6042
J. Phys. Chem. B 2001, 105, 6042-6050
A Thermodynamic Characterization of the Interaction of a Cationic Copolymer with DNA Tatiana Bronich, Alexander V. Kabanov,*,† and Luis A. Marky*,‡ Department of Pharmaceutical Sciences, UniVersity of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025 ReceiVed: December 6, 2000; In Final Form: April 18, 2001
In recent years, the design of non-viral artificial gene delivery systems has been an important trend in the field of gene therapy. Such systems include the use of copolymer-DNA complexes due to the ionic interactions among the participating species. The resulting complexes are stable in aqueous dispersion, despite complete charge neutralization. To optimize the biological activity of these complexes, it is important to have a complete knowledge of their physicochemical properties. In this work, we report on the interaction of a cationic graft copolymer, poly(ethylene oxide)-g-polyethylenimine (PEO-g-PEI) with poly[d(AT)]‚poly[d(AT)] (DNA). A combination of gel electrophoresis, optical, and calorimetric techniques is used to obtain a complete thermodynamic description for both the unfolding of the free and polycation bound DNA, and the interaction of the polycation with DNA. The copolymer-DNA complexes are produced spontaneously resulting from the formation of ion pairs between ionized amino groups of PEI segments of the copolymer and the phosphate groups of DNA. Polycation binding reduces the cooperative unfolding of the DNA without changing the overall conformation of the polynucleotide. The complete thermodynamic profiles show that the interaction of this particular polycation with DNA is generally electrostatic in nature because it exhibits the typical effects induced by increasing the salt concentration. The favorable formation of the polycation-DNA complex is entropy driven and consistent with the observed removal of counterions. The thermodynamic approach taken for this investigation is appropriate, but in order to improve conditions for better DNA delivery systems further investigations of other systems are needed. These systems will have to include variation of the copolymer length, changes in the hydrophilic-hydrophobic balance of the copolymer, as well as the sequence, length, and conformation of DNA.
Introduction In recent years, an important trend in the field of gene therapy is the design of non-viral artificial gene delivery systems.1,2 One promising approach has been to use DNA-polycation complexes formed as a result of ionic interactions between cationic groups of the polycation and negatively charged phosphate groups of DNA.3-5 To improve complex solubility and decrease its aggregation, the use of block and graft copolymers containing polycation and nonionic water-soluble polymer chains have been recently proposed. These copolymers include poly(ethylene oxide)-b-polyspermine,6,7 poly(ethylene oxide)-g-polyethylenimine (PEO-g-PEI),7,8 Pluronic P123-g-polyethylenimine,8 poly(ethylene oxide)-b-poly(L-lysine),9-11 poly(ethylene oxide)-gpoly(L-lysine),12 and dextran-g-poly(L-lysine).13,14 Copolymer binding to DNA results in the formation of micelle-like species containing hydrophobic domains from the neutralized DNA and polycation chains and a hydrophilic corona of nonionic chains. These species are stable in aqueous dispersion, despite complete neutralization of charge, and have sizes in the nanoscale range depending on the physical characteristics of the DNA and copolymer components. Recent studies have evaluated the use of these systems for oligonucleotide and plasmid DNA delivery in vitro.6,9,12,15 Furthermore, there is initial evidence that these systems are promising for gene delivery in vivo.8,16,17 * To whom correspondence should be addressed. † Phone: (402) 559-9364. Fax: (402) 559-9543. E-mail: akabanov@ unmc.edu. ‡ Phone: (402) 559-4628. Fax: (402) 559-9543. E-mail: lmarky@ unmc.edu.
To optimize the biological activity of these complexes, it is important to have a better understanding of their physicochemical properties, as a function of copolymer molecular characteristics and solubility properties, as well as on the copolymer/ DNA ratio. Several articles have recently focused on the characterization of self-assembly, supramolecular structure, and solution behavior of such complexes.7,8,11,18 Much less is known about the thermodynamic behavior of such systems. This work examines the interaction of a cationic graft copolymer, PEO-g-PEI with a synthetic DNA-poly[d(AT)]‚ poly[d(AT)]. A combination of optical and calorimetric techniques was used to obtain a complete thermodynamic description for (i) the unfolding of the free and copolymer bound DNA, and (ii) the interaction of the cationic copolymer with DNA. Materials and Methods Materials. Poly[d(AT)]‚poly[d(AT)], sodium salt, with molecular weight of 1.05 × 106 (∼1620 base pairs in length) was purchased from Pharmacia-LKB Biochemicals. This DNA sample was obtained in lyophilized form and was used without further purification. The buffer solution consisted of 10 mM sodium phosphate at pH 7.0 and was adjusted to the desired ionic strength with NaCl. The concentrations of the poly[d(AT)]‚ poly[d(AT)] stock solutions were determined optically using the following extinction coefficient: 260 ) 6600 M-1cm-1 in moles of phosphates.19 The PEO-g-PEI copolymer was synthesized by conjugation of PEO, Mn ≈ 8000, with randomly branched PEI, Mw ≈ 2000, following a procedure previously
10.1021/jp004395k CCC: $20.00 © 2001 American Chemical Society Published on Web 05/26/2001
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described,7 the two polymers used in this synthesis were obtained from Aldrich Co. Static light scattering measurements yielded an average molecular weight of 16 600 g/mol for the PEO-g-PEI sample, while elemental analysis yielded a total nitrogen content of 2.16 µmol/mg. This corresponds to 1.82.4 PEO segments per PEI chain, in fair agreement with an average PEO/PEI ratio of 1.7, determined by NMR.7 Agarose Gel Electrophoresis. The electrophoretic mobility of the polycation-DNA complexes at several polycation/DNA ratios was analyzed by electrophoresis using 0.8% agarose gels and a buffer consisted of 40 mM Tris-acetate, 1 mM Na2EDTA at pH 7.4. All experiments were run at 60V for 90 min and the DNA was visualized by UV illumination by staining the gels with ethidium bromide (0.5 µg/mL) for 60 min at room temperature. Temperature-Dependent UV Spectroscopy. Absorbance vs temperature profiles (melting curves) in appropriate solution conditions were measured at 260 nm using an AVIV 14DS UV-vis spectrophotometer with a thermoelectrically controlled holder. Heating and cooling curves measurements were performed at 0.2 °C intervals. Melting curves of the free and polycation-bound DNA were recorded at several salt concentrations. Transition temperatures, TM, were evaluated as the temperature at the midpoints of the helix-coil transition of these duplexes, using procedures reported previously.20 The dependence of TM on salt concentration was evaluated from the slopes of the TM vs ln [Na+] lines, which allow us to determine the thermodynamic release of counterions ∆nNa+ accompanying these transitions. The following relationship was used:21,22
∂TM/∂ln [Na+] ) 0.9(RT2M/∆Hcal)∆nNa+
(1)
where ∆nNa+ is simply the difference in the number of bound counterions to the single-stranded state relative to the helical state, the value of 0.9 is a factor for the conversion of activities into concentrations in the 0.01-0.2 M salt range, and the unfolding enthalpy ∆Hcal is measured directly in differential scanning calorimetric experiments. Differential Scanning Calorimetry. The heat of the helixcoil transition for both free and polycation-bound DNA was measured directly with a Microcal MC-2 (Northampton, MA) differential scanning calorimeter (DSC). Typically, a DNA solution with concentration of 1 mM (in phosphates) vs buffer was scanned from 30 °C to 85 °C at a heating rate of 0.75 °C/ min. The two cells were then cooled fast to 30 °C and the whole cycle repeated four additional times. The area under the resulting curve, after subtraction of a buffer vs buffer scan and normalize by the number of moles of solute, is proportional to the transition enthalpy ∆Hcal, which is a model-independent unfolding heat. The instrument was calibrated with a standard electrical pulse. Shape analysis of the heat capacity curves, using the software of the instrument, allows the calculation of two-state transition enthalpies, ∆HvH. Direct comparisons of ∆Hcal with ∆HvH permits to interpret the nature of helix-coil transition.20 To determine thermodynamic parameters for the debinding of polycations at high temperatures, a differential calorimetric experiment was performed by scanning a PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] complex against the solution of the free polydeoxynucleotide with exactly equal concentration. Titration Calorimetry. The interaction heat of PEO-g-PEI with poly[d(AT)]‚poly[d(AT)] at 24 °C was measured with an Omega titration calorimeter from Microcal Inc. (Northampton, MA). A 100 µL syringe was used to inject a PEO-g-PEI solution into the reaction cell containing the DNA sample, stirring of the syringe at 400 rpm mixes the content of this cell effectively.
The concentration of polycation in the syringe was generally 20 times higher than the concentration of the DNA solution in the reaction cell (∼0.7 mM). The reference cell is used as a reference heat reservoir and is filled with distilled water for all titration experiments. The instrument was calibrated by means of a known standard electrical pulse. Typically 10-15 injections of 6 µL each were done in a single titration. The area under the resulting peak, following each injection, is proportional to the heat of interaction Q, which is corrected for the dilution heat of the titrant and normalized by the concentration of added titrant, to yield the molar binding enthalpy ∆Hb. These isothermal calorimetric titrations were used mainly to measure ∆Hb values from the analysis of the initial peaks, which are under conditions below the saturation of DNA. Determination of Binding Affinities and Binding Free Energies. Association constants Kb for the binding of PEO-gPEI to poly[d(AT)]‚poly[d(AT)] were calculated from the observed increase in the thermal stability ∆TM of the copolymerbound duplex relative to free duplex, according to the following relationship:23
∆TM ) (RT°MTM/n∆Hcal) ln(1 + KbaL)
(2)
where T °M and TM are the transition temperatures of the free and saturated bound DNA duplexes, respectively; ∆Hcal is the transition enthalpy of the free duplex and measured directly in DSC experiments; aL is the activity of the free copolymer and assumed equal to half of the total concentration of copolymer in terms of ionized groups; and n is the apparent number of binding sites per duplex, assumed equal to one. The main assumption in using eq 2 to calculate binding affinities is that the binding of copolymer to the single strands is much weaker than to the duplexes. In past years, eq 2 has been used successfully to estimate binding affinities for both small24 and large ligands, which cover up to 20-30 base pairs of DNA.25 The binding affinities obtained in this way refer to high temperatures and are extrapolated to the temperature of interest by the van’t Hoff equation:
∂ln K/∂(1/T) ) -∆Hb(T)/R
(3)
where ∆Hb(T) is the binding enthalpy measured in titration calorimetric experiments, the assumption is made that this enthalpy is independent of temperature i.e., ∆CP is negligible. The binding free energies ∆G°b were calculated from the standard relationship: ∆G°b ) -RT ln Kb, and the entropy changes, ∆Sb, derived using the Gibbs equation. Circular Dichroism (CD). The CD spectra of poly[d(AT)]‚ poly[d(AT)] in the absence and in the presence of PEO-g-PEI were recorded on a Jasco J-710 spectropolarimeter at room temperature using quartz cells with a path length of 1 cm. All spectra were taken between 320 and 200 nm at a scanning speed of 50 nm/min with a wavelength step of 0.5 nm. Electron Microscopy. The transmission electron microscopy (TEM) studies were performed with a Hitachi H-7000 microscope using a negative staining technique. A drop of the sample solution was allowed to settle on a Formvar precoated grid for 1 min. The excess sample was wicked away with filter paper, then a drop of 1% uranyl acetate solution was allowed to contact the sample for 1 min. Results Formation of Complexes. The complexes of poly[d(AT)]‚ poly[d(AT)] with PEO-g-PEI were prepared by mixing buffered solutions of each component at various charge ratios, Z+/- . In
6044 J. Phys. Chem. B, Vol. 105, No. 25, 2001
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Figure 2. Typical UV melting curves of free polynucleotide (1) and PEO-g-PEI/polynucleotide complex (2) in 10 mM sodium phosphate buffer at pH 7.
Figure 1. Agarose gel electrophoresis experiments. A. Free polynucleotide (lane 1) and its complexes with PEO-g-PEI prepared in 10 mM sodium phosphate buffer, pH 7.0, at different Z+/- ratios: 0.5 (lane 2), 0.75 (lane 3), 1.0 (lane 4), 1.5 (lane 5), and 2.0 (lane 6). B. Free polynucleotide (lane 1) and its complexes with PEO-g-PEI prepared in 10 mM sodium phosphate buffer, pH 7.0 in the presence of 0.2 M NaCl, at Z+/- ratios: 0.5 (lane 2), 1.0 (lane 3), 1.5 (lane 4), and 2.0 (lane 5).
this work, Z+/- is the ratio of the concentration of ionizable amino groups of the polyethylenimine segment of the copolymer over the concentration of phosphate groups of DNA. It is important to note that upon mixing no precipitation was observed over the entire range of Z+/- studied and all mixtures remained transparent to the naked eye. The formation of PEOg-PEI/DNA complexes was initially analyzed by gel electrophoresis in low salt concentration, see Figure 1A. The migration of the polynucleotide band is retarded as the amount of PEOg-PEI copolymer is increased. This clearly demonstrates that the cationic segments of the copolymer are neutralizing the negative charges of DNA. Even at conditions where DNA is in
excess (Z+/- < 1), DNA chains are incorporated into the complexes because the band corresponding to the free poly[d(AT)]‚poly[d(AT)] disappears. At Z+/- ) 1, complete retardation takes place (lane 4) suggesting the formation of stoichiometric electroneutral complexes, in which all DNA charges are neutralized by the PEO-g-PEI charges. The additional increase in the PEO-g-PEI content of the mixtures, Z+/- > 1, does not lead to further migration of the complexes, lanes 5 and 6. This suggests that the excess of PEO-g-PEI does not incorporate into the PEO-g-PEI/DNA complex beyond the neutralization point. Despite complete charge neutralization, stoichiometric complexes remain stable in aqueous dispersion. Dynamic light scattering measurements demonstrated that the complexes form small particles with diameter of ca. 90 nm in the Z+/- detectable range of 0.2-2. Furthermore, aggregation was not observed in these mixtures during repeated measurements over a week period. This is in agreement with a previous report on similar complexes with plasmid DNA.8 The electrostatic nature of the interactions of PEO-g-PEI with poly[d(AT)]‚poly[d(AT)] allow us to alter complex formation by changing the ionic strength or salt concentration. Therefore, formation of the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] complexes was also analyzed by gel electrophoresis at higher salt concentrations. The data in 0.2 M NaCl is shown in Figure 1B. As in the case of the low salt conditions, addition of PEO-gPEI to DNA results in the retardation of poly[d(AT)]‚poly[d(AT)]. At Z+/- ) 1, the complexes are still migrating in the gel toward the anode, suggesting that these species are negatively charged, i.e., neutralization of the DNA charges by the copolymer is still incomplete. However, complete retardation of the DNA takes place at Z+/- ) 2. This suggests that at Z+/) 1 only a portion of the cationic copolymer added is bound to DNA, resulting in the formation of a negatively charged nonstoichiometric complex. As the concentration of PEO-g-PEI is increased to Z+/- ) 2, the equilibrium is shifted toward formation of a neutral stoichiometric complex. UV Melting Curves. The fact that PEO-g-PEI/poly[d(AT)]‚ poly[d(AT)] complexes form stable and transparent dispersions allow us their further investigation by conventional optical techniques; specially, the effects of cationic copolymer on the helix-coil transition of the DNA and DNA conformation. The helix-coil transition of poly[d(AT)]‚poly[d(AT)] in the absence and in the presence of PEO-g-PEI was characterized initially by UV-melting curves. Typical melting curves are shown in Figure 2, the unfolding of the free- and copolymer-bound take place in monophasic transitions. It is important to note that the absorbance at 260 nm, which is sensitive to the degree of DNA unfolding, does not change in the presence of polycation. However, the TM and the shape of the melting curve changed
Binding of Polycations to DNA significantly. At low salt concentration, the free poly[d(AT)]‚ poly[d(AT)] unfolds sharply with a TM of 44.5 °C, while the addition of the copolymer to poly[d(AT)]‚poly[d(AT)] shifts the TM toward higher temperatures. At Z+/- ) 1, the unfolding of the copolymer-DNA complex is shifted by 12 °C and the melting curve is much less cooperative, its transition width is broader, see Figure 2. The observed unfolding of the complex is reversible, i.e., the copolymer-DNA complex reassembles upon cooling the mixture yielding a similar melting curve on subsequent heating scans. Differential Scanning Calorimetry. The heat capacity vs temperature profiles for poly[d(AT)]‚poly[d(AT)] and PEO-gPEI/poly[d(AT)]‚poly[d(AT)] complex (Z+/- ) 1) at low salt concentrations are shown in Figure 3a. The unfolding of these macromolecules takes place in monophasic transitions with negligible heat capacity differences between the initial and final states. The TM’s are 44.1 °C for the free polynucleotide and 56.3 °C for the complex, both consistent with the results of UV melting curves discussed above. Figure 3b shows a DSC differential thermogram, i.e., a DSC experiment in which a PEOg-PEI/poly[d(AT)]‚poly[d(AT)] complex in the sample cell is recorded against a solution of free polynucleotide in the reference cell with similar polynucleotide concentration. Two well-defined peaks are obtained: an initial negative peak at low temperatures that corresponds to the transition of free DNA followed by a positive peak corresponding to the unfolding of the copolymer-DNA complex. Since a macromolecule is placed in each cell, excluded volume effects are eliminated in this type of experiment; the water content of each cell is similar yielding baselines with heat capacity values of nearly zero. As expected, the data obtained from the analysis of this scan coincided with the DSC scans of each macromolecule against buffer. The resulting thermodynamic parameters, TM, ∆HvH, and ∆Hcal are shown in Table 1. The comparison of these data indicates that the helix-coil transition of both free and copolymer-bound poly[d(AT)]‚poly[d(AT)] takes place with similar unfolding enthalpies. At low salt concentration, the actual difference is just 0.5 kcal/mol per base pair. However, the cooperativity of the transition is drastically affected and consistent with the observed changes in the shape of the optical melting curves. Inspection of Figure 3a,b shows that the transition curve for the unfolding of the complex is much broader. Specifically, the transition halfwidth is 1.3 °C for the free poly[d(AT)]‚poly[d(AT)] and 6.8 °C for the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] complex. Binding of cationic copolymer induces a decrease in the melting cooperativity of DNA, which is estimated from the ∆HvH/∆Hcal ratio.20 This ratio yields the size of the cooperative melting unit, see last column of Table 1, equal to ∼73 base pairs for the free poly[d(AT)]‚poly[d(AT)] and 16 base pairs for the copolymerDNA complex. The DSC scans in 0.2 M NaCl (Figure 3c) show that the increase of salt also yield less cooperative transitions with cooperative unit size of 29 base pairs and 25 base pairs for the free polynucleotide and copolymer-polynucleotide complex, respectively. Therefore, the addition of salt affects the melting cooperativity of the free polynucleotide to a much lesser extend but in a similar way as the addition of polycation. This is consistent with the electrostatic nature of the interaction of copolymer with DNA. Thermodynamic Release of Counterions. The dependence of TM on salt concentration for the free DNA and PEO-g-PEI/ DNA complex at Z+/- ) 1 is shown in Figure 4. An increase in the concentration of salt results in the characteristic increase of the overall stability of free duplex DNA. At each salt concentration, the differences in TM between the free and
J. Phys. Chem. B, Vol. 105, No. 25, 2001 6045
Figure 3. Differential scanning calorimetry of polynucleotide and PEOg-PEI/polynucleotide complexes (at Z+/- ) 1) in 10 mM sodium phosphate buffer at pH 7. (a) low salt buffer, (b) a scan of the complex versus the free polynucleotide in low salt buffer, and (c) in the same buffer with additional 0.2 M NaCl.
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TABLE 1: Thermodynamic Parameters for the Unfolding of the Unligated Poly[d(AT)]‚poly[d(AT)] and PEO-g-PEI/ poly[d(AT)]‚poly[d(AT)] Complex in Two Salt Concentrationsa [NaCl] (mM) poly[d(AT)]‚poly[d(AT)] 17 217 PEO-g-PEI/ poly[d(AT)]‚poly[d(AT)] 17 217
TM ∆Hcal ∆HvH ∆HvH/∆Hcal (°C) (kcal/mol) (kcal/mol) (no. of bp) 44.1 66.5
7.2 8.5
524 249
73 29
56.3 66.9
6.7 7.3
104 180
16 25
a All measurements were determined in 10 mM sodium phosphate buffer at pH 7.0. The TM’s are within 0.5 °C, ∆Hcal ((3%), and ∆HvH ((10%).
TABLE 2: Equilibrium Parameters for the Interaction of PEO-g-PEI to Poly[d(AT)]‚poly[d(AT)] as a Function of Salt Concentrationa [Na+] (mM) 17 32 61 115 217
TM (°C) 44.6 50.8 56.7 62.3 66.8
T M′ (°C)
Kb(TM) (M-1)
55.8 57.7 60.5 63.4 66.9
1.6 × 10 8.8 × 103 4.5 × 103 1.2 × 103 1.1 × 102
Kb(298) (M-1) 4
d ln Kb/d ln [Na+]
1.4 × 10 7.4 × 103 3.7 × 103 1.0 × 103 0.9 × 102 4
-1.00 ((0.04)
a All values obtained from spectroscopic melting experiments in 10 mM sodium phosphate buffer at pH 7.0, adjusted to the desired NaCl concentration. TM values are within (0.5 °C; Kb values are within (36%; Kb values are calculated using a ∆Hcal ) 7.22 kcal/mol and ∆Hb ) 1.3 kcal/mol, obtained from DSC and calorimetric titration experiments, respectively.
TABLE 3: Standard Thermodynamic Profiles for the Interaction of PEO-g-PEI with Poly[d(AT)]‚poly[d(AT)] at Two Different Concentrations of NaCla [Na+] (mM)
∆G°b (kcal/mol)
∆Hb (kcal/mol)
T∆Sb (kcal/mol)
17 217
-5.6 -2.6
+0.6 +0.1
6.3 2.7
a All thermodynamic parameters determined in 10 mM sodium phosphate buffer at pH 7.0, adjusted to the desired NaCl concentration. The value of ∆Hcal is within (3%, while ∆G°b and T∆Sb values are within (4%.
Figure 4. Dependence of TM on salt concentration for free polynucleotide (circles) and PEO-g-PEI/polynucleotide complexes at Z+/) 1 (triangles).
copolymer-bound DNA reflect the effective binding of polycation to duplex DNA. A linear regression analysis of the TM vs ln [Na+] lines of Figure 4 yielded slopes of 8.8 ((0.3) and 4.3 ((0.2) for the free and copolymer-bound DNA, respectively. A value of ∆nNa+ ) 0.35 (per base pair) is obtained for the free poly[d(AT)]‚poly[d(AT)], which is in excellent agreement with the value of 0.36 predicted for long DNA rods by polyelectrolyte theory.22 On the other hand, it is difficult to calculate the release of counterions accompanying the unfolding of the complex because the increase in salt is favoring complex dissociation. However an upper limit ∆nNa+ value of 0.15 (per base pair) is estimated, which is expected for a complex with a lower charge density. Polycation Binding Affinities. Association constants Kb for the binding of PEO-g-PEI to poly[d(AT)]‚poly[d(AT)] were calculated from the observed increase in the thermal stability ∆TM of the copolymer-bound DNA relative to free DNA, according to eq 2. Table 2 lists the resulting binding affinities at several salt concentrations. At low salt concentration a Kb of ∼104 is obtained while at high salt concentration the Kb value decreases to ∼103. The slope of the line of the ln Kb vs ln [Na+] plot, data not shown, is equal to -1.0, which is close to the theoretical value of -0.78 from polyelectrolyte theory.21,22 This value is proportional to the exchange of ions upon polycation binding, i.e., for every mol of bound polycation (in ionized units) there is a release of 1 mol of sodium ions. Titration Calorimetry. The resulting integral heat for the interaction of PEO-g-PEI with poly[d(AT)]‚poly[d(AT)] as a function of the charge ratio, Z+/-, and salt concentration is
Figure 5. Binding isotherms obtained in isothermal titration calorimetric experiments in 10 mM sodium phosphate buffer at pH 7.0 in low and high salt, as indicated in the curves.
shown in Figure 5. Binding of the cationic copolymer is accompanied by small endothermic heats, which are independent of salt concentration. The resulting ∆Hb values do not exceed 0.6 kcal/mol, which are characteristic for binding processes involving electrostatic interactions. This is consistent with the heat of dissociating the polycation with temperature, which is obtained from the difference in the unfolding heats of the curves of Figure 3b. The overall result is also consistent with previous reports on the electrostatic binding of polylysine, spermine, and Mg2+ to DNA.26 Complete Thermodynamic Profile for PEO-g-PEI Binding. Complete thermodynamic profiles for the binding of PEOg-PEI to poly[d(AT)]‚poly[d(AT)] at two different salt concentrations are presented in Table 3. The measured thermodynamic parameters indicate that the favorable interaction of PEO-gPEI with poly[d(AT)]‚poly[d(AT)] is the result of the typical compensation of an unfavorable enthalpy term with a favorable entropy term. The increase in salt concentration yielded a less
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Figure 7. TEM micrograph of the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] complex at Z+/- ) 1. The bar corresponds to 100 nm.
additional changes of the negative band at 248 nm, see Figure 6. Such changes would have indicated the formation of condensed forms of DNA.27,28 Thus, binding of PEO-g-PEI does not induce major conformational changes in the helical structure of the polynucleotide and it is not causing any condensation. The later observation is further confirmed by electron microscopy, which reveals formation of extended threadlike particles for the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] complex, see Figure 7. Discussion Figure 6. Circular dichroism spectra of polynucleotide (solid) and PEO-g-PEI/polynucleotide complexes in 10 mM sodium phosphate buffer at pH 7.0 and several Z+/- ratios: 0.6 (dotted), 1.1 (dashed), and 1.4 (dashed-dotted). (A) low salt buffer and (B) high salt buffer.
favorable ∆G°b term, which corresponds to a less favorable entropy term, ∆(T∆Sb) of - 3.6 kcal/mol. Circular Dichroism. The observed electrostatic interaction of the cationic copolymer with the polynucleotide raises the following questions. How copolymer binding does affect the structure and conformation of DNA? How effective is the polycation neutralizing the negatively charged phosphates of the DNA? To answer these questions, circular dichroism and electron microscopy techniques have been used to check the conformation and overall macroscopic geometry of the complexes. Figure 6A,B shows the CD spectra of the free poly[d(AT)]‚poly[d(AT)] and PEO-g-PEI bound complexes at several Z+/- ratios and at two salt concentrations. The CD spectrum of the free polynucleotide is typical of a duplex in the “B” conformation, addition of copolymer results in CD spectra with similar shapes. In low salt, the magnitude of the positive band decreases somewhat with increasing the loading of the copolymer, while negligible changes are observed in the magnitude of the negative band. The above changes are negligible at high salt conditions; however, the low salt spectra of the complex at Z+/- values greater than 1 show a small negative band centered at 287 nm, see Figure 6a. An important observation is that the addition of PEO-g-PEI solution to poly[d(AT)]‚poly[d(AT)], at both salt concentrations, is not inducing
Uniform Distribution of Polycation Chains on DNA. The results of the present work show that the interaction of cationic PEI segments of the graft copolymer with the negatively charged phosphate groups of poly[d(AT)]‚poly[d(AT)] leads to the formation of polyelectrolyte complexes. The PEO-g-PEI/DNA complexes form stable aqueous dispersions, even under the conditions of complete charge neutralization of both polymers. This is a good improvement over PEI homopolymers, which form insoluble complexes with DNA. The improved stability of these PEO-g-PEI-polynucleotide complexes in aqueous media is perhaps due to the presence of the hydrophilic PEO chains. In these complexes, the ionized amino groups of PEI neutralize the negative phosphate groups of DNA. This results in the formation of micelle-like species containing a hydrophobic core of neutralized DNA and PEI chains and a hydrophilic corona of PEO segments.29 The gel electrophoresis patterns of the lanes with DNA in excess suggest that the copolymer is being distributed uniformly among the DNA chains, disproportionation of DNA was never observed. This important observation is in contrast with previous findings on the interaction of polylysine, and other basic polypeptides, with DNA, where a nonuniform distribution of polycation chains takes place.27,30,31 On the other hand and under conditions where PEO-g-PEI is in excess, the copolymer is not incorporated into the PEO-g-PEI/DNA particles beyond the neutralization point. This type of behavior has been observed previously in the complexes of PEO-g-PEI with oligonucleotides7 and plasmid DNA.8 Most likely, incorporation of excess copolymer into the complex is prevented by steric repulsion of the PEO chains.
6048 J. Phys. Chem. B, Vol. 105, No. 25, 2001 Effects of Salt Concentration on the Formation of Complexes with Z+/- ) 1. Formation of PEO-g-PEI/poly[d(AT)]‚ poly[d(AT)] complexes is affected by the actual concentration of salt in the solution. Indeed, at a relatively high salt concentration (0.2 M NaCl) the electrophoretic retardation of DNA in the PEO-g-PEI/DNA mixture at Z+/- ) 1 is not complete. This suggests that at this salt concentration only a fraction of the PEO-g-PEI chains binds to DNA while the rest are in equilibrium with the nonstoichiometric polycation/DNA complex having a negative net charge. The increase in the copolymer concentration to Z+/- ) 2 leads to a shift of the equilibrium toward the formation of complex and complete neutralization of DNA is achieved. This behavior can be explained by the partial dissociation of complexes with the increase in salt concentration. This conclusion is also consistent with the lower observed shifts of the thermal stability of the complexes at higher salt concentrations, which lead to a decrease in the binding affinity of the copolymer (Table 2). The overall result is not surprising in view of the known behavior of polyelectrolyte complexes. Specifically, in the case when one of the interacting polyions is relatively short.32 Indeed, the PEOg-PEI copolymer used in this investigation contains rather short PEI segments of ∼46 monomer units. Furthermore, only approximately half of the amino groups of these PEI segments are charged and can participate in the binding reaction at pH 7. In the formation of synthetic polyions complexes, such as polymethacrylate anions and poly(N-ethyl-4-vinylpyridinium) cations, it has been shown that short polycation chains (ca. 40 repeating units) induced a progressive dissociation of the complex as the salt concentration is increased.33 This dissociation is accompanied by removal of the short polycation chains from the complex into solution and, therefore, changes the composition of the initial complex. In contrast, complexes formed by relatively long polycations (ca. 300 repeating units) remain stable under similar conditions.33 Polycation Binding to DNA Increases the Thermal Stability of DNA. UV-melting experiments show that the binding of cationic copolymer to DNA leads to a thermal stabilization of the DNA helix over the entire range of salt concentration studied. This is in good agreement with previous observation that cationic homopolymers and copolymers induce such thermal stabilizations of the DNA.11,14,34 It is well established that low molecular mass counterions (e.g., sodium ions) condensed onto DNA. These condensed counterions and the electrostatic shielding of the counterion atmosphere contribute to the stability of helical structures by reducing the electrostatic repulsions of phosphate groups in the sugar-phosphate backbone of the DNA. In the equilibrium reaction of two strands to form a double helix, a net binding of counterions to the double helical state takes place because of its higher charge density parameter. Therefore, the induced helix-coil transition of DNA with temperature is accompanied by a release of counterions to the surrounding medium. The decrease in the slope of the TM-ln [Na+] plot for the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] compared to the slope for the free poly[d(AT)]‚poly[d(AT)] suggests that in the presence of copolymer the smaller counterion condensation has a smaller contribution to the stabilization of the DNA helix than in the case of the free DNA. Indeed, the formation of salt bridges between the charged amino groups of PEI segment of the copolymer and phosphate groups of DNA results in the simultaneous release of sodium counterions to the surrounding medium. This leads to a reduction of the concentration of counterions in the DNA vicinity. To compensate for the above effects, the bound polyion chains shield the electrostatic
Bronich et al. repulsions of the phosphates groups, which result in the shift to higher temperatures of the helix-coil transition of DNA. Nevertheless, some portion of small counterions might remain bound to DNA due to small defects in the complex structure and/or incomplete saturation of DNA molecules by the cationic copolymer. Moreover, we cannot exclude the possibility that higher temperatures induce complex dissociation, which change both the actual composition of the complex and counterion distribution. All of these factors can contribute to the observed dependence of ionic strength on the thermal stability of the PEOg-PEI/poly[d(AT)]‚poly[d(AT)] complex. Melting Behavior of the Polycation-DNA Complex. Despite the pronounced stabilization of the DNA duplex by the copolymer at low salt concentration, the helix-coil transition of both free polynucleotide and PEO-g-PEI/poly[d(AT)]‚ poly[d(AT)] (Z+/- ) 1) complex are characterized with similar unfolding enthalpies. The actual difference is only 0.5 kcal/ mol per base pair, which corresponds to the dissociation heat of the copolymer at high temperatures. Thus, the observed 12 °C stabilization induced by PEO-g-PEI cannot be attributed to any substantial alteration of base-pairing and base-pair stacking interactions in the polynucleotide duplex. Similar conclusion can be derived from the fact that the absorbance of the free DNA at 260 nm does not change upon binding of the cationic copolymer. In contrast, a decrease in the absorbance of the DNA solution has been previously observed upon binding of polylysine30,35 and dextran-g-poly(L-lysine).14 Such behavior was attributed to a conformational change of the DNA to a dense globular structure triggered by the polycationic backbone14 and/ or to changes in the interior array of stacked base pairs due to the incorporation of oligo(L-lysine) into one of the grooves of DNA.35 In the case of PEO-g-PEI the polycation PEI backbone has a higher conformational flexibility compared to the relatively rigid polylysine molecules. Furthermore, in contrast to polylysine, polyethylenimine does not form intramolecular secondary structures. This might explain why upon binding with the DNA duplex PEO-g-PEI causes less spatial perturbation of the duplex than polylysine-based molecules do. In general, DNA exhibits extended monophasic melting transitions in the presence of small multivalent cations such as spermidine36 or trivalent cations,37 which was attributed to a relatively weak, reversible binding. In contrast, the melting behavior of DNA in the presence of polypeptides is usually biphasic and depends on the methods of preparation of the complexes.27,30,31,38 It has been demonstrated that below saturation conditions the base pairs of DNA exist in two states: complexed and free. These data suggest that binding of long cationic peptides to DNA is tighter. Lately similar behavior was shown for the complexes of DNA with PEO-block-poly(Llysine)11 and dextran-g-poly(L-lysine).14 The monophasic melting transitions observed for the PEO-g-PEI/poly[d(AT)]‚ poly[d(AT)] systems suggests once more that the chains of the cationic copolymer do not bind very tightly to DNA and are uniformly distributed throughout the DNA. Significantly, the spectroscopic and calorimetric data reveal that the binding of PEO-g-PEI to DNA leads to drastic changes in the nature of the helix-coil transition. The comparison of the data in Table 1 indicates that the copolymer-bound polynucleotide duplex melts in less cooperative manner. Similar behavior was observed for the melting of DNA in the presence of sodium counterions. The increase of salt concentration led to an increase of the unfolding enthalpy for poly[d(AT)]‚poly[d(AT)] from 7.2 to 8.5 kcal/mol, see Table 1, which can be attributed to a reinforcement of base-pair staking interactions.39
Binding of Polycations to DNA Similar to the low salt results, binding of PEO-g-PEI to DNA yielded a larger decrease of the transition enthalpy, by 1.2 kcal/ mol, at higher salt concentration. Addition of salt also causes the helix-coil transition to occur in a less cooperative manner. Specifically, in 10 mM phosphate buffer 73 base pairs of poly[d(AT)]‚poly[d(AT)] melt cooperatively while in the presence of 0.2 M NaCl 29 base pairs melt in cooperative manner. In this salt concentration range, the binding of polycation has only a marginal effect on the size of the melting cooperative unit, which decreases from 29 to 25 base pairs. Thus, small cations and polycations exhibit the same trend: both of them induce thermal stabilization of DNA duplex and decrease the cooperativity of the helix-coil transition. However, the effective concentration of simple salt at which the same stabilization effect is achieved in ca. 103 times higher than the effective polycation concentration. This suggests that cationic copolymer provides for a much more effective screening of the DNA phosphates than sodium or that the charged amino groups of the copolymer are binding tighter to DNA. An alternate explanation for the observed broader transition curves of the complexes with salt or polycation is the presence of a distribution of complexes with different composition, structure and melting temperature. This would yield a superposition of melting curves resulting in a broad melting curve. Binding of Polycations to DNA is Entropy Driven. As expected, the binding of polycations to DNA is primarily nonspecific and electrostatic. Since the interaction between cationic graft copolymer and DNA occurs spontaneously, the small ∆Hb values observed in this work confirm once more that this binding phenomenon is primarily entropic in origin. Indeed, the intrinsic free energy change of ion pair formation with the cationic ligand should be small because the nucleotide is already involved in an ionic interaction with sodium ions. However, we cannot exclude the contribution of other interactions like specific H-bonding or van der Waals forces into the complex formation as well as changes in the hydration state of the two polymers involved. Therefore, the small exothermicity term of the binding heat may be the result of both exothermic contributions from van der Waals interactions and endothermic contributions from removal of water molecules of the charged and polar atomic groups participating on binding. The electrostatic contribution to the binding can be determined from the dependence of Kb on salt concentration. The slope of the line from a plot of ln Kb vs ln[Na+] corresponds to the effective number of charges on the ligand that are contributing to the overall stability of the ligand-DNA complex by electrostatic effects.40 The value of -1.00 ((0.03) obtained for PEOg-PEI/poly[d(AT)]‚poly[d(AT)] is indicative that each charged amino group effectively cover not more than one phosphate. It therefore appears that practically all charged amino groups of the copolymer are involved in the formation of salt bonds in the complex. This slope and the magnitude of Kb are both similar to those obtained for the interaction of dipyrandium and dipyrandenium with poly[d(AT)]‚poly[d(AT)]. These small steroid molecules are cationic and bind to DNA by partial insertion and outside respectively and the nature of the driving forces for their binding to DNA is entropic24 because small endothermic enthalpies were measured for the interaction of each ligand with poly[d(AT)]‚poly[d(AT)].41 This comparison suggests that PEO-g-PEI binds outside DNA by covering the DNA double helix. Furthermore, the entropy driven binding reactions discuss here are the net result of favorable entropy contributions from the putative release of both counterions and water
J. Phys. Chem. B, Vol. 105, No. 25, 2001 6049 molecules upon complex formation and the entropy penalty of a bimolecular association. Polycation Binding Does not Induce DNA Condensation. The higher stability of the DNA polynucleotide in the presence of bound PEO-g-PEI is not accompanied by changes of the secondary structure of DNA helix. According to the circular dichroism data, the incorporation of poly[d(AT)]‚poly[d(AT)] helix into the complex does not change its B-like conformation. In the CD spectra, the negative band remains the same while a slight decrease in the positive band takes place. These effects are also observed with increasing salt concentration and reflect a noncooperative winding of the DNA double helix within the family of B conformations.42 However, a similar level of suppression of the positive long-wavelength band of the CD spectra of poly[d(AT)]‚poly[d(AT)] by the polycation can be achieved at much higher concentration of univalent cations.43,44 Thus, this observation suggest that copolymer binding induce minor perturbations in the DNA helical structure. According to Manning,45,46 DNA condensation would occur spontaneously once an effective charge neutralization is reached. A wide variety of multi- and polyvalent cations condensed DNA complexes into very compact forms with toroidal shapes.37,47-50 The collapse of linear DNA proceeds without dramatic changes in the local structure. However, the formation of these condensed DNA phases are accompanied by a large increase in the magnitude of the positive or negative bands of their CD spectrum.27,28 These condensed forms, designated as ψ(+) or ψ(-), depending on the changes of the CD spectrum, are characterized as cholesteric liquid crystalline phases, and suggest long-range ordering in solution.28 The absence of nonconservative negative bands in the CD spectra of PEO-g-PEI/ poly[d(AT)]‚poly[d(AT)] suggests that these condensed DNA structures do not form. This is consistent with our electron microscopy observations of threadlike particles. Previously, we observed the same type of morphology for the complexes of plasmid DNA and PEO-g-PEI.8 Furthermore, formation of rodshaped and wormlike particle of DNA complexes with PEOg-poly(l-lysine) have been reported recently.51 Therefore, it is hypothesized that the steric repulsion of the PEO chains “grafted” to the complex surface may prevent the compaction of polycation-bound DNA into a toroidal form. Conclusion The interaction of the cationic graft copolymer, PEO-g-PEI, with poly[d(AT)]‚poly[d(AT)] results in the spontaneous formation of ion pairs between ionized amino groups of PEI segment of the copolymer and phosphate groups of DNA. Although copolymer binding increases the thermal stability of the DNA incorporated into the complex, it reduces drastically the cooperative unfolding of poly[d(AT)]‚poly[d(AT)] and does not change the overall conformation of the polynucleotide. The complete thermodynamic profiles show that the interaction of this particular cationic copolymer with DNA is generally electrostatic in nature because it exhibits the typical effects induced by increasing the concentration of salt. Copolymer binding to DNA is primarily entropy driven that correlates with the observed removal of counterions. The weak binding of polycations to DNA is needed, if these copolymers would be useful carriers of oligonucleotides to the cell. However, a further understanding of the interaction of cationic polyions with DNA molecules is needed. This includes thermodynamic investigations of several other systems that will include the variation of the length and molecular structure of the cationic segment of the copolymer, as well as the sequence, length, and overall conformation of DNA.
6050 J. Phys. Chem. B, Vol. 105, No. 25, 2001 Acknowledgment. The support of grants BES-9907281 from the NSF (A.V.K.) and GM-42223 from the NIH (L.A.M.) is greatly appreciated. We also thank Supratek Pharma, Inc., for providing the copolymer sample and R. Vaughn for carrying out the electron microscopy experiments. References and Notes (1) Rolland, A.; Felgner, P. AdV. Drug DeliVery. ReV. 1997, 30, 1-227. (2) Kabanov, A. V. Pharm. Sci. Technol. Today 1999, 2, 365-373. (3) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J.-P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297-7301. (4) Kabanov, A. V.; Kabanov, V. A. Bioconjugate Chem. 1995, 6, 7-20. (5) Tang, M. X.; Szoka, F. C. Gene Therapy 1997, 4, 823-832. (6) Kabanov, A. V.; Vinogradov, S. V.; Suzdaltseva, Yu. G.; Alakhov, V. Yu. Bioconjugate Chem. 1995, 6, 639-643. (7) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805-812. (8) Nguyen, H.-K.; Lemieux, P.; Vinogradov, S. V.; Gebhart, C. L.; Guerin, N.; Paradis, G.; Bronich, T. K.; Alakhov, V. Y.; Kabanov, A. V. Gene Therapy 2000, 7, 126-138. (9) Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123-2133. (10) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556-8557. (11) Katayose, S.; Kataoka, K. Bioconjugate Chem. 1997, 8, 702-707. (12) Choi, Y. H.; Liu, F.; Kim, J.-S.; Choi, Y. K.; Park, J. S.; Kim, S. W. J. Controlled Release 1998, 54, 39-48. (13) Maruyama, A.; Kataoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1997, 8, 3-6. (14) Maruyama, A.; Watanabe, H.; Ferdous, A.; Katoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1998, 9, 292-299. (15) Mundigl, O.; Ochoa, G. C.; Slepnev, V. I.; Kabanov, A. V.; DeCamilli, P. J. Neurosci. 1998, 18, 93-103. (16) Ogris, M.; Brunner, S.; Schuller, S.; Kircheis, R.; Wagner, E. Gene Ther. 1999, 6, 595-605. (17) Roy, S.; Zhang, K.; Roth, T.; Vinogradov, S.; Kao, R. S.; Kabanov, A. Nature Biotech. 1999, 17, 476-479. (18) Bronich, T. K.; Ngueyen, H.-K.; Eisenberg, A.; Kabanov, A. V. J. Am. Chem. Soc. 2000, 122, 8339-8343. (19) Marky, L. A.; Macgregor, R. B. Biochemistry 1990, 29, 48054811. (20) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620. (21) Record, M. T., Jr.; Anderson, C. F.; Lohman, T. M. Q. ReV. Biophys. 1978, 11, 103-178. (22) Manning, G. S. Q. ReV. Biophys. 1978, 11, 179-246. (23) Crothers, D. M. Biopolymers 1771, 10, 2147-2160.
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