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Cyclodextrin-based Switchable DNA Condenser Ping Hu1, Yong Chen1,2 and Yu Liu1,2*
Published on 28 May 2015. Downloaded by UNIVERSITY OF NEW ORLEANS on 01/06/2015 00:46:07.
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x 5
Switchable DNA condensers based on β-cyclodextrin derivates bearing cationic imidazolium moieties and hydrolysable ester linkages were synthesized, showing baseresponsive or enzyme-responsive switchable DNA condensation ability under physiological conditions.
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DNA vectors transferring gene into cells have shown their value in studies of gene therapy[1]. One of vital points in the design of a synthetic DNA vector is the ability to pack the large DNA plasmids[2]. To achieve this target, cationic groups are always employed in DNA vectors owing to their electrostatic interaction with anionic phosphate groups of DNA[3]. Based on this strategy, a number of artificial DNA vectors such as lipids[4], surfactants[5] and polymers[6] have been prepared. Cyclodextrins (CDs), a class of cyclic polysaccharides mainly containing 6-8 glucose units linked by 1,4-glucosidic bonds, are widely used in the design of artificial DNA vectors because they are watersoluble, nontoxic, commercially available at low cost. Recently, a number of CD-based DNA vectors[7] including polyrotaxane/ polypseudorotaxane[8], side chain-modified polymers[9], star polymers[10], dentrimers[11] and nanoparticles[12] have been successfully constructed. On the other hand, the accumulation of positive charges induces the inflammation response though several routes [13]. Therefore, some efforts including degradable main structures [14], charge changeable systems[15] and zwitterionic systems[16] were made to minimize such disadvantage. Herein, we design a type of β-CD-based DNA condensers bearing cationic groups and the cleavable ester linkages (Scheme 1). In this system, the polycationic structure of 1 or 2 facilitates their good DNA condensation ability. In the presence of base or enzyme, carboxylate anions resulting from the cleavage of esters can neutralize the imidazolium cations on β-CD to form zwitterions that show lower toxicity towards biotic tissue[16].
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Scheme 1. Structure of β-CD-based DNA condenser. 75
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Possessing a cationic β-CD head and seven hydrophobic tails, 1 or 2 may have the tendency to self-aggregate in water[17]. Therefore, trying to investigate the influence of possible selfaggregation, the CAC value was measured by detecting the transmittance (T%) of 1 at λ = 400 nm in buffer at various concentrations (5 - 750 µM) following a reported method[18]. As is shown in Figure S9, the optical transmittance at 400 nm gradually decreased with increasing the concentration of 1, indicating the formation of aggregates in solution, and an inflection point at 95 µM, referring to the CAC, was observed on the plot of optical transmittance at 400 nm versus the concentration of 1. The T% of 2 at same concentration was detected, and no significant absorbance was found. Considering the concentration of 1 used in the DNA condensation experiments (up to 40 µM), we deduced that most of 1 should exist as monomers under our experimental conditions. Possessing seven positively charged imidazolium groups, the DNA condensation abilities of the “switch-on” β-CD derivatives 1 and 2 were investigated. In the agarose gel electrophoresis experiments (Figure S11), both 1 and 2 could efficiently retain the mobility of pBR322 plasmid DNA. By comparing the lowest concentration needed for DNA to remain in the well, we found that 2 had the higher efficiency to retain DNA in the gel well (15 µM needed to totally retain DNA) than 1 (25 µM needed to totally retain DNA). The possible reason may be rationalized by the two-dimensional NMR experiments, where the ROESY spectrum of 1 showed the significant NOE correlation signals, but the ROESY spectrum of 2 showed the weak NOE correlation signals, between the protons on the hydrophobic substituent and the interiors protons (H3/H5/H6) of β-CD cavity, especially the H5/H6 protons (Figure S5-6). Because the H5/H6 protons are located near the narrow open, while the H3 protons near the wide open of β-CD cavity, these NOE signals indicated that the hydro-
Figure 1. AFM images of pBR322 DNA (left) and its condensate (right) induced by 1.Condition: [DNA] = 1 ng·µL-1 in left graph; [DNA] = 10 ng·µL-1, [1] = 20 µM in right graph.
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phobic substituent of 1 were self-included in the β-CD cavity deeplier than those of 2. Such self-included conformation of 1 would be certainly unfavorable to the interactions of imidazolium cations in 1 with DNA and thus led to the weaker DNA condensation ability. The direct and visible information about the DNA condensation comes from atomic force microscopy (AFM). As shown in Figure 1, the supercoil form of free pBR322 DNA exists as discrete circles or wires, and its height correlates with the width of single DNA chain (about 2 nm)[19]. After the addition of 1, the originally loose DNA circles or wires turn to solid particles with an average diameter of ca. 30 nm. This phenomenon clearly demonstrates the good DNA condensation ability of 1, and the driving force of the DNA condensation may mainly be the electrostatic interactions between the cationic imidazolium groups in 1 and the negatively charged phosphates in DNA. In addition, the pBR322 DNA shows the similar condensation behavior in the presence of 2. It is noteworthy that the ester bonds in 1 and 2 are cleavable, which will consequently change the charge distribution of host βCD. To characterize the properties of the ester-cleaved product, NaOH was added to the solution of 1 (or 2) following a reported method [15b]. As shown in the 1H NMR spectra of 1 (or 2) before and after treated by NaOH (Figure S10), the 1H NMR spectrum of NaOH-treated 1 (or 2) clearly give a quartet peak at δ = 3.553.50 ppm and a triplet peak at δ = 1.07-1.04 ppm, assigned to the -CH2- and -CH3 protons of the hydrolysis product EtOH respectively. This indicates that the ester groups on 1 (or 2) are hydrolyzed to carboxylic anions in a basic environment, which changes the electrostatic nature of 1 (or 2) from a highly cationic status (“cation switch-on” status) to a neutral one (“cation switchoff” status). Interestingly, the mobility of DNA shows little appreciable retaining in the presence of the “switch off”-typed β-CD 1 (or 2) (Figure 2), indicating that the DNA condensation abilities of 1 (or 2) is greatly diminished after the host β-CD is hydrolyzed. In addition, the addition of 1 or 2 could greatly change the zeta potential of DNA from -53.65 mV to +23.97 mV (for DNA + 1) or +24.87 mV (for DNA + 2), but the zeta potential of DNA nearly unchanged with the addition of the ester-cleaved product of 1 (or 2). A possible reason may be that the negative charges resulting from the base-induced hydrolysis prevent the cationic imidazolium groups from interacting with the negatively charged phosphates in DNA, leading to the loss of the DNA condensation ability.
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Figure 2. Agarose gel electrophoresis assay to investigate the DNA condensation of “switch off”-typed 1 and 2. Lane 1, [DNA] = 10 ng µL-1; Lane 2 to 5, DNA + “switch off”-typed 1 (20 - 80 µM); Lane 6 to 9, DNA + “switch off”-typed 2 (20 - 80 µM).
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Figure 3. “On-off” switch of DNA condensation of (a) 1 and (b) 2 in a physiological environment (0.1 M HEPES buffer, pH = 7.3, 37 °C). Lane 1: pBR322 DNA, Lane 2-9: pBR322 DNA + β-CD derivates from 1 to 7 day. ([DNA] = 10 ng·µL-1, [1] = [2] = 20 µM).
Figure 4. “On-off” switch of DNA condensation induced by AChE. Lane 1: free DNA, Lane 2: DNA+2, Lane 3: DNA+2+AChE, Lane 4: DNA+1, Lane 5: DNA+1+AChE. ([DNA] = 10 ng·µL-1, [1] = [2] = 15 µM, [AChE] = 200 U mL-1. 1 or 2 was mixed with AChE for 24 h at 37 oC before gel analysis.
Figure 5. Cell viability of (a) 293T and (b) HeLa cells in the absence or presence of 1.
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Moreover, the “on-off” switch of the DNA condensation can also occur in a physiological environment (pH = 7.3, 37 °C). As shown in Figure 3, 1 turns to a partly “switch-off” status of condensing DNA within 7 days gradually while 2 turns to a “switch-off” status after a second day at a physiological pH and body temperature. In the addition to base and physiological environment, enzymes such as esterase could induce the switch of the DNA condensation abilities. Herein, as a typical esterase, acetylcholinesterase (AChE) was used to cleave the ester bond since its active center is similar to those of many carboxylic esterases. As shown in Figure 4, the DNA condensation ability of 2 could be obviously switched off with the addition of AChE, while 1 showed the obvious resistance to AChE. The possible reason may be that the deep self-inclusion conformation of 1 protects the ester bonds from being attacked to some extent. The cytotoxicity of 1 was investigated using 293T (human embryonic kidney cells) and HeLa cells as model cell lines, and MTT assays was used to detect the cellular viabilities. As shown in Figure 5, owing to the good biocompatibility of the β-CD backbone and the cleavable ester bonds, the cellular viabilities of both cells were higher than 95% even the concentration of 1 rise to 0.1 mM which clearly indicated the low cytotoxicity of these DNA condensers. In addition, the cell transfection experiments into HeLa cell were also tested with pRNAT-H1.1/Shuttle-GFP plasmid DNA, and no visible GFP expression was observed. We think that improvements may be achieved by the further chemical modification of carriers, the appropriate selection of cell and DNA, and the optimization of mixed ratio between carriers and DNA.
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Conclusions With the aim of achieving the DNA condensers with controlled DNA condensation ability and low cytotoxicity, β-CD derivates modified with seven imidazolium and carboxylic ester groups linked by hydrophobic tethers in different lengths were prepared. Significantly, these β-CD derivates present good DNA condensation abilities, and their DNA condensation abilities can be switched by base, enzyme and physiological condition. In addition, the length of hydrophobic tethers is found able to affect not only the DNA condensation ability but also the stability of βCD-based DNA condensers. This property of controllable DNA condensation by adjusting the charge type and distribution of gene carriers may enable its potential application as gene delivery vectors to gain higher efficiency in the future.
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Acknowledgement 45
We thank the 973 Program (2011CB932502) and the NNSFC (91227107, 21432004 and 21272125) for financial support.
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Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, 2Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, P. R. China. Email: [email protected] † Electronic Supplementary Information (ESI) available: synthesis and characterization of β-CD derivates and the experiment details. See DOI: 10.1039/b000000x/
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This article can be cited before page numbers have been issued, to do this please use: P. Hu, Y. Chen and Y. Liu, Chem. Commun., 2015, DOI: 10.1039/C5CC03248B.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Cyclodextrin-based Switchable DNA Condenser Ping Hu1, Yong Chen1,2 and Yu Liu1,2*
Published on 28 May 2015. Downloaded by UNIVERSITY OF NEW ORLEANS on 01/06/2015 00:46:07.
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x 5
Switchable DNA condensers based on β-cyclodextrin derivates bearing cationic imidazolium moieties and hydrolysable ester linkages were synthesized, showing baseresponsive or enzyme-responsive switchable DNA condensation ability under physiological conditions.
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DNA vectors transferring gene into cells have shown their value in studies of gene therapy[1]. One of vital points in the design of a synthetic DNA vector is the ability to pack the large DNA plasmids[2]. To achieve this target, cationic groups are always employed in DNA vectors owing to their electrostatic interaction with anionic phosphate groups of DNA[3]. Based on this strategy, a number of artificial DNA vectors such as lipids[4], surfactants[5] and polymers[6] have been prepared. Cyclodextrins (CDs), a class of cyclic polysaccharides mainly containing 6-8 glucose units linked by 1,4-glucosidic bonds, are widely used in the design of artificial DNA vectors because they are watersoluble, nontoxic, commercially available at low cost. Recently, a number of CD-based DNA vectors[7] including polyrotaxane/ polypseudorotaxane[8], side chain-modified polymers[9], star polymers[10], dentrimers[11] and nanoparticles[12] have been successfully constructed. On the other hand, the accumulation of positive charges induces the inflammation response though several routes [13]. Therefore, some efforts including degradable main structures [14], charge changeable systems[15] and zwitterionic systems[16] were made to minimize such disadvantage. Herein, we design a type of β-CD-based DNA condensers bearing cationic groups and the cleavable ester linkages (Scheme 1). In this system, the polycationic structure of 1 or 2 facilitates their good DNA condensation ability. In the presence of base or enzyme, carboxylate anions resulting from the cleavage of esters can neutralize the imidazolium cations on β-CD to form zwitterions that show lower toxicity towards biotic tissue[16].
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Scheme 1. Structure of β-CD-based DNA condenser. 75
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Possessing a cationic β-CD head and seven hydrophobic tails, 1 or 2 may have the tendency to self-aggregate in water[17]. Therefore, trying to investigate the influence of possible selfaggregation, the CAC value was measured by detecting the transmittance (T%) of 1 at λ = 400 nm in buffer at various concentrations (5 - 750 µM) following a reported method[18]. As is shown in Figure S9, the optical transmittance at 400 nm gradually decreased with increasing the concentration of 1, indicating the formation of aggregates in solution, and an inflection point at 95 µM, referring to the CAC, was observed on the plot of optical transmittance at 400 nm versus the concentration of 1. The T% of 2 at same concentration was detected, and no significant absorbance was found. Considering the concentration of 1 used in the DNA condensation experiments (up to 40 µM), we deduced that most of 1 should exist as monomers under our experimental conditions. Possessing seven positively charged imidazolium groups, the DNA condensation abilities of the “switch-on” β-CD derivatives 1 and 2 were investigated. In the agarose gel electrophoresis experiments (Figure S11), both 1 and 2 could efficiently retain the mobility of pBR322 plasmid DNA. By comparing the lowest concentration needed for DNA to remain in the well, we found that 2 had the higher efficiency to retain DNA in the gel well (15 µM needed to totally retain DNA) than 1 (25 µM needed to totally retain DNA). The possible reason may be rationalized by the two-dimensional NMR experiments, where the ROESY spectrum of 1 showed the significant NOE correlation signals, but the ROESY spectrum of 2 showed the weak NOE correlation signals, between the protons on the hydrophobic substituent and the interiors protons (H3/H5/H6) of β-CD cavity, especially the H5/H6 protons (Figure S5-6). Because the H5/H6 protons are located near the narrow open, while the H3 protons near the wide open of β-CD cavity, these NOE signals indicated that the hydro-
Figure 1. AFM images of pBR322 DNA (left) and its condensate (right) induced by 1.Condition: [DNA] = 1 ng·µL-1 in left graph; [DNA] = 10 ng·µL-1, [1] = 20 µM in right graph.
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phobic substituent of 1 were self-included in the β-CD cavity deeplier than those of 2. Such self-included conformation of 1 would be certainly unfavorable to the interactions of imidazolium cations in 1 with DNA and thus led to the weaker DNA condensation ability. The direct and visible information about the DNA condensation comes from atomic force microscopy (AFM). As shown in Figure 1, the supercoil form of free pBR322 DNA exists as discrete circles or wires, and its height correlates with the width of single DNA chain (about 2 nm)[19]. After the addition of 1, the originally loose DNA circles or wires turn to solid particles with an average diameter of ca. 30 nm. This phenomenon clearly demonstrates the good DNA condensation ability of 1, and the driving force of the DNA condensation may mainly be the electrostatic interactions between the cationic imidazolium groups in 1 and the negatively charged phosphates in DNA. In addition, the pBR322 DNA shows the similar condensation behavior in the presence of 2. It is noteworthy that the ester bonds in 1 and 2 are cleavable, which will consequently change the charge distribution of host βCD. To characterize the properties of the ester-cleaved product, NaOH was added to the solution of 1 (or 2) following a reported method [15b]. As shown in the 1H NMR spectra of 1 (or 2) before and after treated by NaOH (Figure S10), the 1H NMR spectrum of NaOH-treated 1 (or 2) clearly give a quartet peak at δ = 3.553.50 ppm and a triplet peak at δ = 1.07-1.04 ppm, assigned to the -CH2- and -CH3 protons of the hydrolysis product EtOH respectively. This indicates that the ester groups on 1 (or 2) are hydrolyzed to carboxylic anions in a basic environment, which changes the electrostatic nature of 1 (or 2) from a highly cationic status (“cation switch-on” status) to a neutral one (“cation switchoff” status). Interestingly, the mobility of DNA shows little appreciable retaining in the presence of the “switch off”-typed β-CD 1 (or 2) (Figure 2), indicating that the DNA condensation abilities of 1 (or 2) is greatly diminished after the host β-CD is hydrolyzed. In addition, the addition of 1 or 2 could greatly change the zeta potential of DNA from -53.65 mV to +23.97 mV (for DNA + 1) or +24.87 mV (for DNA + 2), but the zeta potential of DNA nearly unchanged with the addition of the ester-cleaved product of 1 (or 2). A possible reason may be that the negative charges resulting from the base-induced hydrolysis prevent the cationic imidazolium groups from interacting with the negatively charged phosphates in DNA, leading to the loss of the DNA condensation ability.
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Figure 2. Agarose gel electrophoresis assay to investigate the DNA condensation of “switch off”-typed 1 and 2. Lane 1, [DNA] = 10 ng µL-1; Lane 2 to 5, DNA + “switch off”-typed 1 (20 - 80 µM); Lane 6 to 9, DNA + “switch off”-typed 2 (20 - 80 µM).
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Figure 3. “On-off” switch of DNA condensation of (a) 1 and (b) 2 in a physiological environment (0.1 M HEPES buffer, pH = 7.3, 37 °C). Lane 1: pBR322 DNA, Lane 2-9: pBR322 DNA + β-CD derivates from 1 to 7 day. ([DNA] = 10 ng·µL-1, [1] = [2] = 20 µM).
Figure 4. “On-off” switch of DNA condensation induced by AChE. Lane 1: free DNA, Lane 2: DNA+2, Lane 3: DNA+2+AChE, Lane 4: DNA+1, Lane 5: DNA+1+AChE. ([DNA] = 10 ng·µL-1, [1] = [2] = 15 µM, [AChE] = 200 U mL-1. 1 or 2 was mixed with AChE for 24 h at 37 oC before gel analysis.
Figure 5. Cell viability of (a) 293T and (b) HeLa cells in the absence or presence of 1.
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Moreover, the “on-off” switch of the DNA condensation can also occur in a physiological environment (pH = 7.3, 37 °C). As shown in Figure 3, 1 turns to a partly “switch-off” status of condensing DNA within 7 days gradually while 2 turns to a “switch-off” status after a second day at a physiological pH and body temperature. In the addition to base and physiological environment, enzymes such as esterase could induce the switch of the DNA condensation abilities. Herein, as a typical esterase, acetylcholinesterase (AChE) was used to cleave the ester bond since its active center is similar to those of many carboxylic esterases. As shown in Figure 4, the DNA condensation ability of 2 could be obviously switched off with the addition of AChE, while 1 showed the obvious resistance to AChE. The possible reason may be that the deep self-inclusion conformation of 1 protects the ester bonds from being attacked to some extent. The cytotoxicity of 1 was investigated using 293T (human embryonic kidney cells) and HeLa cells as model cell lines, and MTT assays was used to detect the cellular viabilities. As shown in Figure 5, owing to the good biocompatibility of the β-CD backbone and the cleavable ester bonds, the cellular viabilities of both cells were higher than 95% even the concentration of 1 rise to 0.1 mM which clearly indicated the low cytotoxicity of these DNA condensers. In addition, the cell transfection experiments into HeLa cell were also tested with pRNAT-H1.1/Shuttle-GFP plasmid DNA, and no visible GFP expression was observed. We think that improvements may be achieved by the further chemical modification of carriers, the appropriate selection of cell and DNA, and the optimization of mixed ratio between carriers and DNA.
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Conclusions With the aim of achieving the DNA condensers with controlled DNA condensation ability and low cytotoxicity, β-CD derivates modified with seven imidazolium and carboxylic ester groups linked by hydrophobic tethers in different lengths were prepared. Significantly, these β-CD derivates present good DNA condensation abilities, and their DNA condensation abilities can be switched by base, enzyme and physiological condition. In addition, the length of hydrophobic tethers is found able to affect not only the DNA condensation ability but also the stability of βCD-based DNA condensers. This property of controllable DNA condensation by adjusting the charge type and distribution of gene carriers may enable its potential application as gene delivery vectors to gain higher efficiency in the future.
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Acknowledgement 45
We thank the 973 Program (2011CB932502) and the NNSFC (91227107, 21432004 and 21272125) for financial support.
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Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, 2Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, P. R. China. Email: [email protected] † Electronic Supplementary Information (ESI) available: synthesis and characterization of β-CD derivates and the experiment details. See DOI: 10.1039/b000000x/
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