3744
Langmuir 2006, 22, 3744-3750
Aggregation Behavior and Thermodynamics of Binding between Poly(ethylene oxide)-block-Poly(2-(diethylamino)ethyl methacrylate) and Plasmid DNA J. F. Tan,† H. P. Too,†,‡ T. A. Hatton,*,†,§ and K. C. Tam*,†,| Department of Biochemistry, National UniVersity of Singapore, Singapore 117576, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and School of Mechanical & Aerospace Engineering, Nanyang Technological UniVersity, 50 Nanyang AVenue, Singapore 639798 ReceiVed September 22, 2005. In Final Form: January 21, 2006 The aggregation behavior and the thermodynamics of binding between poly(ethylene oxide)-block-poly(2(diethylamino)ethyl methacrylate) (PEO-b-PDEAEMA) block copolymers and plasmid DNA were examined. Binding between the polymer and DNA were confirmed by gel electrophoresis. The high affinity between the polymer and DNA was demonstrated through the ethidium bromide (EtBr) displacement assay, and the binding was found to be related to the stoichiometric balance between the amine group of the polymer and the DNA nucleotide molar ratio (N/P molar ratio). The light scattering and TEM results showed that, at low polymer concentration, the hydrodynamic radii (Rh) of the polymer/DNA complexes was around 90 nm; however, at sufficiently high polymer concentration, the complexes condensed to around 35 nm induced by a structural rearrangement of the amphiphilic nature of the block copolymer. The isothermal titration calorimetric results showed that the binding between the polymer and DNA is driven by a large favorable enthalpy.
Introduction Gene delivery has attracted considerable scientific interest for curing genetic deficiencies and for treatment of life threatening diseases.1-3 Delivery through viral vectors in vivo poses safety concerns unlikely to be resolved soon, rendering nonviral delivery systems an attractive alternative.4-6 Although cationic polymers with high charge density exhibit enhanced DNA condensation, this property also contributes to their higher cytotoxicity, as observed for, e.g., in poly(L-lysine).7 The cytotoxicity and solubility of the polymer can be improved by coupling it with poly(ethylene oxide) (PEO). Systems such as Pluronic-b-poly(2-(dimethylamino)ethyl methacrylate) (Pluronic-b-PDMAEMA)8 and PEO-b-polyethylenimine (PEO-b-PEI)9 have been widely studied as potential gene delivery systems. An understanding of the DNA compaction with cationic polymers or surfactants is essential for the optimal design of synthetic vectors and for other applications such as DNA extraction.10,11 Recently, the DNA binding affinity of PEO-b-PEI,9 random copolymer of * Corresponding authors. E-mails:
[email protected] (K.C.T.);
[email protected] (T.A.H.). † Singapore-MIT Alliance. ‡ National University of Singapore. § Massachusetts Institute of Technology. | Nanyang Technological University. (1) Herrmann, F. J. Mol. Med. 1995, 73 (4), 157-163. (2) Partridge, K. A.; Oreffo, R. O. C. Tissue Eng. 2004, 10 (1-2), 295-307. (3) Haider, M.; Megeed, Z.; Ghandehari, H. J. Controlled Release 2004, 95 (1), 1-26. (4) Pannier, A. K.; Shea, L. D. Mol. Ther. 2004, 10 (1), 19-26. (5) Roth, C. M.; Sundaram, S., Annu. ReV. Biomed. Eng. 2004, 6, 397-426. (6) Mulligan, R. C., Science 1993, 260 (5110), 926-932. (7) Choksakulnimitr, S.; Masuda, S.; Tokuda, H.; Takakura, Y.; Hashida, M., J. Controlled Release 1995, 34 (3), 233-241. (8) Bromberg, L.; Deshmukh, S.; Temchenko, M.; Iourtchenko, L.; Alakhov, V.; Alvarez-Lorenzo, C.; Barreiro-Iglesias, R.; Concheiro, A.; Hatton, T. A. Bioconjugate Chem. 2005, 16 (3), 626-633. (9) Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V. J. Am. Chem. Soc. 2000, 122 (35), 8339-8343. (10) Cardenas, M.; Nylander, T.; Thomas, R. K.; Lindman, B. Langmuir 2005, 21 (14), 6495-6502. (11) Cardenas, M.; Dreiss, C. A.; Nylander, T.; Chan, C. P.; Cosgrove, T.; Lindman, B. Langmuir 2005, 21 (8), 3578-3583.
methoxy poly(ethylene glycol) monomethacrylate (MePEGMA),12 PDMAEMA homopolymers,13 and PEO-b-PDMAEMA14 measured using an ethidium bromide (EtBr) displacement assay and light scattering techniques has been reported. The thermodynamics governing the DNA-polycation interaction is also important as it provides information on the structural properties of the polymer complex with DNA and eventually offers interpretations on the biological performance of the complexes. Isothermal titration microcalorimetry (ITC) has been used to provide quantitative information on the thermodynamic parameters that characterize the interactions between DNA and small cationic lipids.15,16 In recent years, researchers have begun to examine the thermodynamics of DNA-polymer complexes using ITC.17,18 In the present study, the PEO-b-PDEAEMA block copolymer shown in Figure 1 was used because of its ability to form pHdependent micelles. At low pH, the amine groups of the DEAMEA are protonated, and the hydrophilic polymer does not aggregate in solution. At around the physiological pH of about 7.4, these groups are partially deprotonated, so that the DEAEMA chains are somewhat hydrophobic and drive the formation of micelles which are stabilized by the hydrophilic PEO segments.19 The remaining cationic DEAEMA segments of the copolymer are able to bind with the negatively charged DNA to form the desired polymer/DNA complexes. We have conducted systematic physicochemical experiments to determine the binding affinities for (12) Nisha, C. K.; Manorama, S. V.; Ganguli, M.; Maiti, S.; Kizhakkedathu, J. N. Langmuir 2004, 20 (6), 2386-2396. (13) Rungsardthong, U.; Ehtezazi, T.; Bailey, L.; Armes, S. P.; Garnett, M. C.; Stolnik, S. Biomacromolecules 2003, 4 (3), 683-690. (14) Deshpande, M. C.; Garnett, M. C.; Vamvakaki, M.; Bailey, L.; Armes, S. P.; Stolnik, S., J. Controlled Release 2002, 81 (1-2), 185-199. (15) Matulis, D.; Rouzina, I.; Bloomfield, V. A., J. Am. Chem. Soc. 2002, 124 (25), 7331-7342. (16) Lobo, B. A.; Davis, A.; Koe, G.; Smith, J. G.; Middaugh, C. R. Arch. Biochem., Biophys. 2001, 386 (1), 95-105. (17) Ehtezazi, T.; Rungsardthong, U.; Stolnik, S. Langmuir 2003, 19 (22), 9387-9394. (18) Bronich, T.; Kabanov, A. V.; Marky, L. A. J. Phys. Chem. B 2001, 105 (25), 6042-6050. (19) Tan, J. F.; Ravi, P.; Too, H. P.; Hatton, T. A.; Tam, K. C. Biomacromolecules 2005, 6 (1), 498-506.
10.1021/la052591i CCC: $33.50 © 2006 American Chemical Society Published on Web 03/17/2006
PEO-b-PDEAEMA and Plasmid DNA Binding
Figure 1. Chemical structure of PEO-b-PDEAEMA.
the PEO-b-PDEAMEA to plasmid DNA using gel electrophoresis and EtBr displacement assays and examined the particle size and morphology of the polymer/DNA complexes using light scattering and TEM methods and the interaction thermodynamics using isothermal titration calorimetry (ITC). We showed that PEOb-PDEAEMA/DNA polyplexes exhibit a rather interesting structural rearrangement during the binding process which was believed to be a result of the amiphiphilic nature of the copolymer. Experimental Section Materials. Ethidium bromide solution was purchased from Biorad, Phosphate Buffers Saline (PBS) and Tris acetate EDTA (TAE) were purchased from Numi Media Preparation. Agarose was obtained from BMA (Rockland, USA). Plasmid. Plasmid hrGFP, a 3.7 kbase pair (kbp) expression vector containing the green fluorescence protein (GFP) driven by CMV promoter, was obtained from Stratagene Inc. The plasmid was amplified using Escherichia coli and purified using the Quantum Plasmid Miniprep kit (Biorad) to remove the bacterial endotoxins. Synthesis of the PEO-b-PDEAEMA. A full description of the synthesis and characterization of the polymers has recently been reported.19 The molecular weight, degree of polymerization, and polydispersity index were determined using 1H NMR to be PEO113DEAEMA70, Mn ) 18 000 Da, and Mw/Mn ) 1.3 (vs polystyrene GPC standard), respectively. Preparation of Polymer/DNA Complexes. The polymer/DNA complexes were prepared by adding polymer solution to plasmid DNA solution in PBS solution (pH 7.4) at the appropriate polymer to DNA ratio (N/P ratio) followed by vortexing and equilibration for 1 h. The N/P ratio was expressed via the ratio of equivalents of DEAEMA units (from NMR results) to the number of nucleotides in DNA. Agarose Gel Electrophoresis. The electrophoretic mobility of the polymer/DNA complexes at different amine group/DNA nucleotide (N/P) molar ratios in PBS buffer at pH 7.4 was determined by gel electrophoresis using 2.0% agarose gel mixed with 20 µg of ethidium bromide in TAE solution. Experiments were run at 100 V for 60 min. DNA was visualized under UV (254 nm) illumination. Ethidium Bromide Displacement Assay. Ethidium bromide (EtBr) (1 µg) was added to 100 µL of PBS solution in each of the three wells of the 96 well microplate and mixed by gentle agitation. Fluorescence was recorded with three readings at an excitation wavelength (λex) of 485 nm and an emission wavelength (λem) of 590 nm in BMG Labtechnologies FLUOstar optima (Biorad). DNA (2.2 µg) was added, and the fluorescence was remeasured. An aliquot of polymer was then titrated into the solution to a predetermined N/P molar ratio. Samples were gently mixed, and readings were taken after 15 min of incubation. The relative fluorescence was calculated as follows:
Langmuir, Vol. 22, No. 8, 2006 3745 polarized 488 nm laser light source, BI-9000AT digital correlator, and other supporting data acquisition and analysis software and accessories. The light scattering experiments were carried out at room temperature. Aliquots of polymer solutions were added to the DNA solution to achieve different N/P ratios. Complexes were mixed by gentle agitation and allowed to equilibrate for 15 min. For dynamic light scattering (DLS), the time correlation function of the scattered intensity was analyzed using the inverse Laplace transformation technique (REPES) to produce the distribution function of decay times. Zeta Potential Measurement. The measurements were carried out using the Brookhaven Zeta PALS (phase analyzer light scattering). The Zeta PALS is an extension of laser electrophoretic light scattering (ELS), which measures the velocity of moving particles that scatter laser light. The ζ potential was calculated via the Smoluchowski model fitting of the mobility data, and thus the stability of the complex and the overall charge of the particle during the course of binding were determined. Transmission Electron Microscopy (TEM). The sample was prepared on a copper grid precoated with carbon and stained with osmium tetraoxide (OsO4) to enhance the contrast between the aggregates and the background. A JEOL JEM-2010 transmission electron microscope operating at 200 kV was used to examine the morphology of the aggregates. Isothermal Titration Calorimetry (ITC). The calorimetric data were obtained using the Microcal isothermal titration calorimeter. This power compensation, differential instrument was previously described in detail by Wiseman et al.20,21 It has a reference cell and a sample cell, each of approximately 1.35 mL, and the cells are both insulated by an adiabatic shield. The titration was carried out at 25.0 ( 0.02 °C, by injecting polymer solution from a 250 µL injection syringe into the sample cell filled with 0.059 mM of DNA solution. The syringe tip acts as a blade-type stirrer to ensure an optimum mixing efficiency at 400 rpm. An injection schedule was automatically carried out using interactive software after setting up the number of injections, volume of each injection, and time between each injection. Analysis of Binding Isotherms. The equilibrium binding parameters, K and ∆H° were determined by applying the nonlinear least-squares routines available within Origin 5.0 (MicroCal Software). Blank titrations (injection of PEO-b-PDEAEMA solution into an aqueous solution that does not contain DNA) were performed. The free energy of complex formation (∆G°) was calculated from ∆G° ) -RT ln K
(1)
and the entropy of binding (∆S°) was determined from ∆S° ) (∆H° - ∆G°)/T
(2)
Results and Discussion
% relative fluorescence ) fluorescence(obs) - fluoresecence(EtBr) fluorescence(DNA + EtBr) - fluorecence(EtBr)
Agarose Gel Electrophoresis. Images of agarose gel electrophoresis of a series of polymer/DNA complexes at different N/P molar ratios are shown in Figure 2. Lane 2 represents the illumination image of DNA intercalated with EtBr. Lanes 3-9 represent the images of polymer/DNA complexes at increasing N/P molar ratios. The results show that the illumination intensity decreases as the polymer concentration increases. For lanes 3-5 (0.3 < N/P molar ratios < 1), an increasing amount of polymer led to increase bound DNA sites and, hence, resulting in fewer available binding sites for intercalating of EtBr into DNA, resulting in a reduced illumination. For lanes 6-9 (N/P molar ratios > 1), no illumination is observed, indicating that the DNA structure is saturated with polymer chains, thereby preventing EtBr intercalation. For lanes 8 and 9 (N/P molar ratios . 1), migration is slightly toward the negative electrode showing that the overall charge on the polymer/DNA complexes may have
Laser Light Scattering. The laser light scattering experiments were conducted using the Brookhaven laser light scattering system which consists of a BI200SM goniometer, an argon ion vertically
(20) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179 (1), 131-137. (21) Jelesarov, I.; Bosshard, H. R. J. Mol. Recognit. 1999, 12 (1), 3-18.
3746 Langmuir, Vol. 22, No. 8, 2006
Figure 2. Gel electrophoresis results for plasmid DNA/PEO-bPDEAEMA complexes at various N/P ratio. (1) marker, (2) DNA only, (3) N/P ratio: 0.30, (4) N/P ratio: 0.61, (5) N/P ratio: 0.91, (6) N/P ratio: 1.52, (7) N/P ratio: 3.05, (8) N/P ratio: 12.2, (9) N/P ratio: 24.4.
Figure 3. Zeta potential for plasmid DNA/PEO-b-PDEAEMA complexes at various N+/P ratio.
been positive in the presence of excess polymer. Besides affecting the binding of EtBr to the DNA, the illuminated bands were also slightly retarded as the N/P ratio increases. As more polymer chains bind to the DNA, the surface of the complexes becomes less negatively charged and the size of the complexes increases. The effect of the surface charge and the size of the complexes will be discussed in subsequent sections. Zeta Potential Measurement. Figure 3 shows the ζ potential of the polymer/DNA complexes in distilled (DI) water solution (DI water was used in this measurement because the ζ potentials were substantially masked by the high salt content in PBS solutions). For DI water, the degree of protonation R, of the amine groups of the polymer is about 30% at pH 7.4;19 hence, for ζ potential measurements, N+/P molar ratios were used to reflect the actual charge ratio. As expected, the naked plasmid DNA possesses a negative ζ potential of about -27 mV. With the addition of increasing amounts of polymer solution, the ζ potential of the complexes becomes less negative due to the neutralization of the negative charges on the DNA by the cationic copolymer. As the N+/P molar ratios approached unity, the ζ potential approached 0 mV, and further increases in the polymer concentration did not cause the ζ potential to increase significantly; it leveled off at about +5-6 mV. Similar ζ potential studies involving the homopolymer PDMAEMA/DNA complexes22 showed that the ζ potentials of the complexes increased to about +30 mV at high polymer/DNA ratios. In contrast, the PEO-b-PDEAEMA/DNA complexes have a much lower positive charge at high N/P ratios, which is probably due to the nonionic PEO segment covering the surface of the complexes. From Figures 2 and 3, it can be concluded that as the N/P molar ratio approached unity, the DNA/EtBr intercalation was blocked as all available binding sites on the DNA are occupied (22) Cherng, J. Y.; van de Wetering, P.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. Pharm. Res. 1996, 13 (7), 1038-1042.
Tan et al.
Figure 4. Ethidium bromide displacement by PEO-b-PDEAEMA interacting with DNA in PBS solution at various N/P ratio.
by the polymer chains, at which point the overall charge of the complex was close to neutral. At high N/P molar ratios, the complexes possessed a slightly positive charge, which caused the complexes to migrate toward the negative electrode in the gel electrophoresis. Ethidium Bromide Displacement Assay. Ethidium bromide (EtBr) binds DNA by intercalating into the DNA base pairs and stretches the double helix of DNA which illuminate under fluorescence.23 In this study, the formation of polymer/DNA complexes was followed by a reduction in fluorescence intensity as EtBr were displaced. As shown in Figure 4, the fluorescence of EtBr/DNA complexes decreased as polymer was added, and this indicates the interaction between the DNA and the polymer is sufficiently strong to displace the EtBr. The reduction in fluorescence intensity with N/P molar ratio is drastic for N/P < 1 and much less pronounced for N/P ratios greater than the stoichiometric ratio of unity. It is observed that the decrease of the fluorescence intensity reached a plateau at about 50%, and this implies that about 50% of the intercalation was not displaced. Rungsardthong13 demonstrated that the extent of the displacement is actually pH dependent. In that study, PDMAEMA homopolymer which is almost completely ionized at pH 4 causes the initial fluorescence to decrease by up to 90%; at pH 8 where the homopolymer is only 20% ionized, the displacement was only about 40%. Similarly, our experiments were conducted at around pH 7.4 and, hence, the reduction in the fluorescence intensity plateau at around 50% of the initial intensity. The result also showed that at high N/P ratio the fluorescence intensity continued to decrease even though the intercalation of EtBr (50%) was not displaced by the polymer. Although electrostatic interactions are essential in the binding between EtBr and DNA, dispersion forces between the aromatic ring systems likely provide the main driving force.15,24 In this respect, the decrease in the fluorescence intensity with higher N/P ratios may be due to the stronger condensation of DNA which yields a very compact structure of an EtBr intercalated double helix structure, disrupting the controlling forces responsible for noncovalent binding.25 Besides that, fluorescence quenching by the polymer may be another reason for the decrease in fluorescence intensity at high N/P ratio.26 Dynamic Light Scattering (DLS). DLS was used to investigate the changes in the particle size of polymer/DNA complexes with polymer concentration. The diffusion coefficient extrapolated (23) Lodish, H.; Berk, A.; Matsudaira, P.; D., B.; Darnell, J. Molecular cell biology, 4th ed.; W. H. Freeman & Company: New York, 2001. (24) Stokes, R. J.; Evans, D. F. Fundamentals of Interfacial Engineering; Wiley-VCH: New York, 1996. (25) Barreleiro, P. C. A.; Lindman, B. J. Phys. Chem. B 2003, 107, 62086213. (26) Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A. E.; Cheng, S. H.; Scheule, R. K. Biochim. Biophys. Acta 1997, 1325, 41-62.
PEO-b-PDEAEMA and Plasmid DNA Binding
Langmuir, Vol. 22, No. 8, 2006 3747
Figure 6. Dependence of the decay rate Γ on the square of the scattering vector (q2) of various particles.
Figure 5. DLS relaxation time distribution for various DNA/polymer complex in PBS solution. (;) PEO-b-PDEAEMA, () PDMAEMA homopolymer.
to zero concentration (D0 ) Γ/q2) for spherical particles is related to the hydrodynamic radius, Rh, by the Stokes-Einstein equation
Rh )
kTq2 6πηΓ
(3)
where k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. The relaxation time distributions (τ) obtained from DLS measurements at a scattering angle of 90° for various polymer/DNA solutions are shown in Figure 5. Figure 5a shows the DLS distribution function for DNA alone. The distribution function is unimodal, with a corresponding value of D0 of about 4.11 × 10-12 m2/s and a value of Rh of about 59.4 nm in agreement with the findings of Fishman27 which gave D0 ∼ 4.3 × 10-12 m2/s and Rh ∼ 56.8 nm for a 3.7k bp DNA. Figure 5b compares the DLS results following the addition of 0.02 mg/mL PDMAEMA homopolymer (]) and PEO-b-PDEAEMA (s) copolymer to the DNA solution. The distributions of both polymer/DNA complexes shift to longer relaxation times, which imply an increase in the particle size with a corresponding Rh of about 90 nm. The increase in the Rh value is probably due to the binding of the polymer to the DNA as observed for various cationic polymer systems, including MePEGMA12 and PEOb-PDMAEMA.14 As expected, complexation of DNA with both PDMAEMA and PEO-b-PDEAEMA polymers resulted in similar Rh values since these polymers are chemically similar. In addition to the slow mode, a small fast mode is also observed which is due to the free polymer chains (∼6 nm) that are not bound to the DNA; since the N/P ratio is greater than 1.0, free unbound polymer chains in solution are expected. Figure 5c shows the DLS results following the addition of a 0.2 mg/mL polymer solution to the DNA solution. For PDMAEMA homopolymer complexes with DNA, the relaxation (27) Fishman, D. M.; Patterson, G. D. Biopolymers 1996, 38 (4), 535-552.
Figure 7. Proposed microstructure and Rh of the DNA/PEO-bPDEAEMA complex at various polymer concentrations in PBS solution.
mode remains about the same at around Rh ∼ 90 nm. However, for the PEO-b-PDEAEMA copolymer, the distribution shifted to a much lower relaxation time, with Rh ∼ 35 nm. This suggests that PEO-b-PDEAEMA/DNA complexes undergo structural rearrangement into a compact structure probably similar to that of free PEO-b-PDEAEMA copolymer micelles, which have relaxation time distributions as shown in Figure 5d. The details on the micellization behavior of PEO-b-PDEAEMA can be obtained from our previous publication.19 The decay rate Γ for various polymer/DNA complexes exhibits a linear relationship with q2, as shown in Figure 6, which confirms that the measured distribution functions were due to the translational diffusion of the individual particles. The Rh values for the polymer/DNA complexes are shown as a function of the polymer concentration and N/P ratio in Figure 7. Rh for the naked DNA is around 56 nm. As polymer solution was added, at concentrations below its critical micelle concentration (CMC), the polymer exists as free cationic unimer which binds to the DNA to form complexes. With the addition of more polymers, the polymer/DNA complexes grew to a size of about 90 nm; above a N/P ratio >1, excess unbound polymeric unimers started to appear (grey region) in the solution. At a polymer concentration of 0.06 mg/mL and above, the polymer/DNA complex undergoes significant structural rearrangement to form aggregates of Rh ∼ 35 nm. This rearrangement was probably due to polymer aggregation above its CMC, when the polymer chains can no longer exist as unimers and aggregate together with the DNA to form a more compact structure in order to minimize its
3748 Langmuir, Vol. 22, No. 8, 2006
Tan et al.
free energy. Dias et al. also observed a similar coil-globule transition behavior of DNA molecules induced by cationic CTAB surfactant.28 These results are in contrast to PDMAEMA homopolymers,13 where the DNA was condensed and precipitated by the PDMAEMA polymer once the number of neutralized charges on the DNA exceeded a critical binding density; on the other hand, the PEO-b-PDEAEMA copolymer system did not cause the DNA to precipitate at a high N/P ratio due to the presence of the hydrophilic PEO segments which stabilized the aggregates. This ability to form smaller compact aggregates at high N/P ratio could mean better cell transfection efficiency.29 A similar structural rearrangement of the DNA at high concentrations of other condensing agents has also been demonstrated for a few systems such as PEO-polylysine30,31 and lipids system.32 The CMC value of the PEO-b-PDEAEMA copolymer was shown previously to be in the range of around 0.002 mg/mL,20 which is significantly lower than the polymer concentration at which the structural changes were observed. The difference can be attributed to two reasons. First, it is due to the presence of DNA. It is well-known that even small amounts of organic molecules can have a significant influence on the CMC of micellar solutions.33 For instance, urea, which forms strong hydrogen bonds, is a common “water structure breaker” that increases the CMC of some surfactants in aqueous solution, especially those based on PEO as the headgroup.34,35 In our case, the DNA also forms strong hydrogen bonds that will ‘break’ the water structure, and reduce the entropy increase associated with the structural rearrangement, in turn causing the CMC to increase. Second, since binding between the polymer and DNA took place once polymer solution was added, there was insufficient unbound polymer chains in the solution to induce micellization at a 0.002 mg/mL polymer concentration, hence shifting the CMC to a higher value. Similar behavior has been observed and discussed for various surfactants and polymeric systems.36 Static Light Scattering (SLS). The z-averaged radii of gyration (Rg) of the polymer/DNA complexes were extracted from the measured static light scattering (SLS) distributions using the Debye equation
KC 1 1 ) 1 + Rg2q2 + 2A2C 3 R(q) Mw
(
)
(4)
where K is an optical parameter (K ) 4π2ntol2(dn/dc)2/NAλ4), ntol is the refractive index of toluene (1.494), dn/dc is the refractive index increment of the DNA (0.168 mL/g), NA is Avogadro’s constant, λ is the wavelength, C is the concentration of the DNA solution, R(q) is the Rayleigh ratio, q is the scattering vector, and A2 is the second virial coefficient. The absolute excess time-averaged scattered intensity, i.e., (28) Dias, R. S.; Innerlohinger, J.; Glatter, O.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 2005, 109 (20), 10458-10463. (29) Merdan, T.; Kopecek, J.; Kissel, T. AdV. Drug DeliVery ReV. 2002, 54 (5), 715-758. (30) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47 (1), 113-131. (31) Nagasaki, Y.; Okada, T.; Scholz, C.; Iijima, M.; Kato, M.; Kataoka, K. Macromolecules 1998, 31 (5), 1473-1479. (32) Vijayanathan, V.; Thomas, T.; Thomas, T. J. Biochemistry 2002, 41 (48), 14085-14094. (33) Hunter, R. J. Foundations of colloid science; Oxford University Press: New York, 1987; pp 564-625. (34) Schick, M. J. Nonionic surfactants; Dekker: New York, 1967; Chapter 15. (35) Schick, M. J. J. Phys. Chem. 1964, 68, 3585-3592. (36) Wang, C.; Tam, K. C. Langmuir 2002, 18 (17), 6484-6490.
Figure 8. KC/R(q) vs q2 for DNA and polymer/DNA complexes in PBS solution. Table 1. Particle Size Characteristics of DNA and DNA/ PEO-b-PDEAEMA Complex DNA DNA + 0.2 mg/mL polymer
Rg (nm)
Rh (nm)
Rg/Rh
88.5 49.7
59.4 35
1.49 1.40
the Rayleighratio R(q), is expressed as
R(q) ) Rtol,90
( )
n 2I - I0 sin θ ntol Itol
(5)
where Rtol,90 is the Rayleigh ratio of toluene at a measuring angle of 90° with a value of 40 × 10-6 cm-1, n is the refractive index of the solvent, I, I0, and Itol are the scattered intensities of the solution, solvent, and toluene, respectively, and θ is the scattering angle. In the present study, KC/R(q) were verified to exhibit a linear relationship with q2 for both DNA and polymer/DNA complexes as shown in Figure 8. Comparison of the Rh and Rg values summarized in Table 1 can provide additional information on the morphology of the particles. The Rg of 88.5 nm for the plasmid DNA is in good agreement with the literature result.27 For random Gaussian chains Rg/Rh ∼ 1.50, which suggests that the plasmid DNA, with Rg/Rh ) 1.49, possesses a random coiled structure. For DNA solution mixed with 0.2 mg/mL PEO-b-PDEAEMA solution, Rg and Rh of the polymer/DNA complexes shrank to 50 and 35 nm, respectively. The reduction of Rg/Rh to 1.4 also implied the possibility of morphological transformation of the complex. However, it is insufficient to elucidate the structural changes on the basis of the light scattering study alone; hence TEM was used to confirm the microstructure of the complex. Transmission Electron Microscopy (TEM) Studies. The TEM images of the DNA/PEO-b-PDEAEMA copolymer complexes at two different polymer concentrations are shown in Figure 9. Figure 9a shows the TEM micrograph of the DNA/polymer complex at a 0.02 mg/mL polymer concentration. The wormlike Guassian structure of the plasmid DNA was observed. Localized dark patches on the DNA backbone were noticed, which might be due to the enhanced contrast caused by the binding of polymer chains onto the DNA. Figure 9b shows the TEM micrograph of the DNA/polymer complex at 0.2 mg/ml polymer concentration. Two types of spherical particles of different sizes were observed and the wormlike DNA structure observed in Figure 9a was absent. The polymer concentration was chosen at 0.2 mg/mL to ensure that it was well above the CMC of the PEO-b-PDEAEMA copolymer.19 The smaller particles in Figure 9b, which were about 20 nm in diameter, correspond to the free PEO-bPDEAEMA copolymer micelles. Since the OsO4 dye only stained the DEAEMA segments (which is the core of the micelles) and
PEO-b-PDEAEMA and Plasmid DNA Binding
Langmuir, Vol. 22, No. 8, 2006 3749
Figure 10. Calorimetric titration of PEO-b-PDEAEMA into DNA solution at 298 K. Top panel: thermogram showing cell feedback (CFB) vs time; bottom panel: differential ethalpic curve vs polymer concentration. Figure 9. TEM micrographs of DNA/PEO-b-PDEAEMA copolymer complex in PBS solution. (a) 0.02 mg/mL polymer solution and (b) 0.2 mg/mL polymer solution.
not the PEO segments, the size of the free micelles appeared to be smaller than the DLS results. The larger particles in Figure 9b, which were about 70-80 nm in diameter were probably the DNA/polymer complexes. The white patches observed in some of these larger particles suggest that these complexes may be somewhat porous. These TEM micrographs confirmed the inferred structural rearrangement of the DNA/polymer complexes at high polymer concentration. In general, the TEM results are in good agreement with the DLS and SLS analyses. On the basis of the experimental evidence from TEM and light scattering techniques, we propose a mechanism describing the microstructural transformation of the aggregates together with the DLS results as shown schematically in Figure 7. In region 1, where the polymer concentration is below the CMC, the individual copolymer chains bind onto the DNA structure, and the complexes maintain their wormlike Guassian structure with an overall increase in size with polymer concentration. As more polymers were added, the binding sites of the wormlike Guassian DNA structure become saturated with the polymer chains, and free unbound polymer chains begin to appear. In region 2, the polymer concentration is greater than the CMC, and the DNA/polymer complexes undergo structural transformation from Gaussian coils to spherical particles (∼80 nm in size) coexisting with excess polymer chains forming smaller free polymeric micelles. Isothermal Titration Calorimetry (ITC). The thermodynamics of the binding of polymer chains to plasmid DNA were investigated using ITC with step-by-step injections of PEO-bPDEAEMA solution into a cell containing 0.059 mM of aqueous DNA solution. The thermogram for the titration of PEO-bPDEAEMA solution into the DNA solution is shown in Figure 10a. Integration of the area under the raw signal curve at each injection gives the differential enthalpy curves shown in Figure 10b. The enthalpy measured in the ITC experiment is a sum of heat from several contributing sources, such as the dissociation of polymeric micelles into free unimers, the dilution effect, and
Table 2. Thermodynamics Parameters of DNA/ PEO-b-PDEAEMA Interaction
DNA/ PEO-b-PDEAEMA DNA/ PDMAEMA13
K (M-1)
∆H° (kJ/mol)
∆G° (kJ/mol)
∆S° (kJ/mol‚K)
4.60 × 107
-464.4
-43.7
-1.4
1.60 × 106
-533.1
-35.3
-1.67
the binding of polymers onto DNA. The enthalpy from the dilution effect was verified to be small compared to that from the binding of polymer to DNA. The differential ITC data shown in Figure 10b indicates that the interaction between the PEO-b-PDEAEMA and DNA was highly favorable enthalpically as a pronounced exothermic heat was observed as polymer solution was titrated into the DNA solution. Just before the heat started to reduce to the baseline, an exothermic peak was observed. This peak was probably due to the structural rearrangement of the polymer/DNA complexes that was observed in the DLS and TEM results (Figures 7 and 9). The fitting curve using the one site-binding model provided with the Origin software was also plotted in Figure 10b. The calculated thermodynamics parameters obtained from curves fitted to the calorimetric experimental data are summarized in Table 2 for the two polymers considered. The thermodynamic parameters shown in Table 2 are in good agreement with similar published results on the titration of cationic poly(bis-acryloylpiperazine-2-methyl-piperazine) P(BMP-2MP)17 and PDMAEMA homopolymers13 into DNA solution. In all cases, the binding is driven by favorable negative enthalpy accompanied by unfavorable negative entropy showing that the binding between the DNA and the cationic polymer is enthalpically driven.
Conclusion The aggregation behavior of PEO-b-PDEAEMA block copolymers with plasmid DNA was examined using electrophoresis and light scattering techniques. The results showed that PEOb-PDEAEMA copolymers can effectively bind to DNA. At low polymer concentration, the polymer/DNA complex had a radius of about 90 nm, similar to that of complexes of DNA with other polycations such as PDMAEMA homopolymer. At a sufficiently
3750 Langmuir, Vol. 22, No. 8, 2006
high polymer concentration, unlike PDMAEMA which will cause the DNA to precipitate, the PEO-b-PDEAEMA formed soluble condensed complexes with DNA of a much smaller radius of about 35 nm due to the amphiphilic nature of the block copolymer. TEM results confirmed the transformation of the DNA/polymer complexes from wormlike Guassian structures to spherical particles at high polymer concentration.
Tan et al.
Acknowledgment. J.F. acknowledges the financial support provided by the Singapore-MIT (SMA) Alliance. In addition, the author thanks Sathish Sadagopan and Theodosia Tan from NUS for the demonstrations of plasmid preparation and Wang Chang from NTU for several useful discussions. LA052591I
