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Langmuir 2006, 22, 3735-3743

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Molecular Mechanism and Thermodynamics Study of Plasmid DNA and Cationic Surfactants Interactions De-Min Zhu* and Robert K. Evans Biologics and Vaccines, Pharmaceutical Research and DeVelopment, Merck Research Laboratories, Merck & Co., Inc., West Point, PennsylVania 19486 ReceiVed August 8, 2005. In Final Form: January 18, 2006 The molecular mechanism and thermodynamics of the interactions between plasmid DNA and cationic surfactants were investigated by isothermal titration calorimetry (ITC), dynamic light scattering, surface tension measurements, and UV spectroscopy. The cationic surfactants studied include benzyldimethyldodecylammonium chloride, benzyldimethyltetradecylammonium chloride, cetylpyridinium chloride, and cetyltrimethylammonium chloride. The results indicate a critical aggregation concentration (cac) of a surfactant: above the cac the surfactant forms aggregates with plasmid DNA; below the cac, however, there is no detectable interaction between DNA and surfactant. Surfactants with longer hydrocarbon chains have smaller cac, indicating that hydrophobic interaction plays a key role in DNAsurfactant complexation. Moreover, an increase in ionic strength (I) increases the cac but decreases the critical micellization concentration (cmc). These opposite effects lead to a critical ionic strength (Ic) at which cac ) cmc; when I < Ic, cac < cmc; when I > Ic, DNA does not form complexes with surfactant micelles. In the interaction DNA exhibits a pseudophase property as the cac is a constant over a wide range of DNA concentrations. ITC data showed that the reaction is solely driven by entropy because both ∆H° (∼2-6 kJ mol-1) and ∆S° (∼70-110 J K-1 mol-1) have positive values. In the complex, the molar ratio of DNA phosphate to surfactant is in the range of 0.63-1.05. The reaction forms sub-micrometer-sized primary particles; those aggregate at high surfactant concentrations. Taken together, the results led to an inference that there is no interaction between surfactant monomers and DNA molecules and demonstrated that DNA-cationic surfactant interactions are mediated by the hydrophobic interactions of surfactant molecules and counterion binding of DNA phosphates to the cationic surfactant aggregates.

Introduction The complexation of DNA with cationic surfactants induces condensation and aggregation of DNA to form sub-micrometersized particles.1-6 These complexes have been implicated as nonviral gene delivery vehicles in various vaccines and gene therapy approaches.6-9 Cationic surfactant induced DNA and RNA precipitation has also been employed for plasmid DNA purification and RNA extraction.10-16 However, the molecular mechanism of the interaction between plasmid DNA and cationic * To whom correspondence should be addressed. Phone: (215) 6524948. E-mail: [email protected]. (1) Mel’nikov, S. M.; Sergryev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401-2408. (2) Mel’nikov, S. M.; Sergryev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951-9956. (3) Dauty, E.; Remy J.-S.; Blessing T. J. Am. Chem. Soc. 2001, 123, 92279234. (4) Spink, C. H.; Chaires J. B. J. Am. Chem. Soc. 1997, 119, 10920-10928. (5) Lleres, D.; Dauty E.; Behr, J.-P.; Me´ly, Y.; Duportail, G. Chem. Phys. Lipids 2001, 111, 59-71. (6) Vijayanathan, V.; Thomas, T.; Thomas, T. J. Biochemistry 2002, 41, 1408514094. (7) Clamme, J. P.; Bernacchi, S.; Vuilleumier, C.; Duporttail, G.; Me´ly, Y. Biochim. Biophys. Acta 2000, 1467, 347-361. (8) Bell, P. C.; bergsma, M.; Dolbnya, I. P.; Brass, W.; Stuart, M. C. A.; Rowan, A. E.; Feiters, M. C.; Engberts, J. B. F. N. J. Am. Chem. Soc. 2003, 125, 1551-1558. (9) Woude, I.; Wagenaar, A.; meekel, A. A. P.; Beest, M. B. A.; Rutters, M. H. J.; Engberts, J. B. F. N.; Hoekstra, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1160-1165. (10) Lander, R. J.; Winters M. A.; Meacle, F. J.; Buckland B. C.; Lee, A. L. Biotechnol. Bioeng. 2002, 79, 776-764. (11) Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. Biotechniques 1991, 11, 298-300. (12) McLoughlin, D. M.; O’Brien, J.; McManus, J. J.; Gorelov, A. V.; Dawson, K. A. Bioseparation 2000, 9, 307-13. (13) Allers, T.; Lichten, M. Nucleic Acids Res. 2000, 28, e6. (14) Macfarlane, D. E.; Dahle, C. E. J. Clin. Lab. Anal. 1997, 11, 132-139. (15) Hamel A. L.; Wasylyshen, M. D.; Nayar, G. P. J. Clin. Microbiol. 1995, 33, 287-291. (16) Macfarlane, D. E.; Dahle, C. E. Nature 1993, 362, 186-188.

surfactants is not well understood. For example, it is not clear whether the interaction is driven by adsorption of cationic surfactant monomers to the phosphate groups of DNA through static charge-charge attraction or by the interaction of multiply charged surfactant aggregates with DNA phosphates. Recently we developed an HIV DNA vaccine formulation for preclinical and clinical studies containing plasmid DNA with HIV-1 gene constructs, a nonionic block copolymer as the adjuvant, and cationic surfactants alkylbenzyldimethylammonium chloride, known as benzalkonium chloride (BAK), to stabilize the particles and to drive the association of plasmid DNA to the polymer particles.17-19 We found that DNA and BAK interactions in the formulation play key roles in vaccine stability and immunogenicity in nonhuman primates.17 These results prompted us to investigate the fundamental mechanism of DNA/cationic surfactant interactions. Our results consistently indicate that there exists a critical aggregation concentration (cac) for the surfactant interaction (17) Evans, R. K.; Zhu, D.-M.;. Casimiro, D. R.; Nawrocki, D. K.; Mach, H.; Troutman, R. D.; Tang, A.; Wu, S.; Chin, S.; Ahn, C.; Isopi, L. A.; Williams, D. M.; Xu, Z.; Shiver, J. W.; Volkin, D. B. J. Pharm. Sci. 2004, 93, 1924-1939. (18) Casimiro, D. R.; Chen, L.; Fu, T.-M.; Evans, R. K.; Caulfield, M. J.; Davies, M.-E.; Tang, A.; Chen, M.; Huang, L.; Harris, V.; Freed, D. C.; Wilson, K. A.; Dubey, S.; Zhu, D-M.; Nawrocki, D.; Mach, H.; Troutman, R.; Isopi, L.; Williams, D.; Hurni, W.; Xu, Z.; Smith, J. G.; Wang, S.; Liu, X.; Guan, L.; Long, R.; Trigona, W.; Heidecker, G. J.; Perry, H. C.; Persaud, N.; Toner, T. J.; Su, Q.; Liang, X.; Youil, R.; Chastain, M.; Bett, A. J.; Volkin, D. B.; Emini, E. A.; Shiver, J. W. J. Virol. 2003, 77, 6305-6313. (19) Shiver, J. W.; Fu, M.; Chen, L.; Casimiro, D. R.; Davies, M.; Evans, R. K.; Zhang, Z.; Simon, A. J.; Trigona, W. L.; Dubey, S. A.; Huang, L.; Harris, V. A.; Long, R. S.; Liang, X.; Handt, L.; Schleif, W. A.; Zhu, L.; Freed, D. C.; Persaud, N. V, Guan, L.; Punt, K. S.; Tang, A.; Chen, M.; Wilson, K. A.; Collins, K. B.; Heidecker, G. J.; Perry, H. C.; Joyce, J. G.; Grimm, K. M.; Cook, J. C.; Keller, P. M.; Kresock, D. S.; Mach, H.; Troutman, R. D.; Isopi, L. A.; Williams, D. M.; Xu, Z.; Bohannon, K. E.; Volkin, D. B.; Montefiori, D. C.; Miura, A.; Krivulka, G. R.; Lifton, M. A.; Kuroda, M. J.; Schmitz, J. E.; Letvin, N. L.; Caulfield, M. J.; Bett, A. J.; Youil, R.; Kaslow, D. C.; Emini, E. A. Nature 2002, 415, 331-335.

10.1021/la052161s CCC: $33.50 © 2006 American Chemical Society Published on Web 03/21/2006

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with DNA, and there is no detectable interaction below the cac. The cac increases with the increase in the ionic strength while the critical micelle concentration (cmc) decreases with the increase in the ionic strength. The opposite responses of the cac and the cmc to the changes of ionic strength resulted in a critical ionic strength (Ic) at which cac ) cmc. When I < Ic, cac < cmc, and when I > Ic, the surfactants form micelles without DNA association. The thermodynamic study revealed that the DNAcationic surfactant interaction is an entropy-driven reaction where hydrophobic interaction of the surfactant molecules plays a key role in the formation of the DNA-surfactant complex. These results for the first time show that DNA/cationic surfactant interactions are mediated by surfactant aggregation and DNA counterion binding and that the surfactant monomers do not form complexes with DNA. Experimental Section Materials. Plasmid DNA of molecular size 6.4 kb used was the same as that reported in refs 17-19. In brief, a synthetic gene for Gag from HIV-1 CAM-1 was constructed using codons frequently used in humans.12,14 The gene was inserted into the V1Jns plasmid (V1Jns-gag) under the control of the human cytomegalovirus (hCMV)/human intron A promoter and bovine growth hormone terminator.2 Benzalkonium chloride (a mixture of several homologues of different hydrophobic chain length) was purchased from Spectrum Chemical (New Brunswick, NJ): benzyldimethyldodecylammonium chloride (BAK12), benzyldimethyltetradecylammonium chloride (BAK14), and cetyltrimethylammonium chloride (CTAC) from Fluka (Milwaukee, WI); cetylpyridinium chloride (CPC) from Zeeland Chemical (Zeeland, MI). Detection of DNA/BAK Interaction by Isothermal Titration Calorimetry (ITC). The interactions of DNA and cationic surfactants were detected by MicroCal VP-ITC (Northampton, MA) at 25 °C. DNA in sodium phosphate buffered saline (PBS, 6 mM phosphate, 150 mM NaCl, pH 7.2) at 0.1 mg/mL was loaded into the sample cell (1.44 mL), and a surfactant solution (5-25 mM) in phosphatebuffered saline (PBS) was loaded into the injector (0.3 mL). In the case of injecting DNA into BAK solutions, a surfactant solution at a designated concentration was loaded into the sample cell, and DNA was loaded into the injector. The injection volume was set at 1-2 µL/injection with the rate of 0.5 µL/s. The titration control was done by injecting surfactant or DNA into PBS in the sample cell. The signal of the control was subtracted to obtain the net thermal response of DNA/BAK reaction. Assay for Free BAK and Free DNA Concentrations in DNABAK Mixture. Free BAK was separated from DNA-bound BAK by centrifugation. A 1 mL aliquot of the DNA-BAK mixture was centrifuged at 25 °C by a Beckman ultracentrifuge for 1 h at 440000g force. DNA with DNA-bound BAK was completely pelleted, while the free BAK was retained in the supernatant. The concentration of BAK was determined by UV absorbance in the range of 230-300 nm combined with second derivative analysis. To determine the free DNA concentration in the mixture, the samples were centrifuged at 21700g force for 30 min to pellet DNA-BAK precipitates and retain the unbound DNA in the supernatant.17 The supernatant was then diluted for 260 nm UV absorbance (A260) determination. A dilution factor (f) was selected to make A260 < 1. Free DNA concentration was calculated by the following equation: CDNA(mg/mL) ) (A260f)/ , where  is the extinction coefficient of DNA which equals 20 mL/mg at 260 nm. Determination of Surface Tension of Surfactant Solutions. The surface tension of the surfactant solutions in the presence and absence of DNA was determined manually using a surface tension drop-volume apparatus at 25 °C.20 The diameter of the pipet used to determine the volumes of the drops was calibrated with distilled water. (20) Zhu, D.-M.; Zhao, G-.X. Colloids Surf. 1990, 49, 269-579.

Zhu and EVans Determination of Particle Size and Light Scattering Intensity (KCps) of the DNA-Surfactant Complex. The hydrodynamic particle size and light scattering intensity of the DNA-surfactant complex was determined by laser dynamic light scattering (Malvern System 4700c) at room temperature at 90°. The argon laser power was set at 0.25-0.4 mW, and the aperture was at 100-300. The mean value of KCps and particle size calculated by Cumulants were from five measurements. Fluorescent Microscopy Imaging of Particles. DNA-surfactant particles were imaged by an Olympus IX71 microscope that was equipped with a Spot digital camera. DNA fluorescent probe PicoGreen (Molecular Probes, Eugene, OR) was added to the mixture (1:1000 (v/v)) to label the DNA. A 3 µL aliquot of sample was placed on a slide and covered by a coverslip sealed with nail polish. A 100× objective and an excitation/emission cube for FITC were used to acquire the fluorescent images of the particles.

Results Critical Aggregation Concentration of Cationic Surfactants To Interact with DNA. We first set out to study the concentration dependence of the interaction of BAK and DNA. Isothermal titration calorimetry (ITC) was used to monitor the interaction. In this experiment, a solution of BAK12 in PBS was placed into the sample cell of the calorimeter, and a DNA solution at 0.5 mg/mL was repetitively injected into the sample cell by the microinjector of the device. Figure 1 shows the thermal responses corresponding to the DNA injections. When the BAK12 concentration in the sample cell was below 0.5 mM, there was no interaction signal. However, a clear thermal signal was seen when the BAK12 concentration was at or higher than 0.6 mM. These results indicated a 0.5-0.6 mM concentration threshold or critical aggregation concentration2,21-23 of BAK12 for its interaction with plasmid DNA To confirm the cac of BAK12 observed from Figure 1, we performed ITC studies by injecting BAK12 into a 0.1 mg/mL solution of plasmid DNA and injecting the same BAK12 solution in PBS as the control. The upper panel of Figure 2 shows the thermal response of each injection of 2 µL of 10 mM BAK12 into the DNA solution; and the lower panel shows the integrated thermal response of each injection. The results showed that there was no net DNA-BAK12 interaction until the cumulative concentration of BAK12 in the sample cell reached 0.55 mM, which is in an excellent agreement with the results in Figure 1. With a closer look, the thermal responses shown in the upper panel of Figure 2 can be divided into three groups along the titration time. In the first group (0-135 min), a transient endothermic signal was seen instantly after BAK was injected, and the signal rapidly converted to exothermic (inset A of Figure 2). The transient endothermic signal is likely due to the reaction between DNA and the “high” concentration of BAK12 at the injection site right after the injection of BAK12. But the BAK12DNA complex formed at the injection site reversibly dissociated with an exothermic signal when BAK12 and the complex diffused throughout the sample cell. The exothermic part of a signal shown in inset A was slightly greater than its endothermic part due to the exothermic contribution from BAK12 dilution to the signal, as it will be seen from the third group. In the second group (135-250 min), the signals were solely endothermic due to the formation of the DNA-BAK12 complex. The third group (after 250 min) featured small negative signals from BAK12 dilution(21) Lindman, B.; Thalberg, K. In Interaction of Surfactants with Polymers and Proteins; Goddard E. D., Ananthapadmamabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; pp 203-276. (22) Guillot, S.; Delsanti, M.; De´sert, S.; Langevin, D. Langmuir 2003, 19, 230-237. (23) Konop, A. J.; Colby, P. H. Langmuir 1999, 15, 58-65.

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Figure 1. Thermal responses of plasmid DNA titration to different concentrations of BAK12 in PBS. DNA concentration in the injector was 0.5 mg/mL. The injection volume was 1 µL/injection with an injection rate at 0.5 µL/s. The concentrations of BAK12 in the sample cell were as follow: (A) 0; (B) 0.5 mM; (C) 0.6 mM; (D) 1.0 mM.

induced responses upon each injection, indicating the depletion of free DNA in the sample cell. We next used ITC to study the interactions of plasmid DNA and the other two cationic surfactants, CPC and CTAC. Figure 3 and Figure 4 show the net thermal responses of CPC and CTAC interaction with DNA when the surfactants were titrated into DNA solution in the sample cell. Surfactant dilution-induced thermal responses are shown in the insets of the figures and are subtracted from the DNA interaction signals. The integration curves exhibited a 0.03 mM cac for CPC (Figure 3) and a 0.055 mM cac for CTAC (Figure 4). Similarly, the titration of DNA into CPC showed that the cac of CPC was in the range of 0.03-0.04 mM (ITC data not shown). Moreover, in Figure 3 and Figure 4 the thermal responses presented a second reaction starting at 0.22 mM CPC and 0.13 mM CTAC cumulative concentrations, respectively. The second reaction was also seen from the integration curve of Figure 2. Particle size studies using dynamic light scattering and fluorescence microscopy found that the second reaction was the aggregation of the primary DNAsurfactant particles (0.1-0.3 µm) to form larger precipitates (>1 µm). The insets of Figure 3 and Figure 4 were the signals from the control titrations of CPC and CTAC into PBS in the absence of DNA. The endothermic signals belonged to the dissociation of the micelles into monomers when the concentrated (5 mM) surfactants were injected into the buffer. After seven to eight injections, the thermal responses reduced back to the baseline, indicating that the later titrated-in micelles stopped dissociating, or the cumulative concentration of the surfactants in the sample cell reached the cmc of the surfactants. Therefore, the cmc of CPC (0.06 mM) and CTAC (0.11 mM) were obtained from those

ITC responses. These values are comparable to the literature values at similar ionic strength.24 We have applied another method combining centrifugation and UV absorbance to confirm the cac of BAK12. To this end, the mixtures of 1 mg/mL DNA and 0-1.5 mM BAK12 were centrifuged (Experimental Section) to completely pellet DNA as well as DNA-associated BAK12. BAK12 retained in the supernatant represented the free BAK12 in the original mixture, and the concentration was determined by UV absorbance. The results showed (Figure 5) that there was no detectable BAK12 association with DNA below 0.5 mM of BAK12, as 100% of BAK12 remained in the supernatant. Remarkably, the free BAK12 level remained constant at 0.6 mM when the total concentration of BAK12 was higher than 0.6 mM. Therefore, this method identified 0.6 mM as the cac of BAK12. To further detect any possible association between DNA and BAK12 below cac, we prepared several mixtures containing 0.5-2 mg/ mL DNA but a constant 0.5 mM BAK12 that was just slightly lower than the cac. Those mixtures were centrifuged to pellet DNA and DNA-associated BAK12. The free BAK12 concentration in the supernatant was determined by UV absorbance. If BAK12 associates with DNA, higher DNA concentration will cost more BAK12 loss from the supernatant. However, ∼100% of BAK12 was detected in all of the supernatants, indicating that no BAK12 was associated with DNA (the inset of Figure 5). (24) van Os, N. M.; Haak, J. R.; Rupert L. A. M. Physico-Chemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier Science: Amsterdam, London, New York, Tokyo, 1993; pp 115, 142. (25) Desnoyers, J. E.; Perron G. In Handbooks of Surface and Colloid Chemistry; Birdi K. S., Eds.; CRC Press: Boca Raton, FL, 1997; p 143.

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Figure 2. Thermal responses of BAK12 titration to plasmid DNA in PBS. BAK12 at 10 mM was loaded into the injector, and 0.1 mg/mL plasmid DNA was loaded into the sample cell. The injection volume was 2 µL/injection at the rate of 0.5 µL/s. Upper panel, the raw thermal signals upon each injection; lower panel, the integrated thermal signals. Inset A, a closer look at the endothermic-exothermic transition of the initial thermal signals; inset B, BAK12 titration to PBS control.

It is known that the surface tension of a surfactant solution is a function of the monomer concentration of the surfactant and that aggregation of the surfactant molecules can be detected from the curve of surface tension vs the concentration of the surfactant. Therefore, we used surface tension measurements to study the interaction of BAK12 and DNA (Figure 6). In the absence of DNA, the transition point at 1.0 mM was the cmc of BAK12. In the presence of 0.1 mg/mL DNA, the cac was observed from a sharp transition of the curve between 0.5 and 0.6 mM BAK12; additional BAK12 above the cac did not further reduce the surface tension but resulted in a constant surface tension (36.5 mN/m), indicating that all the additional BAK12 above the cac interacted with DNA. When BAK12 concentration reached 0.9 mM, the second transition appeared as the surface tension declined again along with the increase in the concentration of BAK12 until the third transition occurred at 1.3 mM. These results indicate that the DNA and BAK12 interaction started at ∼0.55 mM; the free DNA was depleted from the mixture at 0.9 mM BAK12; and single component BAK12 micelles started to form at 1.3 mM BAK12. Interestingly, the 0.1 mg/mL concentration of the DNA used was equivalent to 0.3 mM of phosphates in DNA molecules; it incorporated ∼0.3-0.35 mM BAK12, suggesting an ∼1:1 PO4/BAK12 mole ratio in the complex, that was consistent with the mole ratio obtained from thermodynamic analysis that will be discussed later in this paper. Moreover, before the cac transition of BAK12, the surface tension curves of the solutions with and without 0.1 mg/mL DNA were highly concordant, further suggesting that there is no association of BAK12 to DNA below the cac. The surface tension of the solution

Zhu and EVans

Figure 3. Thermal responses of CPC titration to plasmid DNA in PBS. CPC at 5 mM was loaded into the pipet injector, and 0.1 mg/mL plasmid DNA was loaded into the sample cell. The injection volume was 2 µL/injection at the rate of 0.5 µL/s. Upper panel, the net thermal responses of CPC titration to DNA obtained by subtracting the control (CPC titration to PBS) signals (inset) from the responses; lower panel, integration of the net thermal responses of CPC titration to DNA.

with 0.5 mg/mL DNA also exhibited a transition point between 0.5 and 0.6 mM. However, slightly but noticeably the surface tension curve of the solution with 0.5 mg/mL DNA was underneath the other two curves, suggesting that DNA at 0.5 mg/mL had a measurable effect on the surface tension. Indeed, when free DNA was being depleted along with the addition of BAK12, the surface tension gradually increased until the second transition appeared at 1.9 mM. The slightly lower surface tension caused by higher DNA concentrations could be due to contamination by trace levels of other surfactants in the DNA bulk such as Triton X-100 and cetyltrimethylammonium bromide (CTAB), since these were used in the plasmid DNA purification process.10 The curve also showed that 0.5 mg/mL DNA (or 1.5 mM DNA phosphate) precipitated with 1.35 mM BAK12, again indicating an ∼1:1 PO4/BAK12 mole ratio in the complex. Similarly, with 0.01 mg/mL DNA in the solution, a transition was seen between 0.5 and 0.6 mM of BAK12, but the “flat” part is very narrow because of the low concentration of DNA used (data not shown). In summary, the surface tension data presented a 0.55 mM cac of BAK12. The values of cac and cmc of BAK12, BAK14, CPC, and CTAC are summarized in Table 1. The cac of BAK14, which has a longer hydrocarbon chain than BAK12, is about an order of magnitude lower than the cac of BAK12. It demonstrates that hydrophobic interaction or aggregation of the BAK molecules plays an essential role in DNA/BAK association. The above experiments used PBS to control ionic strength and the pH.

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Figure 5. Free BAK12 concentrations in the mixtures with plasmid DNA in PBS. The free BAK12 concentrations in the mixtures containing 1 mg/mL plasmid DNA and various concentrations of BAK12 were determined by centrifuge combined with UV absorbance second derivative analysis (Experimental Section). Inset, free BAK12 concentrations in the mixture of 0.5 mM BAK12 with DNA at various concentrations. The variability of the data is (3% at 95% CI.

Figure 4. Thermal responses of CTAC titration to plasmid DNA in PBS. CTAC at 5 mM was loaded into the pipet injector, and 0.1 mg/mL plasmid DNA was loaded into the sample cell. The injection volume was 2 µL/injection at the rate of 0.5 µL/s. Upper panel, the net thermal responses of CTAC titration to DNA obtained by subtracting the control (CTAC titration to PBS) signals (inset) from the responses; lower panel, integration of the net thermal responses of CTAC titration to DNA.

However we found that the cac of BAK12 in saline (150 mM NaCl) is identical to the cac in PBS. Therefore, the phosphate ions of the buffer did not effectively compete with DNA phosphate groups for interaction with the surfactant, likely due to the much greater chemical potential of the DNA phosphate backbone caused by the massive negative charge of DNA molecules. Effect of DNA Concentration on cac. The ITC data of DNA titrations into BAK12 (Figure 1) and BAK12 titrations into DNA (Figure 2) resulted in the same cac of BAK12. However, DNA concentrations in the sample cells were very different between the two experiments. For the former, after the first 1.0 µL injection of 0.5 mg/mL DNA into the 1.44 mL of the sample cell containing BAK12 solution, the concentration of DNA in the sample cell was 0.000 347 mg/mL; for the latter, the DNA concentration in the sample cell was 0.1 mg/mL. The 288-fold difference in DNA concentration did not alter the cac of BAK12. The surface tension measurement (Figure 6) also showed that additional 5-fold increase (from 0.1 to 0.5 mg/mL) in DNA concentration had no effect on the cac of BAK12. Those results suggest that DNA behaves like a separate phase in the reaction with the surfactant. Effect of Ionic Strength on cac. The endothermic ITC response in Figures 1-4 indicated that DNA-cationic surfactant interaction has a positive reaction enthalpy. It can be caused by the dissociation of the hydration water molecules and counterions (Na+, Cl-) from DNA and BAK12 when the complex formed.

Figure 6. Surface tension of BAK12 solution in the presence and the absence of plasmid DNA in PBS. Surface tension was determined by drop-volume method (Experimental Section). The average volume from three drops was used to calculate surface tension. The variability of data is (0.2 mN/m. Table 1. cac and cmc of BAK12, CPC, and CTAC in PBS cac (mM) cmc (mM) a

CPC

CTAC

BAK12

BAK14

0.030 0.06a

0.055 0.11a

0.55 1.0b

0.046 Not detected

Results from ITC data. b Results from surface tension measurement.

Therefore, solution ionic strength may affect the cac and the thermal dynamics of the reaction, as both hydration and counterion binding are functions of ionic strength. To study the effects of ionic strength on the cac, we performed an ITC study to determine the thermal response of DNA and BAK12 interaction at 2.1 mM NaClswhich is the lowest NaCl concentration in a 0.1 mg/mL DNA solution prepared from a 7.0 mg/mL DNA stock solution in saline. At 2.1 mM NaCl, the cac of BAK12 identified by ITC was 0.024 mM (ITC signals not shown). To further investigate the effect of ionic strength on the cac of BAK12, the cac at various NaCl concentrations was determined by light scattering. In this experiment, both scattered light intensity

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Zhu and EVans

Table 2. Effects of Ionic Strength on the cac and the cmc of BAK12 I ) 2.1 mM cac from LS (mM) cac from ITC(mM) cmca (mM) a

0.025 0.024 7.1

I ) 10 mM 0.075

I ) 25 mM

I ) 50 mM

0.15

0.25

I ) 150 mM 0.54 0.55 1.0

I ) 400 mM

I ) 500 mM

I ) 1000 mM

0.69

NDb

ND

0.53

0.30

cmc was obtained from the surface tension curve of BAK12 solutions. b ND, no cac was detectable.

Figure 7. Effect of ionic strength on the cac of BAK12. The light scattering intensity of the mixtures of 0.1 mg/mL plasmid DNA with various BAK12 concentrations at the given ionic strength were determined by dynamic light scattering. The light intensity of a mixture was divided by the intensity of the DNA control at the same ionic strength for normalization. Data represent mean ( standard deviation (SD) from five measurements. Inset, the critical ionic strength (Ic) defined by the meeting point of cmc and cac curves.

and particle size of the mixtures of 0.1 mg/mL DNA and different concentrations of BAK12 were determined. The scattered light intensity was used to determine the formation of DNA-BAK12 complex, because of its high sensitivity to the complex formation. Figure 7 shows the plots of the normalized scattered light intensity (SL) vs BAK12 concentrations. The complex formation was indicated by a very sharp increase in the scattered light intensity, and the sharp transition point of the curve defined the cac at given ionic strength. The cac values obtained from ITC and from light scattering experiments, as seen in Table 2, were nearly identical. These results demonstrate that the cac of BAK12 increases along with increasing ionic strength. This property of the cac is opposite to that of the cmc, because the increase in ionic strength always leads to a decrease in the cmc of an ionic surfactant. For example, the cmc of BAK12 declines from 7.1 mM at I ) 2.1 mM to 1.0 mM at I ) 150 mM (Table 2). The opposite effects of ionic strength on the cac and cmc led us to define a critical ionic strength (Ic) at which the cac equals the cmc. For BAK12, Ic, determined by the meeting point of cac and cmc curves vs ionic strength (inset of Figure 7), is 0.4 M. When I < Ic, the cmc is greater than the cac; when I > Ic, the cmc will be lower than an imaginary “cac”. Since the monomer concentration of the surfactant will remain at the cmc when the total concentration is greater than the cmc, the greater-than-cmc imaginary “cac” will not be achieved; i.e., DNA and surfactant micelles will not form complexes at I > Ic. Indeed, when the NaCl concentration was increased to 0.5-1.0 M, light scattering did not detect any DNA-BAK12 aggregates, even when BAK12 concentrations were 30-230% greater than the cmc (Figure 7 and Table 2). In the ionic strength study phosphate buffer was not used so that the lowest 2.1 mM ionic strength can be achieved.

Figure 8. Unbound BAK and DNA (inset) concentrations in the mixtures of plasmid DNA and mixed BAK at various concentrations in PBS. The free BAK and free DNA concentrations in DNA and BAK mixture were determined by centrifuge and UV absorbance, as described at Experimental Section. The variability of the data is (3% at 95% CI.

Interaction of DNA and Mixed BAK. The BAK used for the HIV DNA vaccine formulation17 is a mixture of BAK12, BAK14, and BAK16; therefore, we have studied the interactions between plasmid DNA and a BAK mixture (from Spectrum Chemical) that consisted of 67% BAK12, 24% BAK14, 8% BAK16, and trace amounts of BAK10 and BAK18. The cac of the mixed BAK in PBS in the presence of 0.1 mg/mL DNA determined by dynamic light scattering was 7.5 µM (plots not shown). Further study found that while the free DNA concentration decreases vs the addition of BAK (Figure 8 inset), the free BAK concentration increases along with the increase in the total BAK concentration until the free BAK concentration reaches a 0.5 mM plateau (Figure 8), which is 67-fold greater than the cac of the mixed BAK. This phenomenon is in sharp contrast to the constant free BAK12 concentration at the cac in the DNA-BAK12 mixture when the total concentration of BAK12 is greater than the cac. It raised a question: how could the free BAK concentration be greater than the cac of the mixed BAK? We propose the following mechanism to answer the question. In the BAK mixture, the more hydrophobic components with longer alkyl chains more preferably form aggregates with DNA than the less hydrophobic components. Therefore, when the total BAK concentration increases, the preferable incorporation of more hydrophobic BAK14 and BAK16 into the complexes causes more and more accumulation of free BAK12 in the solution. However, the upper limit of the total accumulation of free BAK will be ecac of pure BAK12, which is 0.55 mM in PBS. To test this hypothesis, three supernatants were prepared from the following three mixtures: (1) 5 mg/mL DNA + 0.85 mM BAK, (2) 5 mg/mL DNA + 0.45 mM BAK, and (3) 1 mg/mL DNA + 0.45 mM BAK. The total BAK concentration in the supernatant (or the free BAK in the original mixtures) was determined, and the compositions of the supernatants were analyzed by reverse-phase HPLC (Table 3). It was found that free BAK in all three supernatants consisted of >96% BAK12,

Plasmid DNA and Cationic Surfactant Interactions

Langmuir, Vol. 22, No. 8, 2006 3741

Table 3. Concentration and Composition of Unbound BAK in the DNA-BAK Mixturea mixture 0.45 mM BAKmix (control) 1 mg/mL DNA + 0.45 mM BAKmix 5 mg/mL DNA + 0.45 mM BAKmix 5 mg/mL DNA + 0.85 mM BAKmix a

free BAK BAK12 BAK14 BAK16 (mM) (%) (%) (%) 0.445 0.27 0.25 0.38

67.4 96.5 96.6 97.6

24.2 2.9 2.9 2.1

8.3 0.6 0.5 0.3

The compositions were determined by reverse phase HPLC.17

the particles in Figure 9 support the size ranges of both the primary particles and the aggregates determined by light scattering. Thermodynamic Analysis. We analyzed the ITC data to explore the stoichiometry of the complex. The standard reaction enthalpy ∆H°ph and ∆H°surf, were defined by the following equations:

∆H°ph ) ∆Hph/(CphV)

(1)

∆H°surf ) ∆Hsurf/((Csurf - cac)V)

(2)

where ∆Hph is the net reaction enthalpy when DNA in the sample cell of ITC is completely complexed by the surfactant titrant; Cph is the equivalent concentration of DNA phosphate and can be obtained by the approach Cph (mM) ) 3CDNA (mg/mL); similarly, ∆Hsurf is the net reaction enthalpy when surfactant in the sample cell is completely complexed by DNA titrant; Csurf is the concentration of the surfactant; and V is the volume of the sample cell of the ITC (1.44 mL). The mole ratio (β) of DNA phosphate (PO4-D) to surfactant (S) in the complex can be calculated by

β ) ∆H°surf/∆H°ph Figure 9. Particle size of the plasmid DNA-surfactant complex in PBS. Particle size was determined by dynamic light scattering (Experimental Section). Insets, fluorescent images of DNA-CPC particles at two CPC concentrations. The value represents the mean ( SD from five measurements.

cac in the presence of NaCl in the solution. In the absence of DNA, γ of cationic micelles is in the range of 0.5-0.8;27 in the presence of DNA, it should be a number ,1, as Cl- ions in the aggregates are largely replaced by DNA phosphate. Therefore, in eq 4, (26) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry; Dekker: New York, 1997; pp 361-362. (27) van Os, N. M.; Haak, J. R.; Rupert, L. A. M. Physicochemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier Science: Amsterdam, London, New York, Tokyo 1993; pp 118-119.

3742 Langmuir, Vol. 22, No. 8, 2006

Zhu and EVans

Table 4. Thermodynamic Parameters of Cationic Surfactant and DNA Interaction and Mole Ratios of DNA Phosphate to the Surfactants in the Complexes

∆H°ph (kJ mol-1) ∆H°surf (kJ mol-1) ∆G° (kJ mol-1) ∆S° (J K-1 mol-1) β a

CPC (I ) 150 mM)

CTAC (I ) 150 mM)

BAK12 (I ) 150 mM)

BAK12 (I ) 2.1 mM)

Mixed BAK (I ) 150 mM)

2.64 2.59 -25.1 92.9 0.98

3.02 1.89 -24.3 87.8 0.63

2.35 1.98 -18.6 69.1 0.84

5.63 5.91 -26.2 107.8 1.05

1.20a

β value in the complex of DNA and the mixed BAK was calculated from the results shown in the Inset of Figure 8.

|γ ln aCl| , |ln cac|; thus, eq 4 is simplified to

∆G°surf ) RT ln cac

(5)

When the interaction of surfactant (S) and DNA phosphate (PO4D) is expressed as

S + β PO4-D ) S‚PO4-Dβ ∆H°, ∆G°, and ∆S° of the reaction are equal to ∆H° and ∆S° surf, respectively; i.e.

(6) , ∆G°

surf

surf,

∆H° ) ∆H°surf

(7)

∆G° ) RT ln cac

(8)

∆S° ) (∆H° - ∆G°)/T

(9)

Table 4 shows the results of the thermodynamic analysis. The positive value of ∆H° demonstrates that enthalpy reduction induced by charge-charge interaction of DNA and surfactant does not overcome the enthalpy increase caused by dissociation of counterions and H2O molecules from DNA and BAK12 during the reaction. However, those dissociations largely increased the entropy of the system; therefore, the interaction of cationic surfactant and DNA is solely an entropy-driven reaction. At lower ionic strength, both DNA and the surfactants have lower counterion binding (or higher ionization) degrees but a greater hydration level; thus, there will be less potential for counterion dissociation but a greater potential for water molecule dissociation when the interaction occurs. The ITC results showed that at lower ionic strength both ∆H° and ∆S° of the DNABAK12 complex are more positive than those at higher ionic strength. For example, when the ionic strength was decreased from 150 to 2.1 mM, ∆H° underwent a 3-fold increase, while ∆S° underwent a 56% increase, resulting in a 22-fold decrease in the cac. It suggests that entropy elevation induced by the dissociation of hydration water molecules from DNA and surfactants dominates the interaction.

Discussions The cac of surfactants is a widely accepted concept to the interactions of ionic surfactants with oppositely charged electrolytic polymers. At the cac the surfactant molecules start to form micelle-like aggregates that associate with polymer molecules.22,28,29 DNA is essentially a negatively charged copolymer of four nucleotides with double helical structures as well as supercoils of plasmid DNA. Some publications report highly cooperative interactions between DNA and surfactants,2,30,31 suggesting the existence of a cac and aggregation of cationic surfactants to interact with DNA, while others have (28) Dutta, P.; Halder A.; Mukherjee, S.; Sen, P.; Sen, S.; Bhattacharyya, K. Langmuir 2002, 18, 7867-7871. (29) Diamant, H.; Andelman, D. Macromolecules 2000, 33, 8050-8061. (30) Hayakawa, K.; Santerre, J. P.; Kwak J. C. T. Biophys. Chem. 1983, 17, 175-181.

proposed that monomers of the surfactant bind to DNA phosphate groups via charge-charge interactions.32,33 It was also proposed that surfactant monomers interact with DNA at concentrations below the cmc and micelles interact with DNA above the cmc.34 Our results obtained from several independent experiments for the first time clearly show that there is no interaction between surfactant and DNA below the cac. The charge-charge interaction between DNA and surfactants during complex formation is preceded by the aggregation of surfactant monomers through hydrophobic interactions. In aqueous solution, the monomers of ionic surfactants are strong electrolytes, while their micelles are partially neutralized by counterions to reduce the electric repulsion between the adjacent ionic groups in the micelles. In the presence of DNA, the phosphate groups of DNA act as counterions to cationic micelles, and a much more competitive counterion than Cl- due to the poly-charge of DNA. The strong interaction between the micelles and DNA phosphates alters the thermodynamics of aggregation, resulting in a lower critical aggregation concentration compared to the cmc of a surfactant. From this point of view, the cac is the cmc of a cationic surfactant with DNA as the counterion. As a polyelectrolyte, DNA also needs counterions, such as Na+, to partially neutralize the phosphate groups to reduce the intramolecular charge-charge repulsion. Upon the increase in NaCl concentration, the competitive association of Na+ to DNA molecules reduces the interaction of DNA with surfactants and thus causes an increase in the cac. However, below the cac no interaction between cationic surfactants and DNA was seen from all of our results of ITC, surface tension measurement, centrifuge/ UV absorbance, and light scattering. These observations lead to an inference that monomers of the surfactants do not interact with DNA phosphate. A natural question is, why do the monomers of a cationic surfactant not interact with DNA phosphates, while Na+ ions do? It seems likely that the highly polarized microenvironment around DNA molecules formed by the phosphate groups and Na+ ion atmosphere is extremely unfavorable to the hydrophobic chains of the surfactant and thus prohibits surfactant monomers from associating to DNA via solely counterion interactions, unless the hydrophobic chains aggregate to minimize the exposure to the polarized microenvironment. Moreover, the critical concentration phenomenon was not seen in solely chargecharge neutralization-derived interactions of DNA with multivalent cations such as cobalt(III) hexamine,35 further supporting the hypothesis that the hydrophobic interaction-mediated aggregation of surfactant molecules is the cause of the cac. Recently, some reports suggested that cationic surfactants interacted with (31) Shirahama. K.; Takashima, K.; Takasawa N. Bull. Chem. Soc. Jpn. 1987, 60, 43-47. (32) Bathaie, S.; Moosavi-Movahedi, A. A.; Saboury, A. A. Nucleic Acids Res. 1999, 27, 1001-1005. (33) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Am. Chem. Soc. 2002, 124, 7331-7342. (34) Wang, Y.; Dubin, P. L.; Zhang, H. Langmuir 2001, 17, 1670-1673. (35) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Mol. Biol. 2000, 296, 10531063.

Plasmid DNA and Cationic Surfactant Interactions

DNA without forming micelles33,34 on the basis that the concentrations of surfactants used in those studies were below the cmc.34 However, an opposite conclusion might be made with the consideration that the cac can be 1-2 orders of magnitude lower than the cmc at low ionic strength. Indeed, critical concentrations were seen in one report33 even though the paper proposed a mechanism of surfactant monomers interacting with DNA with the hydrophobic tails lay down on DNA surface. Zhou et al.36 recently studied the structures of complexes of calf thymus DNA and cationic surfactants using synchrotron smallangle X-ray scattering. They found that surfactant molecules bound by rigid double-stranded DNA formed cylindrical micelles, whereas those bound by more flexible single-stranded DNA could form spherical or short cylindrical micelles. This report demonstrated micelle-like aggregation of surfactant in complexation with DNA. Using 1-anolinonaphthalene-8-sulfonic acid (1,8ANS), a hydrophobic microenvironment sensitive fluorescent molecular probe, we have probed the microenvironment of the aggregates of DNA and BAK12. A transitional increase in 1,8ANS fluorescence intensity was observed around the cac, and similar responses occurred at the cmc of BAK12 in the absence of DNA, suggesting that BAK12 formed micelle-like structures in DNA-BAK12 complexes. It has been reported that the cationic surfactant cetyltrimethylammonium bromide was used for large-scale purification of plasmid DNA from cell lysates.10 The linear genomic DNA and the relaxed open-circle plasmid DNA in the cell lysate exhibited greater affinities to bind CTAB than the supercoiled plasmids, suggesting that the greater flexibility of DNA favors its interaction with cationic surfactant. Interestingly, the plasmid DNA/CTAB precipitate was found completely soluble in 0.65 M NaCl solution10san observation that is entirely consistent with our results that a higher ionic strength causes a higher cac and that DNA dose not interact with micelles when I > Ic. The thermal response signals showed that the reaction of DNA and surfactants is reversible, as the titrated-in high concentration of surfactants rapidly and endothermically formed complexes with DNA at the injection site, followed by exothermic dissociation of the complexes after sufficient mixing. The complex stopped dissociating when the cumulative concentration of surfactants in the sample cell reached the cac. Previously, Lander et al.10 proposed a “nonrandom” reaction model to interpret their experimental results from plasmid DNA and CTAB precipitation: the first CTAB molecule associated to a DNA molecule would promote more CTAB association to the same DNA molecule through hydrophobic interactions until that DNA molecule was precipitated. However, our ITC signals clearly revealed a different mechanism: the so-called “nonrandom” precipitation is actually a mixing process related phenomenons above the cac, the added CTAB micelles will rapidly form precipitates with DNA at the addition site before being homogeneously mixed to randomly interact with DNA in the (36) Zou, S.; Liang, G.; Burger, C.; Yeh, F.; Chu, B. Biomacromolecules 2004, 5, 1256-1261.

Langmuir, Vol. 22, No. 8, 2006 3743

entire container. The “random” reaction may not actually exist given the rapidness of the interaction and the limitations of the mixing process. We previously reported a plasmid DNA-based HIV vaccine containing plasmid DNA, a nonionic block copolymer, and a mixture of cationic surfactants of BAK12, BAK14, and BAK16. This formulation significantly augmented the CTL-mediated cellular immune response in nonhuman primate and provided pronounced protection to the animals against SIV challengeinduced virus infection.17-19 Differential characterization of the formulation demonstrated that there exist significant amounts of free BAK12 (∼0.25 mM) and free DNA (∼4.8 mg/mL) in the formulation, while ∼100% of BAK14 and BAK16 is adsorbed to the polymer particles. But surprisingly there are no detectable DNA-BAK12 binary precipitates in the formulation. The mechanism revealed in this report provided a clear explanation to this finding: the concentration of free BAK12 in the mixture is