Correlation of Amine Number and pDNA Binding Mechanism for

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Langmuir 2008, 24, 8090-8101

Correlation of Amine Number and pDNA Binding Mechanism for Trehalose-Based Polycations Lisa E. Prevette,† Matthew L. Lynch,‡ Karina Kizjakina,† and Theresa M. Reineke*,† UniVersity of Cincinnati, Department of Chemistry, P.O. Box 210172, Cincinnati, Ohio 45221-0172, and The Procter & Gamble Company, Corporate Research DiVision, Miami Valley Laboratories, 11810 East Miami RiVer Road, Cincinnati, Ohio 45252-1038 ReceiVed January 14, 2008. ReVised Manuscript ReceiVed March 13, 2008 Glycopolymers with repeat units comprised of the disaccharide trehalose and an oligoamine of increasing amine have been previously synthesized by our group and shown to efficiently deliver pDNA (plasmid DNA) to HeLa cells while remaining relatively nontoxic. Complexes formed between the most amine-dense of these polycations and pDNA were also found to be relatively stable in serum and have low aggregation, which is desirable for in vivo gene delivery. To lend insight into these interesting results, this study was aimed at investigating the binding strength and mechanism of interaction between these macromolecules, via isothermal titration calorimetry (ITC) and ethidium bromide exclusion assays. The size of these pDNA-polymer complexes, or polyplexes, at various states of formation was determined through light scattering and ζ-potential measurements. Varying degrees of pDNA secondary structure change occurred upon interaction with the polymers, as evidenced by circular dichroism spectra through increasing molar ratios of polymer amine to DNA phosphate, and Fourier transform infrared (FT-IR) results demonstrated stronger electrostatic binding with the phosphate backbone with the least amine-dense of the series. It was concluded that, depending on the number of secondary amines in the repeat unit, these polymers interact with pDNA via different mechanisms with varying extents of electrostatic interaction and hydrogen bonding. These differing mechanisms may affect the ability of trehalose to serve as a deterrent against aggregation in serum conditions and lend insight into the roles of polymer-pDNA binding during the complex transfection process.

Introduction The successful development of modern nucleic acid therapies critically depends on the design of effective vectors for cellular delivery of their cargo. Many synthetic nonviral vehicles, including liposomes, polymers, and dendrimers,1–3 have recently been utilized for these purposes, although the lack of understanding of the fine balance between high transfection efficiency, low cytotoxicity, and vector-nucleic acid complex formation continues to hinder this scientific area. In addition to the issue of biocompatibility, there are other substantial hurdles to overcome, which rely on the stability of binding between the nonviral vector and nucleic acid. For example, in vivo colloidal stability, avoidance of reticuloendothelial system (RES) clearance,4,5 cellular uptake,6,7 endosomal release,8–10 and, for some methods of therapy, intracellular trafficking of the nucleic acid to the nucleus all expose nanoparticle vectors to competitors that * To whom correspondence [email protected]. † University of Cincinnati. ‡ Procter & Gamble Co.

should

be

addressed.

E-mail:

(1) Ma, H.; Diamond, S. L. Curr. Pharm. Biotechnol. 2001, 2, 1–17. (2) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 893–895. (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) Ogris, M.; Brunner, S.; Schuller, S.; Kircheis, R.; Wagner, E. Gene Ther. 1999, 6, 595–605. (5) Chonn, A.; Semple, S. C.; Cullis, P. R. J. Biol. Chem. 1992, 267, 18759– 18765. (6) Manunta, M.; Tan, P. H.; Sagoo, P.; Kashefi, K.; George, A. J. T. Nucleic Acids Res. 2004, 32, 2730–2739. (7) Wiethoff, C. M.; Middaugh, C. R. J. Pharm. Sci. 2003, 92, 203–217. (8) Besterman, J. M.; Low, R. B. Biochem. J. 1983, 210, 1–13. (9) Forrest, M. L.; Meister, G. E.; Koerber, J. T.; Pack, D. W. Pharm. Res. 2004, 21, 365–371. (10) Midoux, P.; Monsigny, M. Bioconjugate Chem. 1999, 10, 406–411. (11) Briane, D.; Lesage, D.; Cao, A.; Coudert, R.; Lievre, N.; Salzmann, J. L.; Taillandier, E. J. Histochem. Cytochem. 2002, 50, 983–991. (12) Wong, A. W.; Scales, S. J.; Reilly, D. E. J. Biol. Chem. 2007, 282, 22953– 22963.

can prematurely destabilize complex binding and delivery.11,12 Indeed, the field of biomaterial design for drug delivery is heavily focused on the understanding the importance of the materials chemistry on the mechanisms of all these steps to develop and introduce more effective vectors. In general, it is thought that longer polymer-based delivery vectors have higher binding cooperativity with nucleic acids, which can increase affinity and delivery efficacy.13–15 Previously, we synthesized glycopolymers in which a disaccharide (diazidotrehalose) and a dialkyne-oligoethyleneamine comonomer were polymerized via copper(I)-catalyzed Huisgen cycloaddition,16–18 commonly referred to as the “click” reaction. This polymerization technique was highly efficient, affording glycopolymers of a variety of lengths (degrees of polymerization, n ) 35-100). The oligoethyleneamine units that were polymerized with the trehalose monomers contained between one and four secondary amines, thus yielding a series of polycations of increasing amine density (Tr1, Tr2, Tr3, and Tr4; Figure 1). The rationale behind designing these new glycopolymers was to create a powerful gene delivery vehicle by incorporating the ionizable amine groups, the disaccharide, and the triazole linker formed by the cycloaddition.16,17 In these studies, slight increases in the ethyleneamine lengths affected pDNA delivery efficiency to a large degree. Polymers Tr3 and Tr4 were found to have very high delivery efficiency, where Tr1 and Tr2 were poor transfection agents. In addition, polymer molecular weight was found to have a large (13) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed. Mater. Res. 1999, 45, 268–275. (14) Sato, T.; Ishii, T.; Okahata, Y. Biomaterials 2001, 22, 2075–2080. (15) Jonsson, M.; Linse, P. J. Chem. Phys. 2001, 115, 3406–3418. (16) Srinivasachari, S.; Liu, Y.; Prevette, L. E.; Reineke, T. M. Biomaterials 2007, 28, 2885–2898. (17) Srinivasachari, S.; Liu, Y.; Zhang, G.; Prevette, L.; Reineke, T. M. J. Am. Chem. Soc. 2006, 128, 8176–8184. (18) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599.

10.1021/la800120q CCC: $40.75  2008 American Chemical Society Published on Web 07/08/2008

Polymer-pDNA Binding Mechanism

Langmuir, Vol. 24, No. 15, 2008 8091 Table 1. Polymer Characterization: Weight-Averaged Molecular Weight (Mw), Degree of Polymerization (n), Polydispersity Index (Mw/Mn), and Mark-Houwink-Sakurada (MHS) r Values (Values between 0.5-0.8 Reflect Random Coil Polymer Solution Structure)

Figure 1. Structure of the trehalose click polymers (Tr1, Tr2, Tr3, and Tr4) examined herein. The number in the name refers to the number of ethyleneamine moieties per repeat unit. All polymers are of similar degree of polymerization, with 53-61 repeat units per chain.16,17

influence on the biological properties where delivery efficiency and toxicity increased with an increase in molecular weight. The polymers with intermediate lengths (n ) 53-61) yielded the optimal efficacy and biocompatibility properties.16,17 The efficient gene delivery observed with the Tr3 and Tr4 agents may be partly due to stronger pDNA binding through hydrogen bonding with the functional groups contained in the polycation repeat unit. Other groups have demonstrated the specific and stable DNA binding of macromolecules possessing heterocyclic moieties, adjacent to amide groups.19–22 We hypothesized that positioning these H-bond donors and acceptors adjacent to each other within the polymer backbone, along with the proper ethyleneamine spacer length, may enable strong hydrogen bond formation with DNA base pairs. In the design of these delivery vehicles, the choice of carbohydrate was also deliberate. Trehalose is an unusual disaccharide, used as a cryo- and lyo-protectant, as it has been shown to prevent protein aggregation and membrane fusion and lysis within biological systems that contain the disaccharide.23–29 This property is thought to be due to its large hydrated volume in solution and its ability to alter the hydration layer of various biomolecules and therefore stabilize them from aggregation.26 We hoped to exploit this remarkable trait by designing a polymer with its own large hydration layer to prevent aggregation of the DNA-polymer complexes or “polyplexes” with serum proteins in vivo, which typically results in reticuloendothelial system clearance before they can reach the targeted cells.30,31 To investigate whether trehalose incorporation encourages this stability, polyplex size was measured by dynamic light scattering at various time intervals in media containing serum. The results showed that although Tr1 and Tr2 formed micrometer-sized particles after 40 min, likely due to protein adsorption, Tr3 and Tr4 reached a maximum diameter of only about 400 nm.16,17 Although the polyplexes seem to swell only slightly, it appeared as though Tr3 and Tr4 are relatively stable from serum-mediated flocculation. The contradicting behavior of the four polymers, differing only in the number of ethyleneamine units, led us to believe more than one DNA binding mechanism may be involved, driving the disparate behavior of similar polycationic vectors. (19) Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12063–12068. (20) Fechter, E. J.; Dervan, P. B. J. Am. Chem. Soc. 2003, 125, 8476–8485. (21) Thomas, J. R.; Liu, X.; Hergenrother, P. J. J. Am. Chem. Soc. 2005, 127, 12434–12435. (22) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468–471. (23) Albertorio, F.; Chapa, V. A.; Chen, X.; Diaz, A. J.; Cremer, P. S. J. Am. Chem. Soc. 2007, 129, 10567–10574. (24) Bardos-Nagy, I.; Galantai, R.; Laberge, M.; Fidy, J. Langmuir 2003, 19, 146–153. (25) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701–703. (26) Engelsen, S. B.; Perez, S. J. Phys. Chem. B 2000, 104, 9301–9311. (27) Lins, R. D.; Pereira, C. S.; Huenenberger, P. H. Proteins: Struct., Funct., Bioinf. 2004, 55, 177–186. (28) Sola-Penna, M.; Meyer-Fernandes, J. R. Arch. Biochem. Biophys. 1998, 360, 10–14. (29) Tanaka, M.; Machida, Y.; Niu, S.; Ikeda, T.; Jana, N. R.; Doi, H.; Kurosawa, M.; Nekooki, M.; Nukina, N. Nat. Med. 2004, 10, 148–154. (30) Leong, K. W. MRS Bull. 2005, 30, 640–646. (31) Davis, M. E. Curr. Opin. Biotechnol. 2002, 13, 128–131.

Tr1 Tr2 Tr3 Tr4

Mw (kDa)

n

Mw/Mn

MHS R

34 39 40 39

56 61 59 53

1.3 1.2 1.2 1.3

0.55 0.74 0.62 0.69

To better understand the physical and biological properties of polyplexes formed from the trehalose “click” polyamide series, particularly the variation in binding and flocculation properties of these polymeric vectors, many characterization methods have been employed herein. Isothermal titration calorimetry (ITC) and ethidium bromide exclusion assays were used to obtain and compare the pDNA binding affinities for Tr1, Tr2, Tr3, and Tr4 (34-40 kDa versions, which revealed high delivery and low toxicity in our previous studies). Polyplex size and charge at various stages of formation were determined via light scattering and ζ-potential measurements to reveal the N/P ratio of nanoparticle flocculation in buffer due to charge neutralization. After performing potentiometric titrations of the trehalose series, we also investigated binding-linked protonation, which revealed differences in the fraction of ionized amines in the four polymers, which certainly can play a role in the biological properties of these vectors. Changes in the specific locations of polymer interaction along the pDNA backbone along with the pDNA secondary structure changes were examined via Fourier transform infrared (FT-IR) spectroscopy and circular dichroism, respectively. All of the findings herein point toward variations in the pDNA binding mechanisms of these analogous polymer structures, which are highly dependent upon amine number. Indeed, these results correlate with the previously studied transfection experiments and reveal that subtle changes in the polymer structure and pDNA binding mechanism can play a large role in the biological properties of nucleic acid delivery vectors.

Methods and Materials Unless otherwise noted, all experiments were performed in triplicate in 10 mM Tris pH 7.4 buffer to maintain consistency in polymer and pDNA dissolution, counterions, and ionization properties of the materials in these studies. Materials. Tris, N-2-(hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), PIPES, MOPS (Aldrich; Milwaukee, WI), and ethidium bromide (Sigma; St. Louis, MO) were used as received. The pCMV-β technical grade DNA (7.2 kb) was purchased from PlasmidFactory (Bielefeld, Germany) and dialyzed extensively into each buffer. The resulting dialysate was used to prepare the polymer solutions. Buffers were prepared to 10 mM using Millipore (18 MΩ) water and titrated to proper pH using 0.1 N HCl or NaOH (Acros; Morris Plain, NJ). The polycations were synthesized as described previously16,17 with the molecular weight (Mw), degree of polymerization (n), and polydispersity index (Mw/Mn) values listed in Table 1. Potentiometric Titration. Tr1-Tr4 were titrated as 0.037 M solutions (in 10 mM Tris buffer pH 7.4) with 0.1 N standardized HCl (Acros; Morris Plain, NJ) at 25 °C. The temperature was maintained throughout each titration using a Fisher Scientific isotherm circulator model 3016 (Pittsburgh, PA). The pH values were recorded with an Accumet pH meter model AB15 (Pittsburgh, PA) equipped with an Orion Ross Semimicro Combination pH electrode (Thermo Electron Corp.; Beverly, MA). Nonlinear regression of the experimental data was accomplished using Graphpad Prism 4.0 (San Diego, CA). Dynamic Light Scattering and Zeta Potential. Polyplex hydrodynamic diameter and electrophoretic mobility were measured

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Figure 2. (a) Titration data plotted as volume (L) of 0.1 N HCl versus pH of the polymer solution in 10 mM Tris pH 7.4 buffer. (b) Percent protonation of the secondary amines in Tr1 (green circle), Tr2 (gold triangle), Tr3 (red square), and Tr4 (blue tilted square) at various pH values as calculated from the titration data above.

at increasing N/P ratios by titrating 1.5 mL of 0.31 mM pDNA solution (calculated for moles of DNA phosphate) in 10 mM Tris buffer pH 7.4 with 20 µL aliquots of 5.5 mM polymer solution (calculated for moles of secondary amines) in the same buffer, using a NanoSeries Zetasizer ZS (Malvern; Worcestershire, U.K.). The instrument employs a 4.0 mW He-Ne laser operating at 633 nm with a 173° scattering angle. Correlation functions were analyzed using a cumulant fit, with sizes reported as the z-average. Three measurements of three runs each were performed after thorough mixing of each sample and a 5 min equilibration time. Isothermal Titration Calorimetry (ITC). Plasmid DNA binding thermodynamics were measured using a Microcal VP-ITC instrument (Northampton, MA) as described previously.32,33 A 1.5 mL solution of 0.31 mM pDNA in 10 mM Tris buffer pH 7.4 was titrated with 10 µL aliquots of 5.5 mM polymer solution in the same buffer at 25 °C. The injections were spaced 150 s apart with a reference power of 10. The pDNA was dialyzed extensively against buffer, and the resulting dialysate was used to prepare the polymer sample in order to reduce pH and/or ionic strength differential artifacts upon titration. Plasmid DNA concentration was quantified after dialysis (32) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L.-N. Anal. Biochem. 1989, 179, 131–137. (33) Prevette, L. E.; Kodger, T. E.; Reineke, T. M.; Lynch, M. L. Langmuir 2007, 23, 9773–9784.

using absorbance at 260 nm, with 1 au ) 50 µg/mL DNA. All samples were degassed prior to use, and measurements were repeated at least thrice. All data were fit using the nonlinear least-squares analysis program Origin 7.0. For the binding-linked protonation study, solutions were prepared exactly as above using the following buffers of increasing ionization enthalpy: PIPES (2.7 kcal/mol), HEPES (4.9 kcal/mol), MOPS (5.3 kcal/mol), and Tris (11.3 kcal/mol) at 10 mM concentration and pH 7.4. All instrument conditions were unchanged. The observed enthalpy of polymer-pDNA binding was taken as the normalized heat per mole of ligand from the second injection, since the first point often deviates due to syringe leakage during the long equilibration time. Observed enthalpy values are the average of three or more trials. For the study to deconvolute the free energy of interaction, 15-300 mM NaCl was added to the 10 mM Tris pH 7.4 buffer prior to solution preparation. Titrations were performed exactly as above, with 5.5 mM Tr1 or Tr4 being titrated into 0.31 mM pDNA, and the observed binding constants were obtained from curve fitting using the one-site model. Ethidium Bromide Exclusion Assay. The fluorescence intensity of ethidium bromide (EB) upon intercalation with DNA (and the subsequent decrease in fluorescence intensity upon EB exclusion

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via polymer binding) was used to support the ITC data by comparing the relative pDNA binding affinities of the trehalose polymers, according to the method of Read et al.34 Fluorescence measurements were acquired using a Varian Cary spectrofluorometer with λex ) 510 nm, λem ) 590 nm, slit width ) 10 nm, and an integration time of 3 s. All solutions were made in 10 mM Tris pH 7.4 buffer. The fluorescence of a 400 ng/mL EB solution (Fbg) and a 10 µg/mL solution of pDNA containing 400 ng/mL of EB (FDNA) were first measured as controls. EB exclusion assays were completed with Tr1, Tr2, Tr3, and Tr4 by titrating 2.0 mL of the FDNA solution with each polymer at 0.5 N/P ratio aliquots (50 µL of a 26 µg/mL polymer solution). After the addition of each 50 µL polymer aliquot, the solution was gently mixed and the reduction in fluorescence (Fx) was measured. The percent relative fluorescence (%F) was determined using the equation below:

%F ) 100 ×

Fx - Fbg FDNA - Fbg

HCl solution, respectively, where [HCl]0 ) 0.1 M. We assumed the amide nitrogen groups, which typically have pKa values much lower than the pH range investigated here [pKa ) -0.51 for acetamide37], and the triazole nitrogens, which have reported pKa values of 1.25,38 are not protonated in this study. Equilibrium constants:

Kt )

[Tris][H+] [TrisH+]

(2)

Kw ) [H+][OH-]

(3)

+

Ka )

[N][H ] [NH+]

(4)

Charge balance:

(1)

Circular Dichroism. Circular dichroism spectra were obtained using a Jasco J-715 spectropolarimeter as an average of three iterations at 25 °C. A 1.5 mL solution of 0.31 mM pDNA in 10 mM Tris pH 7.4 buffer was titrated with various amounts of 5.5 mM trehalose polycation solution to reach N/P ratios of 0-5. These concentrations and titration volumes were used to mimic those of the ITC experiments. A scan rate of 50 nm/min, a resolution of 0.5 nm, and wavelengths from 225-350 nm were chosen. Fourier Transform Infrared Spectroscopy. Measurements were obtained at 25 °C using three replicates of separately prepared samples on a Perkin-Elmer Spectrum One instrument with a horizontal attenuated total reflection (ATR) accessory with a ZnSe 45° crystal of effective path length 12 µm. The pDNA solution was 4 mg/mL in 10 mM Tris buffer pH 7.4, and various N/P ratio (0.25, 0.75, 1.3, and 2.5) polyplex solutions (with constant [pDNA]) were formed using 0.027 M polymer in the same buffer to monitor shifts in the DNA spectral peaks upon interaction. The Tris spectrum was subtracted from each data set (using a flat baseline at 2200 cm-1 as evidence of a sufficient subtraction)35 to obtain the difference spectra for comparison. Polymer-only spectra at 14 mM were also performed as a control.

Results and Discussion Potentiometric Titrations. To determine the protonated fraction of the secondary amines in each of the four trehalosebased polymers, potentiometric titrations of each structure were performed. It is important to understand the degree which each polymer is protonated because electrostatic interactions play a large role in pDNA binding and condensation (and thus the stability of the polyplexes during the transfection process). In addition, the native protonation state of the polymer backbone also greatly affects the buffering capacity of these vectors, since this characteristic is thought to contribute to the “proton sponge” effect, which is theorized to aid endosomal release of the polyplexes.36 Solutions of each polymer in Tris buffer were titrated with HCl, and the pH values were monitored. The data were plotted (Figure 2), and the curves were fit using nonlinear regression with a five-parameter equation modified from that of Suh et al. using the equilibrium constants [Kt for Tris (eq 2), Kw of water (eq 3), and Ka for the polymer amine groups (eq 4)], charge (eq 5), and mass balance relationships (eqs 6–9). In these equations, N stands for a secondary amine group in the polymer, whereas V0 and V represent the initial volume of the polymer solutions (700 µL in Tris buffer) and the volume of the added (34) Read, M. L.; Bettinger, T.; Oupicky, D. In NonViral Vectors for Gene Therapy; Findeis, M. A., Ed.; Humana Press Inc.: Totowa, NJ, 2001; Vol. 65, pp 131-148. (35) Alex, S.; Dupuis, P. Inorg. Chim. Acta 1989, 157, 271–281. (36) Behr, J.-P. Chimia 1997, 51, 34–36.

[TrisH+] + [H+] + [Na+] + [NH+] ) [OH-] + [Cl-] (5) Mass Balance:

Kt[TrisH+] +

+ [TrisH+] )

V0 [TrisH+]0[TrisH+] ) V0 + V

[H ] V0 Ka[NH+] V0 [H+] + [NH ) ] + [TrisH+]0 [N] + + V0 + V V0 + V 0 Kt + [H ] [H ] (6) V0 [H+] [N]0 V0 + V Ka + [H+]

(7)

V0 [Na+]0 V0 + V

(8)

V0 V [HCl]0 + [Cl-]0 V0 + V V0 + V

(9)

[NH+] )

[Na+] ) [Cl-] )

Using the mass balance equations, the charge balance equation can be rewritten as follows:

V0 V0 [H+] + [H+] + [TrisH+]0 [Na+]0 + + V0 + V V0 + V Kt + [H ] Kw V0 [H+] V ) + + [N]0 [HCl]0 + + V0 + V K + [H ] [H ] V0 + V a

V0 [Cl-]0 V0 + V

{(

After rearranging, eq 10 was derived:

V ) V0

[TrisH+]0 [N]0 + + [Na+]0 + [H+] Ka Kt 1+ + 1+ + [H ] [H ] K Kw w + [HCl]0 - [H+] [Cl-]0 - + [H ] [H+]

)

(

)

}

(10)

It is important to note that the secondary amines in these polymers will not have identical Ka values, due to electrostatic suppression (protonation of an amine adjacent to a quaternary ammonium group will be electrostatically hindered). Therefore, to eliminate the need to determine all of the individual ionization constants, (37) Jencks, W. P.; Regenstein, J. In Handbook of Biochemistry and Molecular Biology: Physical and Chemical Data; Fasman, G. D., Ed.; CRC Press: Cleveland, OH, 1975; Vol. 1, pp 306-345.

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a variable Q+ (the ratio of the fraction of unprotonated amine groups to the fraction of protonated amines) was introduced.

Q+ )

fN [N] ) + f [NH ] NH+

(11)

Upon substituting eq 11 for Ka into eq 10, the final titration data fitting function, which describes the protonated fraction of amines as a function of the volume of titrated HCl, was obtained.

{(

V ) V0

[TrisH+]0 [N]0 + + [Na+]0 + [H+] - [Cl-]0 Kt 1 + Q+ 1+ + [H ] Kw Kw + [HCl]0 - [H+] (12) + + [H ] [H ]

)(

)

}

Figure 3. Hydrodynamic diameter of polyplexes formed using 0.31 mM pDNA and 5.5 mM trehalose polymer solution in 10 mM Tris pH 7.4 buffer at 25 °C upon increasing molar ratio of Tr1 (green circle), Tr2 (gold triangle), Tr3 (red square), and Tr4 (blue tilted square).

Since Q+ is a function of the measured pH, it has been defined with a logarithmic relationship of five independent variables (R, β, γ, δ, and ε):

log Q+ ) RpHβ + γpH(β-1) + δpH(β-2) + ε

(13)

Once these five parameters were determined via nonlinear least-squares data analysis (data and fit shown in Figure 2a), the protonated percent of secondary amines in the trehalose-based polymers was obtained and plotted versus pH in Figure 2b. The variation in amine density of the repeat units of these four trehalose-based polymers resulted in their possession of different charged states in Tris pH 7.4 buffer. As previously stated, the existence of a positive charge will lower the pKa of nearby amine groups due to Coulombic suppression. Because the lone amine group in the Tr1 repeat unit does not have immediate neighbors along the polymer chain, Tr1 has a higher fraction of protonated amines at all pH conditions when compared to Tr2-Tr4. Polymers Tr3 and Tr4 have similar charge profiles, and they were shown to possess 12% of the secondary amines protonated at physiological pH (Figure 2b). Tr2 demonstrated the lowest charge fraction (3%) at pH 7.4, likely due to the unfavorable nature of having two immediately adjacent charges in a repeat unit. This titration data lends valuable insight into the differences in strength of electrostatic forces when these trehalose-based polycations are formulated with pDNA. Based on these results, Tr1 is expected to have the highest Coulombic interaction with polyanionic pDNA, Tr2 the weakest, and Tr3 and Tr4 should be similar due to their comparable protonation profiles. One might expect to see these protonation trends reflected in the DNA binding strength; however, compensating interaction forces (hydrogen bonding, hydrophobicity, van der Waals) could be present, warranting a thorough examination of pDNA affinity (Vide infra). It is worth noting that these polymers have a random coil solution structure, as suggested from their Mark-HouwinkSakurada parameters in Table 1 (R ) 0.5-0.8 indicates a random coil structure with a low degree of branching, whereas R ) 0.8-1.0 indicates stiff chains). As a result, the backbone protonation of this series reaches a maximum of about 73% (at pH ) 2.5), likely due to electrostatic suppression of the polymer amine groups by neighboring quaternary ammonium cations from through-space interactions. The information obtained through the potentiometric titrations above reveals that Tr2–Tr4 may have higher buffering capacity within the physiological pH range (5.0-7.4) due to the electrostatic suppression of neighboring amines at pH 7.4 and

Figure 4. ζ-Potential of 0.31 mM pDNA in 10 mM Tris pH 7.4 buffer with 5.5 mM Tr1 (green circle), Tr2 (gold triangle), Tr3 (red square), and Tr4 (blue tilted square) at various N/P ratios.

the ability of the amines to accept protons at pH ) 5. Again, this property is thought to aid endosomal/lysosomal escape within the cellular transfection pathway and aid delivery efficacy. However, to make a valid estimate of the buffering capacity of these polymeric vectors, we must consider this property when the polymer is bound to pDNA in a polyplex. Thus, the bindinglinked protonation must also be investigated (Vide infra) to learn the final charged state of the amines in the form of a polyplex. Before we could perform these experiments, several other parameters required investigation to obtain accurate thermodynamic data to calculate the binding-linked protonation. Particle Size and Zeta Potential. To characterize and understand the size of the polyplexes created from the trehalose polycations and pDNA, light scattering and ζ-potential measurements were acquired at various stages of formation (Figures 3 and 4) within the buffer utilized for the binding experiments. The intensity-averaged hydrodynamic diameter of the polyplexes in Tris buffer pH 7.4 (168-243 nm) was shown to be consistent and independent of polymer amine number at lower N/P ratios. Once a specific N/P ratio was reached (1.2, 1.5, 2.4, and 2.2 for Tr1, Tr2, Tr3, and Tr4, respectively), polyplex-polyplex aggregation occurred due to van der Waals forces enabled by polycation-induced pDNA charge neutralization upon complex formation. It should be noted that this aggregation is distinguished from the polyplex-protein flocculation in biological media mentioned in the introduction, which is a result of the positive surface charge of polyplexes formed at high N/P ratios used for transfection. Since the aggregation in buffer is a result of neutralization (electrostatic repulsion of the polyplexes does not occur in the buffer due to the presence of counterions), the surface charge should correlate with particle size. Therefore, we investigated ζ-potentials at the same N/P ratios used in the light scattering study (Figure 3) for the Tr1, Tr2, Tr3, and Tr4 polyplexes (38) Catalan, J.; Abboud, J. L. M.; Elguero, J. In AdVances in Heterocyclic Chemistry; Katritzky, A. R., Ed.; Academic Press: Orlando, FL, 1987; Vol. 41, pp 187-274.

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(Figure 4). It has been shown, through the potentiometric titration results in Figure 2b, that the four trehalose polymers have dissimilar protonation profiles at pH 7.4 due to the differing amine densities within the repeat unit. Polyplex neutralization occurs through electrostatic interactions between the two oppositely charged macromolecules. In this study, we found that the N/P ratio of zero ζ-potential increased with the repeat unit amine number (1.1, 1.3, 1.5, and 2.1 for Tr1, Tr2, Tr3, and Tr4, respectively). Based on charge fraction alone (Figure 2b), it would be expected that Tr1 polyplexes would possess 0 mV surface charge at the lowest N/P ratio, Tr3 and Tr4 polyplexes at identical intermediate N/P ratios, and Tr2 at the highest N/P ratio. However, this assumes that the interaction between pDNA and the trehalose polycations is only electrostatic in origin, yet H-bonding can also play a large role in the binding affinity. It should also be noted that this observation is based on a constant protonation state of the secondary amines upon binding to pDNA, which is also unlikely, and this parameter is investigated herein. In general, the light scattering and ζ-potential results suggest that the interaction between the trehalose polymers and pDNA is dependent upon the number of secondary amines in the repeat units. The incomplete correlation of these results to the polymer charge fractions at pH 7.4 warranted further study into more complex modes of interaction between these agents and pDNA, and these studies are described below. Plasmid DNA Binding Thermodynamics. Microcalorimetry was performed to obtain binding constants, enthalpy, entropy, and the stoichiometry of the interaction of this series of polycations with pDNA.32,33 The addition of small aliquots of polymer into a dilute solution of plasmid in an adiabatic cell was translated into heat changes per injection, QITC. These heats could be related to the enthalpy (∆H) and stoichiometry (n) of the interaction, using M as the concentration of macromolecule, V as the volume of the cell, and Θ as the fraction of ligand bound to macromolecule by fit to a 1:1 standard model using the following equation:

QITC ) MVnΘ∆H

(14)

One can solve for Θ using the equilibrium equation for the binding constant K (eq 15), with X as the total concentration of ligand and [X] as the free ligand concentration.

Θ (1 - Θ)[X] [X] ) X - MnΘ

K)

(15) (16)

Since the heat signal from the calorimeter likely contains many contributors other than heat of binding, such as polymer and pDNA dilution heat, heat of aggregation of the polyplexes, and conformation change heat, these extraneous factors must be eliminated prior to curve fitting. To account for known aggregation (shown by the light scattering results in Figure 3), we chose to follow the method of Patel and Anchordoquy of removing all points on the thermogram that possibly contain aggregation heats.39 In this case, we removed points past N/P ratios of 1.3, 1.5, 3.0, and 2.3 for Tr1, Tr2, Tr3, and Tr4, respectively (Figure 5), which were determined from the light scattering experiments (Figure 3). Dilution control experiments were also performed (polymer into buffer and buffer into pDNA), and these heat profiles were subtracted from the isotherms before the curve fitting algorithm was applied. The differences in shape of the time versus heat signal thermograms (the upper portion of the ITC graphs shown in Figure 5) for the four polycations are due to differences in stoichiometry of the interactions and polymer (39) Patel, M. M.; Anchordoquy, T. J. Biophys. J. 2005, 88, 2089–2103.

Table 2. Thermodynamic Parameters for the Interaction between 0.31 mM pDNA and 5.5 mM of the Trehalose Polymers in 10 mM Tris pH 7.4 Buffer, as Calculated via ITC Titration Experiments Using a Standard One-Site Langmuir Binding Model

Tr1 Tr2 Tr3 Tr4

K (×10-6 M-1)

∆H° (kcal/mol)

∆S ° (kcal/mol K)

nbind

4.4 ( 0.3 5.4 ( 0.4 2.0 ( 0.2 1.3 ( 0.7

0.73 ( 0.05 1.2 ( 0.1 0.41 ( 0.01 0.71 ( 0.05

0.033 ( 0.001 0.034 ( 0.002 0.030 ( 0.000 0.030 ( 0.000

1.1 ( 0.1 1.3 ( 0.1 2.6 ( 0.0 1.9 ( 0.1

dilution, as evident by the similar postdilution subtraction curves (the bottom portion of the ITC graphs in Figure 5). Because the charge states of these trehalose polymers vary with secondary amine number in the repeat units, the solvent interactions and conformation changes upon dilution into Tris buffer will not be the same for each polymer in the series. Also, higher stoichiometries of association (Table 2, nbind) for the polymer-pDNA binding will shift the binding saturation point to higher N/P ratios, which suggests that Tr3 has a greater nbind value when compared to the other polymer analogues. Nonlinear least-squares data analysis yielded the thermodynamic parameters of interest for the pDNA-polymer interactions, as shown in Table 2. The results revealed some surprising information about the binding strength of these new polymeric vectors, as well as the effect that increasing the amine density has on nucleic acid association constants. All of the polymers have similar Kobs values of 106 M-1, which is on the order of other DNA-binding polycations and proteins.13,33,39,40 The order of pDNA affinity increases very slightly as Tr4 < Tr3 < Tr1 < Tr2 (Table 2), although standard deviation renders the binding strengths of Tr1 from Tr2 and Tr3 fromTr4 difficult to distinguish. The entropy values for the interactions were shown to be very similar (approximately 30 cal/mol K) for each polymer in the trehalose polymer series, likely due to the equal release of counterions and solvent upon association. Differences in the stoichiometry of binding (nbind, calculated in moles of polymer secondary amine to moles of DNA phosphate) demonstrated in Table 2 reflect the different pDNA affinities of these four vectors. For example, Tr1 and Tr2 have slightly higher binding constants and, therefore, slightly lower nbind values necessary to reach binding equilibrium. Understanding the variation in binding enthalpy seen here, where Tr2 has a higher ∆H°, is daunting, and we are unable to decipher all of the contributing heat factors due to the many energetic components that are occurring upon association of these polyions. Electrostatic forces, hydrogen bond making or breaking from the amines and/or the carbohydrate to the phosphate groups and/or base pairs of DNA, proton uptake, disruption of the hydrogen bond network of the solvent, and conformation changes of the macromolecules can all contribute to the complexity of the enthalpic binding term. In speculation, the less favorable (more endothermic) ∆H° values associated with Tr2-pDNA interaction may be a result of breaking hydrogen bonds between the unprotonated secondary amines of the polymer and water molecules of the solvent, with the magnitude being greater due to the initial lower-charged state of Tr2 compared to the other polymers (breaking a hydrogen bond in water corresponds to an enthalpy increase of 1.9 kcal/mol).41 More atomic-level studies are needed to prove the origin of such (40) Nisha, C. K.; Manorama, S. V.; Ganguli, M.; Maiti, S.; Kizhakkedathu, J. N. Langmuir 2004, 20, 2386–2396. (41) Silverstein, K. A. T.; Haymet, A. D. J.; Dill, K. A. J. Am. Chem. Soc. 2000, 122, 8037–8041.

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Figure 5. ITC thermograms of 5.5 mM (a) Tr1, (b) Tr2, (c) Tr3, and (d) Tr4 titrated into 0.31 mM pDNA in 10 mM Tris buffer pH 7.4 at 25 °C. Raw heat per injection is shown on the upper portion of the graph, with normalized data and curve fit below. Aggregation-containing points were subtracted above N/P ratio 1.3 (Tr1), 1.5 (Tr2), 3.0 (Tr3), and 2.4 (Tr4).

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Figure 6. Observed pDNA binding enthalpy versus buffer ionization enthalpy (2.7 kcal/mol for PIPES, 4.7 kcal/mol for HEPES, 5.3 kcal/mol for MOPS, 11.3 kcal/mol for Tris) for 5.5 mM Tr1 (green circle), Tr2 (orange triangle), Tr3 (red square), and Tr4 (blue tilted square) at 25 °C. Table 3. Intrinsic pDNA Binding Enthalpy, ∆Ho, and Moles of Protons per Mole of Secondary Amine, nproton, Uptaken by the Trehalose Polymers upon Binding with pDNAa Tr1 Tr2 Tr3 Tr4

forces due to the release of counterions and solvent molecules upon binding), and these values become more negative as the polymer amine density decreases.39,40,45,46 This proton uptake data, combined with the titration data above, demonstrates the higher pKa values of the secondary amines after polymers Tr1 and Tr2 bind pDNA, which is likely due to their stronger interaction with pDNA as shown in the ITC experiments (Table 2). When these data are compared to the results obtained for Tr3 and Tr4, these systems with higher amine density have a lower proton uptake upon pDNA binding (Figure 6), which is likely caused by the combination of the slightly lower binding affinity with pDNA (Table 2) and the lower percentage of secondary amine protonation due to Coulombic suppression by neighboring protonated amine groups (Figure 2). These data are also supported by the light scattering and ζ-potential results, because although Tr2 starts out as the least protonated system (in pH 7.4 Tris buffer), Tr2 takes up more protons than Tr3 and Tr4 upon pDNA binding. Therefore, the Tr2 polyplexes reach charge neutrality at a lower N/P ratio than Tr3 and Tr4, which again is supported by the ζ-potential experiments (Figure 4).

nproton

∆H0 (kcal/mol)

R2

∆Hobs ) n∆Hbuffer-ionization + ∆H0

0.51 0.39 0.21 0.19

-5.0 -3.3 -2.0 -1.5

1.0 0.99 1.0 0.97

Ethidium Bromide Exclusion Assay. Ethidum bromide (EB) exclusion assays are commonly utilized in this field of study as a qualitative assessment of the relative binding affinity between nonviral vectors and nucleic acids. However, due to many competing interactions, this assay does not represent a true binding affinity or mode. Herein, we chose to perform the EB exclusion assays and compare this very qualitative data with the more quantitative ITC data we have acquired on these systems. An ethidium bromide exclusion assay was performed by titrating each of the four trehalose polymers into pDNA solutions in Tris buffer (Figure 7). In this assay, the fluorescence of the ethidium cation increases upon intercalation with DNA. When a polycation binds to the complex formed between DNA and the ethidium cation, the fluorescence is quenched, possibly due to crowding and/or the displacement of the EB intercalator. The N/P ratio necessary to reduce the fluorescence by 50% (the inhibitory concentration, or IC50 value) is reported in Figure 7b and demonstrates that Tr3 was found to be the most effective at quenching the fluorescence, followed by Tr4, Tr1, and Tr2. These results contradict those obtained through calorimetry and all of the supporting trends shown by light scattering, ζ-potential, and binding-linked protonation data. Indeed, this discrepancy likely lies in the nature of the qualitative EB exclusion assay, where a decrease in fluorescence may not necessarily indicate polymer-pDNA binding (and subsequent EB displacement). The decrease in fluorescence of EB may be the result of polymer-ethidium interactions, molecular crowding, and/or the mechanism of polymer-pDNA binding and compaction, which could certainly differ among various polymers (particularly this polymer series). For example, if Tr3 and Tr4 participate in more hydrogen bonding to the nitrogenous bases, an intercalated molecule may experience a change in environment and, hence, exhibit quenching or displacement. This particular possibility is supported by and further explained through circular dichroism experiments. Relative Hydrophilicity. Hydrophobic effects can play a role in the association between two macromolecules and can also affect the calorimetry and ethidium bromide displacement data. Due to the changes in protonation fraction as the ethyleneamine

a Values are determined from the slope (nproton) and y-intercept (∆H0) of the linear fit of ∆Hobs versus ∆Hbuffer-ionization for each polymer. The R2 values for the linear fit are also provided.

energetic terms; however, the small magnitudes of these enthalpic energetic components and the positive entropy values obtained mimic those commonly seen with polycation-pDNA interactions.42 Binding-Linked Protonation. Through performing the ITC experiments in different buffers, binding-linked protonation was able to be analyzed. This study is important to the interpretation of charge-charge interactions between the polymers and pDNA, since the mechanisms will likely be affected by the trehalose polymer protonation state both before and during the process of binding. Also, these results must be linked to the titration data in Figure 2 to lend insight into the colloidal aggregation behavior of these polyplexes (Figure 3) and the buffering capacity of the polyplexes upon endocytosis as discussed earlier. The slope (nproton) of a line representing the observed pDNA binding enthalpy versus the buffer ionization enthalpy reflects the moles of protons per mole of secondary amine taken up by the polymer upon binding with pDNA (eq 17).43 As shown in Figure 6 and Table 3, the slopes (and thus the proton uptake upon polymer-pDNA binding) increased from 0.19 to 0.51 as the amine density decreased in the polymer series. These data indicate that, upon polyplex formation, the cationic state of the polyplexes follows the order Tr1 > Tr2 > Tr3 > Tr4 and correlates directly with the observed polyplex ζ-potential data (Figure 4). Once the binding-linked protonation was determined for the interaction, the intrinsic enthalpy of binding ∆H0 (y-intercept, Figure 6) can also be obtained for the four polymers. The energy of buffer ionization has been eliminated in this intrinsic enthalpy, although contributions from the enthalpies of the pKa shifts are still included.43,44 Polyplexes formed with Tr1-Tr4 all possess slightly exothermic ∆H0 values (likely indicating entropic driving (42) Ross, P. D.; Shapiro, J. T. Biopolymers 1974, 13, 415–416. (43) Baker, B. M.; Murphy, K. P. Biophys. J. 1996, 71, 2049–2055. (44) Kaul, M.; Barbieri, C. M.; Kerrigan, J. E.; Pilch, D. S. J. Mol. Biol. 2003, 326, 1373–1387.

(17)

(45) Zhou, Y.-L.; Li, Y.-Z. Spectrochim. Acta, Part A 2004, 60A, 377–384. (46) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620–1633.

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Figure 7. (a) Reduction in fluorescence as a result of ethidium exclusion from pDNA by various molar ratios of Tr1 (blue tilted square), Tr2 (gold square), Tr3 (red triangle), and Tr4 (green circle) in 10 mM Tris buffer pH 7.4. (b) Inhibitory concentration (IC50) values from the ethidium bromide exclusion assays.

Figure 8. Thermal gravimetric analysis (TGA) data for Tr1 (green line), Tr2 (gold line), Tr3 (red line), and Tr4 (blue line) being heated from 25 to 420 °C. The range from 25 to 110 °C (inset) reflects water loss and, thus, relative hydrophilicity of the polymers.

number increases per repeat unit in Tr1-Tr4, a study into the relative hydrophobicity of these agents was necessary. To examine this characteristic, the percent water loss of each polyamide was measured via thermal gravimetric analysis (TGA) as the material was heated to the boiling point of water, from 25 to 120 °C. As shown in Figure 8, the four polymers have virtually identical changes in mass loss in this range, revealing very similar hydration states. The heating of polymer Tr1 shows a slightly greater decrease in mass than polymers Tr2-Tr4, which could be a consequence of its lower number of ethylene groups per repeat unit and/or higher native protonation. However, this difference is considered negligible and within error; therefore, we conclude that the four trehalose-based polyamides have very similar hydrophilicity, rendering hydrophobic interactions unlikely contributors to the variations in pDNA binding mechanisms of these vectors. pDNA Secondary Structure Changes. Circular dichroism (CD) can be used to identify DNA secondary structure in various environments. The trehalose polycations do not exhibit an absorbance above 230 nm, so the CD spectra display pDNA molar ellipticity only. Native B-form DNA exhibits a characteristic CD spectrum containing a negative peak at 248 nm and a positive peak at 274 nm (Figure 9, gold line), corresponding to the π f π* transition of the nucleotide bases.47–49 Upon interaction with the trehalose polycations, certain spectral changes occur depending on the particular polymer involved in the pDNA binding. For example, Tr1 and Tr2 exhibited very minor CD (47) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. Physical Chemistry of Nucleic Acids.; Harper & Row Publishers, Inc.: New York, 1974; pp 132147. (48) Gray, D. M.; Ratliff, R. L.; Vaughan, M. R., In Spectroscopic Methods for Analysis of DNA, Academic Press, Inc.: New York, 1992; Vol. 211, pp 389397. (49) Rodger, A.; Norden, B., Circular Dichroism and Linear Dichroism., Oxford University Press: Oxford, 1997; pp 1-131.

spectral alterations upon increasing N/P ratio from 0 to 5.0 (Figure 9), possibly indicating that the interactions of these polymers with pDNA may be more electrostatic in nature (pDNA secondary structure is not altered upon polymer electrostatic interaction with pDNA phosphates). The CD spectrum of pDNA binding to polymer Tr2 demonstrated only a slight red shift of approximately 10 nm; however, no difference in molar ellipticity was noticed over the entire N/P range when compared to naked pDNA. Conversely, Tr1 did not induce a shift in the band wavelength, but this polymer did slightly increase the ellipticity of the positive band for pDNA up to an N/P ratio of 1.0. Yet, as the N/P ratio of Tr1 increased further, the peak ellipticity decreased again from N/P ratios of 1.5-5.0, returning to the original position for naked pDNA. Interestingly, when pDNA was titrated with Tr3 and Tr4, a noticeable red shift and intensity decrease of the positive ellipticity band was observed in the CD spectra, even at the lowest N/P ratio of study (N/P ) 0.50). This result could indicate that Tr3 and Tr4 could associate with pDNA via a mechanism that involves a higher H-bonding component with the base pairs that affects the tilt of the helix. Because these systems have the lowest proton uptake upon pDNA binding and lowest ζ-potential, H-bonding could certainly play a large role in the binding mechanism of these polymers. Therefore, Tr3 and Tr4 could certainly affect EB intercalation even though they do not necessarily bind pDNA with a stronger affinity. All four polymeric vectors are capable of condensing pDNA at the positive N/P ratios examined in the CD spectral studies. Thus, the pDNA secondary structure changes observed in the CD spectra of the Tr3 and Tr4 complexes suggest the change in pDNA conformation to a modified B-form, which has separated base pairs and increased base-helix tilt angle.50 The alteration of pDNA secondary structure could be due to a noncondensing phenomenon, such as the direct interaction of these polymers with the nucleotide bases. Thus, it appears based on the CD results that the polymers that contain a higher amine density in the repeat unit are capable of pDNA base interaction, possibly through the formation of hydrogen bonds. The shorter ethyleneamine spacers between the triazole-amide groups and carbohydrates within the Tr1 and Tr2 structures might hinder effective interaction with the base pairs (in the grooves). These results, along with the higher proton uptake of these polymers upon pDNA binding, suggest that Tr1 and Tr2 may interact with pDNA primarily through electrostatic interactions with the anionic phosphate backbone. The different DNA binding mechanism of Tr3 and Tr4 might also explain the inability of the qualitative ethidium bromide exclusion assay to properly distinguish the relative pDNA binding affinity. As mentioned above, (50) Tunis-Schneider, M. J. B.; Maestre, M. F. J. Mol. Biol. 1970, 52, 521– 541. (51) Taillandier, E.; Liquier, J., In Spectroscopic Methods for Analysis of DNA, Academic Press, Inc.: New York, 1992; Vol. 211, pp 307-335.

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Figure 9. Circular dichroism spectra of 5.5 mM (a) Tr1, (b) Tr2, (c) Tr3, and (d) Tr4 titrated into 0.31 mM pDNA in 10 mM Tris pH 7.4 buffer at N/P ratio ) 0 (gold line), 0.50 (magenta line), 1.0 (red line), 1.5 (maroon line), 2.0 (blue line), 3.0 (green line), 5.0 (navy blue line).

Figure 10. FT-IR spectra of 12 mM pDNA in 10 mM Tris pH 7.4 buffer and 27 mM Tr1 solution in the same buffer at N/P ratio ) 0 (black line), 0.25 (dark green line), 0.75 (maroon line), 1.3 (dark blue line), and 2.5 (magenta line). Polymer only spectrum at 14 mM is shown (light green).

the interaction of these two polycations with the purine and pyrimidine bases of DNA may enhance the quenching or displacement of the intercalated ethidium molecule, rendering these two polymers a falsely high binding affinity when determined using this assay. The indirect nature of such a study should be taken into account when interpreting these qualitative results. Specific Locations of pDNA Binding. The thermodynamics, light scattering, ζ-potential, and circular dichroism spectra suggest that, as the amine number increases in the repeat unit of the trehalose polymers, the pDNA binding mode may change. Thus, it is important to gain some insight into binding sites on the polynucleotide, such as phosphate groups versus base interaction, and so Fourier transform infrared spectroscopy has been chosen to monitor shifts in band frequency and/or intensity upon association between these two macromolecules. The IR spectrum

of pDNA possesses many characteristic stretches: 1715 cm-1 (guanine carbonyl), 1663 cm-1 (thymine carbonyl), 1609 cm-1 (adenine C-N), 1492 cm-1 (guanine/cytosine CdN), 1222 cm-1 (asymmetric phosphate), and 1086 cm-1 (symmetric phosphate).51–54 The changes in these peaks were observed during the formation of the polyplexes at various N/P ratios (Figure 10 shows the overlapping FT-IR spectrum of the pDNA titration with Tr1). Slight absorbance intensity increases were seen during the titration of pDNA with Tr1-Tr4 up to N/P ) 2.5, which is often attributed to alterations in base pair stacking and pairing (52) Taillandier, E.; Liquier, J.; Taboury, J. A. In AdVances in Infrared and Raman Spectroscopy; Heyden & Sons, Inc.: London, 1985; Vol. 12, pp 65-113.. (53) Arakawa, H.; Ahmad, R.; Naoui, M.; Tajmir-Riahi, H.-A. J. Biol. Chem. 2000, 275, 10150–10153. (54) Taillandier, E.; Taboury, J. A.; Adam, S.; Liquier, J. Biochemistry 1984, 23, 5703–5706.

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Figure 11. FT-IR frequency shifts of the (a) guanine carbonyl stretch at 1715 cm-1 and (b) symmetric phosphate stretch at 1086 cm-1 upon Tr1 (green circle), Tr2 (orange triangle), Tr3 (red square), and Tr4 (blue tilted square) interaction with pDNA at various N/P ratios in 10 mM Tris buffer.

upon polymer interaction.51–57 This conclusion correlates with the CD data, which clearly demonstrated base π f π* transition changes also upon polymer binding. The shifts in frequencies of the guanine (1715 cm-1) and symmetric phosphate (1086 cm-1) stretches were monitored and plotted in Figure 11 as a function of N/P ratio to gain insight into the sites of polymer interaction along the pDNA backbone. Other bands were not able to be studied due to overlap from the functional groups on the polymer, particularly from methylene scissoring, N-H bending, and triazole ring stretching from 1490-1550 cm-1.58–61 As the concentration of polymer in the sample is increased, no change in frequency was observed for the guanine carbonyl stretch (Figure 11a), indicating that the polymers do not directly interact with this base along the pDNA backbone. However, this does not eliminate the possibility of an indirect interaction of the polymer with this base, which could occur through water molecules in the solvent. Polymer binding with the phosphate backbone, either through electrostatics or hydrogen bonding, was investigated via the asymmetric and symmetric PO2- stretches at 1222 and 1086 cm-1, respectively. Unfortunately, the asymmetric stretch was complicated by overlap from a polymer triazole ring band at 1260 cm-1;58,59 therefore, we focused this study on the symmetric PO2- stretch. As demonstrated in Figure 11b, Tr1 caused the most noticeable frequency shift, followed by Tr2, and polymersTr3 and Tr4 revealed the smallest shift in the PO2- stretch, indicating a lower electrostatic binding mode. These data directly correlate to all of the previous studies that suggest that polymer Tr1 has the highest electrostatic interaction with the pDNA backbone followed by Tr2. Electrostatic Contribution to the Free Energy of Binding. The previous results suggesting differences in nucleic acid interaction mechanisms led to our interest in the deconvoluted free energy of binding between these new polyamides and pDNA. These studies can give us insight into the electrostatic and nonelectrostatic contributions to the binding mechanism. Man(55) Ouameur, A. A.; Tajmir-Riahi, H.-A. J. Biol. Chem. 2004, 279, 42041– 42054. (56) Benevides, J. M.; Stow, P. L.; Ilag, L. L.; Incardona, N. L.; Thomas, G. J. Biochemistry 1991, 30, 4855–4863. (57) Ruiz-Chica, J.; Medina, M. A.; Sanchez-Jimenez, F.; Ramirez, F. J. Biochem. Biophys. Res. Commun. 2001, 285, 437–446. (58) Shi, W.; Chen, X.-Y.; Xu, N.; Song, H.-B.; Zhao, B.; Cheng, P.; Liao, D.-Z.; Yan, S.-P. Eur. J. Inorg. Chem. 2006, 2006, 4931–4937. (59) Bougeard, D.; Le Calve, N.; Saint Roch, B.; Novak, A. J. Chem. Phys. 1976, 64(12), 5152–5164. (60) Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to Spectroscopy: A Guide for Students of Organic Chemistry; W.B. Saunders Co.: Toronto, 1979. (61) Gunzler, H.; Gremlich, H.-U. IR Spectroscopy: An Introduction; WileyVerlag Chemie: Germany, 2002. (62) Manning, G. S. J. Chem. Phys. 1969, 51, 924–933.

ning’s counterion condensation theory62,63 explains the role that salt ions play in the interactions of macromolecules, and Record et al. extended his theory to develop a linear relationship, described as the term a below, between the binding constant, Kobs, and the concentration of added monovalent salt, [M+].64 Using this relationship, the electrostatic and nonelectrostatic components to the free energy of interaction (∆Ges° and ∆Gns°, respectively) can be determined at a temperature, T, according to

∆Gobs°)-RT ln Kobs

(18)

∆Ges°)-aRT ln[M+]

(19)

∆Gns°)∆Gobs°-∆Ges°

(20)

where

a)

∂ ln Kobs ∂ ln[M+]

(21)

We used ITC to measure the polymer-pDNA binding constants in buffers containing varying concentrations of added NaCl at 298 K for just two of the trehalose-based polymers, Tr1 and Tr4, since they possess very different protonation profiles and demonstrated, via CD spectra, opposite extents of base pair interactions. As shown in Figure 12, the interaction of pDNA and polymer Tr1 demonstrated a stronger salt dependence with a slope (a) of -1.3, compared to -0.74 for Tr4-pDNA binding. This supports all of the previous data and indicates that the pDNA-Tr1 mechanism is more electrostatic in nature than that of Tr4. Substituting the a values into eq 19, the free energies were deconvoluted into their components at both 15 mM and 300 mM NaCl (Table 4). In the case of Tr1 at 15 mM monovalent salt, ∆Ges° was -3.1 kcal/mol with a corresponding ∆Gns° of -5.4 kcal/mol. At the same concentration of NaCl, Tr4 binding induced a ∆Ges° of -1.8 kcal/mol and ∆Gns° of -8.0 kcal/mol. Combining the results for both polymers indicates that Tr1 is indeed capable of enhanced charge-charge association with pDNA (a result of the higher protonated fraction of secondary amines both before and after binding), although the free energy of interaction of both Tr1 and Tr4 with pDNA is predominantly nonelectrostatic. Sources of ∆Gns° are likely hydrogen bonding from the unprotonated secondary amines, the carbohydrate hydroxyl groups, and/or the triazole-amide groups. The electrostatic contribution decreases with an increase in the NaCl concentration (63) Manning, G. S. Q. ReV. Biophys. 1978, 11, 179–246. (64) Record, M. T.; Anderson, C. F.; Lohman, T. M. Q. ReV. Biophys. 1978, 11, 103–178.

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Conclusions

Figure 12. Variation in Kobs for the interaction of (a) Tr1 and (b) Tr4 with pDNA in 10 mM Tris pH 7.4 buffer in the presence of 15-300 mM NaCl. Table 4. Free Energies for pDNA-Polymer Interactions at 15 and 300 mM NaCl in 10 mM Tris pH 7.4 Buffer at 25 °C Tr1 ∆Gobs° (kcal/mol) ∆Ges° (kcal/mol) ∆Gns° (kcal/mol) ∆Gobs° (kcal/mol) ∆Ges° (kcal/mol) ∆Gns° (kcal/mol)

15 mM NaCl -8.6 -3.1 -5.4 300 mM NaCl -6.5 -0.90 -5.6

Tr4 -9.9 -1.8 -8.0 -8.4 -0.53 -7.9

to 300 mM, as expected, while the other component remains constant. This study has revealed the thermodynamic effect of increasing the number of ethyleneamine groups within the repeat units of these trehalose “click” polyamide chains. The longer the spacing between triazole-amide moieties and/or the more unprotonated secondary amines present, the more nonelectrostatic association with plasmid DNA, most likely through hydrogen bonding with the base pairs, as evidenced through the combination of studies performed herein. The differences in the mechanisms of polymer-pDNA binding suggested by the studies herein may help to explain the inherent serum stability of Tr3 and Tr4 polyplexes and the enhanced pDNA delivery efficiency we previously observed.16,17 In the current studies, Tr3 and Tr4 have been found to have a weaker electrostatic component to the binding mechanism. Binding pDNA through a mechanism that involves interactions other than electrostatics (and possible hydrogen bonding) can certainly help aid in stabilizing the polyplexes from the many destructive competitors encountered during both extracellular and intracellular transit. The higher the electrostatic component to the binding, the higher the likelihood that ionic competitors can destabilize the polyplexes, dissociate polymer-pDNA binding, allow nuclease degradation of the pDNA, and significantly decrease transfection efficiency. This is certainly observed in our previous work, which revealed that polyplexes formed with Tr1 and Tr2 aggregate rapidly and become very polydisperse when exposed to cell culture media containing serum.17 Polyplexes Tr3 and Tr4 are more stable from aggregation in serum-containing cell culture media and facilitate very efficient pDNA transfection efficiency in the presence of serum.16,17 In addition, the ability of the oligoethyleneamines in Tr3 and Tr4 to interact (possibly through H-bonding) with the nitrogenous bases suggests pDNA binding in the major and/or minor groove with the oligoethyleneamine linker moieties. Trehalose could then be exposed (with its large hydration radius) on the polyplex surface and also aid in slowing or preventing protein adsorption (thus slowing aggregation) of the polyplexes, which was previously observed.16,17 Further studies to confirm the source of the stability against flocculation with serum proteins, such as NMR structural analysis and molecular modeling, are warranted and being pursued.

There are many factors contributing to the efficacy of polymeric gene delivery vectors, such as colloidal stability, DNA protection from degradation, cellular uptake, cellular trafficking, endosomal escape, and DNA release. The binding and compaction of the DNA by the nonviral vector is the first crucial step in this complex process and has been shown to play a large role in efficacy. Understanding the elusive mechanisms of association is fundamental to this field and can be important toward the design of more efficient vehicles. This study was aimed at determining the factors governing pDNA interaction with a new series of highly effective trehalose-based polycationic vectors and understanding the large disparity in delivery efficiency observed with seemingly similar polymer structures.16,17 Originally, three important characteristics were incorporated into the structure of these polymers: a disaccharide, trehalose, known for its ability to thwart protein adsorption, a triazoleamide linker thought to promote hydrogen bonding and van der Waals interaction to the nucleotide bases, and an oligoethylene unit to facilitate electrostatic association with DNA phosphate groups. The role of the polymer structure in the binding mechanism, the effect of increasing amine number, and the thermodynamic parameters of binding were investigated through many biophysical techniques, such as microcalorimetry, ethidium bromide exclusion assay, light scattering, ζ-potential, potentiometric titration, circular dichroism, and infrared spectroscopy. We have shown that the number of secondary amines in the repeat unit of these polycations slightly alters the association constant but greatly affects the mode of pDNA interaction, which, in turn, greatly affects the biological properties. Tr1 was shown to possess the highest protonation fraction at physiological pH and to have the highest proton uptake upon pDNA binding, which results in this vector interacting with the nucleic acid through a more electrostatic (charge-charge) mechanism. Polymers Tr3 and Tr4, possibly due to longer amine spacers, were shown to incorporate more pDNA base pair interaction. With Tr4, it was demonstrated that direct binding to the pDNA bases likely occurs via hydrogen bonding with the secondary amines and the triazole-amide motif, as was shown with similar N-containing heterocycles adjacent to amides by the Dervan group.19,20 Results of this study suggest that the charge fraction as well as the length of the amine spacer determine the mechanism of interaction between trehalose polycations and plasmid DNA. When the electrostatic component is lower, it appears that hydrogen bonding compensates, but the extent of H-bond formation is highly dependent upon the length of the ethyleneamine spacer (distance between triazole-amide linkers) and the final protonated state of the polymer. Polymers Tr3 and Tr4 may enable trehalose to maintain its large hydration shell and be exposed on the polyplex surface by forming interactions with pDNA through these long spacers, hence providing the polyplexes stability from aggregation and thus higher efficacy.16,17 While some of these conclusions are difficult to prove experimentally, this biophysical study lends valuable insight into the effects that very subtle structural changes (such one ethyleneamine spacer) have on the mechanism of pDNA interaction. This examination also suggests possible explanations for the unanticipated structure-biological property relationships previously observed with this unique and highly effective series of polymeric delivery vehicles.16,17 LA800120Q