Interaction of Astramol Poly (propyleneimine) Dendrimers with DNA

Jun 23, 2014 - Interaction of poly(propyleneimine) dendrimers DAB-dendr-(NH2)x of five generations (x = 4, 8, 16, 32, and 64) with either calf thymus ...
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Interaction of Astramol Poly(propyleneimine) Dendrimers with DNA and Poly(methacrylate) Anion in Water and Water−Salt Solutions Marina V. Zhiryakova† and Vladimir A. Izumrudov*,†,‡ †

Chemistry Department, M.V. Lomonosov Moscow State University, Moscow 119991, Russia A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St. 28, Moscow 119991, Russia

‡

S Supporting Information *

ABSTRACT: Interaction of poly(propyleneimine) dendrimers DAB-dendr-(NH2)x of five generations (x = 4, 8, 16, 32, and 64) with either calf thymus DNA or tagged by pyrenyl groups poly(methacrylate) anion (PMA*) as well as destruction of formed polyelectrolyte complexes by the added sodium chloride were studied by fluorescence quenching techniques. DNA-containing complexes (dendriplexes) were investigated by ethidium bromide assay, whereas formation of PMA* complexes was estimated by fluorescence of the pyrenyl groups that remained free of contact with the dendrimers-quenchers. The ion pairing with DNA phosphate groups was pH-sensitive and accompanied by inaccessibility of a part of the dendrimer amino groups even in slightly acidic media. The growth of the generation number resulted in successive stabilization of the dendriplexes against the added salt. The dendriplexes of all dendrimers except DAB-dendr-(NH2)4 were stable at physiological ionic strength. In contrast to the highly charged cationic polymer poly(N-ethyl-4-vinylpyridinium) bromide of different degrees of polymerization, the dendrimers formed more stable complexes with flexible PMA* rather than with DNA, proving the inaccessibility of a part of the amino groups for the rigid double helix. The revealed regularities appear to be a platform for design of dendriplexes with controllable stability, in particular fulfilling the requirements imposed for gene delivery vehicles.

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regular structure.24 DAB-dendr-(NH2)x of five consecutive generations with x = 4, 8, 16, 32, and 64 are highly soluble in water and water−salt solutions and commercially available. For the sake of simplicity, hereafter the dendrimers will be referred to as G1, G2, G3, G4, and G5, respectively. The structure of G4 is sketched below schematically as twodimensional. (Chart 1). The dendrimer molecule contains x primary amino groups on the surface and (x − 2) tertiary amino groups located in the “core”. According to potentiometry data,25 virtually all amino groups of the dendrimers are able to be protonated and form ion pairs with carboxylate or sulfonate groups of flexible vinyl polyanions, while only part of the protonated amino groups are accessible for ion pairing with phosphate groups of rigid negatively charged nucleic acid. Herein, we report the effect of a generation number of DABdendr-(NH2)x on the stability of the dendriplexes and corresponding PMA* complexes in water−salt solutions. To verify whether the revealed features are common to polyelectrolyte systems, the analogous experiments were conducted with DNA or PMA* complexes formed by the highly charged cationic polymer poly(N-ethyl-4-vinylpyridinium) bromide of different chain lengths. The results of the above comparison studies as well as the model experiments on DNA or PMA* polyelectrolyte complexes allowed us to

INTRODUCTION Interactions of a negatively charged double helix with cationic polymers and dendrimers yield polyplexes and dendriplexes, respectively. Both types of complexes are successfully used for transfection and protection of the cargo nucleic acid from destruction by cell nucleases.1−4 The ion pair formation between the dendrimers and DNA or antisense oligonucleotides provides gene transfection5,6 and antisense efficacy.7,8 Use of the polyamidoamine (PAMAM) dendrimers resulted in significant enhancement in cell uptake of oligonucleotides.9 The structure, physical properties, and applications of dendrimers were discussed in a review,10 and the problems related to the gene delivery were considered in a review.11 In the recent decade, the use of dendrimers as drug carriers and transfection agents was still broadly discussed in the literature.12−22 To give precise control over the stability of dendriplexes, it is essential to ascertain the influence of a generation number of the dendrimer on binding with nucleic acid in water and water−salt media. Of particular interest is to study dendrimers of “earlier” generations in order to reveal dendrimers fulfilling the requirements imposed for a vehicle of oligonucleotide delivery. The vehicle should form with the oligomer of the polyplex which remains stable under physiological conditions being not more than 30 kDa to allow exit from the bloodstream and penetration into cells.23 Astramol poly(propylenimine) dendrimers, DAB-dendr(NH2)x, are treelike highly symmetric cationic molecules of © 2014 American Chemical Society

Received: March 25, 2014 Revised: June 17, 2014 Published: June 23, 2014 8819

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The reaction product was twice precipitated in hexane. Mw determined by light scattering in methanol at 298 K was 1.1 × 105 g·mol−1 (Pw = 700). Measurements. Fluorescence Measurements. The fluorescence intensity of the solutions was determined using a Jobin-Yvon-3CS spectrofluorimeter (France) with a waterthermostatic stirred cell holder. The measurements were made in the capped quartz fluorescence cell upon permanent stirring at 298 K. The excitation and emission wavelengths were set at 535 and 595 nm, respectively, in the case of EB and at 346 and 395 nm, respectively, in case of PMA* solutions. The concentration of EB was 1 × 10−5 mol·L−1 in all experiments. DNA solution was directly mixed with EB in the fluorescence cell. The final concentration of DNA phosphate groups was 4 × 10−5 mol·L−1; i.e., the composition of the formed DNA·EB complex was [EB]/[P] = 0.25. At this ratio corresponding to 1 molecule of intercalated EB per 2 pairs of DNA bases (4 nucleotides), the maximal value I0 of EB fluorescence intensity was achieved.30 The fluorescence measurements were performed in 0.02 M solutions of biological buffers, i.e., MES (pH 6.0 and 6.5), HEPES (7.0, 7.5, and 8.0), TRIZMA (8.5 and 9.0), CHES (9.5), and CAPS (10.0 and 10.5). DAB-dendr-(NH2)x dendriplexes were prepared in the same cells by step-by-step addition of dendrimer stock solution containing all amino groups of the dendrimer in a concentration [N] equal to 0.001 mol·L−1. The titration of DNA·EB and measuring of the fluorescence intensity I were performed with [N]/[P] = 0.1:1 ratio increments at a 2 min time interval. The titration was finished when the ratio [N]/[P] = 2 was achieved. The extra equivalent of amino groups of the dendrimer was added to provide a complete expulsion of EB from the DNA structure.31 Thus, the components of the prepared solutions were dendriplex, DAB-dendr-(NH2)x, and free EB. The same procedure was used for preparation of DNA complexes with other polyamines taking the concentration of all amino groups of the polyamines in the ratio [N]/[P]. Complexes of PMA* with DAB-dendr-(NH2)x were prepared in the fluorescence cells by step-by-step addition of dendrimer stock solution ([N] = 1 × 10−3 mol·L−1) to solution of sodium PMA* containing carboxylic groups in concentration [PMA*] = 4 × 10−5 mol·L−1. The addition was conducted with [N]/ [PMA*] = 0.1:1 increments and a 2 min time interval. The titration was finished when the ratio [N]/[PMA*] = 2 was achieved. The same procedure was done on preparation of PMA* complexes with PEVP taking the concentration of pyridinium groups of PEVP in the ratio [N]/[PMA*]. Destruction of the formed complexes was carried out by successive addition of 4 mol·L−1 NaCl solution in the same cells and measuring the fluorescence intensity I. The time interval between the additions was 5 min. In parallel, the titration of DNA·EB solution or sodium PMA* solution of the same concentration with 4 mol·L−1 NaCl solution was conducted to determine the fluorescence intensity I0. The curves were presented as dependencies of relative fluorescence intensity I/I0 on the salt concentration. Potentiometric titration of aqueous solutions of polyamines and mixtures of polyamines with DNA was performed as described elsewhere.29

Chart 1. Structure of the poly(propyleneimine) dendrimer of fourth generation (G4)

elucidate the role of DNA rigidity and unique structure of the dendrimers in the formation and stability of the dendriplexes in water−salt media.

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EXPERIMENTAL SECTION Materials. DAB-dendr-(NH2)x dendrimers of five generations, i.e., G1 (x = 4), G2 (x = 8), G3 (x = 16), G4 (x = 32), and G5 (x = 64), were purchased from Aldrich. Ethidium bromide (EB) and calf thymus DNA (∼10 000 base pairs) were used as received from Sigma. Concentrations of DNA phosphate groups [P] and EB in solution were determined from UV spectra assuming the molar extinction coefficients ε = 6500 L·mol−1·cm−1 at 260 nm26 and ε = 5600 L·mol−1·cm−1 at 480 nm,27 respectively. A sample of poly(methacrylic acid) (PMA, Mw = 1.6 × 105 g· mol−1) was purchased from Polysciences. Labeled by fluorescence tags, PMA* was prepared as described elsewhere.28 The PMA* sample contained on average one pyrenylmethyl methacrylate group (the tag) per 400 repeat units as it was calculated from the absorbance at 346 nm assuming ε = 50 000 L·mol−1·cm−1. The synthetic polycations poly(N-ethyl-4-vinylpyridinium) bromide (PEVP) and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEM) were synthesized and characterized as described elsewhere.29 PEVP samples of weight-average degrees of polymerization Pw ranging from 70 to 4000 and numberaverage degrees of polymerization of Pn = 10, 20, and 40 and a PDMAEM sample with Pw = 700 were used. A poly-L-lysine hydrobromide (PLL) sample with Pn = 10− 25 was purchased from Sigma and used without purification. Branched poly(ethylenimine) (PEI) was purchased from Scientific Polymer Products as 50 wt % water solution. The Mw of the sample was 5 × 104 g·mol−1; this corresponds to on average 1200 amino groups per molecule. Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEM) was prepared by radical polymerization of monomer (monomer was synthesized and kindly supplied by Institute of Synthetic Rubber, Yaroslavl, Russia) in 50% benzene solution at 313 K under a nitrogen atmosphere for 36 h with AIBN as an initiator.

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RESULTS AND DISCUSSION Both the binding of DNA with DAB-dendr-(NH2)x and dissociation of the dendriplexes in water−salt media were 8820

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studied using the cationic dye ethidium bromide (EB). The intercalation of EB between base pairs of native DNA is accompanied by a significant growth in EB fluorescence intensity which allows monitoring the formation of DNA·EB complexes by fluorescence quenching techniques. The DNA·EB complexes are rather stable (KC is of the order of 106 M−1) and survive even in concentrated salt solutions.30 The binding of the dye results in a large increase in the fluorescence quantum yield of EB (for a review, see ref 32), whereas EB release from DNA·EB is attended with a decrease in the quantum yield. This feature was used for studying the association of short polyamines, e.g., synthetic polypeptides,33,34 analogues of spermine35−37 and histones38 with calf thymus DNA in vitro. Subsequently, we reported31 that the same approach is applicable for investigation of DNA binding with different cationic molecules, in particular linear synthetic polyamines,29,39 basic polypeptides,29 cationic surfactants,40 and dendrimers.41 Moreover, the fluorescence assay proved to be appropriate for elucidating the destruction of DNA-containing complexes by the added salt.29,31,39,42,43 At present, the approach is widely used by many investigators for studying DNA−polycation interaction; for example, see refs 44−47. Formation of Astramol Dendriplexes. The curves of fluorimetric titration of DNA·EB solution with solution of DAB-dendr-(NH2)x are presented as dependencies of fluorescence intensity I on the ratio φ of the molar concentrations of all amino groups of the dendrimer to DNA phosphate groups, φ = [N]/[P]. At pH 9.0 (Figure 1), the successive

Figure 2. Fluorescence intensity of DNA·EB mixtures with the solution of G4 plotted as a function of the ratio φ = [N]/[P] at different pH’s. The other conditions are the same as those in Figure 1.

of the dendrimer remained inaccessible for DNA phosphate groups presumably due to steric hindrance. The point in support of this assumption is linearity of the curves in Figure 2 which are different from typical curves of DNA·EB titration with linear highly charged polycations. In the latter case (Figure 3), the curves are sigmoidal with a sudden

Figure 3. Fluorescence intensity of DNA·EB mixtures with solutions of PEVP, Pw = 120, poly-L-lysine, Pn = 10−25, and dendrimer G4 plotted as a function of the ratio φ = [N]/[P]. The conditions are the same as those in Figure 1.

fall of the intensity in the region close to total neutralization of DNA phosphate groups; cf. the blue curve and red curve corresponding to poly(N-ethyl-4-vinylpyridinium) bromide and poly-L-lysine hydrobromide, respectively. It is well documented that the fall is conditioned by a competitive displacement of the intercalated EB from the double helix as a result of DNA condensation into a stable compact structure. Thus, according to the sedimentation patterns,31 the titration of DNA·EB with PEVP at a charge ratio close to unity was followed by a pronounced accumulation of nonfluorescence dye in solution. In all likelihood, the displacement of the dye is the result of cooperative electrostatic interaction of DNA with cationic polymer. A relatively low affinity of calf thymus DNA to charged oligoamines (analogues of spermine) was reflected by gently sloping curves of the fluorimetric titration.35,36 The short cationic peptide KALA quenched the fluorescence more efficiently but still not sufficient enough to be the powerful DNA-compacting agent, as the curve flattened out and a significant part of the dye was still associated with DNA.34 This curve is closely similar to the red curve in Figure 1 which corresponds to DNA·EB titration with G1 dendrimer.

Figure 1. Fluorescence intensity of DNA·EB mixtures with solutions of G1, G2, G3, G4, and G5 plotted as a function of ratio φ = [N]/[P]. [P] = 4 × 10−5 M, [EB]/[P] = 0.25; pH 9.0, 298 K.

addition of all dendrimers except G1 (x = 4) (red curve) was followed by a practically linear decrease of the fluorescence intensity until φ = 2.2 where the quenching is virtually completed. The same regularity proved to be inherent in DNA·EB mixtures with all dendrimers at different pH’s. For the sake of simplicity, the pH influence is exemplified by the mixtures containing typical dendrimer G4 (x = 32) (Figure 2). As would be expected, the pH decrease was accompanied by a growth in the quenching efficacy due to successive protonation of the amino groups capable of ion pairing with DNA. Nevertheless, even in slightly acidic media, the complete quenching was achieved only at φ = 1.5 but not at φ ≈ 1.0, as occurs on DNA· EB titration with flexible highly charged vinyl polycations.29 This finding implies that a part of the protonated amino groups 8821

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The pH profiles of DNA binding were estimated for different polycations (Figure 5), i.e., PEI (blue), G4 (black), PDMAEM

The smoothing of the curves can be determined by the relatively small number of charges on the short chains and steric hindrances that reduce the affinity. Thus, a monotonic run of the curve was also inherent in DNA·EB titration with poly(ethylenimine) (Figure 4). This branched polyamine is not

Figure 5. Θ−pH profiles of DNA binding with different polycations derived from the fluorescence intensity of the mixtures. Θ−pH dependencies obtained by potentiometric titration of DNA mixtures with the polycations are marked by crosses. φ = [N]/[P] = 1. Figure 4. Fluorescence intensity of DNA·EB mixtures with PEI plotted as a function of the ratio φ = [N]/[P] at different pH’s. The other conditions are the same as those in Figure 1.

(red), and PLL (green). The profiles were derived from the corresponding fluorimetric titration curves by the calculations according to eq 1 (the fluorimetric titration curves not shown). Note that the pH profiles can be derived by potentiometry as described elsewhere,25 but this approach is limited by the initial stage of the binding, Θ < 0.2, where the calculated Θ values remain reliable. In this particular case, the data of potentiometric titration are treated with the use of eq 225,29

able to form with DNA of a highly ordered system of ion pairs because of steric hindrances, and hence, the curves are linear. The efficacy of the quenching in alkaline media, pH 9.5, is minimal and increases with a pH decrease owing to protonation of PEI amino groups. Nevertheless, the sudden fall in the fluorescence intensity does not appear even on the titration at pH 6 (orange curve), where a majority of the ion pairs are formed. Analogously, the titration of DNA·EB with DAB-dendr(NH2)x at x > 4 did not result in appearance of the region of significant quenching at any of the pH’s studied, 6.0 ≤ pH ≤ 10.5. In other words, the smooth shape of curves in Figure 2 suggests inability of G4 dendrimer to squeeze out completely the intercalated dye. Attention is drawn to the fact that the quenching efficacy of DNA·EB fluorescence that is caused by addition of G4 (Figure 2) and, in particular, PEI (Figure 4) is not a monotonically increasing function of pH. This implies that the degree of conversion, Θ, in the ion pairing of DNA with the polycations, which is defined as the ratio of a current number of the ion pairs to the ultimate one,25 depends on the pH and nature of the cationic molecules. The pH dependence of Θ values, i.e., pH profile of the DNA binding, can be estimated on the assumptions that Θ is proportional to the quenching efficiency and a Θ value equal to unity is achieved at neutral pH in a DNA mixture with poly-Llysine hydrobromide at φ = [N]/[P] = 1. In this system, the rather high pKb = 10.4 of PLL provides formation of a great majority of the ion pairs even in slightly alkaline media at pH 9.0, as it follows from coincidence of the curves corresponding to pH 6.0 and pH 9.0 (data not shown). On the basis of the above assumptions, Θ values can be calculated using eq 1: I − In Θ= c Ic − I0 (1)

C Θ= c = C0

(

q V

+

10−14 [H+]

−

C0

10−14 [H+]PC

) (2)

where Cc is the current concentration of the ion pairs, C0 is the initial concentration of polycation PC, q is the number of equivalents of the titrant (HCl) added, V is the volume of the solution, [H+] is the measured total concentration of hydrogen ions, and [H+]PC is the concentration of hydrogen ions in a solution of free polycation. In the range pH > pHPC, [H+]PC can be estimated using eq 3 [H+]PC =

10−14 Kb(1 − Θ)C0

(3)

where Kb is the intrinsic basicity constant of the polycation amino groups. The resulting Θ−pH profiles, that are depicted in Figure 5 by crosses, fit well with the initial parts of the profiles derived from the fluorimetric titration curves. This finding testifies that the fluorescence assay is applicable for studying the DNA binding. Moreover, contrary to the potentiometry, the fluorescence quenching techniques allow monitoring the binding over a wide pH range, 4 < pH < 11, corresponding to a stable DNA·EB complex.30 This statement is clearly demonstrated by a pronounced difference of curves in Figure 5. The nonmonotonic run of the Θ−pH profile (blue curve) is readily explained by steric hindrances on DNA binding with branched polyamine PEI containing three types of amino groups of different pKb. The Θ−pH profiles of dendriplex G4 (black curve) and polyplex PLL (green curve) located in close proximity reflect protonation of the primary amino groups in the alkaline media.

where Ic is the fluorescence intensity of DNA·EB, whereas In and I0 are the intensities of equimolar (φ = 1) DNA mixtures with the polyamine at a given pH and with PLL in neutral media, respectively. 8822

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Figure 6. Fluorescence intensity I0 of DNA·EB solution and the intensity I of DNA·EB mixtures with the dendrimers plotted as a function of salt concentration (A) and the plots which are normalized to the intensity I0 of DNA·EB solution (B). φ = [N]/[P] = 2, and the other conditions are the same as those in Figure 1.

Thus, the unique structure of DAB-dendr-(NH2)x molecules provides efficient electrostatic interaction with DNA presumably by ion-pairing of protonated primary amino groups situated on the dendrimer surface, yet this interaction does not cause the pronounced compaction of an almost fully neutralized double helix owing to steric hindrances. The peculiarities of the DAB-dendr-(NH2)x structure also had a profound impact on the stability of the dendriplexes in water−salt solution. Stability of Astramol Complexes in Water−Salt Media. The fluorescence quenching techniques can also be applied to monitoring the destruction of dendriplexes in water−salt solution. The destruction is accompanied by growth in fluorescence intensity due to EB reintercalation into free sites of the double helix.29,31,43 The titration with salt solution of either a DNA·EB or DNA· EB mixture with DAB-dendr-(NH 2) x (Figure 6A) was conducted as described in the Experimental Section. The decrease in fluorescence intensity I0 of DNA·EB alone is conditioned by conformational changes of the nucleic acid which occur without expulsion of the intercalated EB from the double helix even at high ionic strength.30 At C(NaCl) = 0 (start points of the curves), the values of fluorescence intensity I of the mixtures, x > 4, are negligible, i.e., practically all EB molecules competitively displaced by the added dendrimer. The right-hand branches of the curves reflect dissociation of the dendriplex that proceeds completely at relatively high ionic strength corresponding to coincidence of the curves with the black curve (I0). The same curves are depicted in Figure 6B as the dependencies normalized to the intensity I0 of DNA·EB solution at the same ionic strength. The right-hand branches of the curves evidence the cooperative character of the dissociation of the dendriplexes. For the sake of simplicity, the values of the critical salt concentration CS,d corresponding to I/I0 = 0.6 (eq 4)

CS,d ≡ CS(I /I0 = 0.6)

Figure 7. Dependencies of critical salt concentration CS,d on a number x of primary amino groups of DAB-dendr-(NH2)x in solutions of dendrimer complexes with DNA and PMA*. [P] = [PMA*] = 4 × 10−5 M, φ = [N]/[P] = [N]/[PMA*] = 2. The other conditions are the same as those in Figure 1.

First, all dendrimers except G1 form dendriplexes stable at least in 0.14 M NaCl solution. This strongly suggests that DABdendr-(NH2)x, x > 4, are able to interact with DNA at physiological ionic strength. Second, the higher the generation number, the more stable the dendriplex that is formed. Moreover, for dendrimers of “earlier” generations, the stabilization is more pronounced. This finding is in qualitative agreement with well documented dissociation of complexes formed by oppositely charged linear polyelectrolytes of different degrees of polymerization. As noted below, the same trend proved to be inherent in PMA* complexes with DAB-dendr-(NH2)x. Fluorescence Study on PMA* Complexes with DABdendr-(NH2)x. Primary amino groups of DAB-dendr-(NH2)x are effective quenchers of the fluorescence of pyrenyl groups covalently attached to sodium poly(methacrylate). The quenching of the labeled polyanion PMA* by added DABdendr-(NH2)x is depicted in Figure 8 as a dependence of the fluorescence intensity I on the ratio φ of the molar concentration of all amino groups of the dendrimer to the molar concentration of carboxylic groups, φ = [N]/[PMA*]. It is seen that the successive addition of all dendrimers results in a noticeable and virtually linear decrease of the fluorescence. Note that, in this particular case, the quenching efficacy varies inversely with the dendrimer generation. Thus, the most

(4)

can be used for estimation of the stability of different dendriplexes in water−salt solutions. Thus, from CS,d values plotted versus a number x of primary amino groups in the DAB-dendr-(NH2)x (Figure 7, red curve), two general conclusions can be drawn. 8823

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for assessment stability of PMA*-containing complexes in water−salt solutions. The plot of CS,d, thus defined, versus a number x of primary amino groups of dendrimer is exhibited in Figure 7 (blue curve). The location of the blue curve above the red curve evidences that DAB-dendr-(NH2)x forms more stable complexes with flexible PMA* than with DNA. This finding is in agreement with the above suggestion on the inaccessibility of a part of the inner tertiary amino groups of the dendrimers for the rigid double helix.25 Finally, an added reason for the inaccessibility of the groups is a directly opposite trend in stability of polyelectrolyte complexes formed by DNA and PMA* with poly(N-ethyl-4vinylpyridinium) bromide of different degrees of polymerization (Figure 10). Contrary to DAB-dendr-(NH2)x, this

Figure 8. Dependencies of fluorescence intensity of PMA* mixtures with dendrimers of different generations on the ratio φ = [N]/ [PMA*]. [PMA*] = 4 × 10−5 M. The other conditions are the same as those in Figure 1.

effective quencher proved to be G1 dendrimer. This finding is in direct opposition to the behavior of the dendrimer in DNA· EB solutions where G1 was the weakest quencher (Figure 1). This discrepancy appears to be conditioned by preferential binding of the dendrimers with PMA* segments tagged by pyrenyl groups. It is reasonable to suggest that a driving force of the preferential binding is a gain in Gibbs energy due to both formation of a charge transfer complex between the pyrenyl group (donor) and amino group (acceptor) and hydrophobic interaction of the pyrenyl group with hydrophobic regions formed by the bound partners. It is obvious that at a constant φ the “earlier” the dendrimer generation, the greater number of the dendrimer molecules in solution and, hence, the higher probability of the preferential binding. Figure 9 gives the profiles of the destruction of the complexes by the added salt. The curves are depicted as

Figure 10. Dependencies of critical salt concentration CS,d on the PEVP degree of polymerization in solutions of PEVP complexes with DNA and PMA*. [P] = [PMA*] = 4 × 10−5 M, φ = [N]/[P] = 2. The other conditions are the same as those in Figure 1.

flexible vinyl polycation forms more stable complexes with DNA rather than with PMA*; cf. the mutual arrangement of the titration curves of PMA*−PEVP (blue) and DNA−PEVP (red) mixtures with salt.

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CONCLUSIONS In summary, we report the first comprehensive data on the affinity of poly(propyleneimine) dendrimers DAB-dendr(NH2)x of five generations (x = 4, 8, 16, 32, and 64) to calf thymus DNA in water and water−salt solutions that was estimated by ethidium bromide assay. As it followed from both the slope and linearity of the fluorescence titration curves, the ion pairing with DNA phosphate groups was pH-sensitive and characterized by inaccessibility of a part of the dendrimer amino groups for the nucleic acid even in slightly acidic media. The decrease of the generation number resulted in successive destabilization of the dendriplexes against the destruction action of added salt (NaCl), specifically for dendrimers of “earlier” generations. Nevertheless, all dendrimers except DABdendr-(NH2)4 formed dendriplexes which were stable at physiological ionic strength. The ability of dendrimer amino groups to quench the fluorescence of pyrenyl-tagged poly(methacrylic) acid allowed us to perform comparison studies of salt-induced destruction of the complexes. Thus, in contrast to flexible highly charged polycation-quencher poly(N-ethyl-4vinylpyridinium) bromide of different degrees of polymerization, DAB-dendr-(NH2)x exhibited exactly the opposite effect, forming more stable complexes with flexible PMA* rather than with DNA. The revealed inaccessibility of a part of

Figure 9. Relative fluorescence intensity of PMA* mixtures with dendrimers of different generations plotted as a function of NaCl concentration. φ = [N]/[PMA*] = 2. [PMA*] = 4 × 10−5 M. The other conditions are the same as those in Figure 1.

dependencies of the relative fluorescence intensity I/I0 on the salt concentration, where I and I0 are the fluorescence intensities of the PMA* mixture with DAB-dendr-(NH2)x and PMA* alone, respectively. As expected, the curves have a sigmoid shape with right-hand branches reflecting dissociation of the complex. The dissociation is completed at an ionic strength corresponding to I/I0 = 1. Similarly to assay of dendriplexes, assume the value of the critical salt concentration, CS,d ≡ CS (I/I0 = 0.6) as a parameter 8824

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the dendrimer amino groups for the rigid double helix could be important for practical implementation, specifically on enhancing the proton sponge effect owing to contribution of the protonated nonbound amino groups. Furthermore, the elucidated features of DAB-dendr-(NH2)x may also set a new platform to design dendriplexes with controllable stability, in particular fulfilling the requirements imposed for gene delivery vehicles as potential therapeutic agents.

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ASSOCIATED CONTENT

S Supporting Information *

The values of critical salt concentration corresponding to dissociation of approximately a half of salt bonds in complexes of dendrimer or PEVP with DNA·EB or PMA* are listed in tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +7 (495) 939-3117. Fax: +7 (495) 939-0174. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of Russian Foundation for Basic Research (Grant No. 14-08-01202) is gratefully acknowledged. REFERENCES

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