Multi-targeting Peptide Functionalized Star-shaped

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Multi-targeting Peptide Functionalized Star-shaped Copolymers with Comblike Structure and a POSS-core to Effectively Transfect Endothelial Cells Jun Wang, Syed Saqib Ali Zaidi, Ali Hasnain, Jintang Guo, Xiang-Kui Ren, Shihai Xia, Wencheng Zhang, and Yakai Feng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00235 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Multi-targeting Peptide Functionalized Star-shaped Copolymers with Comb-like Structure and a POSS-core to Effectively Transfect Endothelial Cells Jun Wang1, Syed Saqib Ali Zaidi1, Ali Hasnain1, Jintang Guo1,2, Xiangkui Ren*1,2, Shihai Xia3, Wencheng Zhang4, Yakai Feng*1,2,5

Corresponding Author: Y. Feng, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China Email: [email protected] (Y. Feng) 1 School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, China 2 Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin 300350, China 3 Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital, Logistics University of People’s Armed Police Force, 220 Chenglin Road, Tianjin 300162, China 4 Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force, 220 Chenglin Road, Tianjin 300162, China 5 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, P. R. China

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ABSTRACT: Gene therapy meets one serious bottleneck in clinic application, namely, lack of safe and efficient gene delivery systems. In order to solve this problem, we designed a lowly cytotoxic and highly efficient gene delivery system for the transfection of endothelial cells. Octa(3-ammoniumpropyl)octasilsesquioxane octachloride reacted with 2-bromoisobutyryl bromide under alkaline condition, and sequentially initiated 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) monomethacrylate (PEGMA) via Atom Transfer Radical Polymerization (ATRP) to prepare

the

eight-arm

copolymer

with

a

biocompatible

polyhedral

oligomericsilsesquioxane (POSS). The side chain ends of the comb-like PPEGMA were double-bonded to facilitate the attachment of CAGW or CAG-TAT-NLS functional peptide, thereby enabling the star-shaped copolymers with multifunction. The peptide-functionalized star-shaped copolymers were self-assembled into nanoparticles (NPs) and used to condense pEGFP-ZNF580 (pDNA) to prepare the NPs/pDNA complexes. These complexes had low toxicity as confirmed by MTT. The results of fluorescence microscopy and flow cytometry showed that these multi-targeting functionalized gene complexes could effectively transfect endothelial cells. Their transfection efficiency is higher than the positive control PEI 25 kDa group. Moreover, the western blot test, wound healing assay and in vitro tube formation assay also demonstrated that the transfected cells showed high migration and enhanced angiogenesis. The pDNA was effective delivered and expressed in endothelial cells by these multi-targeting functionalized gene complexes, and

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promoted cell migration and tube formation. These star-shaped copolymers with comb-like structure and a POSS core are a potential gene carrier for gene therapy. Keywords: gene delivery, peptide, POSS, transfection, targeting 1. Introduction Gene therapy has attracted much attention because of its potential application in the treatment of hereditary diseases, malignancies, cardiovascular diseases and so on.1-6 This technique needs to deliver exogenous normal genes and therapeutic genes into target cells to compensate for diseases caused by genetic defect, so as to treat diseases.7-9 Unfortunately, genes cannot pass through the cell membrane barrier.10-12 Therefore, it is necessary to explore and develop an effective gene carrier to deliver the exogenous genes into target cells and express them. Polyhedral oligomeric silsesquioxane (POSS) is an attractive and interesting silicone-containing oligomer material, which presents a cubic cage consisting of a central inorganic Si8O12 core and eight organic groups.13 These organic groups are easy to be chemically modified to introduce functional groups of vinyl, amino, halogen and phenyl groups.14,

15

POSS has unique inorganic and organic hybrid

structure, which enables it to synthesize polymers with a POSS core. Compared with other inorganic or organic nano-building blocks, POSS exhibits several advantages such as easy functionalization, controlled structure, nanometer size, high symmetry and superior thermal stability.16 Especially, POSS has been proved with low toxicity in vivo and in vitro.17-19 Therefore, biomaterials based on POSS core attract more and more attention. POSS modified biodegradable poly(L-lactide) and polycaprolactone

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can degrade more rapidly than their homopolymers.20-22 Functional POSS molecules can be used as polymerizable monomers or initiators to prepare novel nanoscale copolymers. The star-shaped polymers with POSS core are usually prepared via either a arm-first or a core-first method.23 The arm-first method means to synthesize the polymers or block copolymers at first, and then they are connected to the POSS core. For

example,

Gu

et

al.

synthesized

a

L-aspartate)-b-poly(ethylene glycol) copolymer, and

pH-sensitive

poly(benzyl

grafted onto POSS to obtain

star-shaped copolymers.24 This method may not link eight arms onto a POSS core because of steric hindrance, especially when the polymer chains have high molecular weight. While, the core-first method functionalizes POSS firstly, and initiates the polymerization at eight functional groups simultaneously via living/controlled free radical polymerization, such as reversible addition-fragmentation chain transfer polymerization (RAFT) and atom transfer radical polymerization (ATRP). The core-first strategy facilitates the formation of well-defined polymers with accurate arm number and chain length. For example, Ge et al. prepared the quatrefoil-shaped star-cyclic polystyrene copolymers which grew from POSS core by ATRP and click chemistry techniques.25 Liu et al. reported that a series of star-shaped POSS copolymeric gene carriers could form a core-shell-corona micelle in aqueous solution, and showed good drug loading capacity, and strong DNA condensation ability.19 In addition, Xu et al. designed a star-shaped gene carrier composed of a POSS core and eight disulfide-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) arms with different molecular weights.18

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The cationic polymers, such as polyethyleneimine (PEI) and PDMAEMA, are widely used in gene delivery because of their high positive charge and endosomal escape function. Besides, PDMAEMA has been demonstrated to have thermal and pH sensitivities.26-29 However, from the view point of practical application in vivo, highly charged cationic polymers are relatively toxic, therefore they need to be modified with hydrophilic polymers. In our previous studies, poly(ethylene glycol) monomethyl ether (mPEG) and poly(ethylene glycol)-monomethacrylate (PEGMA) were used to modify cationic polymers and biomaterials.30-33 They could effectively shield excessive positive charge and reduce their toxicity. Meanwhile, they could also reduce nonspecific interaction with the reticuloendothelial system so as to extend the blood cycle time of gene complexes. However, PEG modified gene carriers usually affect gene expression efficiency because of low cellular uptake. An effective strategy involves using functional peptides to overcome this problem. Functional peptides are linked onto gene carriers to provide them with multifunction.30, 33, 34 Correspondingly, cell penetrating peptides (CPPs) attract more and more attention.35 In 1988, Frankel et al. reported that human immunodeficiency virus transcription factor (HIV TAT) protein could penetrate the cell membrane barrier and promote the expression of viral genes.36 TAT is a CPP, also known as protein transduction domain. TAT peptide (47-57), which consists of 11 amino acids (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, YGRKKRRQRRR), is the most characteristic fragment of HIV transactivated protein.37, 38 It can directly penetrate the plasma membrane and become one of the most commonly used CPPs.39

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The cell membrane penetrating property is derived from positively charged amino acids, especially arginine, possibly because the guanidine group is critical for promoting cellular uptake.40-42 Nam et al.43 designed the delivery system by introducing primary cardiomyocyte-specific peptide (PCM) and HIV TAT (49-57) into polymer. Their results showed that the cellular uptake and transfection efficiency were significantly improved due to the conjugation of PCM and TAT. Kim et al. successfully synthesized the cystamine bisacrylamide-based SS-poly(amido amine)s (PAA) with low cytotoxicity and efficient gene delivery.44 Moreover, Arg or guanidine group was usually conjugated to various reducible polymers to enhance cellular penetration ability as well as transfection efficiency.45-47 During gene delivery, plasmids should safely enter nucleus, whereas the nuclear localization signals (NLSs) could enhance this transportation.39, 48-50 One of the most widely used NLSs is Pro-Lys-Lys-Lys-Arg-Lys-Val (PKKKRKV) from the large T antigen of the simian virus 40 (SV40). It can be identified by nuclear proteins and can efficiently promote nuclear delivery through the nuclear pore complex.51, 52 However, direct ligation of NLSs and DNA did not significantly enhance transfection.51, 53, 54 NLSs didn’t effectively enhance transfection efficiency when plasmid was not efficiently delivered into cells, because NLSs benefited for nuclear uptake rather than cellular uptake. Combining NLSs with cell membrane targeting peptides and cationic polymers can enhance both cellular uptake and nucleus translocation. Recently, our lab used TAT-NLS peptide to modify PEI-based gene delivery systems which demonstrated high transfection efficiency.55

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In our previous studies, we used REDV, CAG (Cys-Ala-Gly), TAT (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg)

and

NLS

(Pro-Lys-Lys-

Lys-Arg-Lys-Val) peptides to modify gene carriers. These gene carriers demonstrated that high peptide content benefited for high transfection efficiency.55, 56 In this study, we developed a new strategy to prepare gene carriers with high content of functional peptide CAG-TAT-NLS. We synthesized biodegradable POSS star-shaped copolymers with comb-like structure linking many CAG-TAT-NLS peptide sequences. These copolymers could self-assemble to form nanoparticles and deliver gene into endothelial cells (ECs) as shown in Scheme 1. Herein, the cationic polymer PDMAEMA with a POSS-core could efficiently carry plasmids and enhance endosomal escape. The hydrophilic comb-like structure of PPEGMA segment was used to improve carriers’ cytocompatibility. In addition, CAG-TAT-NLS peptide enabled the gene carriers with the active targeting and penetrating of cell membrane, and nuclear localization to ECs. We expected to efficiently deliver genes and express them in order to enhance the proliferation and migration of ECs. To confirm this hypothesis, the transfection efficiency and the expression level of key protein in transfected ECs by the NPs/pDNA complexes were evaluated by cellular uptake, flow cytometry, western blot and in vitro tube formation assay.

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Scheme 1. Self-assembly of NPs/pDNA complexes, and the delivery of pDNA to ECs.

2. Experiments 2.1 Materials Octa(3-ammoniumpropyl)octasilsesquioxane

(POSS-NH3+Cl-),

octachloride

1,1,4,7,10,10-hexamethyltriethylenetetramine

(HMTETA,

2-(dimethylamino)ethyl

98%),

methacrylate

(DMAEMA,

acryloyl

99%), chloride,

2-bromoisobutyryl bromide (BIBB, 98%), 2,2-dimethoxy-2-phenylacetophenone (DMPA), and poly(ethyleneglycol)monomethacrylate (PEGMA, Mn = 360 Da) were purchased from Sigma-Aldrich (St. Louis, USA). Copper(I) bromide (CuBr, 99%) and triethylamine were obtained from Jiang tian Chemicals (Tianjin, China). Cys-Ala-Gly-Trp

(CAGW)

and

Cys-Ala-Gly-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Lys-Lys-Lys-Arg-

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Lys-Val (CAG-TAT-NLS) peptide sequences were purchased from GL Biochem (Shanghai) Ltd. Branched PEI (Mn = 25 kDa) was supplied by Sigma-Aldrich (Beijing, China). BCA protein assay kit was purchased from Solarbio Science and Technology

Co.,

Ltd.

(Beijing,

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

bromide

China). (MTT)

and

dimethyl sulfoxide (DMSO) were purchased from Ding Guo Chang Sheng Biotech Co., Ltd. (Beijing, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Corporation (Carlsbad, CA). Matrigel (Cat. No. 356234) was obtained from Corning Company. Rabbit antihuman ZNF580 polyclonal antibody and goat anti rabbit IgG were purchased from Abcam (HK) Ltd. (Hong Kong, China). Cy5 labeled oligonucleotide (Cy5-oligonucleotide) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai China). EA.hy926 and human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). pDNA plasmids were preserved by department of physiology and pathophysiology, Logistics University of Chinese People’s Armed Police Force. 2.2. Synthesis of POSS-PDMAEMA-PPEGMA-CAG-TAT-NLS 2.2.1 Synthesis of ATRP initiator POSS-(Br)8 POSS-NH3+Cl- (2.5 g, 2.13 mmol) was dissolved in 15 mL highly purified water, and added 4.3 mL of NaOH (4 M L-1) at 0 °C under nitrogen protection. 25 mL chloroform solution of BIBB (9.9 g, 42.9 mmol) and 10.7 mL NaOH solution (4 M L-1) were slowly added to the solution, and stirred for 12 h. The organic layer was

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separated and then extracted with 1 mol L-1 HCl solution, saturated NaCl solution, and purified water. The organic phase was dried with anhydrous Na2SO4, filtered and followed by rotary evaporation. The concentrate was purified by precipitating in diethyl ether for three times, and dried under vacuum at 25 °C until a constant weight. 2.2.2 Synthesis of POSS-PDMAEMA-PPEGMA DMAEMA (5 mL), initiator POSS-(Br)8 (0.15 g), ligand HMTETA (45 µL) were sequentially added into 20 mL methanol in a polymerization tube. After freezing in liquid nitrogen, the tube was evacuated and filled with nitrogen, which was repeated three times. CuBr (5 mg) was added in the tube under nitrogen atmosphere. Then the tube was kept at 60 °C for 24 h. After polymerization, the copper salt was removed from the reaction mixture by a neutral alumina column and colorless liquid was obtained. The liquid was concentrated and precipitated in hexane three times to remove unreacted monomer. The white POSS-PDMAEMA (POSS-D) was obtained by vacuum drying until reaching a constant weight. POSS-PDMAEMA-PPEGMA (POSS-DP) was synthesized from POSS-D and PEGMA by second ATRP reaction with a similar process. 2.2.3 Synthesis of POSS-PDMAEMA-PPEGMA-DAs POSS-DP copolymer (125 mg) was dissolved in 25 mL DMSO in a flask. 5 mL of this solution was transferred into a 50 mL dried three-necked flask, and 0.4 mL triethylamine was added into the solution. Acryloyl chloride/DMSO solution (5 mL) was cautiously added dropwise to the flask at 0 °C. The reaction was continued overnight.

After

dialysis

and

lyophilization

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(freeze-drying),

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POSS-PDMAEMA-PPEGMA-DAs (POSS-DP-DAs) was obtained. 2.2.4

Synthesis

of

POSS-PDMAEMA-PPEGMA-CAGW

and

POSS-PDMAEMA-PPEGMA-CAG-TAT-NLS POSS-DP-DAs was dissolved in purified water and sonicated for 10 min to prepare a 5 mg mL-1 solution. 5 mL of this solution and CAGW peptide (12 mg) were sequentially added into a culture dish. DMPA (4.3 mg) was dissolved in 2 mL DMSO and added into this dish. They were continuously irradiated by an UV lamp for 10 min. The reaction mixture was collected and purified by dialysis and lyophilization to obtain

the

solid

product

of

POSS-PDMAEMA-PPEGMA-CAGW

(POSS-DP-CAGW). CAG-TAT-NLS peptide (38.6 mg) was grafted onto POSS-DP-DAs copolymer (13.3

mg)

to

prepare

POSS-PDMAEMA-PPEGMA-CAG-TAT-NLS

(POSS-DP-CAG-TAT-NLS). The synthesis was carried out by an analogous method described above. 2.3 Preparation of nanoparticles (NPs) 2 mg POSS-D was dissolved in 10 mL aqueous solution, sonicated for 10 min, and obtained micelle solution with a concentration of 0.2 mg mL-1. In addition, the POSS-DP

(0.2

mg

mL-1),

POSS-DP-CAGW

(0.2

mg

mL-1),

and

POSS-CAG-TAT-NLS (0.2 mg mL-1) micelles were also prepared by the same method. 2.5 Preparation of NPs/pDNA complexes pDNA (50 µg mL-1 in PBS buffer (pH 7.4)) was slowly dripped into micelle

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solution by a micro injector. The NPs/pDNA complexes were prepared in accordance with the N/P molar ratios of 0.5, 1, 5, 10 and 20 (N is the N content in copolymer, and P is the P content in pDNA). Before further experiments, the prepared complexes needed to be slowly stirred at room temperature for 2 h. 2.6. Physicochemical characterization of copolymers and NPs 2.6.1 Characterization of copolymers 1

H NMR spectra of the POSS-D and POSS-DP copolymers were recorded with a

Bruker Avance spectrometer (AV-400, Bruker, Karlsruche, Germany) operating at 400 MHz in CDCl3, and the POSS-DP-CAGW and POSS-DP-CAG-TAT-NLS copolymers were dissolved in DMSO-d6. The average molecular weight (Mn, Mw) of POSS-D and POSS-DP copolymers were determined by a Waters 1525 gel permeation chromatography (GPC, Malvern Viscotek, U.K.) using tetrahydrofuran (THF) as eluent solvent. 2.6.2 Characterization of functional peptide content in copolymers In order to quantitatively determine the peptide content in the copolymers, we used fluorescence spectrophotometer (Varian Cary Eclipse fluorescence spectrometer) to detect the fluorescence intensity of micelles. The CAGW peptide containing tryptophan residue was excited by 280 nm light and emitted a fluorescence at the wavelength of 357 nm, while CAG-TAT-NLS peptide was excited at the wavelength of tyrosine residue (275 nm) and determined at 304 nm. The fluorescence intensity of CAGW (2×10-4 ~ 1×10-3 mg mL-1) and CAG-TAT-NLS (2.5×10-3 ~ 4×10-2 mg mL-1) solutions was measured to set as standard curves. The peptide content in the

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copolymers was quantitatively determined from these linear standard curves. 2.6.3 Hydrodynamic diameter and zeta potential of NPs and NPs/pDNA complexes The diameter and zeta potential of NPs and NPs/pDNA complexes with varied N/P molar ratios were measured with a Zetasizer 3000HS (Malvern Instrument, Inc., Worcestershire, U.K.).57 The morphology of dried micelles was characterized by TEM JEM-2100F at 200 kV accelerating voltage. 2.7 Biological characterization of NPs and NPs/pDNA complexes 2.7.1 Agarose gel electrophoresis In order to assess the condensation ability of NPs, agarose gel electrophoresis was performed. The complexes solution (10 µL) was mixed with 6x loading buffer (2 µL) and loaded into a 0.80 wt% agarose gel containing 0.50 µg/mL ethidium bromide. The electrophoresis was performed in 1× TAE buffer at 100 V for 25 min. A UV illuminator was used to indicate the retarded location of pDNA. 2.7.2 In vitro cytotoxicity The cytotoxicity of NPs and NPs/pDNA complexes was evaluated by MTT assay. EA.hy926 cells were seeded in a 96-well plate at a density of 1×104 cells/well and cultured overnight. When the cells reached 80%-90% confluence, they were starved in serum-free medium for 12 h. NPs and NPs/pDNA complexes were added into the medium with different concentrations. After the cells were incubated for 4 h, the medium was replaced by fresh growth medium (10 wt% FBS DMEM). After 48 h, 20 µL of MTT solution (5 mg mL-1) was added into each well, and kept for another 4 h to

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form formazan crystal. Then, the medium was carefully removed. The formed formazan crystals by viable cells were dissolved in DMSO (150 µL/well). The 96-well plate was placed on volatility instrument in dark condition and shook for 10 min at low speed. The optical density (OD) of the formazan solution was estimated with anmicroplate reader (BIO-RAD, iMarkt, USA) at the wavelength of 490 nm. The relative cell viability (%) was calculated using following equation. Relative cell viability(%) =

OD - OD 0 ×100% OD C - OD 0

(1)

where OD is the value of the experimental group, ODC is the value for cells incubated with medium, and OD0 is the value of the medium.

2.7.3 In vitro transfection EA.hy926 cells were seeded in a 24-well plate in a density of 1 × 104 cells/well, cultured until they reached 70%-80% confluence. The cells before transfection were starved for 12 h in serum-free medium. NPs/pDNA complexes containing 3 µg pDNA were added into each well at the N/P molar ratio of 15. After 4 h incubation, the medium was changed to fresh growth medium (10 wt% FBS DMEM) and incubated at 37 °C in an incubator. The expression of green fluorescent protein (GFP) by pDNA in the transfected cells was observed under an inverted fluorescence microscope at 48 h time point. In this experiment, three parallel wells were taken and nine parallel visual fields were randomly observed and analyzed. The expression of GFP was quantitatively determined by flow cytometry method. The transfected cells were washed and centrifuged three times with 0.01 M PBS to remove the growth medium. The transfected cells were distracted in 300 µL PBS to

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detect the expression of GFP by a flow cytometer (Beckman MoFlo XDP).

2.7.4 Wound healing assay The migration ability of transfected EA.hy926 cells was detected by the scratch wound healing assay. EA.hy926 cells were seeded in a 24-well plate, starved for 12 h, and then transfected with NPs/pDNA complexes at the N/P molar ratio of 15. After 24 h, a vertical scratch was made by a sterile 200 µL plastic pipette tip with the help of a sterile ruler. D-Hanks buffer (pH 7.4) was used to wash the cell debris formed during scratches. At 0, 6 and 12 h, the migration process was monitored by using an inverted microscope (OLYMPUS U-RFLT50, microscopy Olympus DP72). The relative migration area was determined using Image J 2.1 software and calculated by following equation.

Migration area (%) =

wounded area - nonrecovered area × 100% wounded area

(2)

2.7.5 Western blot analysis Western blot analysis was used to detect the expression of pDNA at protein level in transfected EA.hy926 cells. The cell lysate RIPA and PMSF were mixed with the volume ratio of 100:1 and kept at low temperature. Cells were washed twice with 0.1 mol/L pre-cooled PBS (pH = 7.4), and lysed for 30 min in 50 µL lysis buffer per well. The total cell proteins were collected by cell scraper and centrifuged for 10 min, then the supernatant protein was quantified by a BCA Protein Assay Kit. By SDS-PAGE electrophoresis, 80 µg of proteins were separated by subunit molecular weight. The proteins were transferred to polyvinylidene fluoride (PVDF) membrane, followed by seal with 8% milk for 1 h and incubation with a rabbit anti-ZNF580 polyclonal

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antibody overnight at 4 °C. Furthermore, PVDF was washed in a shaker for 30 min, incubated with horseradish peroxidase conjugated to goat anti-rabbit IgG to assess the protein loading level, and coated with enhanced chemiluminescent reagents to exposure. The bands were visualized using Image J 2.1, and the β-actin antibody was used as a control.

2.7.6 In vitro endothelial tube formation assay To study angiogenic function of the transfected HUVECs by NPs/pDNA complexes, tube formation assay was performed in vitro. Matrigel was kept at 4 °C overnight, then was spread evenly in a 96-well plate and incubated at 37 °C for 1 h. The transfected cells were seeded onto the surface of solidified matrigel at a density of 4 × 104 cells/well. After 6 h, the photographs of three parallel wells per group were taken by an optical microscope, and the results were analyzed using Image J 2.1.

2.7.7 Cellular uptake of NPs/Cy5-oligonucleotide complexes To estimate cellular uptake, Cy5-oligonucleotide labeled with fluorescence dye was used to transfect EA.hy926 cells and detected by a flow cytometer. After being incubated for 48 h, the transfected cells by NPs/Cy5-oligonucleotide complexes were digested, washed three times with 0.01 M PBS and centrifuged. The cells were finally re-suspended in 300 µL wash buffer and the cellular uptake of the complexes was analyzed using a flow cytometer (Beckman MoFlo XDP, USA). The cells treated by PEI 25 kDa/Cy5-oligonucleotide complexes were used as a positive control.

3. Results 3.1. Synthesis and characterization of star-shaped copolymers with

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comb-like structure and POSS core In order to develop the gene carrier with high transfection efficiency, we designed and synthesized the star-shaped copolymers with comb-like structure and a POSS core. The comb-like structure provided the gene carrier with high content of functional peptide. The star-shaped POSS-DP copolymer and peptide functionalized POSS copolymers were prepared according to the synthesis route as shown in Scheme 2. POSS-NH3+Cl- reacted with NaOH to produce eight-amino groups, followed by amidation with BIBB to obtain a star-shaped initiator POSS-(Br)8 with eight-tertiary C-Br. The star-shaped POSS-DP was synthesized by POSS-(Br)8 initiated two consecutive ATRP reactions of DMAEMA and PEGMA. In order to introduce functional peptides into the star-shaped copolymers, the sulfhydryl group of CAGW peptide was linked to the terminal double bond of comb-like structure by a click reaction. In addition, the CAG-TAT-NLS peptide sequence was attached to the star-shaped copolymer in order to provide the gene carriers with the synergistic effect of multifunctional peptide.

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Scheme 2. Synthesis route of POSS-DP-CAGW and POSS-DP-CAG-TAT-NLS amphiphilic copolymers.

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The number- and weight-average molecular weights of POSS-D and POSS-DP were determined by GPC (Table 1). The number-average molecular weight of POSS-D is 39.1 kDa, which means the number of repeating unit DMAEMA m = 29. POSS-DP copolymer was prepared via a second ATRP. POSS-DP copolymer has a higher number-average molecular weight (66.0 kDa), indicating that POSS-D successfully introduced PPEGMA block with n = 9 repeating units. The narrow molecular-weight distribution (1.10 and 1.17) of the polymers indicated that the ATRP process was well-controlled and formed the eight-armed POSS-D and POSS-DP polymers. 1

H NMR spectra of the copolymers of POSS-D, POSS-DP, POSS-DP-CAGW,

and POSS-DP-CAG-TAT-NLS are shown in Figure S1 (Supporting Information). In Figure S1 A, the POSS-NH3+Cl- characteristic peaks of Si-CH2-CH2-CH2-N (peak a, b, and c,6H) were observed at 0.71 ppm, 1.72 ppm and 2.76 ppm, respectively. The protons of amino group (-NH3+, 3H) showed a strong peak at 8.15 ppm. The eight-armed initiator POSS-(Br)8 was synthesized via acylation of POSS-NH3+Cl- with BIBB under alkaline condition, and its 1H NMR spectrum is shown in Figure S1 B. The characteristic peaks of Si-CH2-CH2-CH2-N were observed at 0.58 ppm, 1.56 ppm and 3.19 ppm, respectively. Especially, the characteristic single peak at chemical shift

δ = 1.88 ppm was ascribed to methyl protons (-COC(CH3)2Br, 6H). The 1H NMR spectra indicated that POSS-NH3+Cl- has been successfully brominated to form POSS-(Br)8. In the 1H NMR spectrum of POSS-D in CDCl3 (Figure S1 C), the broad

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resonances at 0.73 ppm (-CH2CCH3CO-, 3H) and 1.66-2.07 ppm (-CH2CCH3CO-, 2H) were assigned to the PDMAEMA repetitive unit. Additionally, the peaks at 4.06 ppm and 2.57 ppm were ascribed to methylene proton signals (-COO-CH2-CH2-, 4H). The single peak of methyl protons of -N(CH3)2 was observed at 2.27 ppm. The number of PDMAEMA repetitive units in the star-shaped copolymer was calculated by the integral value of the 4.06 ppm peak relative to the peak of 3.73 ppm (-CH2-CH2-CH2-, 2H). The DMAEMA unit was 25. Figure S1 D showed the characteristic peaks of the protons of CH2CH2O in PPEGMA block at 3.63 ppm. The peaks at 5.8 ppm, 6.2 ppm and 6.4 ppm of the CH = CH2 protons proved the successful synthesis of POSS-DP-DAs from POSS-DP (Figure S1 E). Calculating from the peak area of the double bond protons and the characteristic peak area of the PPEGMA block, the hydroxyl groups were functionalized with double bonds up to 30 mol%. After functional peptides were linked onto POSS-DP-DAs, they can be dissolved in DMSO because of the hydrophilic property of the peptides. Their 1H NMR spectra were measured using DMSO-d6 as solvent and shown in Figure S1 F and G. The characteristic peaks of CAGW and CAG-TAT-NLS peptides were observed at 6.8-8.5 ppm, which demonstrated that the star-shaped copolymers had functional peptide sequences.

Table 1 Number- and weight-average molecular weights of POSS-D and POSS-DP polymers by GPC Sample ID

Mn (kDa)

Mw (kDa)

PDI

POSS-D

39.10

43.03

1.10

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POSS-DP

3.2.

66.00

Functional

peptide

content

76.95

in

1.17

POSS-DP-CAGW

and

POSS-DP-CAG-TAT-NLS CAGW and CAG-TAT-NLS peptide sequences were linked onto POSS-DP-DAs in order to increase transfection efficiency of the gene carriers. For POSS-DP-CAGW copolymer, the CAGW peptide content was quantitatively determined by fluorescence analysis of tryptophan (W) residue as reported by previous studies.58, 59 A strong fluorescence emission was observed at 357 nm in the fluorescence spectrum of POSS-DP-CAGW micelle, while POSS-DP micelle did not show any fluorescence peak in Figure 1(1). This confirmed that CAGW peptide had been linked onto POSS-DP successfully. According to the fluorescence standard curve of CAGW, the concentration of CAGW of POSS-DP-CAGW micelle was 0.0008 mg mL-1, meaning the CAGW content in the copolymer was 6.15 wt%. For POSS-DP-CAG-TAT-NLS copolymer, tyrosine residue in TAT was used to determine the content of CAG-TAT-NLS peptide. Tyrosine residue displays a specific fluorescence at 304 nm when it is excited by the light with the wavelength of 275 nm. The content of CAG-TAT-NLS can be calculated on the basis of the fluorescence standard curve of CAG-TAT-NLS. POSS-DP-CAG-TAT-NLS had 22.5 wt% CAG-TAT-NLS peptide content.

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Figure 1. The fluorescence emission spectra of micelles solution and fluorescence standard curves of CAGW and CAG-TAT-NLS peptide solutions. (1) The fluorescence emission spectra of POSS-DP and POSS-DP-CAGW micelles. (2) The

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fluorescence emission spectra of POSS-DP and POSS-DP-CAG-TAT-NLS micelles.

3.3. Hydrodynamic diameter and zeta potential of NPs and NPs/pDNA Complexes POSS-D formed nanosized micelles in aqueous solution, and their shape was relatively regular (Figure S2). The inorganic rigid and hydrophobic POSS core provided them with high structure stability, and the hydrophilic PDMAEMA chains enhanced the stability of the micelles in solution. The star-shaped copolymer of POSS-DP with inorganic rigid POSS could self-assemble into stable micelles in aqueous solution. As a suitable gene carrier, the micelles should have appropriate size and effective positive charge. In general, particle size < 200 nm is beneficial for cell endocytosis and escaping the reticuloendothelial system (RES) clearance.60-62 The size and zeta potential values of the micelles ranged from 128.3 nm to 242.8 nm and from 25.92 mV to 31.23 mV, respectively (Table 2). POSS-D nanoparticles had smallest size (128.3 nm), while POSS-DP showed significantly larger particle size (229.1 nm), because the POSS-DP nanoparticles had a hydrophilic PPEGMA corona layer. PPEGMA chains originated from the polymerization of PEGMA macromonomer, and they had a comb-like PEG structure. This special structure provided the nanoparticles with high hydrophilicity as well as large particle size. But the zeta potential of POSS-DP was lower than POSS-D. The hydrophilic PPEGMA corona layer shielded the PDMAEMA shell so as to decrease the positive charge. POSS-DP-CAGW micelles showed slightly smaller size but higher zeta potential than POSS-DP micelles. The cationic peptide CAG-TAT-NLS contains six lysine residues and seven arginine

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residues in one sequence, which benefits gene carriers with more positive charges. In addition, the CAG-TAT-NLS peptide has a longer peptide sequence than CAGW. Therefore, POSS-DP-CAG-TAT-NLS showed relative large size and high zeta potential among these copolymer micelles. When the micelles loaded pDNA, their particle size and zeta potential showed significant dependence on N/P molar ratio of the NPs/pDNA complexes (Figure 2). When N/P molar ratio increased from 0.5 to 20, the particle size decreased, but the potential increased. When N/P molar ratio was 15, POSS-DP-CAGW/pDNA and POSS-DP-CAG-TAT-NLS/pDNA complexes exhibited small size and positive charge, their values were 101.0 ± 1.6 nm and 112.1 ± 2.8 nm, 15.81 ± 2.19 mV and 16.91 ± 0.89 mV, respectively, thus we used them for following transfection experiments.

Table 2 Size and zeta potential of star-shaped copolymer micelles and peptide modified micelles Sample ID

Size (nm)

PDIa

Zeta potential (mV)

POSS-D

128.3 ± 2.0

0.24

31.23 ± 1.29

POSS-DP

229.1 ± 1.6

0.25

25.92 ± 1.34

POSS-DP-CAGW

215.0 ± 3.1

0.30

26.82 ± 1.63

POSS-DP-CAG-TAT-NLS

242.8 ± 2.9

0.22

30.35 ± 2.17

a

PDI: polydispersity index.

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Figure 2. The size and zeta potential of NPs/pDNA complexes. (A) POSS-DP/pDNA complexes,

(B)

POSS-DP-CAGW/pDNA

complexes,

(C)

POSS-DP-CAG-TAT-NLS/pDNA complexes.

3.4. Agarose gel retardation assay The gene carriers should efficiently load and condense pDNA for perfect binding effect. We evaluated this condensation ability of NPs by agarose gel electrophoresis (Figure 3). In general, naked pDNA with negatively charged phosphate groups can easily migrate from the cathode to the anodic electrophoresis field, but the electrophoretic mobility of the pDNA which is effectively condensed by polycationic carriers will be retarded. Figure 3 showed that pDNA was bounded and fully retarded by POSS-D, POSS-DP, POSS-DP-CAGW, and POSS-DP-CAG-TAT-NLS micelles at N/P molar ratio of 1, 5, 5 and 2, respectively. Thus, we selected the N/P molar ratio of 15 for the following studies.

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Figure 3. Agarose gel retardation assay of NPs/pDNA complexes at various N/P molar ratios. (a) POSS-D/pDNA, (b) POSS-DP/pDNA, (c) POSS-DP-CAGW/pDNA, (d) POSS-DP-CAG-TAT-NLS/pDNA.

3.5 In vitro cytotoxicity The cytotoxicity of NPs and NPs/pDNA complexes against EA.hy926 cells was evaluated by MTT assay. PEI 25 kDa and PEI 25 kDa/pDNA were used as positive control groups. After EA.hy926 cells were treated with the micelles or gene complexes at the N/P molar ratio of 15 for 48 h, the relative cell viability was determined and shown in Figure 4. Compared with NPs groups at same concentration

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of PDMAEMA, the gene complexes groups exhibited high cell activity. The negatively charged pDNA partly neutralized the positive charge of gene carriers, additionally pDNA was beneficial to cell proliferation. More importantly, the NPs and NPs/pDNA showed significantly higher relative cell viability than PEI 25 kDa and PEI 25 kDa/pDNA control groups. In particular, the relative cell viability of the functional peptide modified carrier groups was over 80% even at the concentration of 40 mg mL-1, but PEI 25 kDa control group decreased dramatically to 20%. The concentration of 40 µg mL-1 was selected for further study. These results indicated that the functional peptide modified NPs and their gene complexes had low in vitro cytotoxicity. The good cytocompatibility of gene carriers encouraged us to evaluate their transfection efficiency. A B C D E

140

Relative cell viability(%)

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

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120 100

# #* #

* *# * **

## #

**#* * ## **

# # ## ##** *

** *

80

#

#

# *#*# *#* * *

A/pDNA B/pDNA C/pDNA D/pDNA E/pDNA

#

*#*# # #* * * *#

# #* # # * *#* # *

*

60 40 20 0 5

20

30

40

50

60

-1

Concentration(µg mL ) Figure 4. Relative cell viability of EA.hy926 cells treated with A, B, C, D, E, A/pDNA, B/pDNA, C/pDNA, D/pDNA and E/pDNA at different concentrations of

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PDMAEMA or PEI 25 kDa at 48 h. (A) POSS-D, (B) POSS-DP, (C) POSS-DP-CAGW, (D) POSS-DP-CAG-TAT-NLS, (E) PEI 25 kDa as control groups. The N/P molar ratio of A/pDNA, B/pDNA, C/pDNA, D/pDNA and E/pDNA was 15. ( x ± SD, n = 3, *p < 0.05 vs. PEI 25 kDa group, #p < 0.05 vs. PEI 25 kDa/pDNA group).

3.6 In vitro transfection pDNA was used as a reporter gene to evaluate the transfection efficiency of EA.hy926 cells. The expression level of GFP in transfected EA.hy926 cells was qualitatively evaluated by a fluorescence microscopy and quantified by a flow cytometry. The cells, which were transfected with PEI 25 kDa/pDNA, were used as a positive control, and cells transfected with naked pDNA were served as a negative control. As shown in Figure 5, GFP was not detected in all views in negative group (Figure 5(1) F). Unlike negative group, the gene complexes groups showed obvious GFP in the observed views. Moreover, the functional peptide modified complexes groups had more GFP than POSS-D/pDNA and POSS-DP/pDNA complexes group. The flow cytometry results quantificationally demonstrated their difference. Owing to the targeting function of CAGW peptide to EA.hy926 cells, the transfection efficiency of

POSS-DP-CAGW/pDNA

complexes

group

(4.81%)

was

higher

than

POSS-DP/pDNA complexes group (2.82%) as shown in Figure 5(2). Besides CAG function, CAG-TAT-NLS peptide having NLS peptide is beneficial for nucleus accumulation

of

genes

via

nuclear

localization

effect,

thus

POSS-DP-CAG-TAT-NLS/pDNA complexes group showed the highest transfection

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efficiency (5.79%). Importantly, it was even higher than PEI 25 kDa/pDNA complexes group (5.43%). The above results indicated that POSS-DP-CAG-TAT-NLS can efficiently deliver genes into ECs and express them. The EC transfection efficiency of POSS-D/pDNA complexes was low (2.96%), almost similar to POSS-DP/pDNA complexes group (2.82%). Considering its high cytotoxicity and low transfection efficiency, POSS-D was not used in the following experiments.

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Figure 5. Fluorescence images (dark field and bright field, 1) and transfection efficiency (2) of EA.hy926 cells transfected by NPs/pDNA complexes for 48 h. (A) POSS-D/pDNA

complexes,

POSS-DP-CAGW/pDNA

(B)

complexes,

POSS-DP/pDNA (D)

complexes,

(C)

POSS-DP-CAG-TAT-NLS/pDNA

complexes, (E) PEI 25 kDa/pDNA served as the positive control group, (F) pDNA served as the negative control group.

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3.7 Wound healing assay Wound healing assay was used to evaluate the migration and proliferation ability of the transfected EA.hy926 cells with NPs/pDNA complexes. PEI 25 kDa/pDNA was used as the positive group, and naked pDNA was used as the negative control group. Compared with the negative control group, the experimental groups treated with NPs/pDNA complexes exhibited relatively high migration area after 12 h (Figure 6). In

addition,

POSS-DP-CAGW/pDNA

complexes

and

POSS-DP-CAG-TAT-NLS/pDNA complexes facilitated to enhance the cell migration ability. Their relative recovered area reached about 43.1 ± 1.1% and 58.6 ± 2.2%, respectively, which were higher than PEI 25 kDa/pDNA (32.1 ± 3.1%). Therefore, CAG-TAT-NLS peptide functionalized gene complexes groups could effectively transfect ECs and enhance their migration ability. These results agreed with the fluorescence microscopy and flow cytometry results.

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Figure 6. (1) Migration of transfected EA.hy926 cells at 0, 6, and 12 h, (2) relative recovered area (%) after 12 h calculated by Image-Pro Plus (6.0). (A) POSS-DP/pDNA

complexes,

(B)

POSS-DP-CAGW/pDNA

complexes,

(C)

POSS-DP-CAG-TAT-NLS/pDNA complexes, (D) PEI 25 kDa/pDNA complexes served as the positive control group, (E) naked pDNA served as the negative control group. ( x ± SD, n = 3, *p < 0.05 vs. naked pDNA group, #p < 0.05 vs. PEI 25 kDa/pDNA group).

3.8 In vitro endothelial tube formation assay

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In order to evaluate whether NPs/pDNA complexes could enhance angiogenesis or not, we performed in vitro tube formation assay. We assessed the transfected HUVECs with different gene complexes on matrigel bed, using PEI 25 kDa/pDNA as the control group. The gene complexes could promote angiogenesis in different degree

compared

with

blank

control

group

(Figure

7).

POSS-DP-CAG-TAT-NLS/pDNA complexes group exhibited the highest tube number (40 tubes per field, Figure 7 (2)), even higher than positive control PEI 25 kDa/pDNA group

(34

tubes

per

field).

The

high

transfection

efficiency

of

POSS-DP-CAG-TAT-NLS/pDNA complexes group benefited for high tube number in the tube formation assay. The synergistic effect of CAG peptide, TAT peptide and NLS peptide in CAG-TAT-NLS is favor for high transfection efficiency and angiogenesis. POSS-DP-CAG-TAT-NLS could effectively deliver pDNA into ECs and enhance nucleus accumulation, which can promote EC transfection, endothelialization and angiogenesis. The trend of the above results can be further proved by the total mesh area and number master junctions per field (Figure 7 (3) and (4)).

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Figure 7. In vitro tube formation assay on HUVECs. (1) Microscopy images of HUVECs after incubation on Matrigel for 6 h at 37 °C. The white circles and arrows were used to point the tube ring as an example shown in 1(A). (A) POSS-DP/pDNA complexes,

(B)

POSS-DP-CAGW/pDNA

complexes,

(C)

POSS-DP-CAG-TAT-NLS/pDNA complexes, (D) PEI 25 kDa/pDNA complexes served as the positive control group, (E) naked pDNA served as the blank control group. (2) Tube number per field. (3) Total meshes area per field counted by Image J 2.1. (4) Number master junctions per field counted by Image J 2.1. ( x ± SD, n = 3, *p < 0.05 vs. naked pDNA group).

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3.9 Western blot analysis Western blot analysis is widely used to evaluate the gene expression at the protein level. Herein, the expression of ZNF580 gene in transfected EA.hy926 cells with NPs/pDNA complexes was quantified by this method. PEI 25 kDa/pDNA complexes and naked pDNA were used as positive control group and blank control group, respectively. ZNF580 relative protein level (%) was calculated by the expression of pEGFP-ZNF580 (20.1 kDa) and β-actin gene (42 kDa). Compared with the blank control group (27.8 ± 2.2%), the protein expression in other groups was high with statistical significance (p < 0.05, Figure 8). In addition, the relative protein level of POSS-DP-CAG-TAT-NLS/pDNA complexes group was slightly higher than that of PEI 25 kDa/pDNA group. These results were well corresponded to in vitro fluorescence transfection results, which demonstrated that CAG-TAT-NLS peptide functionalized NPs could be a potential gene delivery carrier for EA.hy926 cells.

Figure 8. Western blot analysis for ZNF580 protein expression in the transfected EA.hy926 cells by different gene complexes, naked pDNA and PEI 25 kDa/pDNA after 48 h. (A) PEI 25 kDa/pDNA complexes served as the positive control group, (B)

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naked pDNA served as the blank control group, (C) POSS-DP/pDNA complexes, (D) POSS-DP-CAGW/pDNA

complexes,

(E)

POSS-DP-CAG-TAT-NLS/pDNA

complexes. (mean ± SD, n = 3, *p < 0.05 vs. naked pDNA group).

3.10 Cellular uptake The cellular uptake of gene complexes is an important factor in gene delivery. For the determination of cellular uptake by flow cytometry, Cy5-oligonucleotide (1 µg per well) with Cy5 fluorescence dye was used to substitute pDNA. All the NPs/Cy5-labeled oligonucleotide complexes groups exhibited high cellular uptake rate close to 100% (Figure 9(2)). However, a marked difference of the mean fluorescence

intensity

(MFI)

was

POSS-DP-CAGW/Cy5-oligonucleotide

found group

for

each

displayed

group higher

(Figure

9(3)).

internalization

efficiency (MFI = 1739) than POSS-DP/Cy5-oligonucleotide group (MFI = 1151), owing to the targeting of CAGW peptide to ECs. When the gene carriers were modified by CAG-TAT-NLS peptide with CAG peptide, the cell-penetrating peptide TAT and the nuclear localization signal NLS, their MFI value (3843) was highest among the cellular uptake tests. The above results demonstrated that the multiple targeting gene delivery system could effectively deliver gene to across EC membrane and acumuate in EC nucleus.

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Figure 9. Flow cytometry data of the transfected EA.hy926 cells by different NPs/Cy5-oligonucleotide complexes. (A) POSS-DP/Cy5-oligonucleotide complexes, (B)

POSS-DP-CAGW/Cy5-oligonucleotide

POSS-DP-CAG-TAT-NLS/Cy5-oligonucleotide

complexes,

complexes,

(D)

(C) PEI

25

kDa/Cy5-oligonucleotide complexes served as the positive control group, (E) naked Cy5-oligonucleotide served as the blank control group. (1) Intracellular fluorescence intensity, (2) percentage of cellular uptake, and (3) mean fluorescence intensity. ( x ± SD, n = 3, *p < 0.05 vs. Cy5-oligonucleotide group, #p < 0.05 vs. PEI 25 kDa/Cy5-oligonucleotide group).

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4 Discussion In this work, we designed and prepared the star-shaped copolymers of POSS-DP-CAGW and POSS-DP-CAG-TAT-NLS with comb-like structure and a POSS core. DMAEMA and PEGMA were sequentially polymerized to form the diblock copolymer of PDMAEMA-PPEGMA via ATRP using POSS-based octa functional ATRP initiator. ATRP acted as a living polymerization to prepare polymers with

low

PDI.

This

copolymer

had

the

star-

and

comb-shaped

PDMAEMA-PPEGMA-grafted POSS, and could form stable micelles in aqueous solution. These stable micelles benefited for gene delivery. Functional peptides of CAGW and CAG-TAT-NLS were linked onto the ends of comb-like PEG chains of POSS-DP

star-shaped

POSS-DP-CAG-TAT-NLS,

copolymer

to

respectively.

prepare This

POSS-DP-CAGW method

could

and

prepare

POSS-DP-CAGW and POSS-DP-CAG-TAT-NLS with high peptide content. This special architecture and high functional peptide content demonstrated strong pDNA condensation ability and could efficiently mediate gene transfection of ECs. These results were well consistent with star-shaped gene carriers.19, 58, 63 The results of 1H NMR and GPC demonstrated the successful synthesis of the POSS-based star-shaped copolymers. Moreover, functional peptide content was quantified by fluorescence spectrophotometer. CAGW peptide was determined at the wavelength of 357 nm after excited at 280 nm. CAG-TAT-NLS peptide fragment was quantified at the wavelength of 304 nm at maximum excitation wavelength of 275 nm due to tyrosine residue in cell-penetrating peptide TAT. POSS-DP-CAGW had CAGW

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content 6.18 wt%, and POSS-DP-CAG-TAT-NLS had 22.5 wt% CAG-TAT-NLS. The high peptide content is attributed to the comb-like structure, which had many double bonds to provide reaction sites. Besides, the synthesis route involving click reaction also benefits for efficiently grafting peptides. Gene carriers with a certain amount of positive charge enable them to load and deliver genes into target cell. The POSS-DP micelles exhibited a zeta potential of 25.92 mV, the functional peptides modified micelles of POSS-DP-CAGW and POSS-DP-CAG-TAT-NLS had high zeta potential of 26.82 and 30.35 mV, respectively. The CAG-TAT-NLS peptide sequence benefits the micelles with high positive charge. All of these micelles could completely load pDNA when N/P molar ratio increased to 5. When N/P molar ratio was 15, the gene complexes showed small size (95.7 nm ~ 112.1 nm) and high positive charge (14.08 mV ~ 16.91 mV), thus we used these complexes for transfection experiments. POSS-PDMAEMA had high zeta potential (31.23 mV), even comparable to the gold standard PEI 25 kDa, but its cytotoxicity was also very high as demonstrated by MTT results. The POSS-DP showed lower cytotoxicity than POSS-PDMAEMA owing to the hydrophilic PPEGMA corona to shield the cationic PDMAEMA chains. Many studies also demonstrated that PEG could effectively shield excessive positive charge and reduce the toxicity of gene carriers.30-33 Interestingly, PEG could also extend the blood cycle time by reducing nonspecific interaction with the reticuloendothelial system. The functional peptide modified copolymers were more cytocompatible beneficial from CAGW and CAG-TAT-NLS peptides. It is worth to

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note that CAGW peptide modified gene carrier efficiently delivered pDNA to ECs and transfected them with high transfection efficiency compared with POSS-DP. Furthermore, the results of fluorescence transfection and western blot showed that POSS-DP-CAG-TAT-NLS/pDNA

complexes

group

had

highest

transfection

efficiency and protein express. They were even better than positive control group of PEI 25 kDa/pDNA. This was attributed to the multifunction of CAG-TAT-NLS peptide in the POSS-DP-CAG-TAT-NLS gene carrier. In addition, we examined the cellular uptake capacity of the POSS-DP/pDNA complexes, POSS-DP-CAGW/pDNA complexes, POSS-DP-CAG-TAT-NLS/pDNA complexes by flow cytometry. The results demonstrated that CAGW peptide and transmembrane

TAT

peptide

POSS-DP-CAG-TAT-NLS/pDNA

could

enhance

complexes

facilitated

endocytosis the

migration

rate. and

proliferation ability of transfected EA.hy926 significantly as demonstrated by scratch wound healing analysis. In vitro angiogenesis assay showed that the transfected HUVECs

by

POSS-DP-CAG-TAT-NLS/pDNA

complexes

enhanced

neovascularization ability. The transfected cells enhanced endothelial tube formation with

high

tube

number

(40

tubes

per

field),

which

is

higher

than

REDV-G4-TAT-G4-NLS/pDNA (25 tubes per field).64 These results demonstrated that the star-shaped POSS copolymers with functional peptides could highly transfect ECs, express related ZNF580 protein, and enhance their migration and angiogenesis. This gene delivery system is potential for endothelialization.

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5 Conclusion In summary, we synthesized the functional peptide modified biocompatible star-/comb-shaped POSS copolymers. These amphiphilic hybrid copolymers consisted of a hydrophobic POSS core, eight cationic PDMAEMA middle blocks, hydrophilic comb-shaped PPEGMA blocks, and many functional CAG-TAT-NLS peptides. They could form micelles in aqueous solution, strongly condensed and efficiently delivered pDNA. They had CAG peptide sequence as specific ECs targeting property, TAT-NLS peptide sequence for high internalization, cationic PDMAEMA block for pDNA condensation and enhancement of endosomal escape, and hydrophilic PEG corona for high biocompatibility. These gene carriers with multifunctional peptides exhibited higher cellular uptake, nuclear internalization and transfection efficiency than PEI 25 kDa. Overall, we believe that this delivery system has preferably prospect of development and application owing to its high efficiency in gene delivery and relatively low cytotoxicity.

Acknowledgements This project was supported by National Key R&D Program of China (grant No. 2016YFC1100300), National Science Foundation of China (Grant No. 51673145 and 31370969), International Science & Technology Cooperation Program of China (Grant No. 2013DFG52040).

References 1.

Yuan, H. M.; Xu, C.; Zhao, Y.; Yu, B. R.; Cheng, G.; Xu, F. J., Well‐Defined

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Protein ‐ Based Supramolecular Nanoparticles with Excellent MRI Abilities for Multifunctional Delivery Systems. Adv. Funct. Mater. 2016, 26 (17), 2855-2865, DOI: 10.1002/adfm.201504980. 2. Novo, L.; Mastrobattista, E.; van Nostrum, C. F.; Lammers, T.; Hennink, W. E., Decationized Polyplexes for Gene Delivery. Expert Opin. Drug Delivery 2015, 12 (4), 507-512, DOI: 10.1517/17425247.2015.988136. 3. Uritu, C. M.; Varganici, C. D.; Ursu, L.; Coroaba, A.; Nicolescu, A.; Dascalu, A. I.; Peptanariu, D.; Stan, D.; Constantinescu, C. A.; Simion, V.; Calin, M.; Maier, S. S.; Pinteala, M.; Barboiu, M., Hybrid Fullerene Conjugates as Vectors for DNA Cell-Delivery. J. Mater. Chem. B 2015, 3 (12), 2433-2446, DOI: 10.1039/c4tb02040e. 4. Greenberg, B.; Butler, J.; Felker, G. M.; Ponikowski, P.; Voors, A. A.; Desai, A. S.; Barnard, D.; Bouchard, A.; Jaski, B.; Lyon, A. R., Calcium Upregulation by Percutaneous Administration of Gene Therapy in Patients with Cardiac Disease (CUPID 2): a Randomised, Multinational, Double-blind, Placebo-controlled, Phase 2b Trial. Lancet 2016, 387 (10024), 1178-1186, DOI: 10.1016/s0140-6736(16)00082-9. 5. Mead, B. P.; Kim, N.; Miller, G. W.; Hodges, D.; Mastorakos, P.; Klibanov, A. L.; Mandell, J. W.; Hirsh, J.; Suk, J. S.; Hanes, J., Novel Focused Ultrasound Gene Therapy Approach Noninvasively Restores Dopaminergic Neuron Function in a Rat Parkinson's Disease Model. Nano Lett. 2017, 17 (6), 3533-3542, DOI: 10.1021/acs.nanolett.7b00616. 6. Duan, S.; Yu, B.; Gao, C.; Yuan, W.; Ma, J.; Xu, F. J., A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich Polycations for Effective Gene Therapy. ACS Appl. Mater. Interfaces 2016, 8 (43), 29334-29342, DOI: 10.1021/acsami.6b11029. 7. Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M., A Review of Stimuli-responsive Nanocarriers for Drug and Gene Delivery. J. Controlled Release 2008, 126 (3), 187-204, DOI: 10.1016/j.jconrel.2007.12.017. 8. Ren, X. K.; Feng, Y. K.; Guo, J. T.; Wang, H. X.; Li, Q.; Yang, J.; Hao, X. F.; Lv, J.; Ma, N.; Li, W. Z., Surface Modification and Endothelialization of Biomaterials as Potential Scaffolds for Vascular Tissue Engineering Applications. Chem. Soc. Rev. 2015, 44 (15), 5680-5742, DOI: 10.1039/c4cs00483c. 9. Zhang, Y.; Zhou, Z.; Zhu, X.; Chen, M., A Smart Gene Delivery Platform: Cationic Oligomer. Eur. J. Pharm. Sci. 2017, 105, 33-40, DOI: 10.1016/j.ejps.2017.05.002. 10. Huang, N. C.; Ji, Q. M.; Ariga, K.; Hsu, S. H., Nanosheet Transfection: Effective Transfer of Naked DNA on Silica Glass. NPG Asia Mater. 2015, 7 (6), 1-8, DOI: 10.1038/am.2015.43. 11. Mintzer, M. A.; Simanek, E. E., Nonviral Vectors for Gene Delivery. Chem. Rev. 2009, 109 (2), 259-302, DOI: 10.1021/cr800409e. 12. Pan, J. J.; Yuan, Y. Q.; Wang, H. W.; Liu, F.; Xiong, X. H.; Chen, H.; Yuan, L., Efficient Transfection by Using PDMAEMA Modified SiNWAs as a Platform for Ca2+-Dependent Gene Delivery. ACS Appl. Mater. Interfaces 2016, 8 (24), 15138-15144, DOI: 10.1021/acsami.6b04689. 13. Cordes, D. B.; Lickiss, P. D.; Rataboul, F., Recent Developments in the Chemistry of Cubic Polyhedral Oligosilsesquioxanes. Chem. Rev. 2010, 110 (4),

ACS Paragon Plus Environment

Page 42 of 48

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

ACS Biomaterials Science & Engineering

2081-2173, DOI: 10.1021/cr900201r. 14. Guerrero, G.; Hägg, M. B.; Kignelman, G.; Simon, C.; Peters, T.; Rival, N.; Denonville, C., Investigation of amino and amidino functionalized Polyhedral Oligomeric SilSesquioxanes (POSS®) nanoparticles in PVA-based hybrid membranes for CO2/N2 separation. J. Membr. Sci. 2017, 544 (15), 161-173, DOI: 10.1016/j.memsci.2017.09.014. 15. He, J. L.; Yue, K.; Liu, Y. Q.; Yu, X. F.; Ni, P. H.; Cavicchi, K. A.; Quirk, R. P.; Chen, E. Q.; Cheng, S. Z. D.; Zhang, W. B., Fluorinated Polyhedral Oligomeric Silsesquioxane-based Shape Amphiphiles: Molecular Design, Topological Variation, and Facile Synthesis. Polym. Chem. 2012, 3 (8), 2112-2120, DOI: 10.1039/c2py20101a. 16. Trastoy, B.; Bonsor, D. A.; Perez-Ojeda, M. E.; Jimeno, M. L.; Mendez-Ardoy, A.; Fernandez, J. M. G.; Sundberg, E. J.; Chiara, J. L., Synthesis and Biophysical Study of Disassembling Nanohybrid Bioconjugates with a Cubic Octasilsesquioxane Core. Adv. Funct. Mater. 2012, 22 (15), 3191-3201, DOI: 10.1002/adfm.201200423. 17. Wang, X.; Yang, Y.; Gao, P.; Li, D.; Yang, F.; Shen, H.; Guo, H.; Xu, F.; Wu, D., POSS Dendrimers Constructed from a 1 → 7 Branching Monomer. Chem. Commun. 2014, 50 (46), 6126-6129, DOI: 10.1039/c4cc01859a. 18. Yang, Y. Y.; Wang, X.; Hu, Y.; Hu, H.; Wu, D. C.; Xu, F. J., Bioreducible POSS-cored Star-shaped Polycation for Efficient Gene Delivery. ACS Appl. Mater. Interfaces 2014, 6 (2), 1044-1052, DOI: 10.1021/am404585d. 19. Li, Y.; Xu, B.; Bai, T.; Liu, W., Co-delivery of Doxorubicin and Tumor-suppressing p53 Gene Using a POSS-based Star-shaped Polymer for Cancer Therapy. Biomaterials 2015, 55 (1), 12-23, DOI: 10.1016/j.biomaterials.2015.03.034 20. Fabritz, S.; Horner, S.; Avrutina, O.; Kolmar, H., Bioconjugation on Cube-octameric Silsesquioxanes. Org. Biomol. Chem. 2013, 11 (14), 2224-2236, DOI: 10.1039/c2ob26807h. 21. Qiu, Z. B.; Pan, H., Preparation, Crystallization and Hydrolytic Degradation of Biodegradable Poly(L-lactide)/Polyhedral Oligomeric Silsesquioxanes Nanocomposite. Compos. Sci. Technol. 2010, 70 (7), 1089-1094, DOI: 10.1016/j.compscitech.2009.11.001. 22. Zhou, Z.; Lu, Z. R., Dendritic Nanoglobules with Polyhedral Oligomeric Silsesquioxane Core and Their Biomedical Applications. Nanomedicine 2014, 9 (15), 2387-2401, DOI: 10.2217/nnm.14.133. 23. Liu, J. Q.; Tao, L.; Xu, J. T.; Jia, Z. F.; Boyer, C.; Davis, T. P., RAFT Controlled Synthesis of Six-armed Biodegradable Star Polymeric Architectures via a ‘Core-first’ Methodology. Polymer 2009, 50 (19), 4455-4463, DOI: 10.1016/j.polymer.2009.07.018. 24. Pu, Y. J.; Zhang, L. G.; Zheng, H.; He, B.; Gu, Z. W., Drug Release of pH-sensitive Poly(L-aspartate)-b-poly(ethylene glycol) Micelles with POSS Cores. Polym. Chem. 2013, 5 (2), 463-470, DOI: 10.1039/c3py00965c. 25. Ge, Z. S.; Wang, D.; Zhou, Y. M.; Liu, H. W.; Liu, S. Y., Synthesis of Organic/Inorganic Hybrid Quatrefoil-Shaped Star-Cyclic Polymer Containing a Polyhedral Oligomeric Silsesquioxane Core. Macromolecules 2009, 42 (8),

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

2903-2910, DOI: 10.1021/ma802585k. 26. Cheng, Q.; Du, L.; Meng, L.; Han, S.; Wei, T.; Wang, X.; Wu, Y.; Song, X.; Zhou, J.; Zheng, S.; Huang, Y.; Liang, X. J.; Cao, H.; Dong, A.; Liang, Z., The Promising Nanocarrier for Doxorubicin and siRNA Co-delivery by PDMAEMA-based Amphiphilic Nanomicelles. ACS Appl. Mater. Interfaces 2016, 8 (7), 4347-4356, DOI: 10.1021/acsami.5b11789. 27. Sun, H.; Chen, X.; Han, X.; Liu, H., Dual-thermoresponsive Aggregation of Schizophrenic PDMAEMA-b-PSBMA Copolymer with an Unrepeatable pH Response and a Recycled CO2/N2 Response. Langmuir 2017, 33 (10), 2646-2654, DOI: 10.1021/acs.langmuir.7b00065. 28. Yuan, T. T.; Dong, J.; Han, G. X.; Wang, G. J., Polymer Nanoparticles Self-assembled from Photo-, pH- and Thermo-responsive Azobenzene-functionalized PDMAEMA. RSC Adv. 2016, 6 (13), 10904-10911, DOI: 10.1039/c5ra26894j. 29. Zhu, C.; Zheng, M.; Meng, F.; Mickler, F. M.; Ruthardt, N.; Zhu, X.; Zhong, Z., Reversibly Shielded DNA Polyplexes Based on Bioreducible PDMAEMA-SS-PEG-SS-PDMAEMA Triblock Copolymers Mediate Markedly Enhanced Nonviral Gene Transfection. Biomacromolecules 2012, 13 (3), 769-778, DOI: 10.1021/bm201693j. 30. Yang, J.; Hao, X. F.; Li, Q.; Akpanyung, M.; Nejjari, A.; Neve, A. L.; Ren, X. K.; Guo, J. T.; Feng, Y. K.; Shi, C. C.; Zhang, W. C., CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo. ACS Appl. Mater. Interfaces 2017, 9 (5), 4485-4497, DOI: 10.1021/acsami.6b14769. 31. Li, Q.; Hao, X. F.; Lv, J.; Ren, X. K.; Zhang, K. Y.; Ullah, I.; Feng, Y. K.; Shi, C. C.; Zhang, W. C., Mixed Micelles Obtained by Co-assembling Comb-like and Grafting Copolymers as Gene Carriers for Efficient Gene Delivery and Expression in Endothelial Cells. J. Mater. Chem. B 2017, 5 (8), 1673-1687, DOI: 10.1039/c6tb02212j. 32. Lv, J.; Hao, X. F.; Li, Q.; Akpanyung, M.; Nejjari, A.; Neve, A. L.; Ren, X. K.; Feng, Y. K.; Shi, C. C.; Zhang, W. C., Star-shaped Copolymer Grafted PEI and REDV as a Gene Carrier to Improve Migration of Endothelial Cells. Biomater. Sci. 2017, 5 (3), 511-522, DOI: 10.1039/c6bm00856a. 33. Wang, H. X.; Li, Q.; Yang, J.; Guo, J. T.; Ren, X. K.; Feng, Y. K.; Zhang, W. C., Comb-shaped Polymer Grafted with REDV Peptide, PEG and PEI as Targeting Gene Carrier for Selective Transfection of Human Endothelial Cells. J. Mater. Chem. B 2017, 5 (7), 1408-1422, DOI: 10.1039/c6tb02379g. 34. Yang, Z.; Jiang, Z.; Cao, Z.; Zhang, C.; Gao, D.; Luo, X.; Zhang, X.; Luo, H.; Jiang, Q.; Liu, J., Multifunctional Non-viral Gene Vectors with Enhanced Stability, Improved Cellular and Nuclear Uptake Capability, and Increased Transfection Efficiency. Nanoscale 2014, 6 (17), 10193-10206, DOI: 10.1039/c4nr02395a. 35. Zheng, N.; Song, Z. Y.; Liu, Y.; Yin, L. C.; Cheng, J. J., Gene Delivery into Isolated Arabidopsis Thaliana Protoplasts and Intact Leaves using Cationic, α-helical Polypeptide. Front. Chem. Sci. Eng. 2017, 11 (4), 521-528, DOI: 10.1007/s11705-017-1612-8.

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Page 44 of 48

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

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36. Frankel, A. D.; Pabo, C. O., Cellular Uptake of the Tat Protein from Human Immunodeficiency Virus. Cell 1988, 55 (6), 1189-1193, DOI: 10.1016/0092-8674(88)90263-2. 37. Gopal, V., Bioinspired Peptides as Versatile Nucleic Acid Delivery Platforms. J. Controlled Release 2013, 167 (3), 323-332, DOI: 10.1016/j.jconrel.2013.02.021. 38. Renigunta, A.; Krasteva, G.; Konig, P.; Rose, F.; Klepetko, W.; Grimminger, F.; Seeger, W.; Hanze, J., DNA Transfer into Human Lung Cells is Improved with Tat-RGD Peptide by Caveoli-mediated Endocytosis. Bioconjugate Chem. 2006, 17 (2), 327-334, DOI: 10.1021/bc050263o. 39. Qu, W.; Qin, S. Y.; Ren, S.; Jiang, X. J.; Zhuo, R. X.; Zhang, X. Z., Peptide-Based Vector of VEGF Plasmid for Efficient Gene Delivery in Vitro and Vessel Formation in Vivo. Bioconjugate Chem. 2013, 24 (6), 960-967, DOI: 10.1021/bc300677n. 40. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B., The Design, Synthesis, and Evaluation of Molecules that Enable or Enhance Cellular Uptake: Peptoid Molecular Transporters. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (24), 13003-13008, DOI: 10.1073/pnas.97.24.13003. 41. Oba, M.; Kato, T.; Furukawa, K.; Tanaka, M., A Cell-Penetrating Peptide with a Guanidinylethyl Amine Structure Directed to Gene Delivery. Sci. Rep. 2016, 6, 19913-19921, DOI: 10.1038/srep19913. 42. Mitchell, D. J.; Steinman, L.; Kim, D. T.; Fathman, C. G.; Rothbard, J. B., Polyarginine Enters Cells More Efficiently than Other Polycationic Homopolymers. Chem. Biol. Drug Des. 2000, 56 (5), 318-325, DOI: 10.1034/j.1399-3011.2000.00723.x. 43. Nam, H. Y.; Kim, J.; Kim, S.; Yockman, J. W.; Kim, S. W.; Bull, D. A., Cell Penetrating Peptide Conjugated Bioreducible Polymer for siRNA Delivery. Biomaterials 2011, 32 (22), 5213-5222, DOI: 10.1016/j.biomaterials.2011.03.058. 44. Kim, S. H.; Jeong, J. H.; Ou, M.; Yockman, J. W.; Kim, S. W.; Bull, D. A., Cardiomyocyte-targeted siRNA Delivery by Prostaglandin E2-Fas siRNA Polyplexes Formulated with Reducible Poly(amido amine) for Preventing Cardiomyocyte Apoptosis. Biomaterials 2008, 29 (33), 4439-4446, DOI: 10.1016/j.biomaterials.2008.07.047. 45. Beloor, J.; Choi, C. S.; Nam, H. Y.; Park, M.; Kim, S. H.; Jackson, A.; Lee, K. Y.; Kim, S. W.; Kumar, P.; Lee, S. K., Arginine-engrafted Biodegradable Polymer for the Systemic Delivery of Therapeutic siRNA. Biomaterials 2012, 33 (5), 1640-1650, DOI: 10.1016/j.biomaterials.2011.11.008. 46. Kim, T. I.; Rothmund, T.; Kissel, T.; Kim, S. W., Bioreducible Polymers with Cell Penetrating and Endosome Buffering Functionality for Gene Delivery Systems. J. Controlled Release 2011, 152 (1), 110-119, DOI: 10.1016/j.jconrel.2011.02.013. 47. Kim, T. I.; Lee, M.; Kim, S. W., A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems. Biomaterials 2010, 31 (7), 1798-1804, DOI: 10.1016/j.biomaterials.2009.10.034. 48. Tammam, S. N.; Azzazy, H. M. E.; Lamprecht, A., The Effect of Nanoparticle Size and NLS Density on Nuclear Targeting in Cancer and Normal Cells; Impaired

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

Nuclear Import and Aberrant Nanoparticle Intracellular Trafficking in Glioma. J. Controlled Release 2017, 253, 30-36, DOI: 10.1016/j.jconrel.2017.02.029. 49. Qu, W. Q., S. Y.; Kuang, Y.; Zhuo, R. X.; Zhang, X. Z., Peptide-based Vectors Mediated by Avidin–biotin Interaction for Tumor Targeted Gene Delivery. J. Mater. Chem. B 2013, 1 (16), 2147-2154, DOI: 10.1039/c3tb00226h. 50. Chen, K.; Guo, L.; Zhang, J.; Chen, Q.; Wang, K.; Li, C.; Li, W.; Qiao, M.; Zhao, X.; Hu, H., A Gene Delivery System Containing Nuclear Localization Signal: Increased Nucleus Import and Transfection Efficiency with the Assistance of RanGAP1. Acta Biomater. 2016, 48, 215-226, DOI: 10.1016/j.actbio.2016.11.004. 51. Ma, V. D. A.; Mastrobattista, E.; Oosting, R. S.; Hennink, W. E.; Koning, G. A.; Crommelin, D. J., The Nuclear Pore Complex: The Gateway to Successful Nonviral Gene Delivery. Pharm. Res. 2006, 23 (3), 447-459, DOI: 10.1007/s11095-005-9445-4. 52. Wang, H. Y.; Chen, J. X.; Sun, Y. X.; Deng, J. Z.; Li, C.; Zhang, X. Z.; Zhuo, R. X., Construction of Cell Penetrating Peptide Vectors with N-terminal Stearylated Nuclear Localization Signal for Targeted Delivery of DNA into the Cell Nuclei. J. Controlled Release 2011, 155 (1), 26-33, DOI: 10.1016/j.jconrel.2010.12.009. 53. Tanimoto, M.; Kamiya, H.; Minakawa, N.; Matsuda, A.; Harashima, H., No Enhancement of Nuclear Entry by Direct Conjugation of a Nuclear Localization Signal Peptide to Linearized DNA. Bioconjugate Chem. 2003, 14 (6), 1197-1202, DOI: 10.1021/bc034075e. 54. Neves, C.; Byk, G.; Scherman, D.; Wils, P., Coupling of a Targeting Peptide to Plasmid DNA by Covalent Triple Helix Formation. FEBS Lett. 1999, 453 (1-2), 41-45, DOI: 10.1016/S0014-5793(99)00674-2. 55. Yang, J.; Li, Q.; Yang, X.; Feng, Y. K.; Ren, X. K.; Shi, C. C.; Zhang, W. C., Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells. Macromol. Rapid Commun. 2016, 37 (23), 1926-1931, DOI: 10.1002/marc.201600345. 56. Yang, J.; Feng, Y. K.; Zhang, L., Biodegradable Carrier/gene Complexes to Mediate the Transfection and Proliferation of Human Vascular Endothelial Cells. Polym. Adv. Technol. 2016, 26 (12), 1370-1377, DOI: 10.1002/pat.3636. 57. Shi, C. C.; Li, Q.; Zhang, W. C.; Feng, Y. K.; Ren, X. K., REDV Peptide Conjugated Nanoparticles/pZNF580 Complexes for Actively Targeting Human Vascular Endothelial Cells. ACS Appl. Mater. Interfaces 2015, 7 (36), 20389-20399, DOI: 10.1021/acsami.5b06286. 58. Duo, X. H.; Wang, J.; Li, Q.; Neve, A. L.; Akpanyung, M.; Nejjari, A.; Ali, Z. S. S.; Feng, Y. K.; Zhang, W. C.; Shi, C. C., CAGW Peptide Modified Biodegradable Cationic Copolymer for Effective Gene Delivery. Polymers 2017, 9 (12), 158-176, DOI: 10.3390/polym9050158. 59. Hao, X. F.; Li, Q.; Lv, J.; Yu, L.; Ren, X. K.; Zhang, L.; Feng, Y. K.; Zhang, W. C., CREDVW-Linked Polymeric Micelles As a Targeting Gene Transfer Vector for Selective Transfection and Proliferation of Endothelial Cells. ACS Appl. Mater. Interfaces 2015, 7 (22), 12128-12140, DOI: 10.1021/acsami.5b02399. 60. Cai, X.; Jin, R.; Wang, J.; Yue, D.; Jiang, Q.; Wu, Y.; Gu, Z., Bioreducible

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Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery. ACS Appl. Mater. Interfaces 2016, 8 (9), 5821-5832, DOI: 10.1021/acsami.5b11545. 61. Ko, N. R., Cheong, J., Noronha, A., Wilds, C. J., Oh, J. K., Reductively-sheddable Cationic Nanocarriers for Dual Chemotherapy and Gene Therapy with Enhanced Release. Colloids Surf., B 2015, 126 (1), 178-187, DOI: 10.1016/j.colsurfb.2014.12.010. 62. Yan, P.; Wang, R.; Zhao, N.; Zhao, H.; Chen, D. F.; Xu, F. J., Polycation-functionalized Gold Nanoparticles with Different Morphologies for Superior Gene Transfection. Nanoscale 2015, 7 (12), 5281-5291, DOI: 10.1039/c5nr00481k. 63. Fan, X. S.; Wang, Z.; He, C. B., “Breathing” Unimolecular Micelles Based on Novel Star-like Amphiphilic Hybrid Copolymer. J. Mater. Chem. B 2015, 3 (23), 4715-4722, DOI: 10.1039/c5tb00415b. 64. Hao, X. F.; Li, Q.; Guo, J. T.; Ren, X. K.; Feng, Y. K.; Shi, C. C.; Zhang, W. C., Multifunctional Gene Carriers with Enhanced Specific Penetration and Nucleus Accumulation to Promote Neovascularization of HUVECs in Vivo. ACS Appl. Mater. Interfaces 2017, 9 (41), 35613-35627, DOI: 10.1021/acsami.7b11615.

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Multi-targeting Peptide Functionalized Star-shaped Copolymers with Comb-like Structure and a POSS-core to Effectively Transfect Endothelial Cells Jun Wang, Syed Saqib Ali Zaidi, Ali Hasnain, Jintang Guo, Xiangkui Ren, Shihai Xia, Wencheng Zhang, Yakai Feng

Corresponding Author: Y. Feng, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China Email: [email protected] (Y. Feng)

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