DNA Condensation Induced by a Star-Shaped Hexameric Cationic

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DNA Condensation Induced by a StarShaped Hexameric Cationic Surfactant Yaxun Fan, Hua Wang, Chengqian He, Fulin Qiao, Shu Wang, and Yilin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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ACS Applied Materials & Interfaces

DNA Condensation Induced by a Star-Shaped Hexameric Cationic Surfactant Yaxun Fan,† Hua Wang,†,‡ Chengqian He,† Fulin Qiao,†,‡ Shu Wang,*,§ and Yilin Wang*,†,‡ †

Key Laboratory of Colloid and Interface Science and §Key Laboratory of Organic Solids, Beijing

National Laboratory for Molecular Sciences (BNLMS), CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT The interactions between a star-shaped hexameric cationic quaternary ammonium surfactant PAHB and calf thymus DNA and induced DNA condensation were investigated by ζ-potential, dynamic light scattering, atomic force microscopy, isothermal titration calorimetry, ethidium bromide exclusion assay, circular dichroism and cytotoxicity assay. With the addition of PAHB, long extended DNA molecules exhibit successive conformational transitions from elongated coil to partially condensed cluster-like aggregate, globules-on-a-string structure, and then to fully condensed globule until the saturation point of interaction between PAHB and DNA, which is slightly above their charge neutralization point. The efficient condensation is mainly produced by the strong attractive electrostatic interaction between the multiple positively charged headgroups of PAHB and negatively charged phosphate groups of DNA, and the hydrophobic interaction among the multiple alkyl chains of PAHB. Moreover the transition of the DNA conformation is also affected by the 1

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transitions of PAHB molecular conformation from star-shaped to claw-like and pyramid-like. Although the DNA conformation is significantly changed by PAHB, the DNA secondary structure does not display obvious variations and the PAHB/DNA mixture does not show cytotoxicity when DNA is partially condensed. These results indicate that star-shaped oligomeric cationic surfactant is a potential condensing agent for gene transfection.

KEYWORDS: DNA condensation, cationic star-shaped hexameric surfactant, DNA/surfactant interaction, conformation transition, cytotoxicity

INTRODUCTION Gene transfection refers to the process of delivering nucleic acids into cells and is the key step for gene therapy. In the process, nucleic acids have to cross several barriers, such as cell membrane and nucleus membrane.1-3 However, the multiple anionic character of DNA limits its permeation across negatively charged cell membranes, and the steric restriction inside the cell hinders its transportation to the nucleous.4 To solve these fundamental problems, DNA has to be condensed into a neutral complex in a compact globular conformation of reduced sizes with the assistant of condensing agents. Virus-based condensing agents have shown high transfection efficiency,5-6 but they can provoke undesirable immune response. Cationic gemini surfactants are one kind of potential alternatives as non-viral condensing agents for gene delivery.7-12 Cationic gemini surfactants,1, 13-18 containing two positive charged amphiphilic moieties connected by a rigid or flexible linker, show more superior properties than traditional monomeric surfactants including significantly reduced critical micelle 2


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concentration (CMC) and enhanced surface activity by the order of magnitude. The unique structures of gemini surfactants endow them with strong binding ability to DNA at very low concentration, low toxicity and enhanced transfection ability. Most of cationic gemini surfactants studied for DNA condensation are based on dicationic quaternary ammonium compounds.19-24 Karlsson and co-workers20 studied the interaction of bacteriophage T4 DNA with a series of cationic gemini surfactants alkanediyl-α,ω-bis-(dimethylalkylammonium bromide) (CsCnCsBr2), and found that the spacer length, valency and tail length of the surfactants determine the aggregation behavior of a gemini surfactant/DNA mixture. Our previous work22-23 studied the interaction of cationic gemini surfactants with different counterions and revealed that the effects of counterions basically follow the Hofmeister series. Moreover with the increase of the chain asymmetry of gemini surfactants, the interaction between the gemini surfactants and DNA becomes more spontaneous.22-23 To further improve the efficiency of surfactants in DNA condensation and reduce the toxicity of cationic gemini surfactants, a great deal of effort has been devoted, such as incorporating natural structural motifs16 or inducing aromatic functional groups similar to DNA bases.21, 25-26 Introducing amino acid in the spacer of cationic serine-based surfactants increases their biocompatibility while introducing amine and ester enhancing their condensation efficiency for DNA.16 Gemini imidazolium surfactants21 show a good DNA binding capability and low cytotoxicity, attributing to the formation of premicellar aggregates at sufficiently low concentration below their CMC values. So gemini surfactants provide great possibilities to build delivery carriers for DNA with designed properties. On the basis of the understanding about the actions of gemini surfactants in DNA delivery, it is expected that increasing the number of amphiphilic moieties in surfactant molecules could be 3



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considered as another alternative strategy to enhance the efficiency of surfactants in DNA condensation and reduce the cytotoxicity. Surfactants with three or more amphiphilic moieties chemically connected by spacer groups can be called as oligomeric surfactants.27-31 We previously studied a series of star-shaped cationic quaternary ammonium oligomeric surfactants bearing amide linkages, and proved the enhancement in aggregation ability with the increase of oligomerization degree attributed to the larger contribution of each hydrocarbon chain in inter- and intramolecular hydrophobic interactions.32-34 In particular, for the tetrameric surfactant (PATC) and hexameric surfactant (PAHB), the stretched star-shaped molecular conformation leads to the formation of large network-like premicellar aggregates at extremely low concentration through intermolecular hydrophobic association between the hydrophobic chains, while small spherical micelles are formed at higher surfactant concentration. Moreover the strong self-assembly ability of oligomeric surfactants can significantly promote their interaction with oppositely charged species. In the mixture of anionic hydrophobically modified polyacrylamide (C12PAM) with trimeric surfactant DTAD or tetrameric surfactant PAHB,35 the surfactants start to interact with the polymer at the concentration of far below their CMC. Both DTAD/C12PAM and PAHB/C12PAM form soluble network-like aggregates, denser crosslinked precipitated aggregates and soluble spherical aggregates with increasing surfactant concentration. In particular, the cationic micelles of oligomeric surfactants can efficiently kill Gram negative E. coli with a very low minimum inhibitory concentration and the antibacterial activity of the oligomeric surfactants increases as the degree of oligomerization increases. However they are nontoxic to mammalian cells at the concentrations used.36 Herein, star-shaped hexameric cationic ammonium surfactant PAHB bearing amide moieties 4


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(Scheme 1) was selected to investigate the effect of oligomeric surfactants on DNA conformations and the PAHB/DNA complex properties. The cooperative behaviors and the related mechanism were studied by ζ-potential, dynamic light scattering (DLS), atomic force microscopy (AFM), isothermal titration microcalorimetry (ITC), ethidium bromide exclusion assay and circular dichroism (CD). The cytotoxicity of PAHB/DNA complex was also evaluated with cancer cells and normal cell. It was found that DNA molecules exhibit multiple successive transitions between coil and globule states by interacting with PAHB, and the secondary structure of DNA does not alter obviously when DNA is completely condensed. The cytotoxicity of PAHB/DNA is dependent on the amount of the free PAHB and the incubation time.

EXPERIMENTAL SECTION Materials. Hexameric cationic quaternary ammonium surfactant PAHB was synthesized and purified according to the method in previous work.34 The structure of PAHB was confirmed by 1H NMR spectroscopy and mass spectroscopy, and the purity was verified by elemental analysis and surface tension measurements. The double-stranded (ds) calf thymus DNA (~ 3 kbp, Mw = 3.9 × 106) was purchased from Fluka and used as received. Its purity was confirmed by taking the ratio A260/280 between the absorbance at 260 nm and at 280 nm on NanoVue Spectrophotometer (GE Healthcare, UK), and the ratio was about 1.8, indicating no protein contamination. Ethidium bromide (EtBr, in Scheme 1) with the best available purity was purchased from Alfa Aesar, and used without further purification. MCF-7 and HaCaT cells were obtained from Center for Cell, Institute of Basic Medical Science in Chinese Academy of Sciences. Dulbecco’s modified eagle medium (DMEM) and 5



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phosphate buffered saline (PBS) were purchased from Hyclone (Beijing, China). Fetal bovine serum (FBS) was obtained from Sijiqing Biological Engineering Materials (Hangzhou, China). Milli-Q water (18 MΩ·cm-1) was used throughout. O

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Scheme 1. Molecular structures of PAHB and ethidium bromide (EtBr). Sample Preparation. Calf thymus DNA and PAHB stock solutions were prepared in 1 mM sodium phosphate buffer (PBS, pH 7.4) at 25 °C by simple dissolving into the desired concentrations. The DNA solutions were freshly prepared and stored at -4.0 °C before use. The final concentration of DNA was fixed at 0.15 mM in nucleotide phosphate groups, which was quantified using a NanoVue Spectrophotometer. ζ-Potential and Particle Size Measurements. The ζ-potential and the particle size measurements were performed at 25 °C by a Malvern Zetasizer Nano-ZS instrument (ZEN3600, Malvern Instruments, Worcestershire, UK) equipped with a 4 mW He-Ne laser at a wavelength of 632.8 nm. A clear disposable capillary cell (DTS1060C) was loaded with ~ 1.0 mL of PAHB/DNA mixed solution with varying PAHB concentrations and used for both measurements. The ζ-potentials were calculated with the Hemholtz-Smoluchowski relationship from the mobility measured by electrophoretic 6


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light-scattering (ELS) experiment. The particle size is derived from a Cumulants analysis of the correlation curve measured by dynamic light scattering (DLS). It reflects the intensity-weighted mean hydrodynamic size of the ensemble collection of particles, and provides the information on the whole changing trend of the particle sizes in the systems. Each data point is reported as the mean and standard deviation of three measurements. Atomic Force Microscopy (AFM). A Multimode Nanoscope IIIa AFM (Digital Instruments, CA) was used for AFM imaging. For ambient imaging, 5-7 μL of PAHB/DNA solution was deposited onto a freshly cleaved piece of mica and left to adhere for 5-10 min. The samples were then briefly rinsed with Milli-Q water and dried with a gentle stream of nitrogen. Probes used were etched silicon probes attached to 125 μm cantilevers with a nominal spring constant of 40 N/m (Digital Instruments, model RTESPW). All the provided morphology images were recorded using a tapping mode at 512 × 512 pixel resolution and a scan speed of 1.0-1.8 Hz. Topographic data were regularly recorded in both trace and retrace to check on scan artifacts. They were shown in the height mode without any image processing except flattening. Analysis of the images was carried out using the Digital Instruments Nanoscope Software (Version 512r2). Isothermal Titration Microcalorimetry (ITC). The calorimetric measurements were taken in a TAM2277-201 microcalorimetric system (Thermometric AB, Järfälla, Sweden) with a stainless steel sample cell of 1 mL at 25.00 ± 0.01 °C. The sample cell of the microcalorimeter was initially loaded with 700 μL of pure water or 0.15 mM DNA solution. Concentrated PAHB solution was consecutively injected into the stirred sample cell in each portion of 6 μL using a 500 μL Hamilton syringe controlled by a Thermometric 612 Lund pump. During the titration process, the system was 7



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stirred at 60 rpm with a gold propeller, and the interval between two injections was sufficiently long for the signal to return to the baseline. The observed enthalpy (ΔHobs) was obtained by integrating the areas of the peaks in the plot of thermal power against time. The reproducibility of experiments was within ± 4%. Ethidium Bromide Exclusion Assay. 0.075 mM Ethidium bromide (EtBr) and 0.15 mM DNA solution (one ethidium bromide per base pair) were mixed and incubated at 25 °C for at least 30 min. Various amounts of PAHB solutions were added to the DNA-EtBr mixture and left to incubate for 30 min. Fluorescence intensity was measured using a Hitachi F-4500 spectrofluorometer. The wavelengths of excitation (λex) and emission (λem) were 480 and 600 nm, respectively. Circular Dichroism (CD). The CD spectra of DNA with or without PAHB were recorded on a JASCO J-815 spectrophotometer using a 5 mm quartz cell at 25 oC. Scans were obtained in a range between 220 and 320 nm by taking points at 0.5 nm, with an integration time of 0.5 s. Five spectra were averaged to improve the signal-to-noise ratio and smoothed using the noise reducing option in the software supplied by the vendor. Cytotoxicity Assay. MCF-7 and HaCaT cells were seeded in 96-well culture plates at a density of 5 × 103 cells/well. After 12 h incubation, the culture medium was replaced by PAHB/DNA mixtures with varying PAHB concentrations in culture medium. Followed by 24 h and 48 h incubation, MTT (5 mg/mL-1 in water, 10 μL/well) was added into each well. After incubation for 4 h at 37 °C, the supernatant was abandoned and 100 μL of DMSO was added into each well to dissolve the produced formazan. After shaking the plates for 5 min, the absorbance values of each well at 570 nm were measured by a microplate reader. 8


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RESULTS AND DISCUSSION DNA Condensation by PAHB. Figure 1 shows the variation of the ζ-potential and size of DNA against the PAHB concentration (CPAHB) and the mixing PAHB/DNA charge ratio (Z+/-). The charge ratio is calculated by the ratio of positive charges of PAHB to negative charges of DNA. The DNA concentration is 0.15 mM in nucleotide phosphate groups in PBS, which is so low that the intermolecular interaction between DNA molecules can be ignored. For DNA itself, the ζ-potential is about -50 mV and the size is about 700 nm, which are consistent with the results reported previously.37 With the initial additions of PAHB solution, the size value starts to decrease while the ζ-potential value increases, indicating that the binding of cationic quaternary ammoniums of PAHB with the bases of DNA takes place at extremely low PAHB concentration due to their strong electrostatic interaction. The critical concentration is determined from the intercept between the linear extrapolations of the rapidly varying portion of the curve and of the almost-horizontal portion at high PAHB concentration, which is ~ 0.013 mM, corresponding to the onset of DNA condensation (marked as C1 in Figure 1). At this point, the aggregate size becomes ~ 300 nm whereas the ζ-potential is -20 mV. This indicates that the DNA chains have been largely condensed into small aggregates with less negative charges by oppositely charged PAHB molecules. Upon further increasing PAHB concentration, both the value of size and ζ-potential significantly change to a constant value, which are about 60 nm and 30 mV respectively. The second critical point is about 0.080 mM (marked as C2 in Figure 1), beyond which either the size value or the ζ-potential value does not change anymore. That is to say, the condensation of DNA has completed and excessive PAHB molecules will not incorporate into the PAHB/DNA aggregates anymore. 9



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0.20

Figure 1. The variation of the ζ-potential (■) and the size (□) of 0.15 mM DNA against the PAHB concentration (CPAHB) and the PAHB/DNA charge ratio (Z+/-). Error bars indicate the deviation of the ζ-potential values. In order to know the variation of the DNA morphology in the process of the PAHB-induced condensation, the morphologies of DNA at different PAHB concentrations from 0 to 0.200 mM were observed by AFM as shown in Figure 2. DNA itself shows an elongated coiled structure with a large contour length and the height is about 6 nm (Figure 2a), which is ascribed to the strong electrostatic repulsion and the rigidity of highly charged DNA chain. With the addition of 0.005 and 0.010 mM PAHB before C1 (Figure 2b and 2c), the elongated coiled structure and the height of the DNA chains almost do not change. When the PAHB concentration is beyond C1, the DNA chains start to be condensed. With 0.015 mM PAHB (Figure 2d), the PAHB/DNA mixture begins to form a compacted structure with coils stretching out, like loose cluster-like aggregates. While further increasing the PAHB concentration to 0.020 mM (Figure 2e), the PAHB/DNA aggregates present a globules-on-a-string structure, which is composed of many small spherical aggregates about 50 nm interconnected by unfolded DNA chains. When the PAHB concentration is equal or larger than 0.030 10


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mM (Figure 2f), only compact spherical aggregates of ~ 100 nm exist. Afterwards the morphology almost does not change anymore (Figure 2g and 2h), and the size only slightly increases possibly because more cationic charged PAHB molecules are involved. The size changing tendency at this stage is slightly different from that observed by DLS (Figure 1) since excess surfactant molecules may exist as free aggregates and reduce the average size in the systems measured by DLS. 2.00 μm

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Figure 2. DNA morphologies observed by AFM for the mixed solution of 0.15 mM DNA with (a) 0, (b) 0.005, (c) 0.010, (d) 0.015, (e) 0.020, (f) 0.030, (g) 0.050 and (h) 0.200 mM PAHB. Combining with all the results above, it can be concluded that the DNA molecules start to interact with PAHB at extremely low concentration, then they are gradually condensed from partially to fully beyond C1 (0.013 mM), and the interaction between PAHB and DNA reaches saturation at C2 (0.080 mM). The C1 and C2 values coincide with the PAHB/DNA charge ratios (Z+/-) of 0.5 and 3.2, respectively. This result indicates that the condensation of the DNA chains already start below the charge neutralization point, suggesting that the PAHB molecules have strong ability to condense DNA. This could be attributed to the strong electrostatic bind of the multi-charged headgroups of

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PAHB with the phosphate groups of DNA as well as the strong hydrophobic interaction among the alkyl chains of PAHB molecules. Thus, the hexameric cationic surfactant PAHB is a high efficiency condensing agent for DNA. Interaction between PAHB and DNA. Isothermal titration microcalorimetry (ITC) was employed to determine the cmc value of PAHB in PBS (0.025 mM) and to investigate the thermodynamic behavior associated with the interaction between PAHB and DNA. Figure 3 illustrates the ITC profile of the observed enthalpy change (ΔHobs) against the final PAHB concentration for the titration of 0.5 mM PAHB into the 0.15 mM DNA solution and the corresponding dilution curve derived from the injection of identical amounts of the PAHB solution into buffer alone. There are two turning points in the PAHB/DNA curve at 0.018 mM and 0.080 mM, which are consistent with C1 and C2, respectively. Before C1, ΔHobs in the PAHB/DNA curve displays endothermic values (~ 26 kJ/mol) and significantly smaller than that (~ 45 kJ/mol) in the PAHB dilution curve. The endothermic values ΔHobs may come from the dissociation of the PAHB aggregates into monomers and the hydration of the PAHB molecules. The value of C1 is slightly lower than but very close to cmc, so the deviation of the PAHB/DNA curve from the PAHB dilution curve mainly results from strong exothermic electrostatic binding between the PAHB monomers and oppositely charged DNA. Beyond C1, both the curves start to decline and the PAHB/DNA curve gradually merges with the PAHB dilute curve. With the addition of more PAHB molecules, the exothermic enthalpy may be attributed not only from the electrostatic interaction between the headgroups of PAHB molecules and the phosphate groups in DNA, but also from the hydrophobic interaction among the alkyl chains of PAHB and between the alkyl chains of PAHB and the bases of 12


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DNA. In this region, the PAHB/DNA complexes undergo a series of structural rearrangements, including loose cluster-like aggregate, globules-on-a-string structure and globule aggregate. However, no significant variation in ΔHobs is reflected in the ITC curve during the transition process, probably because the structural transitions of PAHB/DNA complexes result from the gradual enhancement of hydrophobic interaction and the gradual decrease of electrostatic attraction. Thereafter, when CPAHB > C2, the ΔHobs value declines again, and the PAHB/DNA curve is very close to the dilution curve of PAHB in PBS and the two curves present the same changing situation. This phenomenon further confirms that the interaction between PAHB and DNA has been essentially reduced and reaches saturation, and the ΔHobs value of the PAHB/DNA curve beyond C2 is mainly contributed to the dilution of the PAHB aggregates. 50 45 40

Hobs (kJ/mol)

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Figure 3. The observed enthalpy change (ΔHobs) against the final PAHB concentration in the titrations of 0.5 mM PAHB into 0.15 mM DNA buffer solution (black) and 1 mM PBS buffer (red) at 25 °C. Fluorescence spectroscopy is also used to investigate the interaction of PAHB with DNA by titrating the PAHB solution into the premixed solutions of DNA and ethidium bromide (EtBr). EtBr, 13



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an aromatic planar cationic fluorophore (Scheme 1), is a cationic dye widely used as a probe for native DNA. The fluorescence intensity of the ethidium ion displays a dramatic increase (about 10-fold than EtBr itself) when it intercalates into base pairs of DNA. However, cationic agents added to the EtBr/DNA solutions will displace the intercalated EtBr from DNA/EtBr complexes and condense DNA chains, leading to the quenching of fluorescence intensity. Thus as long as a series of concentrated PAHB solutions are added into the DNA solutions at a fixed concentration, the decrease in probe emission intensity can be used to understand PAHB-DNA interaction and DNA conformation accordingly. Figure 4a shows the emission fluorescence spectra of EtBr with 0.15 mM DNA as a function of PAHB concentration. The emission spectra of free EtBr (black line) and the EtBr/DNA complexes (red line) are also included in this figure. Meanwhile, the maximum fluorescence intensity of the EtBr/DNA complexes in the absence or presence of PAHB as a function of PAHB concentration, and the macroscopic appearances of all the EtBr/DNA/PAHB solutions are showed in Figure 4b. The fluorescence intensity of EtBr starts to decrease with adding a very small amount of PAHB, then declines very sharply after C1 and changes to decrease gradually until C2, and remains constant beyond C2. These two critical points also agree with the C1 and C2 values determined by the other methods above. These results indicate that below C1, a small amount of EtBr initially existing in the DNA helix is displaced into the aqueous bulk by the PAHB monomers, and the PAHB monomers form complexes with unfolded DNA chains. Beyond C1, the electrostatic repulsion of the DNA negative charges is strongly weakened by the binding of cationic PAHB, leading to the DNA condensation. After the DNA chains are fully condensed, the main driving force for the binding of PAHB with the PAHB/DNA complexes becomes the hydrophobic interaction 14


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among the alkyl chains of PAHB, and between the alkyl chains of PAHB and the bases of DNA. Above C2, the emission intensity completely levels off, but does not fall to the initial value of EtBr alone, possibly because some EtBr molecules may still remain inside the DNA helix even though the interaction of the PAHB molecules with DNA has reached the saturation. This is similar to the situation for the cationic amphiphiles/DNA systems.38-39 It is worth noting that EtBr interact with cationic surfactant PAHB weakly, as would be expected for a cationic probe, because there is no obvious increase of the emission in the presence of the significant excessive PAHB. In addition, the maximum emission wavelength is gradually red-shifted from 589 nm to 593 nm below C1, but blue-shifted back to 586 nm beyond C1. The red shift of the emission peak further proves the dissociation of EtBr from the EtBr/DNA complexes by PAHB, while the blue shift of the emission peak demonstrates that the condensed PAHB/DNA structures and PAHB self-assembles provide hydrophobic microenvironment for free EtBr molecules. 22

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Figure 4. (a) Fluorescence emission spectra and (b) the maximum intensity of fluorescence emission of ethidium bromide (EtBr), the EtBr/DNA complex itself, and the EtBr/DNA complexes in the presence of PAHB at different PAHB concentrations. The insets are macroscopic appearances of all the samples. The concentration of DNA was 0.15 mM and the concentrations of PAHB are 0, 0.001, 0.003, 0.005, 0.008, 0.010, 0.020, 0.030, 0.050, 0.080, 0.100, 0.300 and 0.500 mM, respectively. 15



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Considering all the above experimental results and the transitions of PAHB conformations discussed in our previous work,32-35 the mechanism for the condensation process of DNA by PAHB is proposed in Figure 5. Initially, the double-stranded DNA molecules exist in a rather extended coiled structure without PAHB, whereas the PAHB molecules composed of six amphiphilic moieties present a star-shaped stretching molecular conformation at low PAHB concentration due to the rigid spacers and the strong intramolecular electrostatic repulsion among the charged headgroups. When CPAHB < C1, the PAHB monomers bind to the DNA chains due to the strong electrostatic interaction between the highly charged headgroups of PAHB and the phosphate groups of DNA, but they are not enough to change the extended coil structure at this stage. Between C1 and C2, the binding of more PAHB molecules with DNA induces the conformation transition of the DNA chains, and the chains are partially condensed into cluster-like aggregate, then globules-on-a-string structure, and fully condensed globule in final. In this stage, due to the binding of more cationic PAHB molecules with anionic DNA, the DNA chains with weakened charges tend to associate with each other, moreover, the hydrophobic association among the hydrophobic chains of the PAHB molecules already bound on DNA chains further promotes the association of the DNA chains. Firstly, with a less amount of PAHB molecules bound on DNA, the hydrophobic association of the stretched PAHB hydrophobic chains and slightly reduced DNA charges lead to the formation of the loose cluster-like aggregates by cross-linking several DNA chains. While more and more PAHB molecules are bound on the DNA chains, significantly weakened charges of the DNA chains and significantly enhanced hydrophobic interaction among the alkyl chains of PAHB on the DNA chains promote the association of the DNA chains, and may also promote the transition of PAHB conformation from stretched star-shaped to 16


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claw-like conformation and compact pyramid-like conformation. As a result, the PAHB/DNA complexes transfer to globules-on-a-string structure, and then spherical aggregates. Finally the DNA chains are condensed completely. The value of C1 is very close to cmc, indicating that the enhancement of PAHB aggregation ability of greatly promotes the DNA condensation, which is driven by the strong hydrophobic interaction among the alkyl chains of PAHB. Above C2, the size of the condensed spherical DNA aggregates almost does not change anymore, where both the electrostatic interaction between PAHB and DNA and the hydrophobic interaction between the alkyl chains of PAHB and the bases of DNA reach saturation. Poly(amido amine) (PAMAM) dendrimer as a typical star-like polymers has the similar architecture to star-shaped oligomeric surfactant PAHB, and is also widely used to condense DNA molecules.40-41 The process of DNA condensation by the highly charged dendrimer is cooperative via attractive electrostatic interactions and kinetically controlled, and a rich variety of aggregate morphologies strongly depends on the dendrimer generation, i.e. charge density and size. In comparison, the cooperative binding between DNA and PAHB is driven by both attractive electrostatic interaction and hydrophobic interaction. The oppositely charged DNA can greatly promote the transitions in the molecular configuration and aggregate structure of PAHB, inducing the binding to take place at extremely low PAHB concentration. In turn, the strong self-assembly ability of PAHB facilitates the crosslink between the PAHB/DNA complexes and leads to the DNA condensation. Moreover, the aggregation behavior of PAHB can influence the condensation process by adjusting PAHB concentration.

17



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CPAHB > C1

+ DNA

PAHB

N N O N N H

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N

N N O O

O N HN O N

N

Extended DNA Coil

N

N N H

O NH N 6Br

Fully Condensed State

Partially Condensed State

Figure 5. Illustration of the mechanism of PAHB-induced DNA condensation. Effect of PAHB on Secondary Structure and Cytotoxicity of DNA. Corresponding to the different condensation states of DNA by PAHB, the secondary structure of DNA and the cytotoxocity of the PAHB/DNA complexes have also been studied. Figure 6 shows the circular dichroism spectra of pure DNA and the PAHB/DNA complexes at different PAHB concentrations. The CD spectrum of the pure DNA shows a longwave positive band at 278 nm corresponding to π-π base packing, and a shortwave negative band at 243 nm corresponding to helicity. The positive and negative bands intersect at the absorption maximum of 260 nm. The above characteristics belong to the B-form of DNA. Upon the addition of PAHB into the DNA solution, all the CD spectra of the PAHB-bound DNA show similar profiles to the pure DNA, suggesting that the secondary structure of DNA, i.e., B-form, does not change with the condensation of DNA conformation by PAHB. However, as clearly observed in the inset with increasing the PAHB concentration, the magnitude of the positive band firstly starts to decrease beyond 0.020 mM and then increases to a constant value beyond 0.080 mM, while correspondingly the magnitude of the negative band starts to decline and then also keeps a constant. These two breakpoints are also consistent with C1 and C2. The change of positive bands 18


Page 19 of 29

below C1 indicates that before the DNA chain is condensed, the intercalation of PAHB monomers occupies some space in the helical structure to make the π-π base packing of DNA looser. Between C1 and C2, both the positive and negative bands increase obviously, suggesting that the packing of the DNA bases is enhanced and the double helix becomes tighter in the condensed aggregates. When the interaction between PAHB and DNA reach saturation (CPAHB > C2), the DNA chains remain the distance between the bases of DNA and the helical structure of DNA still does not change. Therefore, the DNA secondary structure does not display obvious variations due to DNA condensation. 8

withPAHB 0.001mM 0.003mM 0.005mM 0.010mM 0.020mM 0.050mM 0.080mM 0.100mM 0.200mM 0.500mM

6 4 2 0

1.2

-2

1.1

274 nm 243 nm

Normalized [

CD (mdeg)

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 -6 -8

1.0

0.9

0.8

1E-3

-10 220

240

260

0.01

CPAHB (mM)

280

0.1

300

320

CPAHB (mM)

Figure 6. Circular dichroism spectra of 0.15 mM DNA with different concentrations of PAHB at pH 7.4 and 25 °C. Inset is normalized ellipticity at 274 nm (black) and 243 nm (red) ([θ]free DNA/[θ]complex DNA)

as a function of PAHB concentration.

The in vitro cytotoxicity of PAHB/DNA with different DNA condensation stages was further evaluated against typical human breast tumor cell lines (MCF-7 cells) and immortalized human epidermal cell lines (HaCaT cells) by MTT assay, a general protocol. The concentration of DNA was chosen as 0.03 mM in this section, which is 5 times lower than that discussed above, because the 0.15 mM DNA precipitates in the presence of culture medium containing glucose, amino acid and salts of high concentration. The DNA of lower concentration can also be condensed to ~ 200 nm 19



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when the PAHB/DNA charge ratio Z+/- is 1.0, and the complete condensation can be achieved slightly above neutralization point (Fig. S1). Figure 7a shows the MCF-7 cell viabilities for the PAHB/DNA complexes at 0.03 mM DNA after 24 h and 48 h incubation as a function of Z+/-. After 24 h incubation, the PAHB/DNA mixtures do not display cytotoxicity and can even accelerate the growths of MCF-7 cell lines, no matter if the DNA has been partially or completely condensed over the whole range of the PAHB/DNA charge ratio. Using a longer incubation time, i.e., 48 h, it was found the cytotoxicity becomes larger when charge ratio is above 3.2. It can be stated that below C2 the PAHB/DNA mixtures with incompletely condensed conformation of DNA does not show cytotoxicity, which could be due to the presence of excessive DNA. Meanwhile, the parallel treatment of the complexes to a HaCaT cell for 48 h incubation showed the similar effects on inhibition of viability to the human breast cancer cells (Figure 7b). This excellent property in terms of cytotoxicity is probably because the PAHB concentration required to condense is very low. Moreover, most of the PAHB molecules locate in the stable PAHB/DNA complexes due to the strong interaction between DNA and PAHB, while the concentration of unbound PAHB molecules is extremely low. Especially when Z+/- < 3.0, the theoretical free PAHB concentration is below 0.01 mM, at which PAHB itself is also nontoxic to mammalian cells.36 It may be concluded that the level of cellular damage inflicted by PAHB/DNA complexes depends on the amount of the free PAHB, and the longer incubation time could result in the higher level of cellular uptake and the stronger cytotoxicity.

20


140

MCF-7 24 h MCF-7 48 h

40

ol 0. 2

on tr

6. 0

4. 0

3. 2

2. 0

1. 2

0. 8

0

Charge Ratio (Z+/-)

C

0. 4

0 ol

20

0. 2

20

6. 0

40

60

4. 0

60

80

3. 2

80

100

2. 0

Cell viability (%)

100

on tr

HaCaT 48 h

120

120

C

(b)

1. 2

(a)

0. 8

140

Cell viability (%)

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


0. 4

Page 21 of 29

Charge Ratio (Z+/-)

Figure 7. Cell viability of (a) MCF-7 cells after 24 h (black) and 48 h (red) incubation and (b) HaCaT cells after 48 h incubation with the PAHB/DNA mixed solutions at 0.03 mM DNA and different PAHB/DNA charge ratios (Z+/-).

CONCLUSION The interactions between a star-shaped hexameric cationic ammonium surfactant PAHB and calf thymus DNA and the resultant condensation of DNA have been investigated. Along with the binding between cationic PAHB molecules of multiple amphiphilic units and anionic DNA, the DNA chains with the weakened charges tend to associate with each other, and the hydrocarbon chains of the PAHB molecules bound on DNA chains also tend to associate with each other. These two effects promote the condensation of the DNA chains. As more and more PAHB molecules are bound on the DNA chains, the PAHB/DNA complexes sequentially form loose cluster-like aggregates, globules-on-a-string structure, and spherical aggregates, accompanying with the gradual conformation changes of both DNA and PAHB. Finally the DNA chains are completely condensed. Although the DNA conformation is significantly changed by PAHB, the DNA secondary structure does not display obvious variations and the PAHB/DNA complexes does not show cytotoxicity when 21



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DNA is partially condensed. Therefore the cationic star-shaped oligomeric surfactant is a potential condensing agent for gene transfection. The unique DNA condensation process and excellent property of PAHB/DNA complexes are attributed to the transitions in molecular configuration and aggregate structure of star-shaped oligomeric surfactant driven by the highly cooperative binding between DNA and these special surfactants. These results indicate that the increment of the oligomerization degree in surfactant structures is beneficial to improve the efficiency of surfactants in condensing DNA while weaken the cytotoxicity and the damage of the DNA structures in the condensation. This work should be helpful for designing more effective but less toxicity condensation agents to DNA. ASSOCIATED CONTENT Supporting Information The variation of the ζ-potential and the size of 0.03 mM DNA against the PAHB concentration and the PAHB/DNA charge ratio. This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (S. Wang) * E-mail: [email protected] (Y. Wang) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 22


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are grateful for financial supports from National Natural Science Foundation of China (Grants 21327003, 21603239). REFERENCES (1) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; Söderman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; García Rodríguez, C. L.; Guédat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P. Gemini Surfactants: New Synthetic Vectors for Gene Transfection. Angew. Chem. Int. Ed. 2003, 42, 1448-1457. (2) Chittimalla, C.; Zammut-Italiano, L.; Zuber, G.; Behr, J. Monomolecular DNA Nanoparticles for Intravenous Delivery of Genes. J. Am. Chem. Soc. 2005, 127, 11436-11441. (3) Verma, I. M.; Somia, N. Gene Therapy - Promises, Problems and Prospects. Nature 1997, 389, 239-242. (4) Dowty, M. E.; Williams, P.; Zhang, G.; Hagstrom, J. E.; Wolff, J. A. Plasmid DNA Entry into Postmitotic Nuclei of Primary Rat Myotubes. Proc. Natl. Acad. Sci. USA 1995, 92, 4572-4576. (5) Giacca, M.; Zacchigna, S. Virus-Mediated Gene Delivery for Human Gene Therapy. J. Controlled Release 2012, 161, 377-388. (6) Ibraheem, D.; Elaissari, A.; Fessi, H. Gene Therapy and DNA Delivery Systems. Int. J. Pharm. 2014, 459, 70-83. (7) Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Delivery. Chem. Rev. 2009, 109, 23



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(32) Hou, Y.; Han, Y.; Deng, M.; Xiang, J.; Wang, Y. L. Aggregation Behavior of a Tetrameric Cationic Surfactant in Aqueous Solution. Langmuir 2010, 26, 28-33. (33) Wu, C.; Hou, Y.; Deng, M.; Huang, X.; Yu, D.; Xiang, J.; Liu, Y.; Li, Z.; Wang, Y. L. Molecular Conformation-Controlled Vesicle/Micelle Transition of Cationic Trimeric Surfactants in Aqueous Solution. Langmuir 2010, 26, 7922-7927. (34) Fan, Y.; Hou, Y.; Xiang, J.; Yu, D.; Wu, C.; Tian, M.; Han, Y.; Wang, Y. L. Synthesis and Aggregation Behavior of a Hexameric Quaternary Ammonium Surfactant. Langmuir 2011, 27, 10570-10579. (35) Fan, Y.; Wu, C.; Wang, M.; Wang, Y. L.; Thomas, R. K. Self-Assembled Structures of Anionic Hydrophobically Modified Polyacrylamide with Star-Shaped Trimeric and Hexameric Quaternary Ammonium Surfactants. Langmuir 2014, 30, 6660-6668. (36) Zhou, C.; Wang, F.; Chen, H.; Li, M.; Qiao, F.; Liu, Z.; Hou, Y.; Wu, C.; Fan, Y.; Liu, L.; Wang, S.; Wang, Y. L. Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants. ACS Appl. Mater. Interfaces 2016, 8, 4242-4249. (37) Cao, M.; Deng, M.; Wang, X.; Wang, Y. L. Decompaction of Cationic Gemini Surfactant-Induced DNA Condensates by β-Cyclodextrin or Anionic Surfactant. J. Phys. Chem. B 2008, 112, 13648-13654. (38) Rodríguez-Pulido, A.; Ortega, F.; Llorca, O.; Aicart, E.; Junquera, E. A Physicochemical Characterization of the Interaction between DC-Chol/DOPE Cationic Liposomes and DNA. J. Phys. Chem. B 2008, 112, 12555-12565. 27



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Table of Contents artwork

CPAHB > C1

+ DNA

PAHB

N N O N N H

N

N N O O

O N HN O N

N

Extended DNA Coil

N

N N H

O NH N 6Br

Fully Condensed State

Partially Condensed State

29


DNA Condensation Induced by a Star-Shaped Hexameric Cationic

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