Low Molecular Weight Oligomers with Aromatic Backbone as Efficient

Apr 14, 2016 - First, oligomers containing aromatic rings in the backbone showed ... novel nonviral gene vectors with high efficiency and biocompatibi...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIV LAVAL

Article

Low Molecular Weight Oligomers with Aromatic Backbone as Efficient Non-viral Gene Vectors Chao-Ran Luan, Yan-Hong Liu, Ji Zhang, Qing-Ying Yu, Zheng Huang, Bing Wang, and Xiaoqi Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01561 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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 Applied Materials & Interfaces

Low Molecular Weight Oligomers with Aromatic Backbone as Efficient Non-viral Gene Vectors Chao-Ran Luan, Yan-Hong Liu, Ji Zhang,* Qing-Ying Yu, Zheng Huang, Bing Wang and Xiao-Qi Yu* Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, PR China

Abstract: A series of oligomers were synthesized via ring-opening polymerization. Although the molecular weights of these oligomers are only ~2.5 KDa, they could efficiently bind and condense DNA into nanoparticles. These oligomers gave comparable transfection efficiency (TE) to PEI 25 KDa, while their TE could even increase with the presence of serum, and up to 65 times higher TE than PEI was obtained. The excellent serum tolerance was also confirmed by TEM, Flow cytometry and BSA adsorption assay. Moreover, structure-activity relationship studies revealed some interesting factors. Firstly, oligomers containing aromatic rings in the backbone showed better DNA binding ability. These materials could bring more DNA cargo into the cells, leading to much better TE. Secondly, the isomerism of the disubstituted phenyl group on the oligomer backbone has large effect on the transfection. The ortho-disubstituted ones gave at least one order of magnitude higher TE than meta- or para-disubstituted oligomers. Gel electrophoresis involving DNase and heparin indicated that the difficulty to release DNA might contribute to the lower TE of the latter. Such clues may help us to design novel non-viral gene vectors with high efficiency and biocompatibility. Keywords: Non-viral gene vector, cationic oligomer, aromatic backbone, structure-activity 1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

relationship, serum tolerance

1. INTRODUCTION As a promising therapeutic modality in modern medicine, gene therapy is able to cure diseases through modifying genes, providing new and revolutionary treatments.1, 2 This therapy involves the delivery of a specific gene segment into target cells to cure disease at original level.3, 4 Due to the confined ability of naked DNA to get into cells for expression, studies about suitable vectors for foreign gene show extraordinary importance in gene therapy.5, 6 Although some viral vectors showed remarkable transfection efficiency (TE), many issues such as their immunogenicity, toxicity and oncogenic effects limited their clinical applications.7, 8 As safer alternatives, non-viral gene vectors have received increasing attentions for their easy preparation and modification.9,

10

However, the weak TE of non-viral vectors is the main

barrier lied on their road to clinical applications. To solve this problem, plenty of non-viral systems including cationic liposomes,11 polymers,12 peptides13 and inorganic nanoparticles14 etc. have been developed to improve their TE together with the biocompatibility. Cationic polymers are widely noticed for their remarkable advantages such as good stability and easy preparation.15,

16

Besides, the structures of polymeric materials are easy to be

modified, affording them special properties such as targeting delivery, controlled-release, optimized endocytosis, or excellent biocompatibility.17-19 Previous studies indicated that TE of polymers is related to many aspects including molecular weight, stereochemical structure, charge density, etc.20-22 For instance, the TE of polyethyleneimine (PEI), a commercially available polymeric vector, is related to the degree of polymerization and structural topology.23, 2

ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27

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 Applied Materials & Interfaces

24

High molecular weight PEI displays well TE, but the cytotoxicity is obviously high.25

Decrease of molecular weight might reduce the cytotoxicity, but always results in much lower TE, which could be attributed to inefficient DNA condensation and low cellular uptake.26 So far, polymers with low molecular weights were seldom reported to have comparable TE to 25 KDa PEI, which has been regarded as “golden standard” for polymeric gene vectors.27-30 Our group recently discovered that polymers with rigid aromatic structure in the backbone showed highly increased TE.31 The aromatic rings may largely enhance the DNA condensation ability of cationic polymers, especially those with low molecular weights. On the other hand, we also found that diepoxide ring-opening polymerization is a practical strategy to synthesize polymeric vectors with improved TE and serum tolerance.32-34 The newly formed hydroxyls and oxygen atoms in the backbone may play the roles similar to PEG. Herein, we designed and prepared a series of oligomers with isomeric aromatic linkers in the backbone, and analogs with proper structures were also prepared for comparison. These materials were synthesized through ring-opening polymerization of diepoxides with polyamine. Results reveal that aromatic structure and its isomerism have large influences on the gene transfection at the processes of DNA binding and release, cell endocytosis, and intracellular delivery. The structure-activity relationship studies revealed some interesting factors and may give us clues for the design and synthesis of novel non-viral gene vectors with high efficiency and biocompatibility.

2. EXPERIMENTAL SECTION 2.1. Materials. Unless otherwise stated, all chemicals used in experiments were obtained 3

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 4 of 27

through commercial approach and used without additional purification. Ethanol and epichlorohydrin were dried with proper desiccants and purified under nitrogen and distilled immediately before use. The diepoxide linkage compounds L1-L4 were prepared according to literature

processes.35,

36

25

KDa

branched

PEI

and

MTS

5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

3-(4, were

bought from Sigma-Aldrich (St. Louis, MO, USA). The Dulbecco’s Modified Eagle’s Medium (DMEM) as well as fetal bovine serum was obtained from Invitrogen Corp. The plasmids used herein are pEGFP-N1 (Clontech, Palo Alto, CA, USA) coding for EGFP DNA and pGL-3 (Promega, Madison, WI, USA) coding for luciferase DNA. Luciferase assay kit was bought from Promega (Madison, WI, USA). MicroBCA protein assay kit was obtained from Pierce (Rockford, IL, USA). Cy5TM was purchased from Molecular Probe (Mirus, Madison, WI, USA). Endotoxin free plasmid purification kit was acquired from TIANGEN (Beijing, China). HEK 293 human embryonic kidney cells, A549 human lung cancer cells, HeLa human cervix carcinoma cells, HepG2 human hepatoma cells and HL-7702 human lung cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. If not specified, all other reagents used in the chemical synthesis were obtained from Sigma-Aldrich Co, and used without additional purification. Some experiments, including agarose gel retardation assay, ethidium bromide (EB) exclusion essay, dynamic light scattering (DLS) assay for the measurements of particle size and zeta potential, transmission electron microscopy (TEM), cell culture, in vitro transfection experiments, flow cytometry assay and cytotoxicity assay, were carried out following previously reported procedures except using various cell lines.31-33 Instead of MTT, MTS was 4

ACS Paragon Plus Environment

Page 5 of 27

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 Applied Materials & Interfaces

used in the cytotoxicity assay. 2.2. Synthesis of oligmers. Table 1 lists the chemical names and reaction solvents for preparation of the 12 newly formed oligomers. Diglycidyl ether L1-L4 (1 mmol) was dissolved in the solvent. Then polyamine triethylenetetramine (PA1, TETA) or tetraethylenepentamine (PA2, TEPA) or tri(β-aminoethyl)amine (PA3, TAEA) (1 mmol) was added. Under N2 condition, the reaction mixture was stirred at 80°C for 72 h. After cooling down, the reaction mixture was stirred overnight with the addition of 50 mL of saturated CH3OH/HCl solution at room temperature. After vacuum distillation, the solid was dissolved in 5 mL of deionized water and then purified by dialysis (MWCO 1000 Da) against deionized water for 72 h. The product was obtained after lyophilization. Table 1. Reaction condition of polymers (The detailed structures for the compounds see Scheme 1). Oligomers

Diglycidyl ether

Polyamine

Solvent

Ph-p-TETA Ph-m-TETA Ph-o-TETA Bu-TETA Ph-p-TEPA Ph-m-TEPA Ph-o-TEPA Bu-TEPA Ph-p-TAEA Ph-m-TAEA Ph-o-TAEA Bu-TAEA

L1 L2 L3 L4 L1 L2 L3 L4 L1 L2 L3 L4

PA1 (TETA) PA1 (TETA) PA1 (TETA) PA1 (TETA) PA2 (TEPA) PA2 (TEPA) PA2 (TEPA) PA2 (TEPA) PA3 (TAEA) PA3 (TAEA) PA3 (TAEA) PA3 (TAEA)

DMF (3 mL) DMF (2 mL) C2H5OH (2 mL) DMF (3 mL) DMF (3 mL) DMF (2 mL) C2H5OH (2 mL) DMF (3 mL) DMF (3 mL) DMF (2 mL) C2H5OH (2 mL) DMF (3 mL)

Ph-p-TETA: Yield 17%. 1H NMR (400 MHz, D2O, TMS): δ = 8.35-6.26 (m, 4H, PhH), 4.22-3.59 (m, 3H, -O-CH2-), 3.55-3.11 (m, 3H, -CH-), 3.11-2.82 (m, 2H, -CH2-), 2.82-2.12 (m, 3H, -NH-CH2-). MALDI-TOF-MS: Mw = 2496 Da. Ph-m-TETA: Yield 23%.1H NMR (400 MHz, D2O, TMS): δ = 8.07-6.08 (m, PhH), 4.23-3.52 5

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

(m, 3H, -O-CH2-), 3.52-3 (m, 3H, -CH-), 3-2.72 (m, 2H, -CH2-), 2.72-2 (m, 3H, -NH-CH2-). MALDI-TOF-MS: Mw = 2495 Da. Ph-o-TETA: Yield 33%. 1H NMR (400 MHz, D2O, TMS): δ = 8.17-6.20 (m, PhH), 4.36-3.53 (m, 3H, -O-CH2-), 3.25-2.83 (m, 3H, -CH-), 2.83-2.24 (m, 4H, -CH2-, -NH-CH2-). MALDI-TOF-MS: Mw = 2111 Da. Bu-TETA: Yield 28%. 1H NMR (400 MHz, D2O, TMS): δ = 4.08-3.96 (m, 1H, -O-CH2-), 3.74-3.26 (m, 10H, -CH2-CH2-, -CH-), 3.26-2.91 (m, 5H, HO-CH-CH-N-), 2.91-2.7 (m, 2H, -N-CH2-), 1.72-1.30 (m, -CH2-). MALDI-TOF-MS: Mw = 2500 Da. Ph-p-TEPA: Yield 24%. 1H NMR (400 MHz, D2O, TMS) δ = 8.07-6.52 (m, PhH), 4.35-3.64 (m, 4H, -O-CH2-), 3.36-2.92 (m, 3H, -CH-), 2.92-2.3 (m, 4H, -CH2-, -NH-CH2-). MALDI-TOF-MS: Mw = 2499 Da. Ph-m-TEPA: Yield 24%. 1H NMR (400 MHz, D2O, TMS): δ = 8.31-6.22 (m, PhH), 4.33-3.68 (m, 3H, -O-CH2-), 3.68-3.29 (m, 2H, -CH-), 3.29-2.92 (m, 4H, -CH2-), 2.92-2.3 (m, 4H, -NH-CH2-). MALDI-TOF-MS: Mw = 2741 Da. Ph-o-TEPA: Yield 28%. 1H NMR (400 MHz, D2O, TMS): δ = 7.40-6.45 (m, PhH), 4.41-3.63 (m, 5H, -O-CH2-), 3.29-2.91 (m, 5H, -CH-), 2.9-2.32 (m, 4H, -CH2-, -NH-CH2-). MALDI-TOF-MS: Mw = 2964 Da. Bu-TEPA: Yield 15%. 1H NMR (400 MHz, D2O, TMS): δ = 4.06-3.92 (m, 1H, -O-CH2-), 3.73-3.26 (m, 13H, -CH2-CH2-, -CH-), 3.26-2.89 (m, 5H, HO-CH-CH-N-), 2.89-2.62 (m, 2H, -N-CH2-), 1.65-1.30 (m, -CH2-). MALDI-TOF-MS: Mw = 2517 Da. Ph-p-TAEA: Yield 27%. 1H NMR (400 MHz, D2O, TMS): δ = 8.42-6.40(m, PhH), 4.37-3.64 (m, 3H, -O-CH2-), 3.32-3.06 (m, 3H, -CH-), 3.06-2.92 (m, 1H, -CH2-), 2.89-2.62 (m, 2H, 6

ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27

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 Applied Materials & Interfaces

-NH-CH2-). MALDI-TOF-MS: Mw = 2112 Da. Ph-m-TAEA: Yield 30%. 1H NMR (400 MHz, D2O, TMS): δ = 7.26-6.12 (m, PhH), 4.33-3.64 (m, 5H, -O-CH2-), 3.3-3.05 (m, 1H, -CH-), 3.05-2.92 (m, 1H, -CH2-), 2.92-2.39 (m, 4H, -NH-CH2-). MALDI-TOF-MS: Mw = 2800 Da. Ph-o-TAEA: Yield 22%. 1H NMR (400 MHz, D2O, TMS): δ = 7.10-6.47 (m, PhH), 4.34-3.55 (m, 6H, -O-CH2-), 3.37-2.84 (m, 6H, -CH-), 2.84-2.24 (m, 6H, -CH2-, -NH-CH2-). MALDI-TOF-MS: Mw = 2653 Da. Bu-TAEA: Yield 19%. 1H NMR (400 MHz, D2O, TMS): δ = 4.17-3.89 (m, 2H, -O-CH2-), 3.72-3.25 (m, 11H, -CH2-CH2-, -CH-), 3.25-2.95 (m, 3H, HO-CH-CH-N-), 2.95-2.66 (m, 2H, -N-CH2-), 1.72-1.30 (m, -CH2-). MALDI-TOF-MS: Mw = 1586 Da. 2.3. Circular dichrosim. The circular dichrosim of DNA was testified by Chirascan (Applied Photophysics Ltd). Calf-thymus (CT) DNA (1 mg/mL, 100 µL) was added into purity water (2 mL). After shaking, the circular dichrosim was measured. Then the solution of polymers (1 mg/mL, 20 µL) was added each time and the circular dichrosim was measured. Data were based on three times of accumulation. 2.4. Protein adsorption assay. In brief, 0.2 mL of polymer solution (1 mg/mL) and 0.2 mL of bovine serum albumin (BSA) solution (1 mg/mL) were mixed up and stirred for 15 min. Then 0.6 mL of water was added. After that, the solution was measured utilizing DLS. Data were shown as mean ± standard deviation (SD) based on three independent measurements. 2.5. Confocal laser scanning microscopy (CLSM). HeLa cells were seeded in 35 mm confocal dish (Φ = 15 mm) at a density of 2.5 × 105 cells per well. After 24 h, the medium was replaced with serum-free medium. Then complexes of polymers and Cy5-labeled gene at 7

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

optimized mass ratios (0.8 µg DNA per well) were added. After incubation with 4, 8, and 24 h, the cells were washed with PBS (pH 7.4) and labeled with lysotracker for 30 min at 37°C, and then fixed with 4% paraformaldehyde for 10 min. The intracellular fluorescent signal was detected using confocal laser scanning microscope (CLSM, ZEISS, LSM 780) at excitation wavelengths of 504 nm for lysotracker (green), 633 nm for Cy5 (red), respectively.

3. RESULTS AND DISCUSSION 3.1. Synthesis of target oligomers. The target oligomers were prepared via ring-opening polymerization between diglycidyl ethers L1-L4 as linking moiety and polyamine molecules PA1-PA3 as cationic moiety. As shown in Scheme 1, aromatic rings were introduced into the oligomers by using diepoxides L1-L3, which contain phenyl group with different disubstitution positions (o-, m- and p-). L4 without phenyl group was used for comparison. The chemical names of the 12 newly formed oligomers are listed in Table 1, in which Ph-o-TAEA represents the oligomer formed from o-disubstituted phenyl contained diglycidyl ether (L3) and tri(β-aminoethyl)amine (PA3, TAEA). It’s worth mentioning that during the polymerization, a gel-like material was liable to form. To avoid that, the reaction condition should be controlled carefully. Since the ring-opening polymerization of L1, L2 and L4 in ethanol, which was generally used for such reaction, might lead to gel formation, we performed their polymerization in DMF (Table 1). MALDI-TOF-MS showed that except Bu-TAEA, all oligomers have similar molecular weights of ~2500 Da. Since it’s was well known that polymeric materials with higher molecular weight always show higher cytotoxicity,37, 38 we hope our materials would have lower toxicity while maintain high transfection efficiency (TE). 8

ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27

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 Applied Materials & Interfaces

Scheme 1. Synthetic routes of target oligomers.

3.2. Interaction with plasmid DNA and characterization of the polyplexes. The electrostatic interaction between cationic polymer and negatively charged DNA would avoid the damage of DNA from DNase and negative proteins, also shielding the electrostatic repulsion of cell membrane toward DNA.39 Appropriate DNA binding and condensation ability is a prerequisite for cationic gene vector. Agarose gel retardation assays were carried out to evaluate the condensation ability of the oligomers, and results are shown in Figure 1. All the 12 materials could bind and retard DNA migration completely at oligomer/DNA weight ratio (w/w) of 0.5-2. Each row represents the four oligomers formed from the four different diglycidyl ethers L1-L4 with same polyamine. It could be observed that oligomers with non-aromatic linkage (L4, on the right) showed weaker binding ability, which is consistent with our previous results.31 On the other hand, oligomers formed from TAEA, which has branched polyamine structure, have stronger binding ability than others, and full retardation could be observed at w/w of 0.5-1 (last row). Meanwhile, ethidium bromide (EB) exclusion by fluorescent assay was also processed to further estimate their binding ability.40 Results in 9

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Figure S1 show that all the materials succeeded in quenching the fluorescence intensity of bound EB with the rise of w/w ratio, indicating their good binding ability. Similar to the gel retardation results, oligomers with aromatic groups showed higher fluorescent quenching ability, suggesting their stronger binding with DNA.

Figure 1. Electrophoretic gel retardation assays of polymer/DNA (pUC19) complexes at different w/w ratios.

The better DNA binding ability of aromatic ring-contained oligomers was further confirmed by circular dichroism (CD) analysis. After incubation with oligomers containing aromatic rings, CT DNA gave obviously lower CD signal (Figure 2A and 2B), while the oligomer without aromatic group did not lead to any signal change (Figure 2C). We speculate that the aromatic groups, which may intercalate into the DNA base pairs, contributed to the change of CD signal.41 Such intercalation might lead to better DNA binding and condensation ability of the cationic materials.

10

ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27

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 Applied Materials & Interfaces

Figure 2. Circular dichroism of CT DNA combined with Ph-p-TAEA (A), Ph-o-TAEA (B) and Bu-TAEA (C) at different weight ratios.

Proper particle size and positive surface charge are important in gene delivery. It was reported that complexes within the size range of 50 to several hundred nanometers would receive easier endocytosis.42 DLS measurements showed that the particle sizes of the oligomer/DNA complexes changed dramatically at relatively low w/w ratios (1-2), and became stable at 150-300 nm with the increase of w/w (Figure 3A). The large particles formed at the w/w ratio of 1-2 might be attributed to their neutral zeta-potential, which led to the minimized repulsion and resulting aggregation. Zeta potentials shown in Figure 3B also indicate the charge reversion occurred at w/w of ~1. With the increase of w/w, the positive charge, which was considered to increase their interaction with cell membrane, reached a plateau of +20-40 mV for most of the polyplexes.

Figure 3. Mean particle sizes (A) and zeta-potentials (B) of the polyplexes formed from oligomers and pUC19 DNA at various w/w ratios (DLS at room temperature). Data represent mean ± SD (n = 3). 11

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

To directly visualize the size and morphology of polyplexes, transmission electron microscopy (TEM) was used for the study, and the images are shown in Figure 4. Polyplexes formed from Ph-p-TAEA and Ph-o-TAEA at w/w of 2 appeared as spherical particles with diameters less than 100 nm, which was suitable for endocytosis. Ph-o-TAEA could form smaller polyplex nanoparticles than the other, suggesting that the ortho-disubstitution on the aromatic ring might favor DNA condensation. In addition, the morphology of the polyplexes was hardly affected by serum, and both of the polyplexes kept their sizes below 100 nm in the presence of 10% serum. The results further demonstrate the serum tolerance of the polymers formed by ring-opening polymerization of diepoxides. The discrepancy of the size results between DLS and TEM might be caused by different experimental conditions: DLS examined the particles in aqueous solution, while TEM revealed the morphology of the particles in dehydrated state.43

Figure 4. TEM images of Ph-p-TAEA/DNA (A, C) and Ph-o-TAEA/DNA (B, D) polyplexes at w/w of 2 in deionized water in the absence (A, B) and presence (B, D) of 10% serum.

3.3. In vitro transfection. Luciferase reporter gene was first used to quantitatively evaluate 12

ACS Paragon Plus Environment

Page 12 of 27

Page 13 of 27

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 Applied Materials & Interfaces

the in vitro TE of the oligomers in HeLa cells, and PEI 25 KDa was used as control at its optimal transfection w/w ratio which was admitted as 1.4 (N/P = 10).29, 30 As shown in Figure 5A, except Ph-p-TETA, all oligomers with aromatic backbone gave higher TE than their non-aromatic counterparts (Bu-PAs), further indicating the advantage of aromatic groups in the main chain of cationic polymers.31 Moreover, the isomerism of the disubstituted phenyl group has large effect on the TE. For all three groups of oligomers (divided by polyamine), the ortho-disubstituted ones gave at least one order of magnitude higher TE than meta- or para-disubstituted oligomers. It was well known that for polymeric vectors, higher molecular weight always leads to higher TE.44 Although the molecular weights of these oligomers are only one tenth of PEI 25 KDa, they gave comparable TEs to PEI. To our knowledge, polymeric vectors with such low molecular weight were seldom reported to have comparable TE to 25 KDa PEI.27, 28 We speculate that the good binding and condensation ability contributed by the aromatic groups make the polyplexes more stable and lead to better transfection. This was also illustrated by the delivery in the presence of serum. Since serum components, especially the negatively charged proteins, would interact with the cationic vectors and largely affect the stability of the polyplex, the TE of PEI dramatically decreases in serum-contained medium.45, 46 However, results in Figure 5B show that our oligomers maintained good TE in the presence of serum, Ph-o-TEPA and Ph-o-TAEA gave 65 and 58 times higher TE than PEI, further indicating that these polyplexes are quite stable and are seldom affected by the serum. Besides the aromatic group, the hydroxyls formed from ring-opening polymerization would also contribute to the excellent serum tolerance.33, 34

13

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Figure 5. Luciferase expression transfected by polyplexes at different weight ratios in comparison with 25 KDa bPEI (w/w = 1.4, N/P = 10) in HeLa cells. Data represent mean ± SD (n = 3). (A) Without serum; (B) With serum (10%).

Subsequently, Ph-p-TAEA, Ph-o-TAEA and Bu-TAEA were chosen to further study the transfection in different cell lines and the results are shown in Figure 6. Similar to the results in HeLa cells, these oligomers have much higher serum tolerance than PEI, and the TE in the presence of serum was even higher than that in the serum free transfection. The oligomer including ortho-disubstituted phenyl groups (Ph-o-TAEA) also gave obviously higher TE than the para-disubstituted and non-aromatic counterparts. Moreover, to directly visualize the transfected cells, green fluorescent protein (eGFP) reporter gene was used for the transfection by Ph-p(m,o)-TAEA and PEI. The images shown in Figure 7 indicate the same structure-activity relationship (Bu-TAEA induced almost no green fluorescent signal, data not shown), and unlike PEI, 10% of serum has even positive effect on the TE of the oligomers. For the transfection superiority of oligomers containing ortho-disubstituted phenyl groups, gel electrophoresis involving DNase and heparin may give some clues (Figure S2). Heparin was added to the polyplexes formed from the four oligomers derived from TAEA, and among them, Ph-p-TAEA and Ph-m-TAEA were found unable to release DNA. Since the balance between DNA binding and release is essential for efficient delivery, the lack of DNA release may explain the lower TE of such two oligomers.47 For Bu-TAEA, although heparin-induced DNA 14

ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27

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 Applied Materials & Interfaces

release could also be observed, it can’t protect DNA from degradation by DNase.

Figure 6. Luciferase gene expression transfected by selected polyplexes at different weight ratios in comparison with 25 KDa bPEI (w/w = 1.4) in A549 and HepG2 cells in the absence (A) and presence (B) of serum. Data represent mean ± SD (n = 3).

Figure 7. Fluorescent microscope images of pEGFP-transfected HeLa cells. Ph-p-TAEA, Ph-o-TAEA and Bu-TAEA were at w/w of 4, while PEI was at w/w of 1.4.

3.4. Cellular uptake and intracellular distribution. Besides the DNA condensation and release, the TE of vectors is also related to several factors such as endocytosis, cytoplasm delivery and nuclear entry.48 To clarify the good TE, especially the excellent serum tolerance of the oligomeric materials, flow cytometry was first used to measure the internalization of polyplexes. Ph-p-TAEA, Ph-o-TAEA and Bu-TAEA were chosen again as typical materials. As shown in Figure 8A, all polyplexes including PEI/DNA exhibited high cellular uptake, and more than 80% of cells were positive to Cy5-labelled DNA. However, the fluorescence 15

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

intensity of labelled cells gave an obvious order of Ph-p-TAEA ≈ PEI > Ph-o-TAEA > Bu-TAEA. Such results demonstrate that aromatic group would definitely favor cellular uptake. On the other hand, although Ph-p-TAEA has better cellular uptake, its TE was lower than Ph-o-TAEA, indicating that the former has much lower intracellular gene delivery efficiency, which might largely due to the difficulty of DNA release (Figure S2). Subsequently, for its better TE, we used Ph-o-TAEA to study the influence of serum in cellular uptake. Figure 8B shows that with the increase of serum concentration, the uptake of PEI/DNA polyplexes was severely inhibited. On the contrary, the negative effect on the Ph-o-TAEA mediated cellular uptake was much slighter, especially for the fluorescence intensity. For PEI, compared to the results without serum, 75% serum concentration led to 50% and 90% decrease of internalized cell percentage and fluorescence intensity, respectively. Meanwhile, such decreases were reduced to 25% and 28% by using Ph-o-TAEA with same change of serum condition. Results further demonstrate that polyplexes formed from the oligmers with aromatic group may keep stable from serum-induced aggregation or premature extracellular DNA release. This was also confirmed by bovine serum albumin (BSA) adsorption assay, in which the oligomers showed much less protein adsorption than PEI. In a visualized picture, the addition of serum made the PEI solution turbid, while other solutions remained clear (Figure S3).

16

ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27

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 Applied Materials & Interfaces

Figure 8. Cellular uptake (columns) and fluorescence intensity (dots) of polyplexes (w/w = 2) in comparison with 25 KDa bPEI (w/w = 1.4) without serum (A) or with different concentrations of serum (B) in HeLa cells quantified by flow cytometry analysis. Data represent mean ± SD (n = 3).

The intracellular gene delivery processes mediated by two typical oligomers Ph-o-TAEA and Bu-TAEA were tracked by CLSM (Figure 9.). DNA cargo and lysosome were labelled/stained with red and green fluorescent dyes, respectively. After 4 h incubation, most fluorescent signals were found to be yellow (the last image in each row), indicating that most internalized DNA located in late endosome, which has similar environment to lysosome. With the extension of incubation time, red signals for labeled DNA were observed in Ph-o-TAEA mediated transfection, suggesting the escape of DNA or DNA/oligomer polyplex from the endosome. However, on the contrary, in the transfection using Bu-TAEA, very few red signals was found, and yellow signals remained in the cells. Such results demonstrate that polyplexes formed from aromatic Ph-o-TAEA have better endosomal escape ability than those from non-aromatic vectors. Meanwhile, Ph-o-TAEA induced stronger red signals than Bu-TAEA (the first image in each row), also indicating its better cellular uptake.

17

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Figure 9. CLSM images of HeLa cells transfected with Cy5-labelled DNA by Ph-o-TAEA and Bu-TAEA (w/w = 2) at different time. For each row, from left to right: Cy5-labelled pDNA (red); lysosome stained by lysotracker Green (green); bright field; merged image.

3.5. Cytotoxicity. Cationic polymers with higher molecular weight usually have higher TE, but along with severe cytotoxicity. To avoid forming gel-like materials, we prepared the target compounds under controlled conditions. These oligomers have molecular weights around 2.5 KDa, and previous experiments exhibited that although their molecular weights are relatively low, they could well bind and protect DNA. We hope their small molecular weights may result 18

ACS Paragon Plus Environment

Page 18 of 27

Page 19 of 27

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 Applied Materials & Interfaces

in low toxicity. The cytotoxicities of the polyplexes were studied and compared with that of 25 KDa PEI by choosing five oligomers at various w/w ratios in three tumor (HeLa, A549 and HepG2) and one normal (HL-7702) cell lines via MTS reduction assay. As shown in Figure 10, under the optimal transfection w/w (2 for oligomers and 1.4 for PEI), the viability of the cells treated with oligomers was higher than those treated with PEI in most cases. However, under higher w/w ratios, oligomers may display evident cytotoxicity, which especially those with aromatic groups.

Figure 10. Cytotoxicity of polyplexes at different weight ratios toward HeLa (A), A549 (B), HepG2 (C), and HL-7702 (D) cells. Data represent mean ± SD (n = 3).

4. CONCLUSION A series of oligomers were synthesized via ring-opening polymerization. Although the molecular weights of these oligomers are only one tenth of PEI 25 KDa, they gave comparable 19

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

TEs to PEI. With the presence of serum, the TE of the oligomers even increased (up to 65 times higher TE than PEI). TEM, Flow cytometry and BSA adsorption assay also indicated their excellent serum tolerance and clinical potential. Moreover, structure-activity relationship studies revealed some interesting factors. Firstly, oligomers containing aromatic rings in the backbone showed better DNA binding ability, which was confirmed by gel electrophoresis, EB exclusion and CD analysis. Flow cytometry and CLSM experiments showed that these materials could bring more DNA cargo into the cells, and their polyplexes have better endosomal escape ability. Secondly, the isomerism of the disubstituted phenyl group on the oligomer backbone has large effect on the transfection. The ortho-disubstituted ones gave at least one order of magnitude higher TE than meta- or para-disubstituted oligomers. Gel electrophoresis involving DNase and heparin indicated that unlike the ortho- counterparts, polyplexes formed from meta- or para-disubstituted oligomers are difficult to release DNA, which may largely contribute to their lower TE. The low molecular weight oligomers containing aromatic backbone by ring-opening polymerization give us a new strategy to construct non-viral gene vectors with high efficiency and biocompatibility.



ASSOCIATED CONTENT Supporting Information Fluorescent quenching assay, electrophoretic gel electrophoresis involving DNase and

heparin, BSA adsorption assays, 1H NMR and

13

C spectra of diglycidyl ethers, 1H NMR

spectra of target polymers. These materials are available free of charge via the Internet at http://pubs.acs.org. 20

ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27

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 Applied Materials & Interfaces



AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (X.-Q. Yu); [email protected] (J. Zhang). Tel: (86) 28

85415886. Fax: (86) 28 85415886. Notes The authors declare no competing financial interest.



ACKNOWLEDGEMENTS This work was financially supported by the National Program on Key Basic Research

Project of China (973 Program, 2012CB720603) and the National Science Foundation of China (Nos. 21472131, 21232005).



REFERENCES

(1) Ghosh, P. S.; Kim, C. K.; Han, G.; Forbes, N. S.; Rotello, V. M. Efficient Gene Delivery Vectors by Tuning the Surface Charge Density of Amino Acid-Functionalized Gold Nanoparticles. ACS Nano 2008, 2, 2213–2218. (2) Ma, B.; Zhang, S.; Jiang, H.; Zhao, B.; Lv, H. Lipoplex Morphologies and their Influences on Transfection Efficiency in Gene Delivery. J. Controlled Release 2007, 123, 184–194. (3) Layek, B.; Haldar, M. K.; Sharma, G.; Lipp, L.; Mallik, S.; Singh, J. Hexanoic Acid and Polyethylene Glycol Double Grafted Amphiphilic Chitosan for Enhanced Gene Delivery: Influence of Hydrophobic and Hydrophilic Substitution Degree. Mol. Pharmaceutics 2014, 11, 982–994. 21

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

(4) Opalinska, J.B.; Gewirtz, A. M. Nucleic-Acid Therapeutics: Basic Principles and Recent Applications. Nat. Rev. Drug Discovery 2002, 1, 503–514. (5) Al-Dosari, MS.; Gao, X. Nonviral Gene Delivery: Principle, Limitations, and Recent Progress. AAPS J. 2009, 11, 671–681. (6) Kim, T.; Jiang, H.; Jere, D.; Park, I. K.; Cho, M. H.; Nah, J. W.; Choi, Y. J.; Akaike. T.; Cho, C. S. Chemical Modification of Chitosan as a Gene Carrier In Vitro and In Vivo. Prog. Polym. Sci. 2007, 32, 726–753. (7) Green, J. J.; Langer, R.; Anderson, D. G. A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery. Acc. Chem. Res. 2008, 41, 749–759. (8) McTaggart, S.; Al-Rubeai, M. Retroviral Vectors for Human Gene Delivery. Biotechnol. Adv. 2002, 20, 1–31. (9) Pan, S.; Cao, D.; Yi, W.; Huang, H.; Feng, M. A Biodegradable and Serum-Resistant Gene Delivery Carrier Composed of Polyamidoamine–Poly N,N′-di-(2-aminoethyl) Aminoethyl Glutamine Copolymer. Colloids Surf., B 2013, 104, 294–302. (10) Kim, T. I.; Lee, M.; Kim, S. W. A Guanidinylated Bioreducible Polymer with High Nuclear Localization Ability for Gene Delivery Systems. Biomaterials 2010, 31, 1798–1804. (11) Zhao, Y. N.; Zhang, S. B.; Zhang, Y.; Cui, S. H.; Chen, H. Y.; Zhi, D. F.; Zhen, Y. H.; Zhang, S. F.; Huang, L. Tri-Peptide Cationic Lipids for Gene Delivery. J. Mater. Chem. B 2015, 3, 119–126. (12) Liu, Y.; Lin, C.; Li, J. B.; Qu, Y.; Ren, J. In Vitro and In Vivo Gene Transfection Using Biodegradable and Low Cytotoxic Nanomicelles Based on Dendritic Block Copolymers. J. Mater. Chem. B 2014, 3, 688-699. (13) Bibian, M.; Mangelschots, J.; Gardiner, J.; Waddington, L.; Acevedo, M. M. D.; De Geest, B. G.; Van 22

ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27

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 Applied Materials & Interfaces

Mele, A.; Madder, R.; Hoogenboom, S.; Ballet, S. Rational Design of a Hexapeptide Hydrogelator for Controlled-Release Drug Delivery. J. Mater. Chem. B 2015, 3, 759-765. (14) Chen, J.; Chen, Q.; Gao, C. J.; Zhang, M. L.; Qin, B.; Qiu, H. D. A SiO2 NP-DNA/Silver Nanocluster Sandwich Structure-Enhanced Fluorescence Polarization Biosensor for Amplified Detection of Hepatitis B Virus DNA. J. Mater. Chem. B 2015, 3, 964-967. (15) Hunt, K. K.; Vorburger, S. A. Gene Therapy: Hurdles and Hopes for Cancer Treatment. Science 2002, 297, 415–416. (16) Niidome, T.; Huang, L. Gene Therapy Progress and Prospects: Nonviral Vectors. Gene Ther. 2002, 9, 1647–1652. (17) Jones, C. H.; Chen, C. K.; Ravikrishnan, A.; Rane, S.; Pfeifer, B. A. Mol. Pharmaceutics 2013, 10, 4082–4098. (18) Yang, J. P.; Zhang, Q.; Chang, H.; Cheng, Y. Y. Surface-Engineered Dendrimers in Gene Delivery. Chem. Rev. 2015, 115, 5274-5300. (19) Mohammadifara, E.; Kharat, A. N.; Adeli, M. Poly (Amidoamine) and Polyglycerol; their Linear, Dendritic and Linear-Dendritic Architectures as Anticancer Drug Delivery Systems. J. Mater. Chem. B 2015, 3, 3896-3921. (20) Lin, C.; Zhong, Z. Y.; Lok, M. C.; Jiang, X. Y.; Hennink, W. E.; Feijen, J.; Engbersen, J. F. J. Linear Poly(Amido Amine)s with Secondary and Tertiary Amino Groups and Variable Amounts of Disulfide Linkages: Synthesis and In Vitro Gene Transfer Properties. J. Controlled Release 2006, 116, 130–137. (21) 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, 1044−1052. (22) Deng, R.; Yue, Y. N.; Jin, F.; Chen, Y. C.; Kung, H. F.; Lin, M.; Wu, C. Revisit the Complexation of 23

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

PEI and DNA—How to Make Low Cytotoxic and Highly Efficient PEI Gene Transfection Non-Viral Vectors with a Controllable Chain Length and Structure. J. Controlled Release 2009, 140, 40-46. (23) Hu, Y. L.; Zhou, D.; Li, C. X.; Zhou, H.; Chen, J. T.; Zhang, Z. P.; Guo, T. Y. The Use of Isothermal Titration Calorimetry and Molecular Dynamics to Show Variability in DNA Transfection Performance. Acta Biomater. 2013, 9, 5003-5012. (24) Hu, C.; Peng, Q.; Chen, F.; Zhong, Z.; Zhuo, R. Low Molecular Weight Polyethylenimine Conjugated Gold Nanoparticles as Efficient Gene Vectors. Bioconjugate Chem. 2010, 21, 836-843. (25) Parhamifar, L.; Larsen, A. K.; Hunter, A. C.; Andresen, T. L.; Moghimi, S. M. Polycation Cytotoxicity: a Delicate Matter for Nucleic Acid Therapy—Focus on Polyethylenimine. Soft Matter 2010, 6, 4001-4009. (26) Tripathi, S. K.; Gupta, S.; Gupta, K. C.; Kumar, P. Efficient DNA and siRNA Delivery with Biodegradable Cationic Hyaluronic Acid Conjugates. RSC Adv. 2013, 3, 15687-15697. (27) Yin, H.; Zhao, F.; Zhang, D. H.; Li, J. Hyaluronic Acid Conjugated β-Cyclodextrin-Oligoethylenimine Star Polymer for CD44-Targeted Gene Delivery. Int. J. Pharmacol. 2015, 483, 169–179. (28) Song, Y. Y.; Lou, B.; Zhao, P.; Lin, C. Multifunctional Disulfide-Based Cationic Dextran Conjugates for Intravenous Gene Delivery Targeting Ovarian Cancer Cells. Mol. Pharmaceutics 2014, 11, 2250−2261. (29) Bonetta, L. The Inside Scoopdevaluating Gene Delivery Methods. Nat. Methods 2005, 2, 875-883. (30) Lungwitz, U.; Breunig, M.; Blunk, T.; Gopferich, A. Polyethylenimine-Based Non-Viral Gene Delivery Systems. Eur J. Pharmacokinet. Biopharm. 2005, 60, 247-266. (31) Yi, W. J.; Yu, X. C.; Wang, B.; Zhang, J.; Yu, Q. Y.; Zhou, X. D.; Yu, X. Q. TACN-Based Oligomers with Aromatic Backbones for Efficient Nucleic Acid Delivery. Chem Commun 2014, 50, 6454-6457. (32) Zhang, Q. F.; Yi, W. J.; Wang, B.; Zhang, J.; Ren, L.; Chen, Q. M.; Guo, L. D.; Yu, X. Q. Linear Polycations by Ring-Opening Polymerization as Non-Viral Gene Delivery Vectors. Biomaterials 2013, 34, 24

ACS Paragon Plus Environment

Page 24 of 27

Page 25 of 27

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 Applied Materials & Interfaces

5391-5401. (33) Zhang, Q. F.; Yu, Q. Y.; Geng, Y. Y.; Zhang, J.; Wu, W. X.; Wang, G.; Gu, Z. W.; Yu, X. Q. Ring-Opening Polymerization for Hyperbranched Polycationic Gene Delivery Vectors with Excellent Serum Tolerance. ACS Appl. Mater. Interfaces 2014, 24, 15733-15742. (34) Guo, Q.; Liu, Y. H.; Xun, M. M.; Zhang, J.; Huang, Z.; Zhou, X. D.; Yu, X. Q. Diol Glycidyl Ether-Bridged Low Molecular Weight PEI as Potential Gene Delivery Vehicles. J. Mater. Chem. B 2015, 3, 2660–2670. (35) Suh, J.; Paik, H. J.; Hwang, B. K. Ionization of Poly(ethylenimine) and Poly(allylamine) at Various pH′s. Bioorg. Chem. 1994, 22, 318–327. (36) Zaagsma, J.; Nauta, W. H. In Vitro β-AdrenergicB Locking, Antiarrhythmic, and Local Anesthetic Activities of a New Series of Aromatic Bis(2 -Hydroxy-3-Isopropylaminopropyl)Ethers. J. Med. Chem. 1974, 17, 507-513. (37) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Size Matters: Molecular Weight Affects the Efficiency of Poly(ethylenimine) as a Gene Delivery Vehicle. J. Biomed. Mater. Res. 1999, 45, 268–275. (38) Kunath, K.; Harpe, A. V.; Fischer, D.; Peterson, H.; Bickel, U.; Voigt, K.; Kissel, T. Low-Molecular-Weight Polyethylenimine as a Non-Viral Vector for DNA Delivery: Comparison of Physicochemical Properties, Transfection Efficiency and In Vivo Distribution with High-Molecular-Weight Polyethylenimine. J. Controlled Release 2003, 89, 113–125. (39) Farber, I. Y.; Domb, A. J. Cationic Polysaccharides for Gene Delivery. Mater. Sci. Eng., C 2007, 27, 595-598. (40) Wang, B.; Yi, W. J.; Zhang, J.; Zhang, Q. F.; Xun, M. M.; Yu, X. Q. TACN-Based Cationic Lipids with Amino Acid Backbone and Double Tails: Materials for Non-Viral Gene Delivery. Bioorg. Med. Chem. Lett. 25

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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

Page 26 of 27

2014, 24, 1771-1775. (41) Jia, H. Z.; Luo, X. H.; Cheng, H.; Yang, J.; Li, C.; Liu, C. W.; Feng, J.; Zhang, X. Z.; Zhou, R. X. Extraordinarily Enhanced Gene Transfection and Cellular Uptake by Aromatic Hydrophobicization to PEI25K. J. Mater. Chem. 2012, 22, 24092-24101. (42)

Liu,

Y.

M.;

Reineke,

T.

M.

Hydroxyl

Stereochemistry

and

Amine

Number

within

Poly(Glycoamidoamine)s Affect Intracellular DNA Delivery. J. Am. Chem. Soc. 2005, 127, 3004–3015. (43) Yi, W. .; Zhang, Q. F.; Zhang, J.; Liu, Q.; Ren, L. F.; Chen, Q. M.; Guo, L. D.; Yu, X. Q. Cyclen-Based Lipidic Oligomers as Potential Gene Delivery Vehicles. Acta Biomater. 2014, 10, 1412–1422. (44)

Sarkar,

K.;

Debnath,

M.;

Kundu,

P.

P.

Preparation

of

Low

Toxic

Fluorescent

Chitosan-Graft-Polyethyleneimine Copolymer for Gene Carrier. Carbohydr. Polym. 2013, 92, 2048-57. (45) Dai, F.; Liu, W. Enhanced Gene Transfection and Serum Stability of Polyplexes by PDMAEMA-Polysulfobetaine Diblock Copolymers. Biomaterials 2011, 32, 628–638. (46) He, Y.; Cheng, G.; Xie, L.;Nie, Y.; He, B.; Gu, Z. Polyethyleneimine/DNA Polyplexes with Reduction-Sensitive Hyaluronic Acid Derivatives Shielding for Targeted Gene Delivery. Biomaterials 2013, 34, 1235–1245. (47) Liu, G.; Xie, J.; Zhang, F.; Wang, Z.; Luo, K.; Zhu, L.; Quan, Q. M.; Niu, G. Lee, S.; Ai, H.; Chen, X. Y. N-Alkyl-PEI-Functionalized Iron Oxide Nanoclusters for Efficient siRNA Delivery. Small 2011, 7, 2742-2749. (48) Wang, X.; Shao, N.; Zhang, Q.; Cheng, Y. Mitochondrial Targeting Dendrimer Allows Efficient and Safe Gene Delivery. J. Mater. Chem. B 2014, 2, 2546-2553.

26

ACS Paragon Plus Environment

Page 27 of 27

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 Applied Materials & Interfaces

Table of content entry

27

ACS Paragon Plus Environment