and a marker of cell proliferation expressed in proliferating cells but absent in G0 resting cells.39 In the present study, the treatment with PEI-PBA/siPLK1 rather than PEI-PBA/siNC significantly attenuated Ki67 expression in tumors (Fig. 7D and 7F), suggesting inhibited tumor cell proliferation. Overall, the administration of PEI-PBA/siPLK1 successfully decreased PLK1 gene expression in tumors, which consequently inhibited tumor cell proliferation and led to apoptosis, thereby suppressing tumor growth. 3.8. Safety evaluation Biosafety is always a major concern of gene therapy. Previous studies have shown that the systemic administration of formulated siRNA nanocomplexes was related with systemic toxicity and immunotoxicity.40 Therefore, it is quite necessary to evaluate the biosafety of PEI-PBA/siRNA nanocomplexes in vivo. In the present study, PEI-PBA/siPLK1 nanocomplexes were significantly accumulated in the liver in addition to tumors, hence their effect on liver function must be evaluated. At the end of the experiment, the liver function was evaluated by 19



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measuring serum ALT and AST, which are important liver enzymes and critical biomarker of liver damage.41 As shown in Fig. 8A, the administration of PEI-PBA/siPLK1 or PEI-PBA/siNC did not affect serum concentration of AST and ALT, suggesting that siRNA nanocomplexes did not cause significant liver damage. In some animals, siRNA nanocomplexes were observed to accumulate in the kidney, we therefore evaluated the renal function by measuring plasma BUN and CRE at the end of the experiment. The results showed that neither free siRNA nor siRNA nanocomplexes elevated plasma BUN and CRE, indicating no significant renal toxicity (Fig. 8B). Increasing evidences showed that nanoparticles may stimulate the immune system and cause unwanted immune responses (so called immunotoxicity). Proinflammatory cytokines generally serve as a biomarker of immunotoxicity,42 we therefore measured three proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in the serum. At 24 h after first injection, neither PEI-PBA/siPLK1 nor PEI-PBA/siNC significantly elicited the production of IL-1β, IL-6 and TNF-α in serum (Fig. 8C), suggesting the systemic administration of PEI-PBA/siPLK1 nanocomplexes did not cause significant inflammatory responses in vivo. The potential immunotoxicity of PEI-PBA/siPLK1 nanocomplexes was also evaluated in mouse splenocytes in vitro. Similar to the results of in vitro study, PEI-PBA/siPLK1 nanocomplexes didn’t significantly induce the production of IL-6 or TNF-α at 24 h (Fig. S7), confirming their immunotoxic effect was negligible. The potential toxicity of PEI-PBA formulated siRNA nanocomplexes was further investigated in major organs using H&E staining. As shown in Fig. 8D, the major organs of mice, including heart, liver, spleen, lung and kidney, showed normal histological structure without histopathological abnormalities, lesions or degenerations. And there were no significant histological differences in the major organs between PEI-PBA/siRNA treated and PBS-treated mice. The results further confirmed that PEI-PBA/siRNA nanocomplexes did not induce 20


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significant toxicity in vivo. 4. CONCLUSIONS The present study reported PEI-PBA as a simple and highly efficient nanocarrier, which could interact with aberrantly elevated sialic acid residues on cancer cell membrane, thereby achieving cancer-targeted siRNA delivery. The PEI-PBA/siRNA nanocomplexes not only dramatically enhanced siRNA uptake by cancer cells via the PBA-sialic acid interaction, but also effectively promoted the lysosomal escape of siRNA, thereby successfully decreased target gene expression both in vitro and in vivo. More importantly, the in vivo administration of PEI-PBA/siPLK1 effectively inhibited tumor growth without eliciting significant toxicity or immunotoxicity. Hence, sialic acid-targeted PEI-PBA could be a simple, effective, and safe approach to achieve successful cancer siRNA therapy. 5. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (Grant No. 81371679), Shenzhen Overseas Outstanding Professional Talent (KQCX20140520154115025,), Shenzhen

Science

and

Technology

Program

(JCYJ20150521094519473,

GJHS20140610151856702, CXZZ20130506140505859), Guangdong leading talents program (Antibody/Protein Drugs for Major Diseases), Shenzhen Peacock Next-generation Monoclonal Antibody Drug research and development program

(KQTD201210).

6. SUPPORTING INFORMATION Supporting information included siRNA sequences and additional experimental results. This information is available free of charge via the Internet at http://pubs.acs.org/.

21



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Scheme 1. Synthesis of PEI-PBA conjugates for sialic acid-targeted siRNA delivery.

Fig. 1. Characterization of PEI-PBA conjugates. (A) 1H NMR spectra of PEI-PBA. (B) FT-IR spectra of PBA, PEI1.8k and PEI-PBA. (C) MCF-7 cells were treated with 0-100 µg/ml of PEI-PBA, PEI1.8k, PEI25k for 24 h, and the cell viability was determined using MTT assay.

25



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Fig. 2. Characterization of PEI-PBA/siRNA. (A) Size distribution and TEM image of PEI-PBA nanovectors. (B) Size distribution and TEM image of PEI-PBA/ siRNA nanocomplexes. (C) Size and zeta potential of different nanocomplexes. (D) Agarose gel electrophoresis of siRNA loaded nanocomplexes. (E) RNAase A protection assay of nanocomplexes. (F) Stability of PEI-PBA/siRNA in different solutions. Bars shown are mean ± SE (n=3-4).

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Fig. 3. The cellular binding, uptake and intracellular localization of siRNA in cancer cells. (A-B) MCF-7 cells were incubated with FAM-siRNA or nanocomplexes ± 3-aminophenylboronic acid hydrochloride (PB, 20 µM ) at 37 °C and 4 °C for 2 h. The uptake and binding of siRNA was quantified using flow cytometry. (C-D) The cellular uptake of siRNA by Hela cells and MDA-MB-231 cells. Bars shown are mean ± SE, and differences between siRNA and other groups were analyzed by one-way ANOVA. **: p < 0.01. (E) Confocal images of MCF-7 cells incubated with FAM-siRNA or nanocomplexes for 2 h. Cell nuclei and lysosome were identified by Hoechest 33258 (blue) and Lyso-tracker (red).

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Fig. 4. Gene silencing efficacy of PEI-PBA/ siPLK1 in cancer cells. MCF-7 cells (A-B), Hela cells (C) and MDA-MB-231 cells (D) were treated with 100 nM siPLK1 or nanocomplexes ± 3-aminophenylboronic acid hydrochloride (PB, 20 µM ) for 48 h. PLK1 mRNA was determined by real-time PCR followed by normalization with β-actin mRNA. Bars shown are mean ± SE, and differences between medium and treated groups were analyzed by one-way ANOVA. *: p

Sialic Acid-Targeted Nanovectors with Phenylboronic Acid-Grafted

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