Interactions in DNA CondensationââAn Important Factor for

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Interactions in DNA Condensation——An Important Factor for Improving the Efficacy of Gene Transfection Xuan Nie, Ze Zhang, Chang-Hui Wang, Yun-Shan Fan, Qing-Yong Meng, and Ye-Zi You Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00805 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 15, 2018

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Bioconjugate Chemistry

Interactions in DNA Condensation‒An Important Factor for Improving the Efficacy of Gene Transfection

Xuan Nie†, Ze Zhang†, Chang-Hui Wang‡*, Yun-Shan Fan§, Qing-Yong Meng#* and Ye-Zi You†* †Hefei

National Laboratory for Physical Sciences at the Microscale, CAS Key

Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, P. R. China. Email: [email protected]. ‡Department

of Cardiology, First Affiliated Hospital of Anhui Medical University,

Hefei 230026, P. R. China. Email: [email protected] §Clinical

Nutrition Department, First Affiliated Hospital of Anhui Medical University,

Hefei 230026, P. R. China. #Department

of Radiotherapy, First Affiliated Hospital of Anhui Medical University,

Hefei 230026, P. R. China. Email: [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

KEYWORDS: gene delivery, DNA condensation, cationic polymer. ABSTRACT: The rapid developments of gene therapy are benefit from the construction of efficient gene vectors, which help therapy genes efficiently overcome the barriers in the transport and transfection. Condensing DNA into nanoparticles is crucial role in gene transfection, and the electrostatic interactions of synthetic cationic liposomes and cationic polymers with DNA are generally used for condensing DNA. The recent researches have exhibited that the introduction of the hydrophobic interaction, hydrogen bonding, and coordinative interactions to the gene delivery vectors is also very important for DNA condensation, delivery and transfection. This review focuses on the four types of interactions in the condensed DNA nanoparticles, which could provide a new perspective for improving gene transfection efficacy.


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INTRODUCTION Gene therapy refers to the treatments which transfer genetic molecules into specific cells of a patient for the therapy of diseases.1 It has attracted more and more attention over the past three decades as a potential treatment for curing different diseases, like cystic fibrosis,2 parkinson’s disease,3 inherited retinal diseases,4 hemophilia,5 cardiovascular disease,6 HIV,7 alzheimer disease,8 papillomavirus.9 The first idea of gene therapy was proposed in 1968 by Lederberg, who thought that an attempt could be carried out to transform liver cells of male generations of hemophiliac ancestry by delivering carefully fractioned DNA with the normal hemophiliac gene.10 In the same year, he succeeded in the replication of DNA in a tube, which is viewed as an important milepost in the gene therapy. In 1990, the first gene therapy experiment on treating disease using recombinant DNA was carried out by Martin Cline.11 Since then, gene therapy has developed rapidly until the death of Jesse Gelsinger,12 who died due to the immune response in four days after he received gene therapy. After a period of downturn, gene therapy got another upsurge in the new century. In 2017, three drugs, Kymriah from Novartis Company, Yescarta from Kite Company, and Luxturna from Spark company have got the approval of Food and Drug Administration (FDA). In addition, there are many gene therapy clinical trials, such as SPK-9001, SB-FIX, SB913, BB305, GSK2696274, AVXS-101, AMT-061, EB-101.13 It is worth mentioning that the FDA approved a gene therapy named onpattro in 2018, which is based on nonviral vectors. Gene vectors include viral and non-viral vectors, which are key important for gene



transfection. Recombinant viral vectors are widely used in clinic because of their high delivery and transfection efficacy. However, the lack of large-scale production and potential immunogenicity of viruses hinders their further application.14 The use of cationic polymers can in principle circumvent these problems, and have become the most promising chemical vectors for cell transfection in biotechnology in the past few decades because of their flexibility in their synthesis15 and structural modifications16 for specific biomedical applications. Cationic polymer systems can deliver genes by forming polyplexes with negatively charged deoxyribonucleic acid, which can protect DNA from degradation and facilitates its cellular uptake and intracellular traffic into the nucleus. Up to now, more and more studies have demonstrated that the interactions in DNA condensation can make a great contribution to enhance the efficacy of gene delivery.14,

17-20.

At present, most of the existing vectors do not have strong DNA

binding ability, which results in unstable DNA complex in body fluid, leading to a low transfection efficiency. Therefore, more different interactions have been applied to the design of gene vectors to improve the interaction between vectors and DNA. This review focuses on condensing DNA into nanoparticles by electrostatic interactions, hydrogen bonding, hydrophobic interactions and coordinative interactions (Figure 1).


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Figure 1. The interactions in gene and vectors. Electrostatic Interaction Cationic polymer vectors are widely used in gene delivery, which can interact with phosphate group in DNA backbone through electrostatic interaction, these polymers included PEI,21-24 PAMAM,19, 25-26 PLL,27-29 chitosan, 30-32 etc. However, most of these polymers only can achieve high transfection efficacy using large excess of cationic polymers, which could lead to the higher cytotoxicity. The nitrogen atom has a large electronegativity, and the positive charge of the nitrogen atom is dispersed to the adjacent carbon atom (+0.3 e for each carbon).33 This dispersed positive charge is not strong enough to bind a negatively charged nucleic acid tightly, resulting in a weak binding between DNA and polymers. Due to the weak interaction between DNA and cationic polymers, it takes much excess of positive cationic polymer to fully condense DNA, resulting in the formation of polyplexes with high surface charge and high cytotoxicity. Increasing the affinity between cationic polymer and DNA without reducing the release of DNA after endocytosis can not only achieve effective gene



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delivery with less cationic polymer, but also increase the stability of the complex in the body fluid environment with increasing transfection.34-35 As mentioned above, because the positive charge based on high electronegativity nitrogen is not strong enough to bind DNA, the researchers have developed other cations with more positive changed cation like phosphorus, 33, 36-37 arsenic, 38 and sulfur. 39-40

These atoms, structurally a larger and less electronegative atom than nitrogen,

could form larger cations with different electron density distributions compared with ammonium cations. And the results of ab initio calculations of the charge distribution on the cationic atom and the surrounding carbons have shown that the nitrogen atom had a negative charge with a positive charge on the adjacent carbons while phosphorus and sulfur atoms had a positive charge with a negative charge on the adjacent carbons (as shown in Table 1). Table 1. Charge distribution in different cation compounds. Cation

-CH2

-CH3

Bu4N+

0.5

0.3

41

Bu4P+

+1.1

0.2

41

(ClEt)2S+Me

+0.67

0.02, 0.0002

0.133

Ref

42

Different charge densities influenced DNA binding affinity, and less diffuse positive charge should bind DNA more effectively. For example, the charge of phosphorus in tetrabutylphosphonium cation is +1.1 e, and the charge of adjacent carbon is –0.2 e.41 Under the similar conditions, cationic polymers of terabutylphosphonium can bind to


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DNA much tightly, which can completely condense DNA at +/– ratio of 2 while PEI can fully condense DNA at +/– ratio above 4.33 In the transfection experiment, phosphonium containing polymers have exhibited much higher transfection efficacy than ammonium containing polymer.33 The charge of sulfonium cation is +0.67 e, which can completely condense DNA at +/– ratio of 1. But sulfonium-containing polymers have the moderate transfection efficacy compared with PEI, which may be result from that this sulfonium containing polymer has difficulties in the release of the loaded DNA in cytoplasm due to the strong binding affinity between DNA and sulfonium containing polymers. Therefore, a perfect gene delivery vector can condense DNA very tightly but can efficiently release the loaded DNA after endocytosis.43 And hence, Shen et al.40 reported a new class of polysulfoniums that can degrade into small fragments without any charge in cytoplasm, which highly enhanced DNA release as the disappearance of electrostatic interaction between polymers and DNA (Figure 2). This system can quickly escape from endosome due to the oxidation of the boronic acid by reactive oxygen species (ROS). As for gene transfection experiment, the polysulfonium showed a high transfection efficacy even in serum-containing environment, and it could also effectively deliver the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) gene to kill the cancer cell with a high efficacy in vivo.



Figure 2. The schematic illustration of disintegratable polysulfoniums (a) and the DNA condensation and cell uptake process (b). Reprinted with permission from ref 40.

Another strategy for enhance the interaction of DNA with vectors is increasing the charge density on the surface of nanoparticles.44 You et al. have found that the micellization of cationic polymer could highly increase the surface charge density as shown in Figure 3a, and they have prepared a novel class of bioreducible nanomicelle with a hydrophobic core (Figure 3b). The results have shown that these nanomicelles have high DNA-binding affinity.17, 20, 45-46 DNA could be completely condensed at an


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+/ ratio of 1 by using the formed bioreducible nanomicelles. From the isothermal titration calorimetry (ITC) experiment, they found that the smaller the micelle size was, the stronger the interaction of the vectors and DNA was. The binding constant Ka between DNA and 10 nm micelle is 1.39×109 M1 while the Ka for branch PEI‒25K is only 8.39×107 M1. Therefore, 10 nm micelles are strong DNA binder. In the subsequent transfection experiments, they have found that the transfection efficacy was very high. Especially, the fluorescence intensity of luciferase transfection with 10 nm micelles is 3 orders of magnitude higher than PEI25K in a variety of cell lines at the low N/P radio (Figure 3c).



Figure 3. a) The Zeta potential values of the formed nanomicelles. b) The preparation of bioreducible nanomicelle. c) The GFP transfection in 293T cell of different vectors at different N/P ratio, N/P ratio means the radio of nitrogen in vectors and phosphorus in DNA. Reprinted with permission from ref 44.

Hydrophobic Interaction Conventional cationic polymers interact with DNA through electrostatic


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interaction. However, the DNA and cationic polymer complex are not stable enough in physiological environment because these electrostatic interactions are interfered by various electrolytes in the blood. Therefore, it is very important to develop a nonelectrical interaction mode within the polyplex. Many researches have shown that the induction of hydrophobic interaction into the formed polyplex could improve gene delivery efficacy. The reason is that, on one hand, the hydrophobic property could enhance cellular uptake via lipophilic cell membrane as displayed in Figure 4,47 on the other hand, the hydrophobic moieties can cause a cooperative binding of genetic materials to make contributions in the complex formation48 and enhance the endosomal escape.49

Masotti et al.50 found that the acylation of the PEI could completely

condense DNA into complex with better stability, although alkylation reduces the basicity of PEI, this reduction in basicity did not affect the complexation with DNA. Thermodynamic experiments have exhibited that the hydrophilic modified PEI gave more heat than PEI in the condensation of DNA at the same +/ ratio, which indicates that hydrophobic modified PEI could bind DNA more strongly. When hydrophobic modified polymer was added to the DNA solution, the transition point of charge inversion came at low +/ ratio. Moreover, this transition process of charge inversion depended on the hydrophobicity of polymer.49 Jumbri et al.51 have studied the properties of DNA in ionic liquids. They performed a series of characterizations by using alkylimidazolium as a model compound, and they found that the binding constant increased linearly with the increase of alky chain length of the alkylimidazolium, which indicates that the alky chain contributed to increase the thermal stability of DNA



complex. The CD results have showed that DNA retained its B-form, which also supports that the contribution from the length of alkyl chains played an important role in interaction between DNA and ion liquids.

Figure 4. The function of hydrophobic segment in gene delivery. Reprinted with permission from ref 47. Saltzman et al. 52 prepared a series of high molecular weight terpolymers with low charge density by enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol as shown in Figure 5a. The hydrophobicity of the polymer can be easily varied by using the different lactone with variable ring size. These hydrophobic terpolymers can condense DNA in a low +/ ratio with the slightly positive charge on the surface compared with the hydrophilic cationic polymers (Figure 5b). To a certain extent, the higher the hydrophobicity of the polymer was, the higher the interaction between vectors and DNA was. Based on the transfection experiment in 293T cell and


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Hela cell, it is very clear that hydrophobicity promoted transfection efficacy with lower toxicity (Figure 5c).



Figure 5. a) The structure of hydrophobic terpolymers and different lactones. b) The size and Zeta potential of different vectors with varying hydrophobicity, c) The


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efficacy of different vectors in 293T cell and Hela cell, RLU means relative light unit. Reprinted with permission from ref 52.

Eltoukhy et al.34 reported a class of degradable terpolymers with alkyl side chains, which have a high gene transfection efficacy in different cells. They got the terpolymers by step-polymerization of diacrylate hydrophobic alkylamine and hydrophilic alkylamine. The hydrophobicity of polymer can be regulated by altering the proportion of the monomer. Particularly in this system, the formed polyplex had a high stability under physiological conditions. The high stability resulted from the hydrophobic part in the polymer. One hour after dilution in PBS, the polyplex formed from DNA and the polymer with 20% hydrophobic monomer just got 2>3. Reprinted with permission from ref


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62.

Hydroxyl group is another unit than can form hydrogen bonds with DNA besides Gu+.35,

63-64

Reineke et al. reported that hydroxyl can enhance DNA-polycation

interactions through hydrogen bond between DNA base pairs and vectors (Figure 7). They prepared a library of poly-(glycoamidoamine)s with different stereochemistry of carbohydrate hydroxyl groups. They have found that the hydroxyl group in the polymer is related to the interaction between polymer and DNA, and the cell transfection revealed that the higher the polymer-DNA binding affinity was, the higher DNA delivery efficacy was. Hydroxyl groups provide additional interaction with DNA base pairs compared with the pure electrostatic interaction. To prove their hypothesis that the hydrogen bonding interaction between the polymer and DNA base pairs played an important role in the binding affinity, they used the circular dichroism and FTIR spectral to characterize the different polyplexes. The shape of CD curves is related to the base electronic transition, and they found a shit in the spectrum or loss of ellipticity in the experiment, which reveals that transition is affected by the hydrogen bonding.



Figure 7. Schematic illustration of hydrogen bond enhanced vector and DNA. Reprinted with permission from ref 35.

Similar work was also reported by Allen et al.63. The vinylimidazole homopolymers were alkylated by hydroxyl-containing compound to create a watersoluble, cationic polyelectrolyte. They could tailor the charge density and hydroxyl number by using different hydroxyl. As the result mentioned above, the hydrogen bonding interaction can make contributions to DNA condensation. As the hydroxyl content increase, the interaction between DNA and polymer became more and more strongly. Based on the electrophoretic gel shift assay, it was clear that the hydroxylfree polymer can completely condense DNA above +/– ratio of 10, and the polymers with one hydroxyl could condense DNA at a +/– ratio of 6, while the ratio decreased to 4 for the polymers with two hydroxyl group. When the charge density or hydrogen number increased, the DNA complex became smaller and smaller, and the transfection efficacy increased first and then decreased as the interaction is too strong to accomplish the release of DNA. Coordinative Interaction A wide variety of metal-containing enzymes are found to act an important part in biological process.65 The engineered znic-finger protein can recognize a unique chromosomal site, and the zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between DNA donor and chromosome.66 Metal ions not only interact with proteins to produce functional biomacromolecules,


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but also interact with nucleic acid molecules. Metal ions can interact with the minor and major grooves of DNA through coordination or electrostatic interaction with DNA.67 Some metal ions interact with DNA too strongly to affects DNA replication, transcription and other functions, like antitumor drug cisplatin,68 ruthenium,69 iridium.70 Metal ions, unlike non-metallic positive ions, have more empty orbits and can have stronger coordination with DNA. In general, monovalent metal ions have a weaker effect on DNA, while ions with multivalent states such as Cu2+, Zn2+, Mn2+, and Ca2+ have a strong interaction with DNA through coordination bonds. Because of this strong interaction, DNA could be condensed by multivalent at a low concentration, which makes metal ions promising candidates for gene vectors. 71 The first report of metal complex to condense DNA is trivalent inorganic Co(NH3)63+,72 which can condense DNA into 50 nm. Nevertheless, there are many metal ions that can tightly bind with DNA, few of them are promising to delivery nucleic acid. Some metals ion, such as Cu2+ and Mn2+, can not be used in gene delivery due to their high biological toxicity. Calcium and zinc ions are two kinds of ions used in gene delivery, and there have already been many papers about Ca2+ in gene delivery.73-74

Recently, it was found that zinc ion can not only tightly pack DNA, but

also enhance cell-uptake, escape from endosome, etc.75-77 Therefore it is very promising in gene delivery and transfection. An early paper75 reported that after modified with znic chelate, polylysine had the transfection positive cells increasing to 40% from 1%. Zinc complexes not only increase the interaction with DNA, but also promote endocytosis and endosomal escape. On the other hand, the imidazole ligands of znic in



the vector can be protonated in acid environment, therefore, DNA could be released as the zinc ion is free after the imidazole is protonated in endosome. In recent year, zinc complexes with pyridine ligands have also attracted increasing attention, which first was used in pyrophosphate recognize as the metal center can strongly coordinate with the phosphate group in DNA.78-81 The apparent association constant (Ka) between pyrophosphate anion with znic compound was determined as 6.6 × 108 M1 in 10 mM HEPES buffer (pH = 7.4) at 25 oC. Compared with the imidazole ligands, the pyridine ligands have a large ring, which could enhance the interaction with DNA. Because DNA contains a structure like pyrophosphate, Chen et al.82 used this pyridine chelated zinc in siRNA delivery. Hyaluronic acid modified with znic chelate has a very high gene silencing efficacy compared with the row hyaluronic. The surface of the complex formed by the gene carrier and DNA is negative, mainly because the carrier can form a stable complex with DNA through the coordination bond, which resulted in no excess positive charge on the surface. Nonetheless, hyaluronic acid acts as a tumor targeting unit for cancer cells to ensure endocytosis of the negative complex. Guo et al. 18, 83-84 have used this metal coordinative in DNA delivery (Figure 8). Znic was conjugated to the low molecular weight PEI through disulfide, which has a very high transfection efficacy with a very low weight ratio even in serum-containing environment, even in primary or stem cells. The formed polyplex of DNA and polymer had negative surface charge due to the strongly coordinative interaction, which could avoid the adsorption of serum protein onto polyplex. Since the cell membrane surface is filled with phosphate groups, the polyplex could be effective endocytosed. The above researches


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give a new direction for the design of gene vectors to deal with serum tolerance with low cytotoxicity.

Figure 8.The scheme of znic modified PEI in gene delivery. Reprinted with permission from ref 83.

OUTLOOK There are many processes involved in achieving efficient gene transfection including DNA condensing, endocytosis, endosomal escape, DNA uptaking and nuclear transport. Although many synthetic vetors have been constructed for gene delievry and transfection, it is very difficult for chemeical synthetic vetors to completely overcome all the above obstacles until now while virus vector can do it easily. DNA in a cell is condensed approximately 10000 times by supercoiling with histones during mitosis85-86 while DNA only can be condensed approximately 10 times



using synthetic vectors. The condensed DNA ensures the stability of genetic material. However, for most of the time, DNA is loosely wrapped to achieve physiological processes such as protein translation. It is this transformation of DNA between condensed and decondensed that completes a series of life activities. As for virus vectors, when a virus is assembled, DNA will be pushed inside the capsid by motor protein, the highly condensed DNA is stored with large pressures ~6 MPa.87-88 A hole will be opened by virus to infect the host cell with the decondensed DNA. Scientists are trying to imitate the way of virus condensing and releasing DNA, but no synthetic vectors are as effective as virus vectors.89 So, a good vector can not only condense DNA efficiently to protect DNA from degradation, but also release DNA quickly after endocytosis. It is the gap between synthetic vectors and viral vectors that makes the research of synthetic vectors more challenging. As the devolopment of polymer chemistry, multiple multifunctionalies can be easily integrated into one polymer, polymer structures and functionalies can be precisely controlled, and the synthetic polymer vector can not only efficiently condense DNA, but also facilitate DNA endocytosis, endosomal escape, DNA uptaking and nuclear transport, and achieve similar gene transfection of virus. ACKNOWLEDGMENTS We acknowledge funding support from the National Key R&D Program of China (2017YFA0205601) and the National Natural Science Foundation of China (Grant No. 51625305, 21704095, 21774113 and 21525420). REFERENCES


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Table of Contents Graphic



Figure 1 122x79mm (300 x 300 DPI)


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Table 1



Figure 2 124x47mm (300 x 300 DPI)


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Figure 3 108x93mm (300 x 300 DPI)



Figure 4 81x74mm (300 x 300 DPI)


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Figure 5 120x88mm (300 x 300 DPI)



Figure 6 131x86mm (300 x 300 DPI)


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Figure 7 107x55mm (300 x 300 DPI)



Figure 8 107x70mm (300 x 300 DPI)


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TOC 122x79mm (300 x 300 DPI)


Interactions in DNA CondensationââAn Important Factor for

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