ROS-Response-Induced Zwitterionic Dendrimer for Gene Delivery

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ROS-response Induced Zwitterionic Dendrimer for Gene Delivery Shengran Li, Binggang Chen, Yangchun Qu, Xinxin Yan, Wenliang Wang, Xiaojing Ma, Bo Wang, Sanrong Liu, and Xifei Yu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03758 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 22, 2018

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ROS-response Induced Zwitterionic Dendrimer for Gene Delivery Shengran Li†，‡, Binggang Chen†，‡, Yangchun Qu§, Xinxin Yan†，‡, Wenliang Wang†，‡ , Xiaojing Ma†, Bo Wang†, Sanrong Liu†*, Xifei Yu†，‡,* † Laboratory of Polymer Composites Engineering, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China. ‡ University of Science and Technology of China, Hefei, Anhui 230026 China. § Department of radiology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China.

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ABSTRACT

As one of the most promising therapeutic method, gene therapy has been playing more and more important role in treating disease due to its ultrahigh therapy efficiency. Even if non-viral gene vectors represented by polycation, liposomal, dendrimers, and zwitterionic materials have made great progress in gene complexation, low immunogenicity and biocompatibility, effectively gene release intracellular with low toxicity is still a bottleneck restricting the clinical application of gene therapy. Herein, we designed and synthesized a ROS-responsive dendrimer poly (amido

amine)-N-(4-boronobenzyl)-N,N-diethyl-2-(propionyloxy)ethan-1-aminium

(PAMAM-(B-DEAEP)16) as gene vector whose potential can vary from positive to negative under the elevated reactive oxygen species (H2O2) in cancerous cells. DLS results showed that zeta potential of PAMAM-(B-DEAEP)16 decreased from + 12.3 mV to - 5 mV under 80mM H2O2 in PBS buffer. The 1H-NMR results demonstrated that the intermediate status of PAMAM-(B-DEAEP)16 were zwitterionic in about 6 hours since it consisted the positive quaternary ammonium and negative carboxylic acid simultaneously before the ester bond were completely hydrolyzed. Gel retardation assay showed that PAMAM-(B-DEAEP)16 can condense DNA at above N/P=1, then PAMAM-(B-DEAEP)16 transferred to be zwitterionioc, which began to continuously release DNA with the decrease of the positive charges and increase of the negative charges, and finally to negative charged poly(amido amine)-propionic acid (PAMAM-PAc16) in the

80 mM H2O2. And fluorescence-labeled Cy-5 DNA

indicated that PAMAM-(B-DEAEP)16 can enter into cell completely in about 4 hours. 2 ACS Paragon Plus Environment

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The results showed that this compound we designed exhibited higher gene transfection efficiency and lower cytotoxicity than commercial PEI did. This is the first time that the positive charged dendrimer were transferred into zwitterionic dendrimer under the stimuli of H2O2 and successfully applied to gene delivery. Different from all the previous reports, we did not seek the compromise between the high gene transfection and low toxicity, but find a new avenue to make the gene carrier not only own higher gene transfection efficiency but also exhibit lower toxicity by introducing stimuli-sensitive groups into the positive charged dendrimer to make it capable to adjust the charge property according to the microenvironment. This study not only provided a good method to design materials for gene delivery, but also open a new perspective to understand the process of gene delivery.

KEYWORDS ROS-responsive, zwitterionic dendrimer, gene delivery

1. INTRODUCTION

In recent decades, gene therapy as one of the most promising therapeutic methods played the indispensable role in treating disease. Non-viral gene vectors including cationic polymers, liposomes, dendrimers and polypeptides attracted more and more attention in terms of their highly complexation efficiency, low immunogenicity, biocompatibility and potential for clinical application.1-11 The cationic gene vectors have strong electrostatic interactions with cell membranes and 3 ACS Paragon Plus Environment

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will damage to cell. Therefore, different strategies have proposed to solve this problem.

PEG-modified

dendrimers,7,8

zwitterionic

surface

and

fluorinated

dendrimer9-11 have been proved to greatly improve the serum stability and decrease the toxicity of cationic polymers in gene delivery, protein and drug delivery systems. Among them, zwitterionic materials has been paid more attention since the cationic segments can interact with DNA molecules by electrostatic interactions, and compared with the cationic gene vectors, the zwitterionic molecule exhibited highly gene transfection efficiency with decreased toxicity by forming hydration layer in aqueous environment.12-16 However, they were still limited by the inhibited releases of gene after they arrived at the disease sites, which restrict the clinical application of gene therapy to a certain degree.17, 18 To achieve effectively gene release from vectors in disease sites, many designs have been proposed.19-24. For example, Dr Jiang’s group committed to the application of zwitterionic materials in gene delivery, and they designed a series of polycarboxybetaines (PCBs) modified with degradable ester group which can condense gene by cation and then hydrolyze to zwitterionic with the gene release when PCBs transferred into cells.14-16. Zhang et al. introduced a novel diblock copolymer poly(carboxy betaine methacrylate ethyl ester)-poly(carboxy betaine methacrylate) (PCBMAEE-PCBMA) that possess both hydrolytic and zwitterionic groups, which endow it highly transfection efficiency and effective gene release intracellular.12 Although some advances has been achieved by optimizing the molecular structures to lower the charge density and release DNA, most of them only got a compromise between higher gene transfection efficiency and lower toxicity due 4 ACS Paragon Plus Environment

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to the existence of the cationic segments, which can bind to DNA molecules but also has toxicity to cells. Increased generation of reactive oxygen species (ROS) has been observed in cancer cells, while the signals of ROS is relatively low in normal cells. Recent studies suggested that this biochemical property of cancer cells can be exploited for therapeutic opportunity.20-24 Therefore, there are still huge demands to seek for novel alternative materials with higher transfection efficiency and lower toxicity, and materials specific to cancer cells need to be developed. Herein, we designed and synthesized a reactive oxygen species (ROS)-responsive dendrimer poly(amido

amine)-N-(4-boronobenzyl)-N,N-diethyl-2-

(propionyloxy)

ethan-1-aminium) (PAMAM-(B-DEAEP)16, which exhibited to be positive at the initial stage but could transferred to be zwitterionic and finally to be negative (poly(amido amine)-propionic acid (PAMAM-PAc16)) when they suffered from reactive oxygen species (H2O2) (Scheme 1). Poly (amido amine) (PAMAM), as a universal polycation dendrimer, possessed superior proton sponge activity to escape from endosomal29-32 and were decorated with 2-(acryloyloxy)-N-(4-boronobenzyl)N,N-diethylethan-1-aminium at the end. After the boronic acid group oxidized by H2O2, the boric acid and p-quinone methide left and quaternary ammonium turned to be tertiary amine, subsequently ester bond fast self-catalyzed hydrolyzed to be carboxylic acid, then the gene were released (Figure 1a). PAMAM-(B-DEAEP)16 interacted with DNA when they were positive charged and began to continuously release gene due to the increasement of negative charges and decrease of quaternary ammonium under the stimulation of H2O2. The stimuli-responsive gene vectors are 5 ACS Paragon Plus Environment

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of particular interest because they offer a powerful strategy for delivering and releasing genes at targeted sites efficiently and effectively. In this research, Hela cells was chosen to conduct the biological evaluation. The results demonstrated that the compounds

we

designed

exhibit

high

transfection

efficiency,

excellent

biocompatibility and low cytotoxicities and improved therapeutic efficacy. This is the first time that the zwitterionic dendrimers were induced from the positive charged dendrimer under the stimuli of H2O2 and successfully applied to gene delivery. Different from all the cationic and the previous zwitterionic gene vectors, it is not to seek the compromise between the high gene transfection and low toxicity, but to find a new avenue to make it show higher gene transfection efficiency and lower toxicity at the same time. The material we synthesized can adjust its charge status according to the microenvironment automatically and showed different functions. This study not only provided a new avenue to design the materials for gene delivery but also open a new perspective to understand the process of gene delivery. 2. EXPERIMENTAL PART 2.1. Materials. Ethylenediamine, 4-(Bromomethyl)phenylboronic acid were purchased

from

Sigma-Aldrich

(Shanghai,

China).

Methyl

acrylate,

2-(N,N-Diethylamino)ethyl acrylate and Branched PEI with molecular weight of 25000 Da were bought from Randall (Changchun, China), Aladdin (Shanghai, China) and Sigma-Aldrich (Shanghai, China), respectively. Fetal bovine serum (FBS), trypsin, RPMI 1640 cell culture media, 4’,6-diamidino-2-phenylindole (DAPI), 6 ACS Paragon Plus Environment

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Celltiter-Blue and fluorescein isothiocyanate (FITC) were got from Powertek Biotechnology (Beijing, China). The plasmid DNA that expressed GFP was purchased from Invitrogen (Carlsbad, CA, USA). The plasmids were propagated in Escherichia coli DH5α and extracted using Endo-Free Plasmid Kit (Qiagen, Hilden, Germany). pGL3 luciferase plasmid and luciferase assay system were purchased from Promega (Madison, WI). 2.2. Characterization. The

1H-NMR

spectra were recorded on the 500

NMR(AVANCE III 500HD, Bruker) at room temperature. The size and zeta potential of materials were measured via dynamic light scattering (DLS) using a Zetasizer Nano-ZS from Malvern Instruments with He−Ne laser. The measurements were made with wavelength of 633 nm at 25°C and angle detection at 173°. Transmission electron microscope (TEM) measurement were performed by using JEOL JEM-1011 electron microscope. The GFP expression and cellular uptake trace were monitored by confocal laser scanning microscope (LSM 700 Carl Zeiss Microscopy). The relative light units (RLU) of luciferase plasmid expression were measured by luminometer (Glomax). The DNA bind and release were characterizing by agarose gel retardation assay. The synergy microplate reader (Synergy H1, from Bio Tek) was used to detect the cell viability. 2.3. Synthesis of PAMAM G3.0 dendrons: The synthesis of PAMAM G3.0 was according to the previous researches.33-36 The initiate material ethylenediamine was regarded as the PAMAM G0. Methyl acrylate (5 mL, 0.08 mmol) was added 7 ACS Paragon Plus Environment

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dropwise into a solution of ethylenediamine (1.5 mL, 0.02 mmol)in methanol (20 mL). The reaction was carried out for three days under argon at 30°C. Afterward, the reaction solution was evaporated, and the residue was purified by silica gel column chromatography (Petroleum ether : EtOAc = 2:1) with a yielding of PAMAM G0.5(7.5 g, 95 %) as colorless oil (1H-NMR, Figure S1). The synthesis of PAMAM G1.0 was briefly as follows: a solution of PAMAM G 0.5 (4g, 9.9 mmol) in methanol (20 mL) was added dropwise into the ethylenediamine (2.7 mL, 39.6 mmol), the reaction was carried out for three days at 30 °C under argon. Afterward, the reaction solution was evaporated, and the excess ethylenediamine was removed by azeotropic distillation (toluene/methanol = 9/1) to obtain an amino-terminated dendron of PAMAM G1.0 (5 g, 98 %) as pale viscous oil (1H-NMR, Figure S2). The PAMAM G1.5 was synthesized with PAMAM G1.0 and methyl acrylate as the mentioned procedure above (1H-NMR, Figure S3). The PAMAM G2.0 was synthesized with PAMAM G1.5 and ethylenediamine as the mentioned procedure above (1H-NMR, Figure S4). The PAMAM G2.5 was synthesized with PAMAM G2.0 and methyl acrylate as the mentioned procedure above (1H-NMR, Figure S5). The PAMAM G3.0 was synthesized with PAMAM G2.5 and ethylenediamine as the mentioned procedure above (1H-NMR, Figure S6). 2.4. Synthesis of PAMAM-(B-DEAEP)16: PAMAM G3.0 (1.67 g, 0.51 mmol) was dissolved in 40mL N,N’-dimethylformamide (DMF), which was then added into 8 ACS Paragon Plus Environment

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a 100 mL round-bottom flask with 2-(N,N-Diethylamino)ethyl acrylate(16.32 mmol, 5 mL) and triethylamine(2.28 mL). The reaction system was deoxygenated with argon and reacted for three days at room temperature. Afterward, the solvent was removed under vacuum at 70 °C. Then the residue was dissolved in a little acetone and precipitated in n-hexane three times, then centrifuged to discard the supernatant. The obtained solid was dried under vacuum to give a yellow vicous solid (4.5 g, 96%) (1H-NMR, Figure S7). The obtained product (388 mg, 0.073 mmol) in the above reaction was dissolved in 5 mL DMF and added into a 25 mL round-bottom flask with 4-(Bromomethyl)phenylboronic acid(188 mg, 0.876 mmol). The reaction was carried out for 24 h at room temperature. Afterward, the reaction solution was precipitated in diethyl ether three times and centrifuged to discard the supernatant. The residue was dried under vacuum to give the final product as white solid (400 mg, 80%) (1H-NMR, Figure S8). 2.5.

Preparation

and

characterization

of

PAMAM-(B-DEAEP)16/DNA complex. The PAMAM-(B-DEAEP)

dendrimer 16

and DNA

(shEGFR plasmid) were mixed at different N/P ratio (total terminal amine in materials/phosphates in DNA) and incubated for 30min in PBS buffer (pH = 7.4). The size and zeta potential of complexes were measured by DLS. The morphology of the complexes were observed using TEM. 2.6.

ROS-response

of

dendrimer

PAMAM-(B-DEAEP)16.

PAMAM-(B-DEAEP)16 was dissolved in PBS(pH = 7.4) at a concentration of 3 9 ACS Paragon Plus Environment

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mg/mL, and then H2O2 was added to a concentration of 80mM. The solution was incubated at 37 °C with shaking. Samples were taken every 20 min to measure their zeta potential using DLS. The ROS-response of PAMAM-(B-DEAEP)16 was also monitored through 1H-NMR in D2O with 80 mM H2O2 at different time intervals. 2.7. Agarose gel retardation assay. The PAMAM-(B-DEAEP)16 and DNA (shEGFR plasmid) were mixed at different N/P ratio and incubated for 30 min. The complexes were electrophoresed on a 0.8 % agarose gel at 140V for 20min. DNA bands were visualized by staining with ethidium bromide (EB) excited by UV transillumination. The PAMAM-(B-DEAEP)16 and DNA were mixed at N/P ratio of 10 for 30 min and then incubated with various concentration of H2O2 (0, 0.2 mM, 1 mM, 2 mM, 5 mM) for 3 hours. The complexes were electrophoresed on agarose gel at 140 V for 20 min. The release of DNA from complexes were characterized by visualizing the distribution of DNA bands on gel. 2.8. Gene transfection. For luciferase gene transfection, Hela cell were seeded in 96-well plate at a density of 10000 per well and 80000 per well in 10% FBS-containing RPMI1640 medium and incubated to reach 70 %-80 %. The medium was replaced with fresh medium. Materials/DNA complexes at different N/P ratio (5, 10, 15, 20, 25) were added at a dose of 0.5 μg DNA (pGL3 plasmid) per well and incubated for 4 hours. Branched PEI25k as the control was complexed with DNA then added to the well. The medium was replaced with complete medium and incubated for an additional 44 hours. The determination of luciferase plasmid expression was 10 ACS Paragon Plus Environment

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performed according to the standard protocol described in the manufacture manual (Promega). 50 μl lysate was added to each well and 20 μl lysis solution was mixed with 20 μl substrate after the cells were completely lysised. Protein content of the lysis solution was determined by Bradford protein assay kit. The luciferase activity was normalized with respect to the protein concentration (relative luciferase light units per milligram protein). All data are presented at least three independent measurement. For GFP gene transfection, Hela cells were seeded in 24-well plate at a density of 80000 per well in 10% FBS-containing RPMI1640 medium and incubated to reach 70 %-80 %. Materials/DNA complexes at different N/P ratio (10, 15, 20) were added with a dose of 2 μg DNA (pGL3 plasmid) per well and incubated for 4 hours. Branched PEI25k as the control was complexed with DNA and added to the well. The medium was replaced with complete medium and incubated for additional 44 hours. The GFP transfection images were acquired using a confocal laser scanning microscope (CLSM). 2.9. Cytotoxicity assay. Hela cells were seeded in 96-well plate at a density of 10000 per well and incubated overnight. The medium was removed and fresh medium with different concentration of materials were added. After incubated for 48 hours, 10μl celltiter blue was added each well and incubated for 4 hours. The fluorescence intensity of per well was measured by microplate reader (λex = 560nm, λem = 590nm). The cell viability was calculated based on the formula: 11 ACS Paragon Plus Environment

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cell viability (%) =

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𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑠𝑎𝑚𝑝𝑙𝑒) × 100 (%) 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)

2.10. Cellular uptake. DNA was labeled with Cy-5 before used. Hela cells were seeded in glass-bottom petri dishes at a density of 100000 per dish and incubated overnight. The medium was removed and RPMI1640 medium containing materials/Cy-5 DNA at N/P ratio of 15 with a dose of 2 μg plasmid per dish were added. The cells were incubated for 30 min, 1 h and 4 h, respectively. Afterward, the medium was removed, and cells were washed with PBS three times and fixed with 4 % paraformaldehyde for 30 min at room temperature and then remove it. After that, the cellular nuclei was stained with DAPI at a concentration of 10 μg/μl for 10 min and then washed three times with PBS. The imagines were acquired using confocal laser scanning microscope (CLSM). 3.

RESULTS AND DISCUSSSION 3.1. Synthesis of the dendrimer PAMAM-(B-DEAEP)16. The PAMAM was

firstly synthesized according to the previous reports,33-36 which is a repeating process of Michael addition and amidation reaction. The 1H-NMR images of PAMAM G0.5PAMAM G3.0 were shown in Figure S1-S6, respectively. PAMAM-(B-DEAEP)16 were synthesized by using 2-(acryloyloxy)-N-(4-boronobenzyl)-N,N-diethylethan-1aminium (B-DEAEP) as key groups. Firstly, 2-(diethylamino)ethyl acrylate were attached to the end amine group of PAMAM G3.0 and then connected the p-boronic acid benzyl to tertiary amine by nucleophilic reaction. It can be found from the 1H-NMR

of PAMAM-(B-DEAEP)16 that each end groups of –NH2 on PAMAM G3.0 12 ACS Paragon Plus Environment

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dendrimer was attached with one B-DEAEP and the appearance of B-DEAEP at 4.2-4.3 ppm and 7.2-7.7 ppm indicated the final product was synthesized successfully (Figure S8). 3.2. Charge change process of the dendrimer and ROS-response induced formation of zwitterionic dendrimer. To further identify the charge property of PAMAM-(B-DEAEP)16, DLS measurements were performed to detect the zeta potential and its change. It was found that the charge of PAMAM-(B-DEAEP)16 ranged from + 35 mV to + 45 mV at different concentrations in water (Figure S9). When H2O2 was applied, phenylboronic acid left and the quaternary amine of PAMAM-(B-DEAEP)16 changed into the tertiary amine. Then the ester groups underwent self-catalyzed hydrolysis, leading to the formation of negative charged carboxylic acid. The DLS results showed that the charge decreased from the previous +12.3 mV to zero in 175 min, and finally balanced at -5 mV after 320 min under 80mM H2O2 in PBS buffer (pH = 7.4) (Figure 2). At the same time, 1H-NMR were applied to in-situ monitor the chemical structure change process of the dendrimer under 80 mM H2O2 (Figure1a, b). It was found that the characteristic peaks of a’, b’ and c’ which belonged to 4-(hydroxylmethyl) phenol appeared in 1 hour and reached to maximum after 4 hours. Meanwhile, the peak d’ coming from the 2-(diethylamino) ethanol separated from the intermediates also appeared in 1 hour and reached to maximum in 4 hours, indicating

the quaternary amine changed into tertiary amine in

4 hours and ester bond were rapidly hydrolyzed in 4 hours simultaneously. Combined the 1HNMR with DLS results, it can be found that under the stimulation of H2O2 13 ACS Paragon Plus Environment

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PAMAM-(B-DEAEP)16 successfully changed from positive charged to be zwitterinoic and finally to be negative charged in 6 hours. 3.3. Dendrimer binds and release DNA. It has been known that the size, potential, morphology and complexation efficacy of gene complex are significant to evaluate the materials as gene vectors. The size of the DNA/PAMAM-(B-DEAEP)16 complexes at different N/P ratios were detected by DLS. It can be found from Figure 3a that plasmid DNA could be condensed to around 100nm when N/P was above 5, which was suitable for endocytosis. TEM images showed that the complexes existed as spherical shapes and the diameter of complexes measured to be about 80nm (Figure 3c). Zeta potential of PAMAM-(B-DEAEP)16 after mixed with DNA ranged from

20

mV

to

30

mV

at

different

N/P

in

water,

indicating

that

PAMAM-(B-DEAEP)16 could combine with DNA by electrostatic interaction (Figure 3b). The zeta potential of DNA/PAMAM-(B-DEAEP)16 complex was measured to be positive when N/P ≥ 5, and it was found that it increased with the increasement of N/P ratio and could reach to be about 30 mV at N/P = 25, which indicated that PAMAM-(B-DEAEP)16 can complex DNA strongly due to its highly positive charges (Figure 3b). We then used agarose gel retardation assay to study the gene condensation and release abilities induced by H2O2. As shown in Figure 3d, it can be found that DNA could be completely condensed by PAMAM-(B-DEAEP)16 when the N/P

was

above

3,

which

demonstrated

the

high

binding

affinity

of

PAMAM-(B-DEAEP)16 with DNA. Then the gene release ability of PAMAM-(BDEAEP)16

was

also

investigated

after

H2O2

were

added. 14

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DNA/PAMAM-(B-DEAEP)16 complexes at N/P=10 were used as model and different concentrations of H2O2 were added into and incubated for 3 hours. It has been found that PAMAM-(B-DEAEP)16 completely condense gene before H2O2 were added. After 1mM H2O2 was added to the complexes, some of genes could be released and all the gene could be completely released when the concentration of H2O2 reached to be about 2mM (Figure 3e). Combined with the previous 1H-NMR and DLS results, it can be easily explained why the materials could response to H2O2, and release gene rapidly and efficiently. Because the positive charges in PAMAM-(B-DEAEP)16 endow it the ability to interact with DNA and the PAMAM segments make the whole complex enter into the tumor cells and escape from endosomes successfully. The B-DEAEP segments in the compound are ROS-sensitive, whose ester groups self-catalyzed hydrolyzed and induced the formation of negative charges carboxylic acid. During the process of hydrolysis, the compound consisted both the positive quaternary amine and negative carboxylic acid, which made the compound to be zwitterionic and could release the binding DNA molecules continuously with the decrease of positive charges and increase of negative charges until the final compound showed to be fully negative charged. It is during this process that the DNA was released completely and efficiently. 3.4. Cellular internalization and Gene transfection. Cellular internalization is an important key parameter for eventual transfection efficiency. In this study, complexes of PAMAM-(B-DEAEP)16 and Cy-5 DNA were prepared at N/P ratio of 15. After that, complexes were used to treat Hela cells for 30 min, 1 h and 4 h, 15 ACS Paragon Plus Environment

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respectively. The distribution of complexes in cells was characterized by confocal laser scanning microscopy. As shown in Figure 4, when incubation time reached to be 30min, light red signal could be observed in cytoplasm, which indicated that some PAMAM-(B-DEAEP)16/Cy-5 DNA complexes have transported into Hela cells. After 1h, strong red signal could be observed in cytoplasm and more complexes have transported into cells. The high cellular uptake efficiency should be ascribed to positive charges carried by PAMAM-(B-DEAEP)16 since it could interact with negative charged cell membrane by electrostatic interactions. At the same time, a large amount of protonated amino groups in PAMAM-(B-DEAEP)16 could help complexes escape from endosomes via “proton sponge effect”. It was clearly showed that the uptake of Cy-5 DNA into cells was significantly promoted when incubation time reached 4 h and quite a lot Cy-5 DNA could be found in the nuclei, which indicated that Cy-5 DNA could be released intracellular due to the charge-reversal property of PAMAM-(B-DEAEP)16 under elevated reactive oxygen species(H2O2). These results proved that after transporting into cell, PAMAM-(B-DEAEP)16 help DNA escape from endosomes and then release binding DNA successfully. The transfection efficiency of PAMAM-(B-DEAEP)16 was assessed by luciferase expression and green fluorescence protein (GFP) expression in Hela cells. Based on the results of DLS and gel electrophoresis assay, PAMAM-(B-DEAEP)16 and pGL3 plasmid DNA were condensed at N/P ratios of 3/1, 5/1, 10/1, 15/1, 20/1 and 25/1, respectively. The results showed that PAMAM-(B-DEAEP)16 exhibited great transfection efficiency which was 4.5 fold higher than commercial transfection regent 16 ACS Paragon Plus Environment

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PEI25K at best performed N/P ratio of 20 (Figure 5a). The same results were further verified by GFP expression assay. We chose the complexes at N/P ratios of 5/1, 10/1, 15/1, 20/1 and 25/1 as the models, and PEI25K was still conducted as positive control. As shown in Figure 5c, cells incubated with PAMAM-(B-DEAEP)16/DNA complexes exhibited more bright green fluorescence than cells treated by PEI25k did. Therefore, the higher transfection efficiency of PAMAM-(B-DEAEP)16 may come from the strong gene condensing ability, enhanced cellular internalization and effective gene release capability. 3.5. Cell viability assay. Bio-safety is the prerequisite for gene carrier materials to be furthered applied.37 The Hela cells were incubated with different concentration of PAMAM-(B-DEAEP)16 for 48 hours and then the cell cytotoxicity was determined by Celltiter-Blue cell viability assay. As shown in Figure 5b, the viability of Hela cells treated by PAMAM-(B-DEAEP)16 from 10 to 100 μg/mL all remained higher than 90% based on the control. The low cell cytotoxicity may be ascribed to the charge transition of PAMAM-(B-DEAEP)16 from positive to negative accounting for the quite decrease of positive charges. This result indicated that the materials we synthesized own low cell cytotoxicity and good biocompatibility which ensured feasibility for further application. 4.

CONCLUSIONS In this study, a ROS-responsive dendrimer PAMAM-(B-DEAEP)16 was

fabricated to be used as gene vector. The results showed that it can bind with DNA 17 ACS Paragon Plus Environment

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molecules and be internalized by the cells due to the electrostatic interactions. The most interesting thing is that under the stimulation of reactive oxygen species (H2O2) intracellular, PAMAM-(B-DEAEP)16 can vary from positively charged to zwitterionic and finally to be negatively charged accompanying the gene effectively packed with dendrimer and eventually release intracellular. Among the previous studies of zwitterionic materials for gene delivery, most researches have been trying to get a compromise between the high gene transfection efficiency and low cytotoxicity since the cationic groups gave ease to binding to DNA while also brought high toxicity at the same time. Different from those, the gene vectors we designed here are ROS-sensitive and it will change its charge properties according to the microenvironment it encountered. Therefore, the DNA can be efficiently carried and released while the gene vectors showed good biocompatibility simultaneously. Our results strongly demonstrated that this stimuli-responsive PAMAM-(B-DEAEP)16 have highly gene transfection efficiency and low cell cytotoxicity compared with the traditional PAMAM dendrimer gene vectors. It is believed that the method we provided here is instructive and promising on the road of exploring gene therapy.

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Scheme 1. The schematic illustration of ROS-responsive charge-reversal process of PAMAM-(B-DEAEP)16.

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Figure 1. The H2O2-response process of PAMAM-(B-DEAEP)16: (a) The H2O2-triggered chemical structure changes of PAMAM-(B-DEAEP)16; (b) The 1H-NMR

trace of PAMAM-(B-DEAEP)16 with a concentration of 80mM H2O2 in

D2O at different times.

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Figure 2. The dynamic changes of zeta potential in a solution of 3mg/mL PAMAM-(B-DEAEP)16 with 80 mM H2O2 in PBS ( pH=7.4) at room temperature.

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Figure

3.

The

diameter

(a)

and

zeta

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potential

(b)

of

complexes

of

DNA(shEGFR)/PAMAM-(B-DEAEP)16 at different N/P ratios in water; (c) The TEM image of DNA(shEGFR)/PAMAM-(B-DEAEP)16 complexes at N/P ratio of 15; (d) Agarose

gel

retardation

assay

of

binding

affinity

of

DNA(shEGFR)/PAMAM-(B-DEAEP)16 complexes at different N/P ratios; (e) Agarose

gel

retardation

assay

of

DNA

release

from

DNA(shEGFR)/PAMAM-(B-DEAEP)16 complexes (N/P=10) at different H2O2 concentrations.

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Figure 4. Confocal laser scanning microscopy images of Hela cells cultured with PAMAM-(B-DEAEP)16/Cy-5 DNA complexes in serum-free medium for 30min, 1h and 4h, respectively. Cy5-DNA is shown in red, and the cell nucleus stained with DAPI are in blue. All scale bars are 20µm.

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Figure 5. (a) Luciferase gene transfection of DNA(pGL3)/PAMAM-(B-DEAEP)16 at different N/P ratios in Hela cells, PEI25K/DNA (N/P=10) as the control; (b) The cell viability

of

Hela

PAMAM-(B-DEAEP)16;

cells (c)

incubated The

with GFP

various

concentrations

transfection

images

of of 24

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DNA(pGL3)/PAMAM-(B-DEAEP)16 at different N/P ratios, PEI25K/DNA (N/P=10) as the control.

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ASSOCIATED CONTENT Supporting Information. The 1H-NMR spectroscopies of PAMAM G0.5-G3.0, PAMAM-(DEAEP)16 and PAMAM-(B-DEAEP)16, as well as the zeta potential of PAMAM-(B-DEAEP)16 at different concentrations in water were listed in the supporting information. This information is available free of charge at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (Mr. Sanrong Liu); [email protected] (Prof. Dr. Xifei Yu) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The financial support from the Nation Natural Science Foundation of China (21674109 and 21603214) is acknowledged.

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Table of content

ROS-response Induced Zwitterionic Dendrimer for Gene Delivery

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