Complexes of Poly(ethylene glycol)-Based Cationic Random

Feb 18, 2004 - Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with. RCP. .... are rather limited.41-50 This may b...
0 downloads 0 Views 241KB Size
Download PDF
2386

Langmuir 2004, 20, 2386-2396

Complexes of Poly(ethylene glycol)-Based Cationic Random Copolymer and Calf Thymus DNA: A Complete Biophysical Characterization C. K. Nisha and Sunkara V. Manorama Materials Science Group, Inorganic and Physical Chemistry Division, Indian Institute of Chemical Technology, CSIR, Hyderabad 500007, India

Munia Ganguli and Souvik Maiti* Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India

Jayachandran N. Kizhakkedathu Department of Pathology and Lab Medicine, 2211 Wesbrook Mall, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada Received September 16, 2003. In Final Form: December 12, 2003

Complete biophysical characterization of complexes (polyplexes) of cationic polymers and DNA is needed to understand the mechanism underlying nonviral therapeutic gene transfer. In this article, we propose a new series of synthesized random cationic polymers (RCPs) from methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride with different mole ratios (32:68, 11:89, and 6:94) which could be used as a model system to address and answer the basic questions relating to the mechanism of the interaction of calf thymus DNA (CT-DNA) and cationic polymers. The solubility of the complexes of CT-DNA and RCP was followed by turbidity measurements. It has been observed that complexes of RCP with 68 mol % MePEGMA precipitate near the charge neutralization point, whereas complexes of the other two polymers are water-soluble and stable at all compositions. Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with RCP. Ethidium bromide exclusion and gel electrophoretic mobility show that both polymers are capable of binding with CT-DNA. Atomic force microscopy images in conjunction with light scattering experiments showed that the complexes are spherical in nature and 75-100 nm in diameter. Circular dichroism spectroscopy studies indicated that the secondary structure of DNA in the complexes is not perturbed due to the presence of poly(ethylene glycol) segments in the polymer. Furthermore, we used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the helix-coil transition of CT-DNA in the complexes. UV and differential scanning calorimetry melting experiments revealed that DNA in the complexes is more stable than in the free state and the extent of stability depends on the polymer composition. Isothermal titration calorimetry experiments showed that the binding of these RCPs to CT-DNA is associated with small exothermic enthalpy changes. A complete thermodynamic profile showed that the RCP/DNA complex formation is entropically favorable. Much broader opportunities to vary the architecture of the polymers studied here make these systems promising in addressing various basic and practical problems in gene delivery systems.

Introduction Successful delivery of therapeutic genes to intended targets remains a challenge for researchers. Viral vectors efficient in gene transfection in vivo pose safety concerns unlikely to abate soon, rendering nonviral delivery systems an attractive alternative.1-10 One promising approach is to use DNA-polycation complexes (polyplexes) formed as * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +91-11-2766-6156. Fax: +9111-2766-7471. (1) Vijayanathan, V.; Thomas, T.; Thomas, T. J. Biochemistry 2002, 41, 14085-14091. (2) Mulligan, R. C. Science 1993, 260, 926-932. (3) Anderson, W. F. Science 1992, 256, 808-813. (4) Hanania, E. G.; Kavanagh, J.; Hortobagyi, G.; Giles, R. E.; Champlin, R.; Deisseroth, A. B. Am. J. Med. 1995, 99, 537-552. (5) Nishikawa, M.; Huang, L. Hum. Gene Ther. 2001, 12, 861-870. (6) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33-37. (7) Zuber, G.; Dauty, E.; Nothisen, M.; Belguise, P.; Behr, J. P. Adv. Drug Delivery Rev. 2001, 52, 245-253. (8) Maurer, N.; Fenske, D. B.; Cullis, P. R. Expert Opin. Biol. Ther. 2001, 1, 923-947.

a result of ionic interactions between the cationic groups of the polymer and the negatively charged phosphate groups of DNA.11-16 Common cationic polymers such as polylysine, polyethylenimine, and polyamidoamine dendrimers have been extensively used for this purpose.17-24 Electroneutral polyplexes that contain equivalent amounts (9) Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. J. Biol. Chem. 1994, 269, 2550-2561. (10) Hafez, I. M.; Maurer, N.; Cullis, P. R. Gene Ther. 2001, 8, 11881196. (11) Kwon, G. S.; Kataoka, K. Adv. Drug Delivery Rev. 1995, 16, 295-309. (12) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131. (13) Kakizawa, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2002, 54, 203-222. (14) Kabanov, A. V.; Lemieux, P.; Vinogradov, S.; Alakhov, V. Adv. Drug Delivery Rev. 2002, 54, 223-233. (15) Kabanov, A. V. Pharm. Sci. Technol. Today 1999, 2, 365-372. (16) Smedt, S. De.; Demeester, J.; Hennink, W. Pharm. Res. 2000, 5, 1425-1433. (17) Tang, M. X.; Szoka, F. C. Gene Ther. 1997, 4, 823-832.

10.1021/la035737r CCC: $27.50 © 2004 American Chemical Society Published on Web 02/18/2004

Polyplexes from CT-DNA/Random Copolymer

of polyion units and negatively charged phosphate groups of DNA are insoluble in water and hence unsuitable for application as gene delivery vehicles. Conjugation of the cationic polymer to hydrophilic segments of poly(ethylene glycol) (PEG), dextran (Dx), and other glucose molecules markedly improves the solubility of the polyplexes.25-40 For example, grafting of poly(L-lysine) (PLL) with PEG results in the formation of soluble polyplexes in a 40 µg/ mL DNA solution, while nonsoluble aggregated polyplexes form when a PLL homopolymer is used.16 These polyplexes are expected to form micelle-like structures with a nanometric core of cationic units neutralized by the phosphate anions surrounded by a shell of hydrophilic segments (Scheme 1). The hydrophilic shell at the exterior of the polyplexes not only enhances the solubility of the polyplexes but also prevents aggregation due to steric repulsion and reduces nonspecific interactions with biomolecules. Physicochemical properties including charge, dissociation behavior, and the shape and size of the polyplexes could play a significant role in efficient gene transfer in vitro and in vivo.15 A few isolated studies have been reported to this effect. For example, Pollard et al. suggested that formation of spherical particles by compaction of plasmid DNA is more of a determining factor for a nuclear trafficking mechanism than the ionic charge of the DNA complexes.41 Even though the interaction between the polymeric cations and DNA is electrostatic in origin, geometric and chemical structures of the polymer also play a significant role in the complex formation. Even the number of positive charges and the spacing of cationic (18) Boussif, O.; Lezoualch, 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. (19) Chan, C. K.; Jans, D. A. Hum. Gene Ther. 1999, 10, 1695-1702. (20) Chan, C. K.; Senden, T.; Jans, D. A. Gene Ther. 2000, 19, 16901697. (21) Remy, J. S.; Abdallah, B.; Zanta, M. A.; Boussif, O.; Behr, J. P. Adv. Drug Delivery Rev. 1998, 30, 85-95. (22) Maksimenko, A. V.; Mandrouguine, V.; Gottikh, M. B.; Bertrand, J. R.; Majoral, J. P.; Malvy, C. J. Gene Med. 2003, 5, 61-71. (23) Abdelmoez, W.; Yasuda, M.; Ogino, H.; Ishimi, K.; Ishikawa, H. Biotechnol. Prog. 2002, 18, 706-712. (24) Dennig, J.; Duncan, E. J. Biotechnol. 2002, 90, 339-347. (25) Li, S.; Huang, L. Gene Ther. 1997, 4, 891-900. (26) Kursa, M.; Walker, G. F.; Roessler, V.; Ogris, M.; Roedl, W.; Kircheis, R.; Wagner, E. Bioconjugate Chem. 2003, 14, 222-231. (27) Kwon, G. S.; Suwa, S.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Controlled Release 1994, 29, 17-23. (28) Kabanov, A. V.; Vinogradov, S. V.; Suzdaltseva, Yu. G.; Alakhov, V. Yu. Bioconjugate Chem. 1995, 6, 639-643. (29) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805-812. (30) 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 Ther. 2000, 7, 126-138. (31) Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123-2133. (32) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556-8557. (33) Harada, A.; Kataoka, K. Science 1999, 283, 65-67. (34) Choi, Y. H.; Liu, F.; Kim, J.-S.; Choi, Y. K.; Park, J. S.; Kim, S. W. J. Controlled Release 1998, 54, 39-48. (35) Bronich, T. K.; Ngueyen, H.-K.; Eisenberg, A.; Kabanov, A. V. J. Am. Chem. Soc. 2000, 122, 8339-8343. (36) Maruyama, A.; Ishihara, T.; Kim, J.-S.; Kim, S. W.; Akaike, T. Bioconjugate Chem. 1997, 8, 735-742. (37) Kim, W. J.; Akaike, T.; Maruyama, A. J. Am. Chem. Soc. 2002, 124, 12676-12677. (38) Maruyama, A.; Katoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1997, 8, 3-6. (39) Maruyama, A.; Watanabe, H.; Ferdous, A.; Katoh, M.; Ishihara, T.; Akaike, T. Bioconjugate Chem. 1998, 9, 292-299. (40) Asayama, S.; Maruyama, A.; Cho, C. S.; Akaike, T. Bioconjugate Chem. 1997, 8, 833-838. (41) Pollard, H.; Remy, J. S.; Loussouarn, G.; Demolombe, S.; Behr, J. P.; Escande, D. J. Biol. Chem. 1998, 273, 7507-7511.

Langmuir, Vol. 20, No. 6, 2004 2387 Scheme 1. Conceptual Illustration of Polyplex Formation from Block Copolymer and Random Copolymer

charges within the polymer have an effect on the size of DNA nanoparticles formed by polycations.15 In view of this complex scenario, designing attractive polymers becomes a necessity. While a number of copolymers consisting of cationic polymers of polylysine, polyethylenimine, or polyamidoamine dendrimers and hydrophilic polymers such as PEG have been developed and their in vitro and in vivo performance as carrier candidates has been tested,25-35 studies on the biophysical characterization of these polyplexes or the fate of DNA in them are rather limited.41-50 This may be due to the lack of a proper model polymer available to biophysical chemists. Polymers typically used in preparing polyplexes are mainly based on block copolymers that are neither commercially available nor easy to synthesize. Moreover, the possibility of structural variations in these block copolymers is also limited. Recently we have reported a series of random copolymers prepared using methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTAC) with different mole ratios ranging from 10 to 94 mol % of MePEGMA (Chart 1). We believe this series could be used as a model system to address and answer some basic questions relating to the mechanism of the interaction of DNA and cationic polymers.51 These polymers can be synthesized by simple free radical polymerization techniques. Functionalization of the outer surface (shell) of a delivery vehicle by a hydrophilic polymer (such as PEG) to modify its physicochemical and biological properties can be of immense value for increased transfection efficiency. An optimum thickness of the shell (which can (42) Torigoe, H.; Ferdous, A.; Watanabe, H.; Akaike, T.; Maruyama, A. J. Biol. Chem. 1999, 274, 6161-6167. (43) Vijayanathan, V.; Thomas, T.; Shirahata, A.; Thomas, T. J. Biochemistry 2001, 40, 13644-13651. (44) Saminathan, M.; Thomas, T.; Shirahata, A.; Pillai, C. K. S.; Thomas, T. J. Nucleic Acids Res. 2002, 30, 3722-3731. (45) Thomas, T.; Balabhadrapathruni, S.; Gallo, M. A.; Thomas, T. J. Oncol. Res. 2002, 13, 123-135. (46) Ramirez, F. J.; Thomas, T. J.; Antony, T.; Ruiz-Chica, J.; Thomas, T. Biopolymers 2002, 65, 148-157. (47) Park, S. Y.; Harries, D.; Gelbart, W. M. Biophys. J. 1998, 75, 714-720. (48) Golan, R.; Pietrasanta, L. I.; Hsieh, W.; Hansma, H. G. Biochemistry 1999, 38, 14069-14076. (49) Krishnamoorthy, G.; Duportail, G.; Mely, Y. Biochemistry 2002, 41, 15277-15287. (50) Dunlap, D. D.; Maggi, A.; Soria, M. R.; Monaco, L. Nucleic Acids Res. 1997, 25, 3095-3101. (51) Nisha, C. K.; Basak, P.; Manorama, S. V.; Maiti, S.; Jayachandran, K. N. Langmuir 2003, 19, 2947-2955.

2388

Langmuir, Vol. 20, No. 6, 2004

Chart 1. Structure of the Random Copolymer Used in This Study

Nisha et al. Table 1. Characteristics of the RCP in Terms of Final Composition, Molecular Weight (Mw), Number of Cationic Units per Polymer Chain (N+), and Radius of Gyration (Rg)a polymer composition (mol %) polymer MePEGMA MAPTAC N+ RCP-f32 RCP-f11 RCP-f6 a

68.2 88.9 94.1

31.8 11.1 5.9

Mw (g/mol)

Rg (nm)

A2 (mol mL/ 2 g × 105)

465 1.8 × 106 77 ( 3 8.2 ( 0.6 476 3.5 × 106 103 ( 5 5.3 ( 0.6

Obtained from ref 51.

small exothermic enthalpy changes and the overall binding process is entropically favorable. Materials and Methods be varied by simply varying the chain length of the PEG segment) and density of the shell (which can be varied by varying the quantity of the same) are necessary for the best in vivo performance of these carriers. Copolymerization of an ionic monomer with a PEG-based macromonomer has the advantage of being able to vary the morphology of the copolymers (thus of the polyplexes) in both directions, either by varying the chain length of the PEG macromonomer or by changing the feed ratio of the monomers. There is also the option of modifying the charge density (distance between two adjacent charges) along the polymer chain by introducing a third monomer having the same relative amount of ionic units and PEG segments in the copolymer. Further, biophysical studies could serve to improve the design of new gene transfer vectors. In this work, we report a complete biophysical characterization of the polyplexes obtained from cationic random copolymers (RCPs) of MePEGMA and MAPTAC monomers with calf thymus DNA (CT-DNA) (Chart 1). Beginning with a study on the structural properties of the polyplexes, we seek to understand the effect of polymer architecture on the DNA/cationic polymer interaction at the molecular level in terms of DNA stability, conformation, size, shape, binding affinity, and complete thermodynamics of the interaction. Many experiments have been performed to this end. While turbidity measurements demonstrate the effect of PEG in improving the solubility of the polyplexes, the ability of the two polymers to form stable complexes in an aqueous medium is followed by ethidium bromide exclusion and gel retardation experiments. The accessibility of DNA to nuclease enzymes has also been assessed. The nature of the charges of the polyplexes, conformational changes, stability of the DNA in the polyplexes, and the size and shape of the polyplexes are characterized by different techniques such as ξ-potential measurements, circular dichroism (CD), temperature-dependent UV, differential scanning calorimetry (DSC), atomic force microscopy (AFM), and light scattering. The overall thermodynamical parameters of the binding event of these polymers with CT-DNA has been followed by isothermal titration calorimetry (ITC). The data reveal that the complexes of the polymer with 68 mol % PEG content are insoluble in water, whereas complexes of the polymer with 89 and 94 mol % PEG content are water-soluble and stable. The complexes are in the range of 75-100 nm in size and are spherical in nature. The binding parameters show that the polymers with higher PEG content bind to CT-DNA with lower affinity. Binding of these RCPs to DNA enhances the enthalpy of the helixcoil transition by reinforcing the base-pair stacking interactions of the DNA. Comparison of the thermodynamic parameters revealed that binding is associated with

Materials. Ethidium bromide (Aldrich), Dnase 1 (Sigma), and highly sonicated CT-DNA (Bangalore Genie, India) were used as received. The size of CT-DNA is between 2 and 2.5 kbp as determined by gel electrophoresis. Milli-Q water was used for all the experiments. Synthesis of the RCPs. A full description of the synthesis and characterization of the polymers has recently been reported.51 Briefly, the copolymers were synthesized by free radical polymerization of MePEGMA and MAPTAC in water (150 mL, total monomer concentration ∼ 4 wt %) using 2,2′-azobis(2-amidinopropane) dihydrochloride (1 mol % with respect to monomer) as an initiator at 50 °C. Sodium chloride (0.089 g) was added to the monomer solution to obtain a salt concentration of 10 mM. The monomer solution was purged with argon for 30 min. Polymerization was then carried out under an argon atmosphere for 18 h. The reaction mixture was cooled and dialyzed against distilled water using cellulose membranes (cutoff value of 10 kDa) for 2 weeks. The purified copolymer solution was freezedried and analyzed by 1H NMR for the final composition. The characteristics of the RCPs are given in Table 1. Gel Electrophoresis. The electrophoretic mobility of the RCP/ CT-DNA complexes at different polycation/DNA ratios was determined by gel electrophoresis using 1.0% agarose gel in a buffer consisting of 45 mM Tris-borate and 1 mM EDTA at pH 8.0. Experiments were run at 80 V for 90 min. DNA was visualized under UV illumination by staining the gels with ethidium bromide overnight at room temperature. Nuclease Resistance of RCP/CT-DNA Complexes. CTDNA (35 µM) and an equimolar amount of RCP in 10 mM phosphate buffer (pH 7.0) containing 5 mM magnesium sulfate were mixed directly to obtain the RCP/DNA complex with Z+/) 2.0. After addition of 10 units (10 µL) of Dnase I to 1 mL of RCP/DNA complex at 25 °C, the absorbance change at 260 nm was monitored in order to follow DNA degradation by Dnase I. Ethidium Bromide Exclusion Assay. Ethidium bromide (5 µM) and 10 µM (one ethidium bromide per base pair) DNA solution were mixed in 10 mM phosphate buffer and allowed to incubate at 25 °C for 10 min. Various amounts of RCP were added to the DNA-ethidium bromide mixture and then incubated for 30 min. Fluorescence intensity was measured using a spectrofluorometer (FluoroMax-3, Spex) after diluting to 2 mL with 10 mM phosphate buffer. The excitation (λex) and emission (λem) wavelengths were 480 and 600 nm, respectively. The fluorescence of the DNA solutions in 10 mM phosphate buffer with ethidium bromide was set to 0% ethidium bromide released. ζ-Potential. ζ-Potential measurements were performed at 25 °C using a ZetaPlus zeta potential analyzer (Brookhaven Instrument Co.) equipped with a 15 mV solid-state laser operating at a wavelength of 635 nm. Typically, a solution of 35 µM CTDNA in 10 mM phosphate buffer was titrated with the appropriate RCP solution in the same buffer, by stepwise addition of 3-5 µL aliquots of this solution. CD Spectroscopy. The conformation of the polymer-DNA complexes was derived by the simple inspection of their CD spectra. The CD (JASO 700, Japan) spectra were obtained at 25 °C. Typically, a solution of 35 µM CT-DNA in 10 mM phosphate buffer was titrated with the appropriate RCP solution in the same buffer, by stepwise addition of 3-5 µL aliquots of this solution.

Polyplexes from CT-DNA/Random Copolymer Atomic Force Microscopy. A solution of 15 µM CT-DNA in 10 mM phosphate buffer and appropriate RCP solution in the same buffer were mixed together to obtain polyplex solution. The polyplex solution (2-3 µL) was deposited on a freshly split untreated mica strip (Molecular Imaging, Tempe, AZ) and allowed to adsorb for 5 min at room temperature. The mica surface was then imaged using a PicoSPM system (Molecular Imaging) operating in MAC mode. A 225 µm long magnetically coated cantilever (MAC lever) with a spring constant of 2.8 N/m and a resonance frequency of 65 kHz was used. The cantilever is made to oscillate due to the magnetic force resulting from the solenoid placed under the sample plate. The image is generated by the change in amplitude of the free oscillation of the cantilever as it interacts with the sample. The height differences on the surface are indicated by the color code: lighter regions indicate higher heights. Dynamic Light Scattering. Dynamic light scattering (DLS) data were recorded using a DLS-700 instrument (Otsuka Electronics Co. Ltd., Japan) fitted with a 5 mW He-Ne laser, operating at 632.8 nm, by placing the sample tube in the temperature-controlled chamber of the goniometer. All measurements were done at 90°. The DLS intensity data were processed using the software provided with the instrument to obtain the hydrodynamic diameter, the polydispersity index, and the diffusion coefficient of the samples. Typically, a solution of 35 µM CT-DNA in 10 mM phosphate buffer was titrated with the appropriate RCP solution in the same buffer, by stepwise addition of 3-5 µL aliquots of this solution. Measurement of the Melting Curve of RCP/DNA Complexes. CT-DNA was dissolved in 10 mM sodium phosphate buffer (pH 7.0) to a final concentration of 35 µM. Different volumes of RCP solutions were separately added to a constant volume of DNA solution to obtain RCP/DNA complexes of different charge ratios. After 2 h of incubation at room temperature, the melting profiles of the complexes were obtained by monitoring the absorbance of the complexes at 260 nm as a function of temperature. The samples were heated from 40 to 90 °C at a scanning rate of 1.0 °C/min. Transition temperatures (TM) have been calculated from the melting curves in order to gain insight into the helix-coil transition. Differential Scanning Calorimetry. The unfolding heats of CT-DNA in the free state or in polyplexes were measured with the MC-2 differential scanning calorimeter from Microcal Inc. (Northampton, MA). Typically, a solution of 250 µM DNA solution in 10 mM sodium phosphate buffer (pH 7.0) and CT-DNA/RCP solution of Z+/- ) 2.0 in the same buffer were scanned against a buffer solution from 40 to 100 °C at a heating rate of 1.0 °C/ min. A buffer versus buffer scan was subtracted and normalized by the effective number of base pair moles used. Integration of the resulting curve (∫∆Cp dT) yields model-independent enthalpies of unfolding, ∆Hcal. The entropy of unfolding (∆Scal) was obtained by a similar integration (∫(∆Cp/T) dT) from the ∆Cp/T versus T plot. For the calculation of these terms, it is assumed that the duplex and random coil states have similar heat capacities in both the free state and the polyplexes. The free energy at any temperature T is obtained from the Gibbs relationship: ∆G°cal(T) ) ∆Hcal - T∆Scal ) ∆Hcal(1 - T/TM). Model-dependent enthalpy changes, van’t Hoff enthalpy (∆HvH), were obtained from shape analysis of the melting curves assuming a two-state transition. Furthermore, the nature of the helix-coil transition has been studied from the ratio ∆HvH/∆Hcal; a value close to 1 indicates a two-state transition. Isothermal Titration Calorimetry. A Microcal Omega instrument was used to measure the heat evolved during the interaction of RCP with DNA at 25 °C. RCP solution was injected by a 100 µL syringe into the reaction cell containing the DNA solution with constant stirring at 400 rpm. The concentration of the DNA solution in the reaction cell was approximately 0.2 mM, expressed in terms of phosphate, and a 15 times concentrated solution of the RCP was used to titrate this solution. Typically, 15-20 injections of 5 µL each were done in a single experiment. The instrument was calibrated with a known electrical pulse, and its overall sensitivity is ∼1 µcal. Raw data were converted into injection heats using Microcal Origin 5.0 software provided by MicroCal. The dilution heat was estimated from the “beyondthe-end-point” part of the calorimetric profile, a procedure verified

Langmuir, Vol. 20, No. 6, 2004 2389

Figure 1. Variation of the turbidity as a function of the charge ratio in the mixture, Z+/-, for (2) RCP-f32, (9) RCP-f11, and (b) RCP-f6. The CT-DNA concentration was kept constant at 15 µM in phosphate. Turbidity measurements, reported as (100 - %T)/100, where T is the transmittance, were carried out at 420 nm using a CARY 400 Bio(Varian) spectrophotometer. The turbidity values were recorded after the values became stable (about 30 min). in numerous separate dilution runs. The dilution heat was subtracted, and the heat of complex formation obtained was integrated over the titration range.

Results Solubility of the CT-DNA/RCP Complexes. The solubilities of DNA/RCP complexes (polyplexes) with different compositions were tested by turbidity measurement experiments. Figure 1 shows the variation of turbidity of different DNA/RCP systems, as a function of Z+/- (where Z+/- is the ratio of the concentration of cationic units of the polymers to the concentration of negative charges present in CT-DNA). As shown in Figure 1, macroscopic phase separation was observed for the complexes from the DNA/RCP-f32 system, whereas no such phase separation was observed for DNA/RCP-f11 and DNA/RCP-f6 systems up to Z+/- ) 5. In the latter complexes, it is expected that the cationic units of the polymer segment will form ion-pairs with the anionic phosphate groups of DNA yielding charge-neutralized complexes. The complexes, however, will remain soluble in contrast with regular DNA/cationic polymer systems, even at complete charge neutralization points, due to the lyophilizing effect of the PEG segments of the copolymer. Hence, the stability of such complexes in an aqueous medium should obviously depend on the relative amount of PEG segments present in the polymer. In the case of RCP-f32/DNA, where the PEG content is 68 mol %, the lyophilizing effect of PEG segments is not high enough to make these complexes stable in the aqueous medium. In contrast, for the other two RCPs, RCP-f11 and RCP-f6, this lyophilizing effect is sufficient to hold the complexes in water even at Z+/- ) 1. Gel Electrophoresis Studies. Gel electrophoresis studies allow visualization of the interaction of DNA and polycations. Polyplexes of CT-DNA with RCP-f11 and RCPf6 were formed at different charge ratios, and agarose gel electrophoresis was subsequently performed. Representative images for both polymers are presented in Figure 2. While free DNA or incompletely neutralized DNA migrates in the electric field toward the anode, full retardation occurred at and above Z+/- ) 1.5 in the case of both polymers. This study clearly demonstrates that the cationic segments of the copolymer are neutralizing the negative charges of DNA, thereby resulting in the formation of stable polyplexes.

2390

Langmuir, Vol. 20, No. 6, 2004

Nisha et al.

Figure 4. Dnase 1 digestion assays for (2) DNA/RCP-f32, (9) DNA/RCP-f11, and (b) DNA/RCP-f6 complexes.

Figure 2. Electrophoretic mobility shift assay on agarose gel (1%) for polyplexes of (a) RCP-f11 and (b) RCP-f6. Polyplexes were prepared by mixing of RCP and CT-DNA at different charge ratios. The experiments were run in a buffer consisting of 45 mM Tris-borate and 1 mM EDTA at pH 8.0 at 80 V for 90 min. The gels were visualized under UV illumination by staining the gels with ethidium bromide overnight at room temperature. Figure 5. Variation of ζ-potential as a function of the charge ratio in the mixture, Z+/-, for (9) CT-DNA/RCP-f11 and (b) CTDNA/RCP-f6 systems. The CT-DNA concentration was kept constant at 50 µM in phosphate. Measurements were performed at 25 °C using a ZetaPlus zeta potential analyzer (Brookhaven Instrument Co.) equipped with a 15 mV solid-state laser operated at a wavelength of 635 nm.

Figure 3. Percent of ethidium bromide exclusion due to the binding of (9) RCP-f11 and (b) RCP-f6 as a function of Z+/- at 25 °C. The excitation (λex) and emission (λem) wavelengths were 480 and 600 nm, respectively. The fluorescence of the DNA solutions in 10 mM phosphate buffer with ethidium bromide was set to 0% ethidium bromide released.

Ethidium Bromide Exclusion Assay. The ability to form complexes with DNA was inferred from the ethidium bromide exclusion assay results. Figure 3 shows the amount of ethidium bromide released from DNA upon complex formation with RCP. In both complexes of RCPf11 and RCP-f6, the percentage of ethidium bromide exclusion increases by increasing the charge ratio (Z+/-) to 1.4 and 1.8, respectively. Nuclease Resistance of DNA/RCP Complexes. The stability of DNA in the polyplex was further studied from the viewpoint of nuclease resistance.52 While addition of Dnase I to native DNA solution immediately increases the absorbance due to the fragmentation of DNA, no (52) Katayose, S.; Kataoka, K. J. Pharm. Sci. 1998, 87, 160-163.

substantial increase in the absorbance was observed for the polyplex system (Figure 4). High nuclease resistance ability of the polyplex system indicates the stable and inert nature of DNA when it complexes with both the RCPs, implying that the complex formation inhibits exposure of DNA to chemical perturbations present in the system. No substantial difference in Dnase activity was observed for RCP-f11 and RCP-f6. ζ-Potential Measurements. Soluble complexes of cationic random copolymers and CT-DNA were characterized using the laser microelectrophoresis technique. Figure 5 presents the ζ-potential of the particles as a function of Z+/-. In all cases, the addition of copolymer to the CT-DNA solution resulted in an increase in ζ-potential. A further increase in the concentration of copolymer caused the ζ-potential to level off at small negative values, ca. -2.0 and -1 to -1.5 for RCP-f11 and RCP-f6, respectively, indicating that the particles are essentially electroneutral. Since no changes in ζ-potential were observed at Z+/- > 1.5 for RCP-f11 and at Z+/- > 1.9 up to Z+/- ) 4 for RCP-f6, it can be concluded that the excess polycation does not incorporate into the complex above these Z+/- ratios. Similar observations have been reported in the literature.53,54 CD Spectroscopy Study. Circular dichroism spectroscopy has been used to detect possible conformation (53) Lim, Y. B.; Choi, Y. H.; Park, J. S. J. Am. Chem. Soc. 1999, 121, 5633-5639. (54) Choi, J. S.; Joo, D.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480.

Polyplexes from CT-DNA/Random Copolymer

Langmuir, Vol. 20, No. 6, 2004 2391

Figure 6. Circular dichroism spectrum of CT-DNA (100 µM in phosphate) of polyplexes with varying amounts of (a) RCPf11, Z+/- ) 0-4, and (b) RCP-f6, Z+/- ) 0-4, in 10 mM sodium phosphate buffer pH 7 at 25 °C. The down-headed arrow indicates the increment of Z+/-.

changes of DNA upon formation of complexes with RCPs. Figure 6 shows the CD spectra of the free CT-DNA and RCP bound complexes at several Z+/- ratios. The CD spectrum of the free CT-DNA is typical of a duplex in the B conformation, and addition of copolymer results in CD spectra with similar shapes. In the case of RCP-f11, the magnitude of the positive band decreases somewhat with increasing Z+/-, while in the case of RCP-f6, negligible changes are observed. The negative bands in the CD spectrum are unaffected in the case of both of the RCPs. Similar observations have been reported when cationic graft copolymers poly(L-lysine)-graft-dextran and poly(ethylene oxide)-g-polyethylenimine (PEO-g-PEI) interact with poly(dA)‚poly(dT) and poly[d(AT)]‚poly[d(AT)], respectively.38,55 A decrease in the magnitude of the band at around 270 nm has also been observed for free DNA with an increase in salt concentration.56,57 Though noticeable changes were observed in the case of RCP-f11, such changes were totally absent when DNA interacts with RCP-f6. The overall study suggests that binding of RCP to phosphate groups of the DNA induces only minor perturbations in the DNA helical structure. Even this perturbation can be prevented if the polymer contains sufficient amounts of PEG. Atomic Force Microscopy. Atomic force microscopy was used to determine the size and shape of the polyplexes. Figure 7 shows graphs detailing the height and diameter of polyplexes formed from two RCPs at two Z+/- values. The elongated shape and broad size distribution are evidenced in these images at a charge ratio of 1 for both polymers. At this charge ratio of 1.0, both of the polymers could only partially bind to DNA. The extent of complexation increased on increasing the charge ratio. A complete complex was observed at Z+/- ) 2 for both of the polymers. It is also noticed from the images that the complexes are more irregular in shape at Z+/- ) 1 and become spherical in shape at higher Z+/- values where the entire DNA are bound in the polyplexes. The size of the isolated particles obtained from AFM images were found to be 80 ( 10 nm and 90 ( 10 nm for the complexes of RCP-f11 and RCP-f6 with Z+/- ) 2, respectively. (55) Bronich, T.; Kabanov, A. V.; Marky, L. A. J. Phys. Chem. B 2001, 105, 6042-6050. (56) Burckhardt, G.; Zimmer, C.; Luck, G. FEBS Lett. 1973, 15, 3539. (57) Vorlickova, M.; Kypr, J.; Kleinwachter, V.; Palecek, E. Nucleic Acids Res. 1980, 9, 3965-3973.

Figure 7. Atomic force microscopy images of polyplexes of (a) RCP-f11, Z+/- ) 1, (b) RCP-f6, Z+/- ) 1, (c) RCP-f11, Z+/- ) 2, and (d) RCP-f6, Z+/- ) 2.

Figure 8. Variation of apparent hydrodynamic diameter (dap) as a function of the charge ratio in the mixture, Z+/-, for (9) RCP-f11 and (b) RCP-f6. The CT-DNA concentration was kept constant at 50 µM in phosphate. Hydrodynamic diameter measurements were done on a Beckman Coulter N4 Plus particle size analyzer.

Size Measurements by Light Scattering. The sizes of the polyplexes were also determined by light scattering techniques. Figure 8 presents the dependence of the effective diameters of RCP/CT-DNA complexes on Z+/-. Addition of the polymer in the range 0.25 < Z+/- < 1 resulted in a profound increase in the particle size, apparently caused by the RCP-induced interaction with DNA. Further increase of RCP concentration results in the decrease of the particle size, and above a certain Z+/value (1.5 for RCP-f11 and 1.7 for RCP-f6), the sizes of the particles do not change. It can be concluded that for RCPf11, particle formation is completed at Z+/- ) 1.5 and the size of the particle is 80 ( 10 nm. In the case of RCP-f6, a complete particle was obtained at Z+/- ) 1.8 with the size of 95 ( 10 nm. Figure 9 shows the particle size distribution (polydispersity index) as a function of Z+/for both polymers. As seen from Figure 9, the particles are polydisperse at lower Z+/- values but become quite monodisperse at higher Z+/-. This is consistent with our AFM findings. No size change was observed after storing these systems in solution for very long periods of time, which indicates that particles do not aggregate on storage.

2392

Langmuir, Vol. 20, No. 6, 2004

Figure 9. Change in polydispersity index of the polyplexes as a function of Z+/- for (9) RCP-f11 and (b) RCP-f6.

Nisha et al.

Figure 11. Differential scanning calorimetry of (2) free CTDNA, (9) the polyplex of RCP-f11, and (b) the polyplex of RCP-f6 (at Z+/- ) 2) in 10 mM sodium phosphate buffer at pH 7. Table 2. Thermodynamic Parameters for the Unfolding of CT-DNA and DNA/RCP Complexes at Z+/- ) 2a complex free DNA RCP-f11 RCP-f6

∆HvH ∆Hcal TM (°C) TM (°C) (kcal/mol) (kcal/mol) ∆HvH/∆Hcal 73 73 73

93 81

8.2 9.2 8.9

738.2 279.4 311.5

90 30 35

a All measurements were done in 10 mM sodium phosphate buffer at pH 7.0. The TM’s, ∆Hcal, and ∆HvH are within (1 °C, (3%, and (10%, respectively.

Figure 10. UV melting curves of polyplexes of (a) RCP-f11 and (b) RCP-f6 as a function of Z+/- in 5 mM sodium phosphate buffer; the curves correspond to Z+/- values of (2) 0, (9) 0.5, (b) 1.0, and (() 2.

Measurement of Melting Curve of RCP/CT-DNA Polyplexes. The stability of the polyplexes was studied from the melting curves obtained by UV spectroscopy. This method has been widely used for the characterization of other cationic polymer/CT-DNA complexes. Melting is conveniently monitored by an increase in the absorbance (hyperchromic effect) that results from the disruption of base stacking in double-stranded DNA due to the breakage of hydrogen bonds.58 Figure 10 shows the melting profiles of RCP/CT-DNA complexes at different charge ratios in 5 mM sodium phosphate buffer. Melting curves show a biphasic behavior with the main transition at 73 °C (which is primarily due to the melting of free DNA, TM°), decreasing in size as the charge ratio increases, while a second transition emerges at higher temperature. Similar biphasic melting behavior has been observed for the complexes of DNA with PEO-block-poly(L-lysine) and dextran-g-poly(L-lysine),25,38 whereas monophasic melting transitions have been observed for the PEO-g-PEI/poly[d(AT)]‚poly[d(AT)] systems.55 The second transition at higher temperature is due to the helix-coil transition of complexed DNA (TM), and it was at ∼93 and 81 °C for RCP-f11 and RCP-f6, respectively. The melting temperature shift upon complex formation (∆TM ) TM - TM°) is higher in the case of RCP-f11 (20 °C) than in the case of RCP-f6 (8 °C). This indicates that DNA in the polyplex of RCP-f11 is more stable than DNA in the polyplex of RCP-f6. (58) Breslauer, K. J. Methods Enzymol. 1995, 259, 221-242.

DSC Measurements of RCP/CT-DNA Polyplexes. As demonstrated by the melting curve experiments described above, stabilization of the double-stranded helical structure of DNA complexed with RCP-f11 or RCPf6 was achieved through the neutralization of the negative charge on the phosphate group by the -N+(CH3)3 group of the cationic unit present in the polymer. To measure the true enthalpy changes associated with the helix-coil transition of DNA in the polyplex, differential scanning calorimetry was performed. The DNA concentration used (250 µM) was approximately 8 times higher than that in the UV melting studies (∼35 µM in base pair) in order to have a measurable signal in the DSC instrument. The DSC curve of native CT-DNA showed an endothermic peak at 73 °C (Figure 11), while that for RCP/CT-DNA with Z+/- ) 2 showed similar peaks but at higher temperatures of 82 and 94 °C for RCP-f11 and RCP-f6, respectively. All melting temperatures, that is, of free CT-DNA and the complexed DNA, obtained from this experiment are consistent with the previous UV melting experiments and are within experimental error. The true enthalpy changes, ∆Hcal, associated with the helix-coil transition were obtained from the integrated area under the DSC curves. The enthalpy change for the helix-coil transition of free DNA was 8.2 kcal/mol of base pair. The enthalpy changes for DNA complexed with RCP-f11 and RCP-f6 were estimated to be 9.3 and 8.9 kcal/mol of base pair, respectively. It has been observed that the unfolding enthalpy of CT-DNA in the polyplex is more than that of free CT-DNA. The van’t Hoff enthalpy changes, ∆HvH, involved in this transition were obtained from the shape analysis assuming a two-state model and are presented in Table 2. The ratio between ∆HvH and ∆Hcal (∆HvH/∆Hcal) measures the cooperative melting unit of DNA.55 The obtained cooperative melting units are ∼90, 30, and 35 for DNA in the free state, in polyplexes of RCP-f11, and in polyplexes of RCP-f6, respectively. This is consistent with the electrostatic nature of the interaction of random copolymer with DNA.55

Polyplexes from CT-DNA/Random Copolymer

Langmuir, Vol. 20, No. 6, 2004 2393 Table 3. Standard Thermodynamic Profiles for the Interaction of RCP with CT-DNA at 25 °Ca

RCP RCP-f11 RCP-f6

Kb Kb (EB exclusion) (melting) ∆Hb ∆Gb° T∆Sb -1 (M ) (kcal/mol) (kcal/mol) (kcal/mol) (M-1) 9 × 104 7 × 104

2.5 × 104 0.8 × 104

-6 -5.3

-0.90 -0.60

5.1 4.7

a All thermodynamic parameters were determined in 10 mM sodium phosphate buffer at pH 7.0. The values of ∆Gb°, ∆Hcal, and T∆Sb are within (10%, (3%, and (7%, respectively.

stranded DNA. In the equation, TM° and TM are the transition temperatures of the free and saturated bound DNA duplexes, respectively; ∆Hcal is the transition enthalpy of the free duplex obtained from DSC experiments; aL is the activity of the free copolymer and is assumed to be equal to half of the total concentration of copolymer in terms of cationic units; and n is the apparent number of binding sites per duplex, assumed to be equal to 1. The binding affinities obtained from this equation are at the melting temperatures of the DNA in the polyplex and were extrapolated to the temperature of interest using the van’t Hoff equation: Figure 12. Isothermal titration calorimetry experiments. (a) Sample raw data for the titration of RCP-f11 into CT-DNA duplex at 25 °C in 10 mM sodium phosphate buffer. Each peak shows the heat produced by a serial injection of an aliquot of polymer solution (10 µL of 1.092 mM in cationic units) into the DNA solution. (b) Variation of enthalpy changes obtained from integration with respect to time, with appropriate molar correction (see the text), vs Z+/-.

Binding Enthalpy from Isothermal Titration Calorimetry. To investigate the energetics of binding in more detail, titrations of DNA samples with RCP were performed using isothermal calorimetry. Typical raw experimental data are presented in the upper panel of Figure 12. The lower panel shows the integrated heats of reaction plotted against Z+/- after the correction for dilution effects of the polymer (see the experimental section). In general, exothermic heats accompany the binding of RCP to CTDNA. The enthalpies at 25 °C are -900 cal/mol for RCPf11 and -600 cal/mol for RCP-f6. This is in contrast to the finding by Bronich et al. where they have observed that binding of PEG-PEI copolymer with DNA is accompanied by small endothermic enthalpy changes.55 A large exothermic enthalpy change (-4.76 kcal/mol) and a small endothermic enthalpy change (0.76 kcal/mol) were observed when cationic surfactant binds to isolated and continuous phosphate sites of DNA, respectively.59 In other studies on DNA-lipid interaction, a small endothermic binding enthalpy (960 cal/mol) was observed.60 The low enthalpy changes for both polymers are consistent with the previous reports on the electrostatic binding that is involved with very small enthalpy changes.55 Determination of Binding Affinities. The observed increase in the thermal stability, ∆TM, of the polymerbound DNA was used to calculate the binding affinities, Kb, for the binding of RCPs to CT-DNA according to the following equation:61

∆TM ) (RTM°TM/n∆Hcal) ln(1 + KbaL)

(1)

assuming that the binding of polymer to single-stranded DNA is much weaker than the binding with double(59) Spink, C. H.; Chaires, J. B. J. Am. Chem. Soc. 1997, 119, 1092010928. (60) Pozharski, E.; MacDonald, R. C. Biophys. J. 2002, 83, 556-565. (61) Crothers, D. M. Biopolymers 1971, 10, 2147-2160.

δ ln K/δ(1/T) ) -∆Hb(T)/R

(2)

where ∆Hb(T) is the binding enthalpy measured in titration calorimetric experiments and used in this calculation. The calculated binding affinities are 2.5 × 104 and 7.5 × 103 M-1 (in terms of cationic charge concentration) for RCP-f11 and RCP-f6. This again reinforces that RCP-f11 binds with CT-DNA with a higher affinity than RCP-f6. Another approach to evaluate the binding affinities from the ethidium bromide exclusion study was investigated. The charge ratio of the complexes of RCP-f11 copolymer with DNA needed to achieve 50% (CRCP-f11-50%) release of bound ethidium (CRCP-f6-50%) is less than the charge ratio needed for RCP-f6 copolymer. This critical concentration of the copolymer needed to exclude 50% ethidium bromide can provide a qualitative comparison between the binding affinities of the copolymers with DNA following the equation62

KEBCEB ) KRCP-f11CRCP-f11-50% ) KRCP-f6CRCP-f6-50% (3) where KEB is the binding affinity of ethidium bromide for CT-DNA and was determined from independent fluorescence experiments by titrating a known amount of ethidium bromide by successive addition of concentrated CT-DNA solution under the same conditions. The obtained KEB from a Scatchard plot (data not shown) was found to be 2.8 × 105 M-1 at 25 °C. KRCP-f11 and KRCP-f6 are the binding affinities of RCP-f11 and RCP-f6, respectively. CRCP-f11-50% and CRCP-f6-50% are the concentrations needed to exclude 50% of bound ethidium bromide (CEB-50%). The obtained KRCP-f11 and KRCP-f6 are presented in Table 3. Again, we observed that RCP-f11 binds with higher affinity to CT-DNA than RCP-f6. This is due to the presence of a greater number of hydrophilic PEG segments in RCP-f6 which causes more steric hindrance than in RCP-f11 to further the electrostatic polyionic interaction of free copolymers than in RCP-f11. The binding affinity of the cationic amphiphiles to DNA has been extensively studied. It has been observed that cationic lipids exhibit strong binding to DNA even at (62) Morgan, A. R.; Lee, J. S.; Pulleyblank, D. F.; Murray, N. L.; Evans, D. H. Nucleic Acids Res. 1979, 7, 547-569.

2394

Langmuir, Vol. 20, No. 6, 2004

Figure 13. A comparison of the enthalpic and entropic contributions to the binding free energy at 25 °C, where ∆G° ) ∆H - T∆S: (a) RCP-f11 and (b) RCP-f6.

moderate ionic strength, in contrast to DNA condensation by polyvalent cations. The difference is due to the lamellar organization of the lipid with binding sites for an entire DNA double strand as a single unit. Though an effort has been made to understanding the biophysics of DNAcationic polymer interaction of therapeutic interest, less attention has been paid to finding the binding affinity. Recently, Bronich et al. have reported the binding affinity between PEG-PEI block copolymer and polydA‚polydT and found it to be in the order of 103 M-1 in terms of cationic units.55 In this study, we have observed the binding affinity of these random copolymers to be in the order of 104 M-1 in terms of cationic units. Complete Thermodynamic Parameters. The thermodynamic profiles for RCPs binding with CT-DNA were determined at 25 °C by combining the general thermodynamic relations ∆Gb° ) -RT ln K and ∆Gb° ) ∆Hb° -T∆Sb° with the experimental K values obtained from UV melting curves and ∆Hb values obtained from the corresponding calorimetric titrations. In these calculations, it was assumed that ∆Hb does not depend on the concentration and therefore may be equated with ∆Hb°. The obtained parameters are presented in Table 3. Figure 13 compares entropy-enthalpy compensation, and inspection of Figure 13 also shows that the binding of RCP is primarily an enthalpy-driven process. Low exothermic ∆Hb values result very likely from local, short-range, nonspecific and electrostatic interactions between the cationic units of the polymer and the negatively charged phosphate groups. Discussion The research described here represents a complete biophysical characterization of the interactions between two newly synthesized random cationic copolymers and the nucleic acid CT-DNA. Such a thorough study is needed for a better understanding of the various factors involved in the polyplex formation, which in turn imparts more detailed insight into designing novel polyplexes. From turbidity studies (Figure 1), it has been observed that among the three copolymers, RCPs with PEG content of 89 and 94% form soluble polyplexes when they bind with CT-DNA. RCPs bind with CT-DNA through electrostatic interactions among the cationic charges in the polymers and negatively charged phosphate groups of CT-DNA. This interaction causes charge neutralization of the phosphate groups of the DNA backbone. RCP-f11 and RCP-f6 contain sufficient amounts of water-soluble polymer, PEG, which allow the polyplexes to be soluble in water due to PEG’s lyophilizing effects. In the case of RCP-f32, which contains

Nisha et al.

only 68% PEG, the lyophilizing effect arising from the PEG segments is not sufficient to make the polyplexes water-soluble. This result clearly indicates that the solubility of the polyplexes depends on the polymer composition and can be fine-tuned by just varying the PEG content of the cationic polymers, thereby making it rather attractive from an application point of view. Gel electrophoresis (Figure 2) and ethidium bromide exclusion studies (Figure 3) of the soluble polyplexes of RCPs with 89 and 94% PEG content indicate that both polymers are capable of binding with CT-DNA completely at a charge ratio of 1.4 ( 0.1 and 1.8 ( 0.1, respectively. One could expect that the binding event will be completed at Z+/- ) 1. However, AFM images (Figure 7) also confirm that the binding events are completed at higher Z+/values, which indicates that the polyplexes obtained from RCPs are nonstoichiometric. DNA present in these polyplexes is inaccessible for Dnase 1, which clearly indicates that the PEG segments present in the outer part of the polyplexes protect the DNA present inside the polyplexes. The high resistance of DNA to such nuclease attack is surely an advantage of using these polyplexes as a reservoir for DNA under physiological conditions. ζPotential measurements indicate that the polyplexes obtained from both the RCPs are electroneutral in nature (Figure 5). Introduction of further RCP to the polyplexes does not cause any change in the ζ-potential of the polyplex. This indicates that above Z+/- ) 1.5 for RCP-f11 and Z+/) 1.9 for RCP-f6, the polymer does not get incorporated into the polyplexes. Steric repulsion arising from the PEG segments could prevent further incorporation of RCP into the polyplexes. In CD studies, a minor decrease in the CD signal at 273 nm was observed when RCP-f11 binds to CT-DNA (Figure 6); no such decrease was observed in the case of the RCPf6 binding. In addition, the CD signal at the negative band (246 nm) was unaltered with both polymers. It is known that a wide variety of multi- and polyvalent cations condense DNA into very compact forms. The characteristics in CD spectra of such condensed DNA are very much dependent on the size (length) and shape (linear or circular) of the DNA. Damaschun et al. showed that addition of spermidine to 750 bp long DNA causes changes in the CD spectrum which is attributed to the BfΨ transition,63 while Gosule and Schellman saw no changes in the CD spectrum of unsonicated T7 DNA.64 Another study on the influence of DNA length on spermine-induced condensation by electric dichroism measurements demonstrates that 258 and 436 bp DNA condensed into rodlike particles while 748 bp or longer DNA condensed into torusshaped particles.65 In the present case, we do not see any major changes in the CD spectrum, probably due to the longer length of the CT-DNA. But it is clear that binding of polycations causes DNA condensation and leads to the formation of compact particles as observed in AFM images (Figure 7). The sizes of the polyplexes obtained from AFM images are in the range of 80-90 nm. At lower charge ratios, polyplexes are quite polydisperse and irregular in shape but become regular in size and shape when complete binding is achieved (Figure 7). This phenomenon is also evidenced by light scattering experiments (Figure 8 and Figure 9). Polyplexes of RCP-f11 are smaller than polyplexes of RCP-f6 (Figure 7). This is probably due to better compaction of DNA with RCP-f11 owing to its higher charge (63) Damaschun, H.; Damaschun, G.; Becker, M.; Buder, E.; Misselwitz, R. Nucleic Acids Res. 1978, 10, 3801-3809. (64) Gosule, L. C.; Schellman, J. A. J. Mol. Biol. 1978, 121, 311-326. (65) Marquet, R.; Wyart, A.; Houssier, C. Biochim. Biophys. Acta 1987, 909, 165-172.

Polyplexes from CT-DNA/Random Copolymer

Langmuir, Vol. 20, No. 6, 2004 2395

density along the polymer chain forming a more compact structure. These results also confirm that polyplex morphology can be controlled by tailoring the polymer architecture. UV melting (Figure 10) and DSC studies (Figure 11) indicate that DNA present in the polyplexes is more stable than the free state. This is consistent with previous studies of a variety of cationic polymers including homopolymers and copolymers that stabilize DNA upon binding to it.25,38,55 The binding of monovalent and multivalent (e.g., Na+, Mg2+, etc.) counterions to DNA reduces the electrostatic repulsion among the phosphate groups of the DNA backbone and induces the stability of the helical structures of DNA.66 As a result, the helix-coil transition occurs at higher temperatures at high salt concentrations. In cationic polymers, the binding of cationic units to DNA phosphate groups induces stability in a similar way. It has been observed that the melting temperature of poly[dA-dT] was increased by 6 °C when changing the medium from 1 M NaCl to 1 M Me4N+Cl.67 This has been interpreted in terms of the increased stability of the helix due to stronger binding of the simple tetraalkylammonium ions compared to the Na+ to the duplex. Since the cationic units of the RCPs studied here are tetraalkylammonium ions, it is quite likely that the polymer also stabilizes DNA to a higher extent due to stronger binding of the cationic units to the phosphate groups of the DNA backbone. RCPf11 stabilizes DNA better than RCP-f6 due to the difference in the PEG content. RCP-f11 contains less PEG per cationic unit (8 and 15.5 PEG segments per cationic unit for RCPf11 and RCP-f6, respectively) and allows the cationic charge of the polymer and the negative phosphate groups of DNA to come closer together. The electrostatic interaction between the cationic unit of RCP-f11 and DNA phosphate groups is stronger than that formed by the cationic unit of RCP-f6. As a result, DNA in the polyplex of RCP-f11 is more stable and melts at higher temperatures. The biphasic melting behavior indicates the coexistence of completely neutralized and partially neutralized DNA. This result is in agreement with the gel electrophoresis mobility studies, where we observed two bands below Z+/) 1. Enthalpy changes for helix-coil transitions obtained from DSC studies are endothermic in all cases but are more endothermic in nature when DNA is present in the polyplexes. These results show that binding of RCP to DNA reinforces the base-pair stacking interactions of DNA. Similar base-pair stacking interactions of DNA have been observed when DNA binds to other cationic charged units.55 More endothermic enthalpy for RCP-f11 indicates that the extent of reinforcement is greater for RCP-f11 than RCP-f6, confirming that RCP-f11 binds more tightly than RCP-f6. Further inspection of the melting behavior of CT-DNA in the polyplexes showed that DNA in polyplexes melts in a less cooperative manner. Binding of RCPs to CT-DNA is associated with small exothermic enthalpy changes as shown by ITC experiments (Figure 12). The small enthalpy of binding is consistent with the electrostatic interactions similar to those observed in other such systems. In contrast to our study, a small endothermic enthalpy change was observed when PEG-PEI binds with poly[d(AT)]‚poly[d(AT)].55 In these binding events, the observed enthalpy changes are the sum of the enthalpy changes associated with the following events which occur during the binding processes: (1) exothermic contribution from electrostatic

interactions, (2) exothermic contribution from van dar Waals interactions, (3) endothermic contribution from the removal of bound water from the charged and polar groups, (4) exothermic contribution from the rehydration of the polyplexes (which depends on the PEG content of binding polymers), (5) endothermic contribution from the conformational changes of DNA and the polymers, and so on.68 The nature of enthalpy changes, exothermic versus endothermic, will definitely depend on the polymer architecture as the contributions stated in points 2, 4, and 5, which directly depend on the polymer architecture. The difference in the binding enthalpy between RCP-f11 and RCP-f6 confirms this statement, as these two polymers are different only in terms of their composition. Though the affinity obtained in this study is apparent and contains large error, still it may give us a qualitative idea to answer the question: how strongly do these polymers bind with DNA compared to other reported cationic agents? Moreover, it will help us to estimate the complete thermodynamic parameters involved in the binding process (this will be discussed in the following paragraph). Binding affinities obtained by two different approaches indicate that they are in the order of 104 M-1 (cationic unit of the polymers), and this is in good agreement with the results obtained by Bronich et al.55 Indeed, the apparent binding constant, Kapp, is given by Kapp ) N exp(-N∆G°/RT), in which N is the number of cationic charges per polymer molecule and ∆G° is the free energy change upon binding of an individual charge. With N ) 465 for RCP-f11 and N ) 476 for RCP-f6 (Table 1), the apparent binding affinity would be in the micromolar range even though ∆G° is as small as 0.04RT. Evaluation of the complete thermodynamic parameters (∆G°, ∆H, and T∆S) allows us to have detailed insight into this binding process. Comparing these three parameters (Figure 13), it is clear that the binding process is primarily entropy driven. The spontaneity of the binding process and small exothermic enthalpy changes confirm this statement. The increment of entropy in the system mainly arises from the release of monovalent counterions from the DNA phosphate groups and is supported by the results of the thermodynamic analysis of the osmotic stress-induced condensation of DNA in the presence of multivalent cations.69

(66) Wolf, B.; Hanlon, S. Biochemistry 1975, 14, 1661-1670. (67) Marky, L. A.; Patel, D.; Breslauer, K. J. Biochemistry 1981, 20, 1427-1431.

(68) Chaires, J. B. Biophys. Chem. 1997, 64, 15-23. (69) Parsegian, V. A.; Rand, R. P.; Rau, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3987-3992.

Conclusions The polymers used in this study to synthesize the polyplexes are random copolymers of MePEGMA and MAPTAC and can easily be synthesized by free radical polymerization reaction and controlling the PEG content. Though all the copolymers bind with CT-DNA to yield polyplexes, the polyplexes of the polymers with PEG contents of 89 and 94 mol % are stable and water-soluble. The polyplexes are in the range of 75-100 nm in size and are spherical in shape. These polyplexes have micellelike structures with nanometric hydrophobic domains of cationic units neutralized by phosphate anions surrounded by a sheath of hydrophilic segments. The thermal stability of DNA in the polyplexes is higher than that of free DNA, and the extent of stability depends on the PEG content of the polymers. Polymers with lower PEG content bind with higher affinity, and the binding process is mainly entropically driven. The ease of preparation and broader choice of polymer architecture make these systems attractive with potential theoretical and practical significance in vehicle-based gene therapy research.

2396

Langmuir, Vol. 20, No. 6, 2004

Acknowledgment. Financial support for this work from the Department of Science and Technology, Government of India, New Delhi (fast track young scientist grant to S.M, Project Ref. No. SR/FTP/PS-34/2001), and from the Council of Scientific and Industrial Research, Government of India, New Delhi (senior research fellowship

Nisha et al.

to C.K.N.), is gratefully acknowledged. S.M. gratefully acknowledges Professor Luis A. Marky from the University of Nebraska Medical Center, Omaha, NE, for fruitful collaboration in the initial stages of the project. LA035737R