Cell-Targeting Cationic Gene Delivery System Based on a Modular

May 18, 2016 - Cell-Targeting Cationic Gene Delivery System Based on a Modular Design Rationale. Jia Liu†, Luming Xu†, Yang Jin‡, Chao Qi†, Qi...
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A cell-targeting cationic gene delivery system based on a modular design rationale Jia Liu, Luming Xu, Yang Jin, Chao Qi, Qilin Li, Yunti Zhang, Xuling Jiang, Guobin Wang, Zheng Wang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04462 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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A cell-targeting cationic gene delivery system based on a modular design rationale

Jia Liu†a, Luming Xu†a, Yang Jin†b, Chao Qia, Qilin Lia, Yunti Zhangc, Xulin Jiangc, Guobin Wang*d, Zheng Wang*a,d, Lin Wang*a,e

a. Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022 b. Department of Respiration, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022 c. Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, China 430072 d. Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022; Email: [email protected], [email protected]; Tel: +86-27-85726612 e. Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022; Email: [email protected]; Tel: +86-27-85726612 †, These authors contributed equally to this work. * Correspondence to: Lin Wang, Phone: 86-27-85726612. E-mail: [email protected] Zheng Wang, Phone: 86-27-85726612. E-mail: [email protected] Or to:Guobin Wang, Phone: 86-27-85726612. E-mail: [email protected]

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ABSTRACT En route to target cells, a gene carrier faces multiple extra- and intracellular hurdles that would affect delivery efficacy. Although diverse strategies have been proposed to functionalize gene carriers for individually overcoming these barriers, it is challenging to generate a single multi-functional gene carrier capable of surmounting all these barriers. Aiming at this challenge, we have developed a supramolecular modular approach to fabricate a multi-functional cationic gene delivery system. It consists of two pre-functionalized modules: (1) a host module: a polymer (PCD-SS-PDMAEMA) composed of poly(β-cyclodextrin) backbone and disulfide-linked PDMAEMA arms, expectedly acting to compact DNA and release DNA upon cleavage of disulfide linkers in reductive microenvironment; and (2) a guest module: adamantyl and folate terminated PEG (Ad-PEG-FA), expectedly functioning to reduce non-specific interactions, improve biocompatibility, and provide folate-mediated cellular targeting specificity. Through the host-guest interaction between β-cyclodextrin units of the “host” module and adamantyl groups of the “guest” module, the PCD-SS-PDMAEMA-1 (host) and Ad-PEG-FA (guest) self-assemble

forming

a

supramolecular

pseudo-copolymer

(PCD-SS-PDMAEMA-1/PEG-FA). Our comprehensive analyses demonstrate that the functions pre-assigned to the two building modules are well realized. The gene carrier effectively compacts DNA into stable nano-sized polyplexes resistant to enzymatic digestion, triggers DNA release in reducing environment, possesses significantly

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improved hemocompatibility, and specifically targets folate-receptor positive cells. Most importantly,

endowed

with

PCD-SS-PDMAEMA-1/PEG-FA

these

supramolecular

pre-designed gene

carrier

functions, exhibits

the

excellent

transfection efficacy for both pDNA and siRNA. Thus, this work represents a proof-of-concept example showing the efficiency and convenience of an adaptable, modular approach for conferring multiple functions to a single supramolecular gene carrier towards effective in vivo delivery of therapeutic nucleic acids.

KEYWORDS: gene delivery, disulfide bond, poly(ethylene glycol), cyclodextrin, folate, host-guest interaction.

INTRODUCTION The recent emergence of revolutionary CRISPR-Cas9 genome-editing techniques has ignited unprecedented research interests in gene therapy.1, 2 Gene therapy is thought to be a promising approach for treating diseases, such as genetic disorders, acquired disorders and cancer.3 However, the same challenges facing all gene therapy including the powerful CRISPR-Cas9 genome editing technique are bio-safety and delivery efficacy of carriers that transfer therapeutic nucleic acids to target sites.4-7 Cationic polymers are one of the major types of non-viral gene carriers with the features of easy fabrication and good bio-safety.8 Various polycations, including -3  

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poly(ethyleneimine)

(PEI),9,

10

poly(2-(dimethylamino)

ethyl

methacrylate)

(PDMAEMA),11-14 and polyamidoamine (PAMAM)15 dendrimers, have been explored as gene carriers. Nevertheless, cationic polymeric carriers are often suffered from their low transfection efficiency, due to the various extra- and intracellular barriers existing in gene delivery routes,16 such as enzyme-mediated degradation of carried DNA in extracellular microenvironment and endosomal compartments, and rapid clearance of gene carriers by reticuloendothelial system (RES) in vivo.17, 18 To cope with these obstacles, diverse strategies have been developed. For instance, reduction-degradable cationic polymers can release nucleic acids via cleavage of disulfide bonds triggered by intracellular glutathione (GSH);19-25 poly(ethylene glycol) (PEG) was used to decrease non-specific interactions with serum proteins in order to avoid in vivo RES clearance; antibodies or target ligands were introduced to gene carriers helping enhance receptor-mediated cellular uptake by specific tissue or cells.26-28 However, most of these strategies often concern just one aspect of gene delivery. A gene carrier possessing the functions required to surmount all the aforementioned barriers would be ideal but lacks, partly because the fabrication and preparation of such a multi-functional gene carrier involve technically challenging, complicated multi-step chemical reactions and verbose purification procedures. A potential solution comes from supramolecular chemistry, an effective, convenient way of generating macromolecular architectures by assembling individual

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molecular building blocks via host-guest interactions.29, 30 A variety of supramolecular functional materials have been studied for biomedical applications, such as β-cyclodextrin (β-CD), a cyclic oligosaccharide composed of 7 α (1→4)-linked glucose units.31-33 β-CD has a basket-shaped structure with a hydrophobic cavity that can spatially accommodate adamantane (Ad) to form the inclusion complexes with a strong binding ability (Ka is about 105 M-1 in water).34, 35 Its cyclodextrin unit acts as the “host” moiety bridge-linking different functional building components. Diverse shapes of β-CD based polycations (CDCPs), including linear, star and dendritic shapes, have been developed as a “host” to accommodate “guest” molecules with different topological structures towards desired functionality.36-46 Taking advantages of this host-guest interaction of supramolecular chemistry, we hope to develop a modular approach for fabricating multi-functional gene delivery system using the building blocks with pre-built-in functions. The “host” module we intend to generate would have two functions: (1) effective compacting of therapeutic nucleic acids; (2) reductive-triggered degradation of the carrier for releasing nucleic acids. To integrate these functions into host molecules, we consider fabricating a disulfide-linker-containing brush-shaped cationic polymer with a β-cyclodextrin-based polymer (PCD) backbone. The cationic nature of this host would efficiently compact nucleic acids, while their release only occurs upon the cleavage of disulfide bonds specifically triggered by intracellular reductive environment. Meanwhile, the “guest” building module would be assigned to possess another two functions: (1)

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“stealth” effect avoiding rapid clearance by RES; (2) cell targeting activity allowing cell specific delivery. To realize this design, we consider fabricating an adamantyl-terminated PEG with folate groups (Ad-PEG-FA), whose PEG polymers could provide “stealth” effect and conjugated folate could achieve carrier’s specific cellular uptake mediated by folate-receptors (FR).47-49 With this design rationale, we have generated such a multi-functional gene carrier system. By atom transfer radical polymerization (ATRP), the host module has been synthesized

to

be

the

reducible

brush-shaped

PDMAEMA

derivatives

(PCD-SS-PDMAEMA), which contain poly(β-cyclodextrin) as the backbone and disulfide-linked PDMAEMA as the arms (Figure 1). The host PCD-SS-PDMAEMA and the guest Ad-PEG-FA are self-assembled to be a supramolecular pseudo-copolymer (PCD-SS-PDMAEMA/Ad-PEG-FA), which binds DNA/RNA forming a nano-sized polyplexes (Figure 1). The comprehensive physical, chemical and biomedical characterizations demonstrate that this gene carrier system possesses high gene condensation capability, reducible sensitivity, low cytotoxicity, specific cellular uptake and high transfection efficacy. Therefore, this supramolecular modular approach might be a promising way of customizing a versatile gene delivery system for biomedical applications.

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Figure 1. Schematic illustration of the host-guest self-assembly process, receptor-mediated specific cellular uptake, and glutathione-triggered intracellular gene release from the PSD/PEG-FA supramolecular gene delivery system.

EXPERIMENTAL SECTION Materials N,N'-carbonyldiimidazole (CDI), cystamine dihydrochloride, 2-bromoisobutyryl bromide, 4-dimethylaminopyridine (DMAP), 2-(dimethylamino)ethyl methacrylate (DMAEMA), 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA), 25 kDa branched polyethylenimine (PEI) were purchased from Sigma–Aldrich. Triethylamine (TEA) was purchased from Sinopharm chemical regent Co., Ltd (Shanghai, China). Methoxy

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poly(ethylene glycol) amine (mPEG-NH2, Mw: 3.5 kDa) was obtained from JenKem technology (Beijing, China). Adamantyl and folate terminated poly(ethylene glycol) (Ad-PEG-FA, Mw: 3.4 kDa) was synthesized in previous work.50 YOYO-1 and Lipofectamine 2000 was obtained from Invitrogen. Plasmid pcDNA3_Luc in TE buffer at a concentration of 5.0 mg/mL was from Plasmid Factory (Germany). EGFP targeting siRNA

(5’-pACCCUGAAGUUCAUCUGCACCACcg-3’,

3’-ACUGGGACUUCAAGUAGACGUGGUGGC-5’) and negative control siRNA were synthesized by GenePharma (China).

Synthesis of disulfide-containing ATRP initiator (PCD-SS-BIB) Mono bromisobutylryl functionalized cystamine (Cyst-BIB) were synthesized from cystamine and 2-bromoisobutyryl bromide. First, cystamine dihydrochloride was neutralized by NaOH solution and extracted with dichloromethane to obtain fresh cystamine. Then cystamine (5.0 g, 32.5 mmol) and triethylamine (1.95 mL, 14 mmol) were dissolved in dichloromethane (100 mL) at an ice bath for 30 minutes, subsequently 2-bromoisobutyryl bromide (1.86 g, 8.1 mmol) solution in dichloromethane (20 mL) was added dropwise within 30 minutes. After reaction 24 hours at room temperature, the resulting white suspension was filtered and the filtrate was rotary evaporated to remove dichloromethane and triethylamine. The resulting raw product was purified by gel column chromatography with an ethyl acetate/methanol mixture (3/1, v/v) as eluent to

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obtain Cyst-BIB. Poly(β-cyclodextrins) (PCD, Mw: 60.4 kDa, PDI: 2.02) was synthesized at epichlorohydrin/cyclodextrin molar ratio of 4/1 as previous reported51 and described in supporting information. The ATRP macroinitiator (PCD-SS-BIB) was synthesized by two steps: (1) activating the hydroxyl groups of PCD with N,N'-carbonyldiimidazole (CDI) and (2) reaction of activated hydroxyl groups with amine groups of Cyst-BIB. In detail, PCD (0.3 g) was dissolved in DMSO (10 mL), subsequently CDI (0.47 g, 2.9 mmol) solution in DMSO (4 mL) was added dropwise. After stirring overnight, the resulting solution was poured into a THF/diethyl ether (2/3, v/v) mixture to precipitate the product, and the product was further purified by precipitating three more times in THF/diethyl ether mixture. Next, the resulting solid (0.15 g) and DMAP (0.15 g, 1.2 mmol) were dissolved in DMSO (10 mL), and then Cyst-BIB (0.36 g, 1.2 mmol) was added dropwise to the mixture and stirred at room temperature for 24 hours. The reaction solution was precipitated in THF/diethyl ether (2/3, v/v) mixture to obtain the formed polymer, which was purified by repeating precipitation two more times and drying under vacuum. The resulting polymer PCD-SS-BIB was characterized by 1H NMR.

Synthesis of PCD-SS-PDMAEMA via ATRP For preparation of PCD-SS-PDMAEMA polymers (termed as PSD) via ATRP, the feed ratios of monomer (DMAEMA) and initiator site of PCD-SS-BIBA were 20/1, 40/1

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and 60/1, respectively. The feed CuBr and PMDETA were 1.5 times the mole of the initiator site. In short, macroinitiator PCD-SS-BIB (50 mg, 0.08 mmol initiator site), monomer DMAEMA (276 µL, 1.6 mmol) and ligand PMDETA (25 µL, 0.12 mmol) were introduced into a flask containing 6 mL of DMSO, and the solution was deoxygenated by three vacuum-nitrogen cycles. The catalyst CuBr (17.2 mg, 0.12 mmol) was subsequently added under a nitrogen atmosphere. Polymerization was performed at 55oC for 3 hours. The formed polymer was purified by dialysis (MWCO: 12~14 kDa) against EDTA-2Na solution (0.1 mM) for 2 days and then against H2O for 2 days. The final product was obtained by lyophilization and its molecular weight was determined by aqueous size exclusion chromatography with multi-angle laser light scatter (SEC-MALLS).

Synthesis of adamantyl functionalized PEG (Ad-PEG) 1-Adamantanecarbonylchloride (0.4 g, 2.01 mmol) was dissolved in anhydrous dichloromethane (15 mL) at an ice bath, and then mPEG-NH2 (0.7 g, 0.2 mmol) and triethylamine (0.22 g, 2.2 mmol) solution in dichloromethane (20 mL) were added dropwise within 30 minutes. The reaction mixture was stirred at room temperature for overnight, then the dichloromethane and triethylamine were removed by vacuum and the obtained product was dissolved in 10 mL water. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was dialyzed against water (MWCO: 1000 Da). The adamantyl-terminated PEG (Ad-PEG) was obtained by lyophilization, and analyzed by

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H NMR.

Formation of PSD-1/PEG and PSD-1/PEG-FA supramolecular copolymer PSD-1 was dissolved in ddH2O to prepare a solution with a concentration of 1 mg/mL. Then, different amounts of Ad-PEG or Ad-PEG-FA in DMSO were added dropwise into PSD-1 solution with moderate stirring. The mixture solutions were stirred at room temperature for 24 hours, and dialyzed against water (MWCO: 12~14 kDa) for 2 days. The PSD-1/PEG and PSD-1/PEG-FA supramolecular pseudo-brushed copolymer were obtained by lyophilization, and the copolymers were abbreviated as PSD-1/PEG and PSD-1/PEG-FA. The host-guest interactions of PSD-1/PEG-FA were analyzed using 2D 1

H nuclear Overhouser effect spectroscopy (NOESY) in D2O as previously described.51-53

Characterizations 1

H nuclear magnetic resonance (NMR) spectra were measured using Mercury 300

MHz spectrometer (Varian Associates Inc. NMR instruments, Palo Alto, CA) with CDCl3, D2O or DMSO as solvent. 2D 1H nuclear Overhouser effect spectroscopy (NOESY) spectra were obtained with a Bruker Avance III-600 MHz spectrometer (Bruker Biospin, Germany). The molecular weights of the cationic polymers were measured by SEC-MALLS using a Waters 2690D separation module, a Waters 2414 refractive index detector and a Wyatt DAWN EOS MALLS detector. Two chromatographic columns

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(Shodex OHpak SB-803 and SB-802.5) with a pre-column (Shodex SB-G) were used in series. A sodium acetate solution (0.3 M, pH 4.4) was used as eluent at a flow rate of 0.6 mL/min.

Cell culture Human cervix carcinoma cells (HeLa), SV-40 transformed African green monkey kidney cells (COS-7) and human nasopharyngeal cells (KB) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin at 37oC under an atmosphere of 95% air and 5% CO2. KB and COS-7 cells stably expressing enhanced green fluorescent protein (KB_EGFP and COS-7_EGFP) were generated by infection with the EGFP expressing lentivirus (LV).54

Formation and characterization of polymer/pDNA polyplexes Formulation of polyplexes: The cationic polymers in HBS buffer (20 mmol Hepes, 130 mmol NaCl, pH 7.4) were diluted to appropriate concentrations in HBS buffer, and added into pcDNA3_Luc plasmid DNA (pDNA) solution (50 ng/µL, in HBS), following with vortex (5 seconds) and incubation (30 minutes) at room temperature to obtain polymer/DNA polyplexes. The siRNA polyplexes were prepared with the same protocol. Agarose gel electrophoresis retardation assay: The formed polyplexes were

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analyzed by electrophoresis in a 1% (w/v) agarose gel containing ethidium bromide (EB, 0.25 mg/mL) in Tris–acetate (TAE) buffer at 90 V for 45 minutes. DNA bands were visualized with an UV (254 nm) illuminator and photographed with a Gel Doc™ XR+ imaging system (Bio-Rad, USA). The reduction triggered DNA release of PCD-SS-PDMAEMA based polyplexes in the presence of DTT was also evaluated by agarose gel retardation assay. The PSDs/DNA and PSD-1/PEG-FA/DNA polyplexes prepared at different weight ratios were incubated with 10 mM DTT for 30 minutes at room temperature, and analyzed by electrophoresis at 90 V for 45 minutes. DNase I protection assay: The PSD-1/DNA and PSD-1/PEG-FA/DNA polyplexes at various weight ratio were incubated with DNase I (5 units for 1 µg pDNA, TAKARA Corp., DaLian, China) for 30 minutes at 37oC. The digestion was stopped by the addition of EDTA to a final concentration of 10 mM. To dissociate the pDNA molecules from polyplexes, heparin was added to a final concentration of 2 mg/mL, the mixtures were further incubated for 30 minutes at 37oC, and analyzed by agarose gel electrophoresis at 90 V for 45 minutes. Measurement of particle size and zeta potential: The polymer/DNA polyplexes were prepared at various w/w ratios by adding appropriate concentrations of polymer solutions (960 µL in HBS) to 40 µL of plasmid DNA solution (50 ng/µL, in HBS). After incubation at room temperature for 30 minutes, particle size and zeta-potential were measured by dynamic light scattering (DLS) using a Nano-ZS ZEN3600 (Malvern

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Instruments) at 25oC. The stability of PSDs/DNA and PSD-1/PEG-FA/DNA polyplexes (w/w ratio of 20) were evaluated by monitoring the particle size over 7 days at 25oC. The morphology of PSD-1/DNA and PSD-1/PEG-FA/DNA polyplexes was observed using a transmission electron microscope (JEM-2100 HR, JEOL). The DTT triggered destabilization of PSD-1/DNA polyplexes was also studied by DLS. The PSD-1/DNA polyplexes at w/w ratio of 20 were incubated with 10 mM DTT at 25oC, and the particle size was monitored over time. The size of the polyplexes incubated in HBS without DTT was measured as a control. BSA adsorption assay: Polyplexes of PSD-1, PSD-1/PEG, PSD-1/PEG-FA and PEI with pDNA at w/w ratio of 20 were prepared in HBS buffer (pH 7.4). After incubation for 30 minutes, 0.1 mL bovine serum albumin (BSA) solution (2.5 mg/mL in water) was added to 0.9 mL of the polyplex dispersions (1.0 µg/mL of pDNA). These dispersions were shook at 200 rpm for 60 minutes at 37°C and subsequently centrifuged at 8,000 rpm for 3 minutes. The supernatants were collected and the BSA concentrations were determined using a BCA protein assay kit (Beyotime, China). The amount of BSA adsorbed per milligram polyplexes was then calculated using the following equation: BSA adsorbed/mg polyplexes =

BSA0 -BSAs Wpolyplexes

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In vitro cytotoxicity assay The cytotoxicity of the different polymers was studied using the CCK8 assay on HeLa cells and COS-7 cells.55 The cells were seeded in a 96-well plate (6×103 cells/well) in 100 µL DMEM culture medium with 10% FBS at an humidified atmosphere of 95% air and 5% CO2. After incubation for 24 hours, the cultured medium was removed and 200 µL polymer solutions (concentrations ranging from 20 to 400 µg/mL in DMEM) was added. After incubation for 48 hours, the medium was replaced by 100 µL fresh medium and 10 µL CCK8 reagent (Dojindo, Japan) was added to each well for further 2 hours incubation at 37oC. The absorbance at 450 nm was recorded with a reference at 630 nm using a microplate reader (TECAN Infinite® F50).

Hemolysis assay Red blood cells (RBCs) were isolated from 5 mL of human blood sample (from a healthy volunteer) anticoagulated with EDTAK2 and collected by centrifugation at 1000 rpm for 5 minutes, then washed with PBS until the supernatant became clear. Subsequently the erythrocytes were diluted in a PBS solution to 2% (v/v). 1 mL of RBCs suspension was added into 1 mL of polymer solution (PSD-1, PSD-1/PEG, PSD-1/PEG-FA or PEI) with concentration from 50 to 200 µg/mL. Then, the suspensions were shook (100 rpm) at 37oC for 4 hours. Finally, these dispersions were centrifuged at

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3000 rpm for 10 minutes and the absorbance of supernatant containing released hemoglobin was determined at 545 nm using a microplate reader (TECAN Infinite® F50). Triton X-100 and PBS were used as the positive and negative control, respectively. The percentage of hemolysis was calculated as follows: hemolysis%=

ODsample -ODnegative ×100% ODpositive -ODnegative

where ODsample, ODnegative, and ODpositive are the absorbance values of the test sample, negative control (PBS), and positive control (Triton X-100), respectively. All of the hemolysis experiments were carried out in triplicate. This study was approved by the review board and Ethics Committee of Huazhong University of Science and Technology, Wuhan, China.

In vitro pDNA transfection activity assay Transfection activity of pcDNA3-Luc plasmid based polyplexes was studied in HeLa, KB and COS-7 cells. The FR-positive HeLa and KB cells were used to evaluate the folate-mediated targeting transfection efficiency, while the FR-negative COS-7 cells were selected to study the nonspecific transfection. The cells were cultured at a density of 6×103 cells per well in 96-well plates and incubated in an atmosphere of 5% CO2 at 37oC for 24 hours. Next, the medium in each well was replaced with 0.1 mL of DMEM with 10% FBS (folate free or containing 1 mM folate), and then the polymer/DNA polyplexes (0.25 µg pDNA in 25 µL) were added and incubated for 4 hours at 37°C. After that, the - 16  

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medium was replaced by fresh DMEM, and the cells were incubated for 44 hours at 37°C. Thereafter, the cells were washed with PBS and lysed with 1× Reporter Lysis buffer (50 µL, Promega). The luciferase expression was measured using a luminometer (GloMax® 20/20, Promega, USA). The total protein was measured using a BCA protein assay kit (Beyotime, China). Luciferase activity is expressed as RLU/mg protein.

Cellular uptake Confocal microscopy and flow cytometry were used to measure the cellular uptake efficiency of polymer/pDNA polyplexes in HeLa cells. The HeLa cells were seeded in 15 mm glass bottom cell culture dish at a density of 2×104 cells/well, and incubated at 37oC for 24 hours. Next the medium was replaced by 0.9 mL fresh DMEM with 10% FBS (folate free or containing 1 mM folate), and 0.1 mL polyplexes at w/w ratio of 20 containing 2 µg pcDNA3-Luc plasmid (labeled with YOYO-1) were added to each well. For fluorescent imaging, after incubation for 4 hours, the medium with polyplexes was removed and the cells were washed thrice with PBS. Subsequently the cells were fixed with 500 µL of 4% paraformaldehyde solution for 10 minutes at room temperature. Next, the cells were washed thrice with PBS and 500 µL of DAPI solution (2 µg/mL) was added to the dish. After incubation at 37oC for 15 minutes, the DAPI solution was removed and the cells were washed thrice with PBS. Fluorescent images were acquired by Nikon Ti-U microscope equipped with a CSU-X1 spinning-disk confocal unit

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(Yokogawa) and an EM-CCD camera (iXon+; Andor). For flow cytometry, after incubation for 4 hours, the cells were washed thrice with PBS, trypsinized and harvested. Then the cells were quantified using a BD LSRFortessaTM X-20 flow cytometer.

In vitro siRNA transfection activity assay Transfection of anti-EGFP siRNA mediated by PSD-1, PSD-1/PEG and PSD-1/PEG-FA was studied in EGFP stably expressed KB_EGFP and COS_EGFP cells. Cells were seeded in 6-well plates (3×105 cells per well) and incubated for 24 hours. The medium was replaced with 1 mL fresh DMEM, then 0.1 mL polymer/siRNA polyplexes (150 pmol siRNA) was added and incubated for 4 hours at 37oC. After that, the medium was replaced by fresh DMEM, and the cells were incubated for 72 hours at 37°C. Then the cells were imaged using Olympus IX71 fluorescence microscope and the mean fluorescence intensity of cells was quantified using flow cytometry. EGFP fluorescence of cells treated with polyplexes loaded with scrambled siRNA was set as the control. The relative mean fluorescence was calculated as follows: Mean fluorescence  % =

MFIsample   ×100%, MFIcontrol

where MFIsample was the mean fluorescence intensity of the cells treated with the polyplexes loaded with anti-EGFP siRNA, and MFIcontrol was the mean fluorescence intensity of the cells treated with the polyplexes loaded with scrambled siRNA. - 18  

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Statistical analysis The statistical analysis was carried out using student’s t-tests. *P