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Jan 10, 2016 - ABSTRACT: The thermoresponsive structural transition of poly(N-isopropylacrylamide) (PNIPAAm)-b-DNA copoly- mers was explored...
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Effects of Complementary DNA and Salt on the Thermoresponsiveness of Poly(N‑isopropylacrylamide)‑b‑DNA Masahiro Fujita,*,† Hayato Hiramine,‡ Pengju Pan,† Takaaki Hikima,§ and Mizuo Maeda*,†,‡ †

Bioengineering Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan Department of Advanced Materials Science, School of Frontier Science, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa-shi, Chiba 277-8561, Japan § RIKEN SPring-8 Center, Advanced Photon Technology Division, Kouto 1-1-1, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan ‡

ABSTRACT: The thermoresponsive structural transition of poly(N-isopropylacrylamide) (PNIPAAm)-b-DNA copolymers was explored. Molecular assembly of the block copolymers was facilitated by adding salt, and this assembly was not nucleated by the association between DNA strands but by the coil−globule transition of PNIPAAm blocks. Below the lower critical solution temperature (LCST) of PNIPAAm, the copolymer solution remained transparent even at high salt concentrations, regardless of whether DNA was hybridized with its complementary partner to form a double-strand (or single-strand) structure. At the LCST, the hybridized copolymer assembled in spherical nanoparticles, surrounded by double-stranded DNA; subsequently, the non-cross-linking aggregation occurred, while the nanoparticles were dispersed if the salt concentration was low or DNA blocks were unhybridized. When the DNA duplex was denatured to a single-stranded state by heating, the aggregated nanoparticles redispersed owing to the recovery of the steric repulsion of the DNA strands. The changes in the steric and electrostatic effects by hybridization and the addition of salt did not result in any specific attraction between DNA strands but merely decreased the repulsive interactions. The van der Waals attraction between the nanoparticles overcame such repulsive interactions so that the non-cross-linking aggregation of the micellar particles was mediated.

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INTRODUCTION DNA has been recognized as a versatile tool for directing the bottom-up self-assembly of materials because of its sequencespecific interactions, such as base pairing of A-T and G-C. DNA modified with various functional groups can attach chemically to metal, inorganic, and organic nanoparticles. The resulting DNA-functionalized nanoparticles can be programmed to assemble into well-ordered three-dimensional structures according to the design of the DNA sequence and length.1−8 In some cases, this process can cause a change in optical properties, permitting the wide application of these structures as highly sensitive biosensors.9 Compared to the large number of investigations on DNA-encoded metal and inorganic nanostructures, research into the DNA-programmed selfassembly of soft materials such as polymeric micellar particles has been relatively scarce. This is likely because such research requires a more complicated preparation procedure. However, the morphology of soft nanoparticles can be tailored by varying the molecular weight, primary structure, molecular topology, and so on, making it feasible that their properties and functions could be tuned, even though the chemical compositions are the same.10−14 Poly(N-isopropylacrylamide) (PNIPAAm) grafted with single-stranded DNA (ssDNA) is a DNA-encoded polymeric material and shows an interesting thermoresponsiveness.15−19 © XXXX American Chemical Society

The copolymer is water-soluble at room temperature but undergoes dehydration of the PNIPAAm component above its lower critical solution temperature (LCST, ∼32 °C). Because the negatively charged DNA is still hydrophilic, the molecular chains assemble in a core−shell-type spherical particle that consists of a PNIPAAm hydrophobic core surrounded by ssDNA, unless the PNIPAAm fraction in the copolymer chain is too large or small. Even at high salt concentrations, the micellar particles disperse stably owing to steric repulsion of the DNA strands. Accordingly, the copolymer dispersion remains transparent above the LCST. The hybridization with fully complementary DNA, however, induces the particles to aggregate, resulting in the clouding of the dispersion. It is noted that this particle aggregation differs entirely from bridging by DNA base pairing, i.e., molecular cross-linking.1−8 This behavior is hence referred to as non-cross-linking aggregation. However, if the particles are covered with singlebase-mismatched dsDNA, then the aggregation is not induced. The DNA-encoded soft nanoparticles are thus expected to be developed as functional materials, e.g., for detecting slight differences such as single nucleotide polymorphisms.15−17 Received: November 9, 2015 Revised: December 22, 2015

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including 0.1 or 0.5 M NaNO3 were prepared. When adding the complementary DNA, the concentration was 1 equiv with respect to the block polymer. The temperature-dependent absorbance was measured on a UV-2550 UV−vis spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance at 500 nm for an aqueous solution of polymer sample (0.5 g L−1) was monitored upon heating at 1 °C min−1. The DSC measurements were performed with a VP-DSC microcalorimeter (MicroCal Inc., Northampton, MA, USA). An aqueous solution of polymer sample (0.5 g L−1) was heated at 1 °C min−1. Three or more repeated measurements were performed for each sample. Solution SAXS measurements were carried out at BL45XU RIKEN Structural Biology Beamline I of SPring-8 (Harima, Japan).27 A PNIPAAm-b-ssDNA solution (1.0 g L−1) in 10 mM PB (pH 7) including 0.1 M NaNO3 was prepared. In the case of PNIPAAm-bdsDNA solution, complementary DNA was added to the polymer solution at 25 °C before measurement. The final concentration of complementary DNA was 1 equiv with respect to the block polymer. For the measurements at a high salt concentration, NaNO3 was adjusted to 0.5 M. The SAXS images were taken with a Pilatus 300 KW detector during heating from 25 to 60 °C. The image data were converted into one-dimensional intensity data as a function of scattering vector q = (4π/λ) sin θ, where 2θ is the scattering angle and λ is the wavelength. The intensity data I(q) is proportional to the product of the form factor P(q) and the structure factor S(q) associated with the interference between scattering objects, as shown below.28

The graft copolymer, however, has a broad molecular weight distribution because of the low controllability of the conventional radical polymerization. Well-defined polymers with precisely designed chemical structures and nanostructured morphologies are helpful in better understanding the structure−property relationship. Therefore, we previously developed a method for making block copolymers, PNIPAAm-b-ssDNAs, with controlled molecular length, composition, and topology.20,21 The micellar structures were significantly dependent on the composition of the copolymers and topology, allowing for the fine-tuning of particle properties and functions through the molecular design. The increases in size and density of the PNIPAAm core were found to facilitate the non-cross-linking aggregation of the particles. The mechanism of the non-cross-linking aggregation interaction has not yet been completely explained. The role of the end-to-end stacking interaction between DNA duplexes in the non-cross-linking aggregation has been a controversial subject.22−24 Furthermore, it was reported that the association of DNA strands, except via end-to-end adhesion, plays an important role in the molecular assembly of PNIPAAm-DNA chains.25,26 According to these reports, an aqueous solution of PNIPAAm grafted with ssDNA rather than with dsDNA was easily clouded by heating. It has been considered that the charge density of a single strand is lower so that the copolymer chains with ssDNA are more susceptible to assembly by the addition of salt. This behavior appears to be the opposite of that in our previously reported colloidal system of PNIPAAmDNA.15−21 In this study, we aimed to reveal the roles of complementary DNA and its associated electrostatic property in the thermal behavior of PNIPAAm-b-DNA. Synchrotron radiation smallangle X-ray scattering (SAXS) was conducted principally in order to follow the thermoresponsiveness of the block copolymer with a precisely controlled molecular weight. The structural evolution from the molecular assembly to the dispersion/aggregation of the resulting micellar particles was analyzed. The mechanism of PNIPAAm-DNA colloidal stability was discussed, along with the results for the thermal properties and turbidity.

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I(q) ∝ P(q) S(q)

(1)

If the objects disperse stably without interfering with each other, then S(q) is unity. P(q) is given by squaring the scattering amplitude of the object. The scattering amplitude of a core−shell particle having two graded interfaces between the core and shell (inner) and between the shell and solvent (outer) was assumed for the present colloidal system. A detailed description of the scattering amplitude is found elsewhere.19,21 Briefly, the density profile of the graded interface with a thickness of 2σ is based on a parabolic shape.28 The resulting scattering amplitude A(q) is expressed by A(q) = (Δρc − Δρs )Vc Φc(q , R in , σin) + Δρs VsΦs(q , R out , σout) (2) where Δρc and Δρs are the differences in electron density between the core and solvent and between the shell and solvent and Vc and Vs are the volumes of the core and shell. In this equation, Rin = Wc + σin and Rout = Wc + 2σin + Ws + σout, where Wc and Ws are the dimensions of the central parts of the core and shell and σin and σout are half thicknesses of the inner and outer interfaces, respectively. In addition, Φ(q, R, σ) is the Fourier transformation of the radial density profile, which was given previously by Berndt et al.29 A Gaussian distribution was used to described the polydispersity of particle size, and the contribution of the spatial fluctuation of the molecular chains to I(q) at high q was considered.30 For the analysis of colloidal aggregates, a hard-sphere model with an adhesive surface, proposed by Baxter,31 was applied in this study, as in our previous work.19 In this model, the interaction potential between two micellar particles, U(r), is expressed by

EXPERIMENTAL SECTION

Materials. A linear diblock (AB-type) copolymer was made by atom-transfer radical polymerization of N-isopropylacrylamide and then coupling with 9-base ssDNA by click chemistry, according to our previous reports.20,21 The sequence of the ssDNA was 5′-GCC ACC AGC-3′. The ssDNA with an amine at its 5′ ends and its complement were purchased from Eurofins Genomics (Tokyo, Japan). The other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and Sigma-Aldrich (St. Louis, MO, USA). The desired degree of polymerization of PNIPAAm was set in the range of 250 to 400 to provide adequate amphiphilicity. This range was chosen because if the PNIPAAm block is too short or too long then the block copolymers cannot form micellar nanoparticles: the former does not undergo a coil−globule transition over a wide temperature range, and the latter leads to a gel-like structure above the LCST.21 Characterization. The weight- and number-averaged molecular weights (Mw and Mn) of PNIPAAm-b-ssDNA were measured with a gel-permeation chromatograph (Shimadzu, Japan) equipped with a multiangle laser light scattering detector (DAWN8+, Wyatt Technologies, Santa Barbara, CA, USA) (GPC-MALS). The details of the measurements are given in previous reports.20,21 For the turbidity and DSC measurements, PNIPAAm-b-ssDNA solutions (0.5 g L−1) in 10 mM phosphate buffer (PB) (pH 7)

⎧+∞ r < DHS ⎪ U (r ) = ⎨ ln[12τ(δ − DHS)/DHS] DHS < r < δ ⎪ ⎩0 r>δ

(3)

where r is the distance between two particles, DHS is the hard -sphere diameter, and δ − DHS is the width of the square-well attraction potential. In this expression, the reciprocal of parameter τ is called the stickiness parameter, which represents the strength of adhesion. The structure factor has the form

S(q) = [1 − C(q)]−1 B

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Langmuir The function C(q) is the Fourier transformation of the direct correlation function, expressed by

C(q) = − 24η[αf1 (x) + βf2 (x) + 0.5αηf3 (x)] − 2λ 2η2f4 (x) + 2ληf5 (x)

(5)

with α=

(1 + 2η − μ)2 , 4 (1 − η)

3η(2 + η)2 − 2μ(1 + 7η + η2) + μ2 (2 + η) , 2(1 − η)4 η(1 + 0.5η) μ = λη(1 − η), γ = , 3(1 − η)2 η 6 ε=τ+ , λ = {ε − (ε 2 − γ )0.5 } η 1−η β=−

(6)

and f1 (x) = x −3(sin x − x cos x) f2 (x) = x −4{2x sin x − (x 2 − 2)cos x − 2} f3 (x) = x −6{(4x 3 − 24x)sin x − (x 4 − 12x 2 + 24)cos x + 24}

Figure 1. (a) Absorbance of 1P289-1ssD9 (dotted line) and -1dsD9 (solid line) in 10 mM PB at the indicated concentrations of NaNO3. The absorbance was monitored at 500 nm during heating (1 °C min−1). The copolymer concentration was 0.5 g L−1. (b) DSC thermograms of 1P289-1ssD9 (dotted line) and -1dsD9 (solid line) in 10 mM PB at the indicated concentrations during heating (1 °C min−1). The copolymer concentration was 0.5 g L−1. In both parts, the blue and red lines represent the data in the presence of 0.1 and 0.5 M NaNO3, respectively.

f4 (x) = x −2(1 − cos x), f5 (x) = x −1 sin x (7) where η is the volume fraction and x = qDHS. The hard-sphere diameter DHS is considered to be an effective diameter for a colloidal particle with a diffuse surface so that DHS may be smaller than the overall particle size.32−34 The analysis using this model allows the particles with the diffuse surface to interpenetrate.

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RESULTS AND DISCUSSION Thermal Behavior of PNIPAAm-b-ssDNA. PNIPAAm-bssDNA copolymers were well-defined with narrow molecular weight distributions. The block copolymer with Mn = 34 400 and Mw/Mn = 1.1 was used here. This AB-type block copolymer is hereafter represented as 1P289-1ssD9, where P denotes the PNIPAAm block and its subscript corresponds to the degree of polymerization and D9 denotes nine-base DNA. The aqueous solution of the copolymer in 10 mM PB (pH 7) including 0.1 M NaNO3 remained transparent over the wide temperature range investigated here, as shown in Figure 1a. This is in contrast to the finding that the aqueous solution of PNIPAAm homopolymers (1P289) prior to the coupling reaction with DNA suddenly became cloudy at 34−35 °C. In the DSC of the copolymer solution (Figure 1b), however, an endothermic peak at ∼37 °C, indicating the coil−globule transition of the PNIPAAm block, was observed. Because of the hydrophilic nature of DNA, the LCST of the block copolymer was slightly higher than those of the homopolymers. These results suggested that block copolymer chains with adequate amphiphilicity assemble in nanosize micelles at the LCST. As in our previous reports,19,21 this micellization was then followed by SAXS. The SAXS profiles of 1P289-1ssD9 in the presence of 0.1 M NaNO3 are shown in Figure 2a. The SAXS data were acquired at elevated temperatures. The micellization was detected as a sudden increase in I(q) at low q. In Figure 2a, the increase was clearly observed at 35 °C. The increase in apparent molecular weight by the process of the assembly of the block copolymer was responsible for the increase in SAXS intensity. Using Guinier analysis, we can estimate the increase in the apparent molecular weight, or the number of copolymer chains in one micelle, and the size of an individual micelle. In

fact, the scattering intensity I(q) in the vicinity of q = 0 can be approximated as I(0) exp(−q2Rg2/3)28 so that the association number Nass (I(0) ∝ Nass) and the radius of gyration Rg are derived from the curve fitting.21 The values of Nass and Rg are plotted against temperature in Figure 3. The sudden increase in Rg at the LCST is definitely due to the process of molecular assembly, i.e., micellization. More than 100 molecular chains assembled to form 1 micelle with a radius of ca. 20 nm at 40 °C. The scattering data above the LCST were analyzed by curve fitting with the theoretical intensity on the basis of the core− shell sphere model, which is expressed by eq 2. The experimental data were well fitted with the theoretical intensities (solid lines), as shown in Figure 2a. From the best-fit curves, we estimated the mean core radius Rc = Rin and shell thickness Ls = Rout − Rin of the micelles. The values are plotted against temperature in Figure 4. The value of Ls was almost constant at 4 to 5 nm, which was comparable to the length of nine-base ssDNA (∼3.4 nm), and the core radius Rc was 15−20 nm above the temperature region around the transition; all of these findings were similar to the results of our previous report.21 Clearly, the micellar particle is composed of a PNIPAAm core surrounded by an ssDNA layer. Next, the thermoresponsiveness at a higher salt concentration of 0.5 M NaNO3 was explored. In the DSC, the endothermic peak was observed at a lower temperature of ∼34 °C. This is presumably because the salt facilitated the disruption of the network structure of water around the hydrophobic isopropyl group and, as a result, the LCST of PNIPAAm was lowered as the salt concentration increased (we confirmed that the LCST of 1P289 decreases by about 3 °C).35 In the same way, the micellization at this temperature was recognized by SAXS, and the scattering data above the LCST C

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Figure 2. SAXS intensity data of unhybridized and hybridized copolymers in 10 mM PB including the indicated concentrations of NaNO3, taken at elevated temperatures from 25 to 60 °C: (a, b) 1P289-1ssD9 at 0.1 and 0.5 M NaNO3 and (c, d) 1P289-1dsD9 at 0.1 and 0.5 M NaNO3, respectively. The copolymer concentration was 1.0 g L−1. The data are shifted vertically by an increment factor for clarity. The blue and red lines indicate the fitting curves for the dispersed and aggregated micellar particles, respectively.

Figure 3. Radius of gyration, Rg, and the association number, Nass, of 1P289-1ssD9 and -1dsD9 plotted against temperature, as estimated from Guinier analysis. The open and solid symbols indicate the values at 0.1 and 0.5 M NaNO3, respectively. The plots of aggregated particles are missing because the analysis was applied only to the scattering data of dispersed objects.

Figure 4. Mean core radius, Rc, and shell thickness, Ls, of 1P289-1ssD9 and -1dsD9 core−shell particles at 0.1 and 0.5 M NaNO3, plotted against temperature. These values were calculated according to the best-fit parameters in curve fitting. The open and solid symbols indicate the values at 0.1 and 0.5 M NaNO3, respectively.

almost independent of the salt concentration. In contrast, the core radius showed a tendency to depend on the salt concentration. The same behavior was observed in our previous

were well described by the core−shell model (Figure 2b). The shell thickness was also comparable to the length of ssDNA and D

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Langmuir studies.19,21 The size of the micellar particle was found to be affected by charged DNA strands. The molecular assembly might be facilitated by screening the electrostatic repulsion between DNA strands.19,36 Thermal Behavior of PNIPAAm-b-dsDNA. We subsequently analyzed the SAXS data of the block copolymer in which the ssDNA block was hybridized with the complementary DNA, PNIPAAm-b-dsDNA, which were taken at temperatures higher than room temperature. Figure 2c shows the SAXS profiles of 1P289-1dsD9 at 0.1 M NaNO3. Similar to the case of unhybridized copolymer, an increase in intensity in the vicinity of q = 0 nm−1 was observed at around 35 °C. Above this temperature, the association number Nass was estimated to be greater than 100 (Figure 3). The temperature dependence of the absorbance and the DSC thermogram in Figure 1 support the idea that the hybridized copolymer chains assemble in nanomicelles at this temperature by the coil−globule transition of PNIPAAm blocks. The SAXS data of 1P289-1dsD9 above the LCST were also well fitted with the theoretical intensity of the core−shell spherical particle. The nanomicelle was found to consist of a PNIPAAm core with a radius of 15−20 nm and a dsDNA layer with a thickness of 4 to 5 nm, as shown in Figure 4, which were almost the same structure and size as for the unhybridized copolymer. The core−shell particles dispersed stably at the low salt concentration, irrespective of the single- or double-stranded state. The effective charge densities of ss- and dsDNA are considered to be almost the same as each other because of compensation by countercation condensation.37 The electrostatic repulsion of the negatively charged dsDNA strands is considered to be sufficient for colloidal stability. We have reported so far that hybridization with the complementary DNA after micellization (e.g., at 40 °C) at high salt concentrations results in the aggregation of the micellar nanoparticles.15−17,19,21 The non-cross-linking aggregation superficially appeared to be induced by some attraction between DNA duplexes. The point in question is whether the non-cross-linking aggregation is caused by such an attractive interaction, e.g., the end-to-end stacking interaction between DNA duplexes.22−24 If it is caused by such an interaction, then the association between DNA strands would mediate the formation of some kind of aggregated structure, regardless of the coil−globule transition of the PNIPAAm block. It would thus be of significant interest to investigate the molecular assembly and the structural evolution of the hybridized copolymer at a high salt concentration during heating. Figure 2d demonstrates the SAXS profiles of 1P289-1dsD9 at 0.5 M NaNO3, taken at elevated temperatures. On the basis of this SAXS analysis, the structural change in the hybridized copolymer during this heating process is schematically illustrated in Figure 5. In spite of the hybridization with the complement and the high salt concentration, no remarkable change of the scattering intensity in the vicinity of q = 0 nm−1 was observed below the LCST. There was no significant difference from the case of the unhybridized copolymer. This suggests that both the unhybridized and hybridized copolymer chains are isolated stably without the association between DNA strands even at the high salt concentration, as shown in Figure 5a. The distinct change in the intensity profile was observed at around 30−35 °C, where the DSC thermogram showed an endothermic peak. There can be little doubt that the change in the SAXS intensity profile in this temperature range was not due to the association between DNA strands but rather to the micellization by the coil−globule transition of PNIPAAm

Figure 5. Schematic illustration of the thermoresponsive structural transition of PNIPAAm-b-dsDNA at high salt concentrations: (a) PNIPAAm-b-dsDNA below the LCST, (b) micellization of PNIPAAm-b-dsDNA at the LCST, (c) non-cross-linking aggregation of micellar nanoparticles, and (d) redispersion of micellar nanoparticles by DNA melting.

blocks. The intensity profile just after micellization was described by a spherical core−shell model (Figure 5b). The core size and shell thickness were 15−20 nm and 4−5 nm, respectively, which were almost the same as those of other samples (Figure 4). At slightly higher temperatures, however, the difference between the intensity profiles of hybridized and unhybridized copolymers was clearly recognized. As demonstrated by the red lines in Figure 2d, the scattering data at 40− 50 °C were described by Baxter’s model (eqs 3−7). In this temperature range, the absorbance of the copolymer solution increased to over 1.0 (Figure 1a). The clouding of the solution was due to the aggregation of particles. The Rc and Ls of the nanoparticles remained unchanged at about 20 nm and 4 to 5 nm, respectively. It is thus reasonable to consider that the hybridized copolymer first assembles in spherical particles by the coil−globule transition of PNIPAAm blocks at the LCST and then the resulting micellar particles aggregate immediately, as shown in Figure 5c, as a result of both the low steric repulsion of the DNA strand and the low electrostatic repulsion. The curve fitting of the SAXS data also showed that the effective DHS is smaller than the diameter of the particle, indicating that the particles aggregate while DNA shells overlap with each other. The degree of penetration was estimated to be ca. 6 nm, which was comparable to the shell thickness. The graft density of DNA (Nass/4πRc2) was calculated to be ca. 0.03 strands/nm2, similar to the values reported so far.16,19,21 There is enough space for DNA strands to interdigitate fully. From these results, the end-to-end stacking between dsDNA strands does not seem to be operative in the non-cross-linking aggregation of the particles.19 The SAXS data of the unhybridized copolymer at 40−50 °C can still be explained by the dispersed core−shell model. Although there was a slight increase in absorbance at the temperature used, as shown in Figure 1a, the change was smaller than in the case of the hybridized copolymer, in contrast to the result in ref 25. The assembly of the PNIPAAm blocks undoubtedly occurred at this temperature, as can be seen in the corresponding DSC measurement (Figure 1b). Most copolymer chains micellize to the core−shell nanoE

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non-cross-linking aggregation of the nanoparticles is observed as the clouding of the suspension. The micellar particles disperse if the DNA remains single-stranded and the salt concentration is low, and the denaturing of DNA by heating leads to the redispersion of nanoparticles.

particles, although some of the copolymer chains might assemble to form a complicated structure. Further heating caused the SAXS intensity profiles of the hybridized copolymer to change. The data above 55 °C were entirely different from those at 40−50 °C, as shown in Figure 2d. The dispersed core−shell model provided a better approximation than did the sticky hard-sphere model. It is worth noting that the turbidity of the solution began to decrease around ∼55 °C, as shown in Figure 1a. The behavior can thus be explained by the redispersion of the aggregated particles (Figure 5d) because of the denaturing of rigid dsDNA into flexible ssDNA (the melting temperature of the 9-mer dsDNA at [total DNA strand] = 50 μM and [Na+] = 0.5 M was determined to be about 55 °C from the results of the UV absorbance measurement). That is to say, the revival of steric repulsion by DNA denaturing provides colloidal stability to the particles. The core size of the particles slightly increased after the redispersion (Figure 4). Although the reason is unclear at present, the micellar particles, which were forced to aggregate just after the micellization at LCST, might reorganize into a more stable structure at high temperatures. As mentioned above, the thermoresponsiveness of the PNIPAAm-DNA solution is triggered by the coil−globule transition of the PNIPAAm segment. When the copolymer has adequate amphiphilicity through control of the molecular composition, the molecular chains assemble in spherical nanoparticles surrounded by DNA strands, irrespective of whether DNA hybridization occurs, at the LCST. The clouding of this suspension must be further accompanied by a depression in steric and electrostatic repulsions. The hybridization of the complementary DNA and the electrostatic shielding do not bring about an attractive interaction, such as the end-to-end stacking between DNA strands or other hydrophobic interactions. The stability of nanoparticles after micellization is determined by the balance of the van der Waals attractive interaction between the micelle cores and the steric repulsion attributable to DNA mobility and flexibility under the conditions where the electrostatic repulsion is suppressed.19,21 If the PNIPAAm fraction is too large, then the van der Waals attraction between the resulting micelles easily overcomes the repulsions, and the micelles can aggregate instantly. In this case, the clouding of the copolymer solution would occur regardless of the DNA structure and ionic strength. Accordingly, it is important to control the fraction of PNIPAAm in the copolymer in order to design a turbidimetric method for detecting slight differences in the DNA structure.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address

(P.P.) State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was financially supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant Number 25220204). The synchrotron radiation SAXS experiment was performed at BL45XU in SPring-8 with the approval of RIKEN (proposals 20130031 and 20140022). We thank Dr. Wei-Yang Ooi and Mr. Yuya Morita, RIKEN, for their help with the polymer synthesis.

(1) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based Method for Rationally Assembling Nanoparticles into Macroscopic Materials. Nature 1996, 382, 607−609. (2) Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ‘Nanocrystal Molecules’ Using DNA. Nature 1996, 382, 609−611. (3) Macfarlane, R. J.; Lee, B.; Jones, M. R.; Harris, N.; Schatz, G. C.; Mirkin, C. A. Nanoparticle Superlattice Engineering with DNA. Science 2011, 334, 204−208. (4) O’Brien, M. N.; Jones, M. R.; Lee, B.; Mirkin, C. A. Anisotropic Nanoparticle Complementarity in DNA-mediated Co-crystallization. Nat. Mater. 2015, 14, 833−839. (5) Zhang, Y.; Pal, S.; Srinivasan, B.; Vo, T.; Kumar, S.; Gang, O. Selective Transformations between Nanoparticle Superlattices via the Reprogramming of DNA-mediated Interactions. Nat. Mater. 2015, 14, 840−847. (6) Soto, C. M.; Srinivasan, A.; Ratna, B. R. Controlled Assembly of Mesoscale Structures Using DNA as Molecular Bridges. J. Am. Chem. Soc. 2002, 124, 8508−8509. (7) Kim, A. J.; Scarlett, R.; Biancaniello, P. L.; Sinno, T.; Crocker, J. C. Probing Interfacial Equilibration in Microsphere Crystals Formed by DNA-directed Assembly. Nat. Mater. 2009, 8, 52−55. (8) Hong, B. J.; Eryazici, I.; Bleher, R.; Thaner, R. V.; Mirkin, C. A.; Nguyen, S. T. Directed Assembly of Nucleic Acid-Based Polymeric Nanoparticles from Molecular Tetravalent Cores. J. Am. Chem. Soc. 2015, 137, 8184−8191. (9) Rosi, N. L.; Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105, 1547−1562. (10) Riess, G. Micellization of Block Copolymers. Prog. Polym. Sci. 2003, 28, 1107−1170. (11) Rodríguez-Hernán dez, J.; Chéc ot, F.; Gnanou, Y.; Lecommandoux, S. Toward ‘Smart’ Nano-objects by Self-assembly of Block Copolymers in Solution. Prog. Polym. Sci. 2005, 30, 691−724. (12) Chien, M. P.; Rush, A. M.; Thompson, M. P.; Gianneschi, N. C. Programmable Shape-shifting Micelles. Angew. Chem., Int. Ed. 2010, 49, 5076−5080.

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CONCLUSIONS In this study, we have studied the structural transition of PNIPAAm-b-DNA block copolymers during the heating process. The effects of salt and complementary DNA on the thermal behavior were explored. Salt facilitated the molecular assembly of the block copolymers. The assembly process was not triggered by the association between DNA strands but by the coil−globule transition of PNIPAAm blocks. If the copolymer has an amphiphilicity, then the molecular chains assemble in spherical nanoparticles. Although the non-crosslinking aggregation superficially appears to be mediated by some specific attraction between DNA strands, in fact the hybridization and addition of salt merely decrease the repulsive interactions. When the van der Waals attraction between the nanoparticles that increased as a result of micellization overcomes the steric and electrostatic repulsions of DNA, the F

DOI: 10.1021/acs.langmuir.5b04141 Langmuir XXXX, XXX, XXX−XXX

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NOTE ADDED AFTER ASAP PUBLICATION Equation 7 was corrected after ASAP publication date on January 22, 2016.

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DOI: 10.1021/acs.langmuir.5b04141 Langmuir XXXX, XXX, XXX−XXX