Thermoresponsive Micellization and Micellar Stability of Poly(N

Article pubs.acs.org/Langmuir

Thermoresponsive Micellization and Micellar Stability of Poly(N‑isopropylacrylamide)‑b‑DNA Diblock and Miktoarm Star Polymers Pengju Pan,†,⊥ Masahiro Fujita,*,† Wei-Yang Ooi,†,‡ Kumar Sudesh,‡ Tohru Takarada,† Atsushi Goto,§ and Mizuo Maeda*,† †

Bioengineering Laboratory, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan Ecobiomaterial Research Laboratory, School of Biological Science, Universiti Sains Malaysia, Minden, 11800, Penang, Malaysia § Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan ‡

ABSTRACT: Linear and miktoarm star-shaped diblock copolymers consisting of single-stranded DNA and poly(Nisopropylacrylamide) (PNIPAAm) with various compositions were synthesized via atom transfer radical polymerization and click chemistry. The temperature-responsive phase transition behavior, micellization, was systematically examined using UV−vis spectrometry, high-sensitivity differential scanning calorimetry, dynamic light scattering, and small-angle X-ray scattering. The lower critical solution temperature (LCST) increased, and its enthalpy decreased with decreasing PNIPAAm content. The copolymers self-assembled into well-defined nanoparticles having a core composed of PNIPAAm and a coronal layer of DNA above LCST. The particle size and micellar aggregation number of copolymer chains depended on the macromolecular composition and chain architecture. On the other hand, regardless of their factors, the surface area occupied by one DNA strand was found to be almost unchanged. The hybridization of DNA on the nanoparticles with fully complementary one induced the aggregation of the particles in a non-cross-linking configuration. The nanoparticle composed of miktoarm star copolymer showed a quicker DNA-hybridization response in this non-cross-linking aggregation compared with the case of a linear analogue.

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INTRODUCTION

create the DNA-encoded soft nanoparticles. In this method, the structure and properties of colloidal nanoparticles can be tuned from the macromolecular chemical structures and micellization conditions. The informational nature of DNA would confer these polymeric nanoparticles with novel biofunctions such as targeted drug/gene deliveries and biodiagnostics. So far, we have developed DNA-encoded soft nanoparticles by self-assembly of poly(N-isopropylacrylamide) (PNIPAAm) grafted with single-stranded (ss) DNA above its lower critical solution temperature (LCST).6,7 The nanoparticles with DNA are applicable to functional materials for biosensing, which is based on DNA hybridization-induced colloidal destabilization (non-cross-linking aggregation).6,8,9 The graft copolymers,

Amphiphilic block copolymers have received sustained interest because of their ability to form organized assemblies in solution, which have potential numerous applications such as drug delivery, nanoreactors, and nanotemplates.1 The morphologies of assemblies, closely related to their functions and applications, can be tailored from the chemical natures of blocks, their lengths and connectivity (chain architecture), and micellization conditions.2 DNA has been recognized as a versatile building block for such molecular self-assemblies in virtue of its unique structure and self-recognition property. The programmable assembly of DNA-functionalized inorganic nanoparticles has been extensively studied because of its potential applications in diagnostics.3 The studies on the selfassembly behavior of DNA-encoded soft nanoparticles are, however, quite scarce.4,5 The micellization of amphiphilic copolymers bearing DNA blocks would be a feasible method to © 2012 American Chemical Society

Received: August 1, 2012 Revised: September 10, 2012 Published: September 26, 2012 14347

dx.doi.org/10.1021/la303128y | Langmuir 2012, 28, 14347−14356

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Scheme 1. Schematic Illustration of the Syntheses of Linear Diblock and 4-Miktoarm Star PNIPAAm-b-ssDNA Conjugates via Click Chemistry

(CH2)6-5′-GCC ACC AGC-3′) and 15 (NH2-(CH2)6-5′-TAC GCC ACC AGC TCC-3′) bases and, as target DNAs for the 9-base DNA, fully complementary (5′-GCT GGT GGC-3′) and terminally one-base mutated (5′-CCT GGT GGC-3′) ones were purchased from Operon Biotechnologies (Tokyo, Japan). 5′-Alkyne-modified oligonucleotides (see Scheme 1) were prepared from the amino-terminated oligonucleotides by a reaction with 4-pentynoic acid succinimidyl ester.11 The azide-functionalized linear, 2-azidoethyl 2-bromoisobutyrate (1N3-1iBuBr), and 3-arm, 1,1,1-tris(2-bromoisobutyryloxymethyl)-2-azidoethane (1N3-3iBuBr), initiators were synthesized according to our previous report.11 Polymerization of PNIPAAm Homopolymers. The feed ratios of initiator, catalyst, and ligand ([I]0/[CuBr]/[Me6TREN]) were 1/1/ 1.2 and 1/3/3.6 for the synthesis of linear and 3-arm star PNIPAAm homopolymers, respectively. The target degree of polymerization (DP) and molecular weight were controlled by changing the feed ratio of NIPAAm to initiator ([M]0/[I]0) (see Table 1). As a representative

however, have broad molecular weight distribution because of the low controllability of conventional radical polymerization. The synthesis of copolymers with respect to well-defined molecular weight, composition, and architecture, which are the crucial factors in self-assembly,1 is essential for better understanding the structure−property relationship between DNA-encoded micelle and its DNA hybridization-induced colloidal destabilization. We have therefore tried to develop synthetic routes for PNIPAAm-b-ssDNA conjugates.10,11 In a previous report, we have provided an approach to prepare PNIPAAm-b-ssDNA with the tunable and well-defined architectures.11 In this study, a series of AB type linear and AB3 type miktoarm star PNIPAAm-b-ssDNA conjugates with various block lengths and precisely controlled chain architectures were synthesized and characterized. The effects of block length and chain architecture on the micellization of DNA-conjugated amphiphilic copolymers were examined by UV−vis spectrometry, high-sensitivity differential scanning calorimetry (DSC), and dynamic light scattering (DLS). The synchrotron radiation solution small-angle X-ray scattering (SAXS) technique was used to obtain detailed structural information because this technique is a powerful method to follow dynamical behaviors such as micellization in addition to obtain the static structural insight.12−19 On the basis of the SAXS data, the structural characteristics of the DNA-encoded soft nanoparticles were unveiled. Furthermore, an application of the DNA-encoded soft nanoparticle as turbidimetric DNA detection was examined. The DNA-hybridization response was proven to be controllable by tuning the molecular structure.

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Table 1. Target DP, Yield, and Molecular Weights of Linear and 3-Arm Star PNIPAAm Homopolymers code

target DPa

yield (%)

Mn,thb (kDa)

Mnc (kDa)

Mw/Mnc

1P35 1P79 1P151 1P280 1P399 3P69 3P119 3P252 3P370

50/1 100/1 200/1 400/1 800/1 100/1 150/1 300/1 600/1

52 53 56 53 44 47 62 69 63

3.18 6.23 13.0 24.2 40.1 5.92 11.1 24.0 43.4

3.93 8.91 17.1 31.7 45.2 7.78 13.5 28.6 41.8

1.12 1.12 1.11 1.08 1.13 1.12 1.09 1.07 1.13

a

Overall target DP, i.e., feed ratio (mol/mol) of NIPAAm monomer ([M]0) to initiator ([I]0). bCalculated molecular weight, Mn,th = Mn,initiator + ([M]0/[I]0) × yield × 113.16. cMn and Mw were measured by GPC-MALLS using DMF with 0.1 M LiBr as eluent.

EXPERIMENTAL SECTION

Materials. The reagents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and used without further purification unless otherwise specified. N-Isopropylacrylamide (NIPAAm) was purified by recrystallization from a mixture of toluene and n-hexane (1/1 (v/v)). The ligand tris[2-(dimethylamino)ethyl]amine (Me6TREN) was prepared according to the method in ref 20. Copper(I) bromide (CuBr) was stirred in glacial acetic acid overnight, filtered, and rinsed with ethanol and diethyl ether. The solid was then dried under vacuum at room temperature for 24 h. Tris(benzyltriazolylmethyl)amine (TBTA), a ligand for copper-catalyzed azide−alkyne click chemistry (CuAAC), was purchased from SigmaAldrich (St. Louis, MO). The amine-reactive alkyne modifier, 4pentynoic acid succinimidyl ester, was synthesized according to the literature.21 5′-Aminohexyl-terminated synthetic DNAs of 9 (NH2-

example, the polymerization of an azide-functionalized 3-arm star PNIPAAm homopolymer with an overall target DP of 600 is described here. NIPAAm (2.00 g, 17.7 mmol), 1N3-3iBuBr (17.9 mg, 0.0295 mmol), Me6TREN (24.4 mg, 0.106 mmol), and isopropanol (4.00 g) were mixed in a Schlenk flask and degassed using three freeze−pump− thaw cycles. While the mixture was frozen, CuBr (12.7 mg, 0.088 mmol) was added. The flask was then filled with argon, and the mixture was left to dissolve at room temperature. The reaction mixture was stirred for 5 h at 25 °C. After the polymerization, the mixture was passed through an alumina column using THF as eluent. The resulting polymer was purified by precipitation from THF into hexane four times and then dried under vacuum to yield 1.26 g (yield: 63%) of PNIPAAm as white solid. Here, the linear and 3-arm star PNIPAAm 14348


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homopolymers are marked as 1Pm and 3Pm, respectively, where P denotes PNIPAAm block and the subscript m corresponds to the overall degree of polymerization. Preparation of PNIPAAm-b-ssDNA Conjugates. Scheme 1 illustrates the synthesis of linear diblock and 4-miktoarm star PNIPAAm-b-ssDNA conjugates via the click reaction between the azide-functionalized PNIPAAm and alkyne-modified oligonucleotide. A typical synthetic procedure for the 4-miktoarm star PNIPAAm-bssDNA conjugate containing a 3-arm PNIPAAm block with the overall DP = 370 (3P370) and 9-base ssDNA is described here. A solution of alkyne-modified ssDNA (100 nmol) in 2.5 mL of 20 mM phosphate buffer (PB) (pH 7.2) and 3P370 (41.8 mg, 1.00 μmol) were mixed in a 10 mL plastic culture cube. A stock solution (0.1 mL) of catalyst/ ligand (1.0 μmol of CuSO4/1.1 μmol of TBTA) in DMSO/water (1/1 (v/v)) and 4.0 μmol (0.79 mg) of sodium ascorbate were added. The reaction mixture was shaken at 15 °C for 20 h. Then, the mixture was purified using the anionic exchange chromatography and gel permeation chromatography, dialyzed against water (MWCO = 3500), and lyophilized to yield the product as white solid. The yield was 64% based on the amount of ssDNA, which was determined from the absorption at 260 nm using a UV−vis spectrophotometer. The linear and 4-miktoarm star-shaped PNIPAAm-b-ssDNA conjugates are marked as 1Pm-1ssDn and 3Pm-1ssDn, respectively, where ssD denotes ssDNA. The numerals before P and ssD denote the numbers of PNIPAAm and ssDNA blocks in one molecule, respectively. The subscripts m and n correspond to the DPs of PNIPAAm (DPPNIPAAm) and ssDNA (DPDNA, i.e., the number of base) blocks, respectively. GPC-MALLS Measurements. The weight- and number-averaged molecular weights (M w and Mn , respectively) of PNIPAAm homopolymers were determined by gel permeation chromatography (Shimadzu, Japan) equipped with a multiangle laser light scattering detector (DAWN8+, Wyatt Technologies, Santa Barbara, CA) (GPCMALLS), using DMF containing 0.1 M LiBr as eluent. The flow rate of eluent was 0.3 mL/min, and the column temperature was 30 °C. A Shodex OHpak SB-803 HQ column (Showa Denko, Japan) was used. The refractive index increment (dn/dc) of PNIPAAm homopolymer was determined to be 0.0671 mL/g in DMF containing 0.1 M LiBr at 30 °C using a refractometer (OPTILAB DSP, Wyatt Technologies, Santa Barbara, CA).11 The Mw and Mn of PNIPAAm-b-ssDNA conjugates were examined by GPC-MALLS measurements using 10 mM tris-HCl buffer (pH 7.4) containing 0.1 M NaCl as eluent. The column temperature was set to 10 °C, and the flow rate of eluent was 0.5 mL/min. The local temperature of MALLS detector was ca. 25 °C. The dn/dc values of the conjugates were estimated from the additive equation, dn/dc = WDNA(dn/dc)DNA + WPNIPAAm(dn/dc)PNIPAAm, where W represents the weight fraction.22 The dn/dc’s of ssDNA and the PNIPAAm homopolymers were taken as 0.247 and 0.167 mL/g, respectively.6,23 UV−vis Spectroscopy. To measure the cloud point (Tcp) of PNIPAAm homopolymer and the turbidimetric change of PNIPAAmb-DNA micelle solution in the presence of target DNA, the temperature- and time-dependent transmittances were measured on a UV-2550 UV−vis spectrophotometer (Shimadzu, Kyoto, Japan) equipped with a temperature-controlled sample holder. The transmittance at 500 nm for an aqueous solution of polymer sample (0.5 g/ L) was monitored upon heating from 10 to 70 at 1 °C/min. When examining the colloidal stability of the micelles covered with doublestranded (ds) DNA, the copolymer solution was heated to 40 °C and held for 10 min. Then 1 mol equiv of fully cDNA (5′-GCT GGT GGC-3′) or one-base mutant DNA (5′-CCT GGT GGC-3′) for duplex formation was added to the micelle solution at the temperature. After the addition of NaNO3, the transmittance of the solution was measured at 500 nm. For the time-dependent transmittance, the NaNO3 concentration was adjusted to 500 mM. High-Sensitivity DSC. DSC measurements were performed with a VP-DSC microcalorimeter (MicroCal Inc., Northampton, MA). To examine the thermo-triggered phase transition, an aqueous solution of polymer sample (0.5 g/L) was heated from 15 to 70 at 1 °C/min after holding at 15 °C for 15 min to remove the thermal history. Three or more repeated measurements were performed for each sample. The

temperature of phase transition was regarded as the peak temperature (Tp) in the DSC curve, yielding the maximum specific heating capacity (Cp) during transition. The enthalpy of transition (ΔH) was determined from the peak area. DLS. The hydrodynamic radius, Rh, of PNIPAAm-b-ssDNA assembly was measured using a Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, UK) with a 633 nm He−Ne ion laser light set at a scattering angle of 173°. The sample solution of conjugate (0.5 or 2.0 g/L) in 10 mM PB (pH 7.4) was passed through a syringe-driven filter unit (0.22 μm, Millex-GV) prior to the measurements. Synchrotron Radiation Solution SAXS. Solution SAXS measurements were carried out at the BL45XU RIKEN Structural Biology Beamline I of SPring-8 (Harima, Japan) using an incident Xray with a wavelength of λ = 0.09 nm. Two-dimensional (2-D) SAXS patterns were detected using a CCD camera (C4880, Hamamatsu Photonics, Hamamatsu, Japan) coupled with an X-ray image intensifier (V5445P MOD, Hamamatsu Photonics, Hamamatsu, Japan). The PNIPAAm-b-ssDNA solution (2.0 g/L) in 10 mM PB (pH 7.4) was measured in the temperature region of 25−60 °C. The 2-D SAXS data were converted into the one-dimensional intensity I(q) as a function of the scattering vector q (q = 4π sin θ/λ, where 2θ is the scattering angle) by circularly averaging. The SAXS data in the vicinity of q = 0 can be analyzed by the Guinier approximation, which is independent of the particle shape.24 The exponential form of Guinier law is written as

⎛ 1 ⎞ I(q) ≈ I(0) exp⎜ − q2R g 2⎟ ⎝ 3 ⎠

(1)

where I(q) is the scattering intensity, Rg is the radius of gyration, and I(0) is the forward scattering intensity estimated by extrapolation of I(q) to q → 0. According to the logarithmic form of eq 1, Rg and I(0) can be derived from the linear fitting of ln[I(q)] vs q2 (Guinier plot) at qRg < 1. For the PNIPAAm-b-ssDNA conjugates, the micellar aggregation number (Nagg) above LCST was evaluated by comparing the I(0) to that measured below LCST.11 The SAXS data above LCST were further analyzed to obtain the information on internal structure of colloidal particle. A core−shell model with two graded interfaces between core and shell characterized by σin and between shell and solvent characterized by σout, was employed in this study.25 Similarly to the case reported by Berndt et al., the radial density profile of particle, ρ(r), with a symmetric form based on a parabolic shape was adopted.25 The formulas of the profile and its Fourier transformation, Φ(q,R,σ), are described in their papers in detail. The dimensions of the central parts of core and shell are denoted as Wcore and Wshell. The scattering amplitude of a core−shell particle is thus described by A(q) = (Δρe‐core − Δρe‐shell )Vcore Φcore(q , R in , σin) + Δρe‐shell Vshell Φshell (q , R out , σout)

(2)

where Δρe‑core and Δρe‑shell are the contrast of electron densities of core and shell and Vcore and Vshell the volumes of core and shell. In this equation, Rin = Wcore + σin and Rout = Wcore + 2σin + Wshell + σout. In a dilute system, the SAXS intensity is proportional to squared scattering amplitude of particle A(q)2. The polydispersity in particle size was assumed to be a Gaussian distribution.

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RESULTS AND DISCUSSION PNIPAAm Homopolymers. The linear and 3-arm star PNIPAAm homopolymers were synthesized by a typical ATRP procedure from the azide-functionalized initiators using Cu(I) as catalyst and isopropanol as solvent. It has been shown recently that the Cu(I)-mediated ATRP of NIPAAm in a polar solvent such as isopropanol actually proceeds via a single electron transfer mechanism.26 The chemical structure of azidefunctionalized PNIPAAm homopolymer is illustrated in Scheme 1. Table 1 summarizes the target DP, yield, and molecular weights calculated theoretically (Mn,th) and attained 14349


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temperature dependence of transmittance at 500 nm and Cp for the linear and 3-arm star PNIPAAm homopolymers with different molecular weights. As can be seen in this figure, the transmittance decreased dramatically, and single endothermic DSC peak appeared upon heating at 35−45 °C, ascribing to the dehydration of PNIPAAm at LCST. As the molecular weight increased, the DSC peak became sharp and shifted to low temperatures. The cloud point, Tcp, corresponding to the temperature with 50% transmittance,27 was evaluated on the basis of the result shown in the parts a and b of Figure 2. The molecular weight dependences of Tcp for both the linear and star homopolymers are shown in Figure 3. In addition, the Tp and ΔH evaluated

experimentally by GPC-MALLS measurements (Mn and Mw). Both the linear and star PNIPAAm homopolymers showed relatively high yield. The GPC-MALLS chromatograms of linear and 3-arm star PNIPAAm homopolymers showed sharp peaks with Mw/Mn ∼ 1.1 (Figure 1), and the observed

Figure 1. GPC-MALLS chromatograms of PNIPAAm linear (a) and 3-arm star (b) homopolymers and PNIPAAm-b-ssDNA linear (c) and miktoarm star (d) conjugates. Figure 3. Cloud points of linear and 3-arm star PNIPAAm homopolymers as a function of degree of polymerization.

molecular weights were close to the theoretical ones. The molecular weight of azide-functionalized PNIPAAm can be manipulated within a wide range by changing the feed ratio of monomer to initiator. The thermoresponsive phase transition of PNIPAAm homopolymers with various molecular weights and chain architectures was examined by temperature-controlled UV− vis spectroscopy and high-sensitivity DSC. Figure 2 shows the

from the DSC measurements are plotted as a function of DPPNIPAAm shown in parts a and b of Figure 4. Both Tcp and Tp decreased with increasing the molecular weight as expected theoretically.28 The molecular weight dependence of LCST was less pronounced for PNIPAAm larger than DPPNIPAAm = 300.29 The ΔH of PNIPAAm was 40−60 J/g, in agreement with the

Figure 2. Transmittance at a wavelength of 500 nm (a, b) and DSC thermograms recorded upon heating (1 °C/min) (c, d) of linear (a, c) and 3arm star (b, d) PNIPAAm homopolymers in water. The polymer concentration was 0.5 g/L. 14350


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Figure 4. DSC peak temperature (a) and enthalpy of transition (b) for PNIPAAm homopolymers and their bioconjugates derived from the DSC heating curve.

literature values,30,31 and showed an increase with molecular weight, as reported previously.19,32 The phase transition was affected by the chain architecture. The 3-arm star PNIPAAm exhibited slightly lower LCST and smaller ΔH compared with their linear counterparts. The lower LCST of star PNIPAAm may be a result of the presence of hydrophobic initiator core at the center of star and more bromine groups at the chain ends.33 Phase Transition of PNIPAAm-b-ssDNA Conjugates. The linear and 4-miktoarm star PNIPAAm-b-ssDNA conjugates were made via the click reaction between the azidefunctionalized PNIPAAm and alkyne-terminated ssDNA. The chemical structures of the DNA conjugates are also illustrated in Scheme 1. The products were obtained in 60−90% yield. The yield decreased with increasing the length and the arm number of PNIPAAm chain. This might be because of the decrease in end functional density and the steric hindrance. Table 2 lists the molecular weights (Mn,th, Mn, and Mw) and

Although the fractionation might also cause Mn to decrease apparently for some of the copolymers, within experimental error, the molecular weights measured by GPC-MALLS agreed with those calculated theoretically. The phase transition of PNIPAAm-b-ssDNA conjugates was also investigated by high-sensitivity DSC. The temperature dependences of Cp for the linear and miktoarm star PNIPAAmb-ssDNA in aqueous solutions are shown in Figure 5. Every PNIPAAm-b-ssDNA conjugate exhibited a broader endothermic peak than that of the original PNIPAAm homopolymer demonstrated in the parts c and d of Figure 2. In addition, the DSC endotherm decreased in magnitude with decreasing DPPNIPAAm. The peak was no longer recognized in the conjugate with the lowest molecular weight such as 1P35-1ssD9 and 3P691ssD9. The values of Tp and ΔH evaluated from the DSC data for the conjugates are plotted in Figure 4 together with those of PNIPAAm homopolymers for comparison. The effect of ssDNA block on Tp and ΔH can be clearly seen. The hydrophilic ssDNA block causes to increase in an affinity of copolymer to water, leading to an increase in Tp and a decrease in ΔH.34 In contrast to the architectural dependence of LCST for the PNIPAAm homopolymers, the miktoarm star PNIPAAm-b-ssDNA conjugates showed almost the same as or slightly higher Tp than their linear diblock counterparts with the similar molecular weight and composition. This might be indicative of the effect of DNA position. The DNA binding to PNIPAAm segment at the center rather than at the end of the segment might solubilize the conjugate more effectively. Thermo-Triggered Micellization of PNIPAAm-bssDNA Conjugate. The thermo-triggered micellization of PNIPAAm-b-ssDNA conjugates was examined by DLS. The conjugates with the shortest PNIPAAm segments such as 1P351ssD9 and 3P69-1ssD9 did not assemble to form particles in the temperature region investigated (10−70 °C), as expected from the aforementioned DSC results (Figure 5), where no endothermic peak was observed upon heating. For the conjugates with larger DPPNIPAAm, the formation of colloidal particle in the order of tens of nanometers was observed. The size distribution of particle was very narrow (polydispersity index (PDI) less than 0.1) compared with the case of PNIPAAm-g-ssDNA.35 We confirmed previously that the critical micelle concentration values of the block copolymers are around 15 mg/L.11 Figure 6a shows the plot of hydrodynamic radius Rh against DPPNIPAAm for the nanoparticles formed from the linear and miktoarm star conjugates with 9-base ssDNA. The data were taken at 60 °C. The Rh varied from 15 to 29 nm for the linear conjugates and from 12

Table 2. Molecular Weights and Compositions of Linear and Miktoarm Star PNIPAAm-b-ssDNA Conjugates code

Mn,tha (kDa)

Mnb (kDa)

Mw/Mnb

1P35-1ssD9 1P79-1ssD9 1P151-1ssD9 1P280-1ssD9 1P399-1ssD9 1P280-1ssD15 1P399-1ssD15 3P69-1ssD9 3P119-1ssD9 3P252-1ssD9 3P370-1ssD9 3P252-1ssD15 3P370-1ssD15

6.11 9.16 15.9 27.1 43.0 28.9 44.8 8.32 14.0 26.9 46.3 31.6 51.0

7.35 12.6 22.6 29.2 45.8 33.2 45.7 8.85 12.9 26.0 46.6 31.1 47.4

1.03 1.05 1.08 1.07 1.16 1.03 1.05 1.04 1.01 1.02 1.10 1.01 1.03

a Calculated molecular weight, Mn,th = Mn,th(PNIPAAm) + Mn,DNA. Mn,DNA is 2.93 kDa for 9-base ssDNA and 4.72 kDa for 15-base ssDNA. bMn and Mw were measured by aqueous GPC-MALLS using 10 mM tris-HCl buffer (pH 7.4) with 0.1 M NaCl as eluent.

compositions of the conjugates. The measurement ascertained that all the conjugates show narrow molecular weight distributions, as can bee seen in the parts c and d of Figure 1. In most cases, the Mw/Mn values of block copolymers were slightly lower than those of homopolymers. This is probably because the samples are subjected to further fractionation during the click reaction and subsequent purification processes. 14351


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Figure 5. DSC thermograms of linear (a) and miktoarm star (b) PNIPAAm-b-ssDNA conjugates in water. The conjugate concentration was 0.5 g/L.

Figure 6. Hydrodynamic radius of micelles formed from linear and miktoarm star PNIPAAm-b-ssDNA conjugates plotted as a function of DPPNIPAAm for the conjugates with 9-base ssDNA (a) and DPDNA (b). The measurements were carried out for the conjugate solution (0.5 g/L) in 10 mM PB (pH 7.4) at 60 °C.

Figure 7. SAXS profiles collected at various temperatures for solution of 1P399-1ssD9 (a) and 3P370-1ssD9 (b) conjugates in 10 mM PB (pH 7.4). For clarity, the data are shifted vertically with an increment factor. The dotted lines indicate the positions of the form factor minima for the profiles above LCST. The concentration of conjugate was 2.0 g/L.

to 20 nm for the miktoarm star ones, as DPPNIPAAm increased from about 100 to 400. The miktoarm star conjugates assembled into smaller particles than their linear counterparts. In addition, the conjugates with the longer DNA block were found to form into larger particles than the case of shorter DNA, as shown in part b of Figure 6. The compositional and chain-architectural dependences of particle size will be further discussed in the following parts. Internal Structure of Colloidal Nanoparticles. The structures of colloidal nanoparticles formed from PNIPAAm-b-

ssDNA conjugates were analyzed in more detail by synchrotron radiation solution SAXS. Figure 7 shows representative SAXS profiles for 2.0 g/L 1P399-1ssD9 and 3P370-1ssD9 conjugates in 10 mM PB (pH 7.4), taken at elevated temperatures from 25 to 60 °C. Below LCST, the scattering intensity from the random coil nature of conjugate, which showed no characteristic peak or minima, was observed.24 As the temperature increased up to around LCST, the scattering intensity at lower scattering angles increased abruptly. This is attributable to the assembly of conjugates at the phase transition. 14352


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Figure 8. Guinier plots for linear and miktoarm star PNIPAAm-b-ssDNA conjugates in 10 mM PB (pH 7.4) at 60 °C. The concentration of conjugate was 2.0 g/L. For clarity, the data are shifted vertically with an increment factor. The solid lines indicate the linear fits in the small q region.

Figure 9. Temperature dependences of the radius of gyration (Rg) (a) and aggregation number (Nagg) (b) of 1P399-1ssD9 and 3P370-1ssD9 in 10 mM PB (pH 7.4). The concentration of conjugate was 2.0 g/L.

Table 3. Structural Characteristics of Micelles Formed from PNIPAAm-b-ssDNA Conjugates at 60 °C in 10 mM PB (pH 7.4) with a Conjugate Concentration of 2.0 g/L code

Rh (nm)

Rg (nm)

Rg/Rh

Nagg (chains)

Wcore (nm)

σin (nm)

Wshell (nm)

σout (nm)

σpolya (nm)

Rcore (nm)

Lshell (nm)

Rtotalb (nm)

ρcorec (g/cm3)

SDNA (nm2/chain)

1P151-1ssD9 1P280-1ssD9 1P399-1ssD9 1P399-1ssD15 3P119-1ssD9 3P252-1ssD9 3P370-1ssD9 3P370-1ssD15

19.2 24.3 28.6 35.5 12.3 15.2 20.5 23.2

13.5 17.2 20.4 21.2 8.6 11.1 16.2 15.7

0.70 0.71 0.71 0.60 0.70 0.73 0.79 0.67

101 137 187 76 21 67 126 52

13.8 16.7 20.7 18.8 5.9 9.6 14.7 10.7

0.01 0.01 0.03 0.3 0.8 0.8 0.4 0.3

3.0 3.0 3.1 6.1 2.4 2.9 3.4 6.0

1.4 1.4 1.2 0.4 0.07 0.06 0.03 0.05

2.0 2.1 3.0 2.3 1.3 1.7 2.0 1.5

13.8 16.7 20.7 19.1 6.7 10.4 15.1 11.0

5.8 5.8 5.5 7.2 3.3 3.8 3.9 6.4

19.6 22.5 26.3 26.3 10.0 14.2 19.0 17.4

0.26 0.36 0.37 0.19 0.38 0.67 0.61 0.65

23.7 25.8 29.0 60.7 26.8 20.4 22.8 29.3

a σpoly is the standard deviation for the structural parameter of Rout. bRtotal is defined as Rout + σout = Rcore + Lshell. cρcore was calculated as Nagg × Mn,PNIPAAm/(4/3πRin3NA) where NA is Avogadro’s number.

A sudden increase in Nagg was indeed recognized at around LCST, as shown in Figure 9b. The values of Rg and Nagg evaluated for all the colloidal nanoparticles of PNIPAAm-b-ssDNA conjugates are listed in Table 3. The Rg/Rh ratio is a typical indicator for the particle morphology. As seen in Table 3, the Rg/Rh ratios of the particles formed from the linear and star conjugates were close to the theoretical value of hard sphere (0.775). The value of Nagg ranged from ca. 100 to 190 molecules for the linear copolymers and from ca. 20 to 130 molecules for the miktoarm ones with 9-base ssDNA as DPPNIPAAm increased, and as a result, the particle size increased. The particle size (Rg and Rh) also depended on the length of DNA block. The conjugates with 15-base ssDNA block showed lower values of Rg/Rh. This might be because the outer shell of particle becomes loose.36,37

The SAXS data at low q were analyzed according to the Guinier approximation. Figure 8 demonstrates the representative Guinier plots for SAXS profiles of PNIPAAm-b-ssDNA taken at 60 °C, and their approximate curves expressed by eq 1. From the Guinier analysis, the radius of gyration, Rg, for chain and particle, and the micellar aggregation number of polymer chains, Nagg, were evaluated. These values are plotted as a function of temperature in Figure 9. The Rg increased abruptly at 35−40 °C. This behavior was obviously due to the micellization of PNIPAAm-b-ssDNA at LCST. The decrease in Rg upon subsequent heating might be attributable to further progress of dehydration of PNIPAAm segments. The micellization, the assembling of molecular chains, accompanies the increase in apparent molecular weight of scattering object. 14353


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Figure 10. SAXS profiles of linear and miktoarm star PNIPAAm-b-ssDNA conjugates in 10 mM PB (pH 7.4) at 60 °C. The concentration of conjugate was 2.0 g/L. For clarity, the data are shifted vertically with an increment factor. The solid lines indicate the fitting curve. The arrows indicate the positions of the form factor minima.

might be an important factor to regulate the overall particle size in micellization or micelle aggregation number. For the miktoarm star conjugates, the PNIPAAm segments are likely susceptible to the intramolecular or interarm assembly prior to intermolecular aggregation, so that the compact colloidal particles with a higher packing density of ρcore = 0.6−0.7 g/ cm3 formed against the repulsions, whereas the particles of linear PNIPAAm-b-ssDNA conjugates showed no more than ρcore = 0.2−0.4 g/cm3, which was consistent with the results reported for PNIPAAm-co-poly(ethylene oxide) by Qiu and Wu.22 Turbidimetric Detection of DNA. We further studied the application of PNIPAAm-b-ssDNA conjugates for the turbidimetric detection of DNA. Figure 11 shows the time-dependent

In the scattering intensity distributions taken above LCST, the form factor minima up to second order were clearly observed as indicated with the dotted lines, e.g., at q = 0.19 and 0.33 nm−1 for 1P399-1ssD9 and q = 0.25 and 0.43 nm−1 for 3P370-1ssD9, as shown in Figure 7, manifesting the formation of well-defined colloidal particle with a narrow size distribution.24 It has been considered that the nanoparticle consists of hydrophobic PNIPAAm core surrounded by hydrophilic DNA. This was also supported by the encapsulation behavior of a hydrophobic regent during micellization in our previous report.11 Therefore, the present SAXS data were analyzed according to the core−shell structural model expressed by eq 2 in order to extract detailed structural information on the colloidal nanoparticles. Panels a and b of Figure 10 depict the SAXS data taken at 60 °C for the linear and 4-miktoarm star conjugates and the corresponding theoretical curves, respectively. The experimental scattering distributions were fitted well with the theoretical curves. The best-fit parameters are summarized in Table 3. The analysis proved that the PNIPAAm-b-ssDNA conjugates form into well-defined core− shell type spherical particles with fairly narrow size distributions. The overall radius of particle including the outer interface, Rtotal = Rout + σout, was reasonably comparable with Rh evaluated by DLS. For the longer 15-base ssDNA block, however, there was a clear difference between Rtotal and Rh. This is because the swelling of the coronal layer, as discussed above. The core radius of particle, Rcore, increased with increasing DPPNIPAAm. The shell thickness, Lshell = σin + Wshell + 2σout, was approximately consistent with the length of DNA block. Especially, the values of Lshell for the miktoarm star conjugates were much closer to the theoretical length of DNA strand (ca. 3.4 and 6.0 nm for 9- and 15-base DNAs, respectively), assuming that the rise per one base of ssDNA is 0.43 nm.38 This indicated that more uniform coronal layer consisting of DNA blocks surrounds the core part formed by assembling the miktoarm conjugates. On the basis of the structural parameters extracted from the curve fitting, the surface area occupied by one ssDNA block was calculated as SDNA = 4πRcore2/Nagg. As shown in Table 3, SDNA was almost constant at 20−30 nm2/strand, regardless of the PNIPAAm length and chain architecture. Interestingly, for the conjugates with the longer DNA, the calculation suggested that the DNA block occupies a larger space. This is likely due to steric and electrostatic repulsions between neighboring DNA blocks. Such repulsive interaction between the DNA blocks

Figure 11. Time-dependent transmittance for micelle solutions of 1P280-1dsD9, 1P399-1dsD9, 3P252-1dsD9, and 3P370-1dsD9 in 10 mM PB (pH 7.4) with 500 mM NaNO3 at 40 °C after adding fully complementary and terminally one-base mutated DNA. The concentration of conjugate was 0.5 g/L.

transmittance change of the PNIPAAm-b-DNA micelle solutions after the addition of fully complementary or terminally one-base mutated DNA in the presence of 500 mM NaNO3 at 40 °C. For the hybridization with the terminally one-base mutated DNA, the micelle solution remained transparent. On the other hand, the hybridization with the complementary target DNA brought about a decrease in 14354


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Interface Science” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The synchrotron radiation SAXS experiment was performed at BL45XU in SPring-8 with the approval of RIKEN (Proposals 20100040 and 20110025). We thank Dr. Takaaki Hikima, RIKEN SPring-8 Center, for his help with the SAXS experiment at SPring-8.

transmittance for both linear and star-shaped PNIPAAm-bDNA conjugates, indicating the non-cross-linking aggregation of micelle particles, which is analogous to that reported for the PNIPAAm-g-DNA copolymer.6,7,35 It is noteworthy that the change of the transmittance depends on molecular architecture in addition to copolymer composition. As manifested by the present SAXS analysis (see Table 3), the miktoarm star copolymers form into the particles with higher core density. It is reasonable that the increase in particle density as well as particle size accelerates more dramatically the aggregation of particles because the dominant attraction in the non-crosslinking aggregation is considered to be van der Waals interaction between particle cores.35,39 The responsivity of DNA-encoded soft nanoparticle to DNA hybridization is controllable by tuning the molecular composition and architecture.

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(1) Blanazs, A.; Armes, S. P.; Ryan, A. J. Self-Assembled Block Copolymer Aggregates: From Micelles to Vesicles and their Biological Applications. Macromol. Rapid Commun. 2009, 30, 267−277. (2) Riess, G. Micellization of Block Copolymers. Prog. Polym. Sci. 2003, 28, 1107−1170. (3) Rosi, N. L.; Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105, 1547−1562. (4) Ding, K.; Alemdaroglu, F. E.; Borsch, M.; Berger, R.; Herrmann, A. Engineering the Structural Properties of DNA Block Copolymer Micelles by Molecular Recognition. Angew. Chem., Int. Ed. 2007, 46, 1172−1175. (5) Chien, M. P.; Rush, A. M.; Thompson, M. P.; Gianneschi, N. C. Programmable Shape-Shifting Micelles. Angew. Chem., Int. Ed. 2010, 49, 5076−5080. (6) Mori, T.; Maeda, M. Stability Change of DNA-Carrying Colloidal Particle Induced by Hybridization with Target DNA. Polym. J. 2002, 34, 624−628. (7) Mori, T.; Maeda, M. Temperature-Responsive Formation of Colloidal Nanoparticles from Poly(N-isopropylacrylamide) Grafted with Single-Stranded DNA. Langmuir 2004, 20, 313−319. (8) Miyamoto, D.; Tang, Z.; Takarada, T.; Maeda, M. Turbidimetric Detection of ATP Using Polymeric Micelles and DNA Aptamers. Chem. Commun. 2007, 4743−4745. (9) Tang, Z. L.; Takarada, T.; Sato, Y.; Maeda, M. Colloidal Nanoparticles from Poly(N-isopropylacrylamide)-graft-DNA for Single Nucleotide Discrimination Based on Salt-induced Aggregation: Extension to Long Target DNA. Chem. Lett. 2004, 33, 1602−1603. (10) Isoda, K.; Kanayama, N.; Miyamoto, D.; Takarada, T.; Maeda, M. RAFT-Generated Poly(N-isopropylacrylamide)−DNA Block Copolymers for Temperature-Responsive Formation of Polymer Micelles. React. Funct. Polym. 2011, 71, 367−371. (11) Pan, P.; Fujita, M.; Ooi, W.-Y.; Sudesh, K.; Takarada, T.; Goto, A.; Maeda, M. DNA-Functionalized Thermoresponsive Bioconjugates Synthesized via ATRP and Click Chemistry. Polymer 2011, 52, 895− 900. (12) Nakano, M.; Matsumoto, K.; Matsuoka, H.; Yamaoka, H. Characterization of Micellization Behavior of Amphiphilic Polymer Having Octadecyl Group by Small-Angle X-ray and Neutron Scattering. Macromolecules 1999, 32, 4023−4029. (13) Nakano, M.; Deguchi, M.; Matsumoto, K.; Matsuoka, H.; Yamaoka, H. Self-Assembly of Poly(1,1-diethylsilabutane)-block-poly(2-hydroxyethyl methacrylate) Block Copolymer. 1. Micelle Formation and Micelle-Unimer-Reversed Micelle Transition by Solvent Composition. Macromolecules 1999, 32, 7437−7443. (14) Choi, S.-H.; Bates, F. S.; Lodge, T. P. Structure of Poly(styreneb-ethylene-alt-propylene) Diblock Copolymer Micelles in Squalane. J. Phys. Chem. B 2009, 113, 13840−13848. (15) Sommer, C.; Pedersen, J. S.; Garamus, V. M. Structure and Interactions of Block Copolymer Micelles of Brij 700 Studied by Combining Small-Angle X-ray and Neutron Scattering. Langmuir 2005, 21, 2137−2149. (16) Pedersen, J. S.; Svaneborg, C.; Almdal, K.; Hamley, I. W.; Young, R. N. A Small-Angle Neutron and X-ray Contrast Variation Scattering Study of the Structure of Block Copolymer Micelles: Corona Shape and Excluded Volume Interactions. Macromolecules 2003, 36, 416−433. (17) Pedersen, J. S.; Hamley, I. W.; Ryu, C. Y.; Lodge, T. P. Contrast Variation Small-Angle Neutron Scattering Study of the Structure of Block Copolymer Micelles in a Slightly Selective Solvent at Semidilute Concentrations. Macromolecules 2000, 33, 542−550.

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CONCLUDING REMARKS In the present work, we have systematically investigated the phase transition, micellization, and internal micelle structure of thermoresponsive PNIPAAm-b-ssDNA conjugates with different compositions and chain architectures generated by ATRP and click chemistry. With the PNIPAAm content in the conjugate increased, their LCST decreased and ΔH increased. The conjugates formed a well-defined core−shell type micelle with a very narrow distribution in size above LCST. The effects of compositions and molecular architectures on the internal structure of PNIPAAm-b-ssDNA micelles were clarified. Both the linear and miktoarm star-shaped conjugates self-assembled into well-defined core−shell type nanoparticles having a core composed of PNIPAAm and a coronal layer of DNA. The structural parameters of Rg, Rcore, and Nagg for the micelles formed from the linear conjugates increased with the length of PNIPAAm block. The increase of ssDNA length brought about the increase of particle size, whereas Nagg decreased. The molecular architecture also affects the micelle morphology. As compared with their linear analogues, the particles formed from the miktoarm star conjugates had smaller Nagg, and smaller particle size, but larger ρcore. A more remarkable fact is that the formation of high-density nanoparticle results in a quick response of turbidimetric detection of DNA based on noncross-linking aggregation. The present study demonstrates for the first time that the DNA-hybridization response is controllable by tuning the molecular structure. The results obtained here will serve as the basis for designing the DNAfunctionalized soft particles with tunable structure and optimized functions.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (M.F.), [email protected] (M.M.); Tel +81-48-467-9312; Fax +81-48-462-4658. Present Address ⊥

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 This work was financially supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Molecular Soft14355


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