Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles for

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles for Colloidal Self-Assembly Eun Young Hwang, Jae Sang Lee, and Dong Woo Lim Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04002 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles for Colloidal SelfAssembly

Eun Young Hwang, Jae Sang Lee and Dong Woo Lim*

Department of Bionano Engineering and Bionanotechnology, College of Engineering Sciences, Hanyang University, Ansan, Republic of Korea.

* Department of Bionano Engineering and Bionanotechnology, College of Engineering Sciences, Hanyang University, Ansan, Republic of Korea; Email: [email protected]

Keywords: Opposite charge, Stimuli responsiveness, Anisotropic nanoparticles, Colloidal selfassembly, Drug Delivery systems

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Abstract Anisotropic nanoparticles (ANPs) composed of distinct compartments are of interest as advanced materials because they offer unique physicochemical properties controlled by the polymer composition, distribution of functional groups, and stimuli-responsiveness of each compartment. Furthermore, colloidal self-assembly of ANPs via noncovalent interactions between compartments can create superstructures with additional functionality. In this study, ANPs with two compartments composed of oppositely charged and thermally responsive ternary copolymers were prepared using electrohydrodynamic (EHD) co-jetting. One compartment was composed of poly(N-isopropylacylamide-co-stearyl acrylate-co-allylamine), which is positively charged in aqueous solution at pH 7, and the other compartment was composed of poly(Nisopropylacylamide-co-stearyl acrylate-co-acrylic acid), which is negatively charged. The ANPs were stabilized in aqueous solution by physical crosslinking due to hydrophobic interactions between the 18-carbon alkyl chains of their stearyl acrylate moieties, and self-assembled into supracolloidal nanostructures via electrostatic interactions. Colloidal self-assembly and thermal responsiveness were controlled by compartment charge density and solution ionic strength. The supracolloidal nanostructures exhibited both the intrinsic temperature-responsive properties of the ANPs and collective properties from self-assembly. These multifunctional, stimuli-responsive nanostructures will be useful in a variety of applications, including switchable displays, drug delivery carriers, and ion-sensitive gels.


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Introduction Anisotropic nanoparticles (ANPs) are of growing interest for use in biomedical and industrial applications, including multiplexed biosensing and cellular imaging,1-2 electronic paper devices,3 switchable displays,4 and colloidal stabilizers.5 ANPs composed of distinct compartments have an asymmetric distribution of functional groups, unlike isotropic nanoparticles (INPs), resulting in unique physicochemical and colloidal properties. ANPs have been used as advanced nanomaterials such as colloidal surfactants,6 Janus catalysts,7 and three-dimensional fiber scaffolds.8 Furthermore, colloidal self-assembly of ANPs induced by external electromagnetic forces and interparticle interactions can result in hierarchical nanostructures.9-10 Self-assembled

ANP

nanostructures

have

been

developed

for

display

devices,11

electromechanical systems,12 three-dimensional hydrogel superstructures,13 and transfer agents,14 exploiting the intrinsic properties of the individual particles as well as new collective properties derived from their self-assembly.15-18 Driving forces that induce ANP self-assembly include electrostatic interactions between oppositely charged particles, hydrophobic interactions between amphipathic particles, external electromagnetic fields, and changes in environmental stimuli. 19-24 For example, spherical particles with electric dipolar hemispheres prepared using directional coating formed controlled clusters with a defined shape due to electrostatic interactions.19 In addition, Janus particles with oppositely-charged polyelectrolytes on the surface of each compartment, which were prepared using “grafting from” and “grafting to” polymer synthesis approaches, showed aggregation controlled by solution pH.20 Amphiphilic binary and ternary particles composed of hydrophilic and hydrophobic compartments showed self-assembly in watermethanol mixtures depending on the aspect ratio of hydrophobic and hydrophilic compartments.21 Thus, clustering due to aggregation of hydrophobic compartments was controlled by the volume 3 ACS Paragon Plus Environment

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ratio of hydrophilic-to-hydrophobic compartments in the particle. Furthermore, magnetic and fluorescent Janus particles synthesized by Fe3O4 particle embedding and surface modification showed magnetic field-induced assembly and alignment.22 In response to stimuli such as temperature and pH, Janus nanoparticles with a pH-dependent polymer on one side and a temperature-dependent polymer on the other side can self-assemble either at low pH or high temperature.9 These illustrate the degree to which colloidal self-assembly of ANPs can be controlled and used to create defined superstructures with multiple properties and functionalities.23-24 However, many ANPs, including the Janus particles described above, show only surface anisotropy, originating from immobilization of ionic surfactants or polymers onto the surface of each ANP compartment. In this respect, the advanced ANPs would allow controlled incorporation of functional nanomaterials or biomolecules (DNA, RNA, and protein) within each compartment. Methods for preparing anisotropic micro- or nanostructures include Pickering emulsions,25 selective deposition,26 lithography,27 microfluidic photopolymerization,28 self-assembly,29 and electrohydrodynamic (EHD) co-jetting.30 Of these platform technologies, EHD co-jetting best facilitates formation of ANPs with multifunctionality because it can encapsulate inorganic nanomaterials within compartments by adjusting parameters such as jetting solvent, polymer concentration, flow rate, applied voltage, and jetting distance.31 Multi-compartmental ANPs prepared using EHD co-jetting have incorporated inorganic nanomaterials and therapeutic drugs in compartments for biomedical use.30, 32 The other techniques listed above generally synthesize micron-sized particles with anisotropy, but have difficulty forming multiple compartments and incorporating functional materials.18, 33-35 ANPs prepared by EHD co-jetting are potentially useful


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as switchable devices, functional nanomaterials for biological sensors and drug delivery, interface stabilizers, and clusters for self-assembly. Introduction of stimuli-responsiveness to ANPs is intriguing because it offers unique properties derived from the asymmetric distribution of stimuli-responsive compartments.9, 20 Their stimuliresponsive properties are determined by polymer chain dimensions, molecular weight, secondary structure, solubility, and changes in intra- and intermolecular interactions in response to changes in environmental conditions.36 Stimuli-responsive polymers have been developed that are sensitive to changes in temperature,37 pH,38 ionic strength,39 light,40 redox,41 and biological ligands.42 For example, thermally-responsive nanofiber matrices were prepared by electrospinning of poly(Nisopropylacylamide-co-stearyl acrylate) (poly(NIPAM-co-SA)) and were physically crosslinked due to the hydrophobic SA moieties. They exhibited rapid and reversible dimensional changes due to their swollen/collapsed behavior as a function of temperature.43 Furthermore, thermallyresponsive electrospun nanofibers prepared from poly((2-(dimethylamino)ethyl methacrylate)-coSA-(9,9-di-hexyl-2-(4-vinylpenyl)-9H-fluorene)

(poly(DMAEMA-co-SA-StFl))

showed

reversible changes in dimension and photoluminescence in response to changes in temperature due to both poly(DMAEMA) and poly(StFl).44 These polymers have fast responses and reversible transitions with temperature, pH, and ionic strength due to conformational changes, allowing controllable actions such as target binding45 and injectable gel formation.46 Recently, we reported anisotropic nanofiber scaffolds composed of physically crossslinked poly(NIPAM-co-SA) compartments with thermal responsiveness, and chemically crosslinked poly(ethylene glycol) compartments, for use in advanced biomedical applications.32 In response to a thermal stimulus, these nanofibers showed anisotropic actuations induced by collapse of thermally responsive compartment. 5 ACS Paragon Plus Environment

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There have been no previous reports of ANPs which combine stimuli-responsive and oppositely charged compartments for controlled colloidal self-assembly. In this study, we hypothesized that colloidal self-assembly of ANPs composed of oppositely charged, stimuli-responsive compartments could be spontaneously induced by electrostatic interactions under aqueous conditions, and the resulting colloids would have the intrinsic properties of the individual ANPs and additional properties resulting from the self-assembled nanostructures. The ANPs were prepared by EHD co-jetting of two thermally-responsive ternary copolymers synthesized by free radical polymerization. One compartment was composed of poly(N-isopropylacylamide-costearyl acrylate-co-allyamine) (poly(NIPAM-co-SA-co-AAm)), which is positively charged in aqueous solution at pH 7, and the other compartment consisted of negatively charged poly(Nisopropylacylamide-co-stearyl acrylate-co-acrylic acid) (poly(NIPAM-co-SA-co-AAc)). These oppositely charged compartments were stabilized in an aqueous solution by physical crosslinking via hydrophobic interactions between the long (18 carbon) alkyl chains of the SA moieties of both copolymers, and formed self-assembled supracolloidal nanostructures induced by electrostatic interactions. The degree of both self-assembly and thermal responsiveness was controlled by the charge density, which was a function of the apparent molar ratio of AAc and AAm in each compartment, and by the solution ionic strength. These self-assembled supracolloidal nanostructures have versatility due to their compositional anisotropy, which goes beyond a simple anisotropic distribution of surface functional groups, and will be useful as drug delivery carriers, colloidal crystals for switchable display, and building blocks for ion-sensitive gels.

Experimental section Materials 6 ACS Paragon Plus Environment

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N-isopropylacrylamide (NIPAM, 97%) obtained from Tokyo Chemical Industry Co. Ltd. (TCI, Japan) and stearyl acrylate (SA) from Sigma-Aldrich (St Louis, MO, USA) were purified by recrystallization from n-hexane and ethanol, respectively. The thermal radical initiator, 2,2’azobis(2-methylpropionitrile) (also known as azobisisobutyronitrile, or AIBN), was purchased from Acros (Thermo Fisher Scientific, Waltham, MA, USA) and was purified by recrystallization from methanol. Acrylic acid (AAc) from Sigma-Aldrich was distilled under reduced pressure before use. Allylamine hydrochloride (AAm), ethanol, n-hexane, dimethylformamide (DMF), chloroform, Nile red, fluorescein isothiocyanate (FITC) isomer 1, bicarbonate buffer, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich. Deionized water purified by Milli-Q (Millipore Water Purification Systems; EMD Millipore, Bedford, MA, USA) was used. Synthesis of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) Poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) were synthesized by free radical polymerization of three monomers (NIPAM, SA, and AAc or AAm). NIPAM, SA, and AAc were purified prior to synthesis; AAm was used without purification. NIPAM was dissolved in n-hexane at 40 C and recrystallized below 4 C to remove impurities including inhibitors. Ten grams of NIPAM was dissolved in 200 ml of n-hexane in a beaker to produce a concentration of 5 w/v%. Crystallized NIPAM was formed at low temperature, and the solution was filtered through filter paper (Whatman® qualitative filter paper grade 1) using an aspirator (EYELA 1000S, US). The product was dried in a vacuum to remove n-hexane. Similarly, SA was recrystallized from ethanol. Two grams of SA was dissolved in 50 ml of ethanol to produce a concentration of 4 w/v%. The solution was filtered after recrystallization and the product was dried in a vacuum to evaporate


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ethanol. AAc was purified to remove polymerization inhibitors by distillation at 40 C under reduced pressure. Poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) with different feed molar ratios were synthesized by free radical copolymerization of the monomers in ethanol using AIBN as a free radical initiator. Two different feed molar ratios of monomers – NIPAM:SA:AAc or AAm at 96.5:3:0.5 and 96:3:1 – were used to introduce different charge densities for each polymer chain. The monomers were dissolved in pure ethanol (75 wt%) and degassed with nitrogen for 30 minutes prior to polymerization. AIBN at 0.005 wt% of the total monomer weight was added into the reaction mixture for thermally-triggered initiation of polymerization. The polymerization process proceeded with mechanical stirring at 83 C for 16 hours. The resulting mixture was cooled to room temperature and poured into deionized water to purify the copolymers. Unreacted NIPAM, AAc, and AAm monomers remained dissolved in deionized water, while SA monomers precipitated due to the hydrophobicity of their alkyl side chain. Finally, a series of copolymers were filtered and freeze-dried using a lyophilizer (MCFD8508; Ilshin Lab Co., Ltd., Korea) under vacuum. Polymer characterization The chemical compositions of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-coAAm) were analyzed by 1H nuclear magnetic resonance (1H NMR) using a Bruker Avance III 400 MHz NMR spectrometer (Bruker BioSpin AG, Falländen, Switzerland), using dimethyl sulfoxide (d-6) as a solvent. Apparent molar ratios of monomers within the ternary copolymers were obtained by comparing relative peak signals of the corresponding protons from each monomer. To determine number-average molecular weight, weight-average molecular weight, and polydispersity index of the ternary copolymers, gel-permeation chromatography (GPC) was 8 ACS Paragon Plus Environment

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performed using a high-performance liquid chromatography (HPLC) 1260 series instrument (Agilent Technologies, Palo Alto, CA, USA) with a Shodex GPC column KF-803 (Shodex GPC system-21; Showa Denko Co., Tokyo, Japan). Tetrahydrofuran (THF) was used as mobile phase at a flow rate of 1.0 ml/min, and polystyrene in the range of 1,270 to 139,000 g/mol was used as a standard. A potentiometric titration for copolymers was performed to determine pKa values with a pH meter (pH-200L, Neomet, Korea) by dropwise addition of 0.1 M NaOH or HCl into poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) solution. Thermal properties of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) were confirmed by measuring the UV absorbance of the copolymer solutions and the hydrodynamic radii of their micellar structures in phosphate buffer as a function of temperature. Prior to measurement of UV absorbance, each copolymer was dissolved in 3 mM phosphate buffer at pH 5.6, which was composed of 0.0041 w/v % of sodium phosphate dibasic heptahydrate and 0.0393 w/v % of sodium phosphate monobasic monohydrate to give a final concentration at 0.1 w/v % of copolymer solutions. The low critical solution temperature (LCST) of the copolymers was determined by monitoring the absorbance of polymer solutions at 350 nm using a UV-Vis spectrometer (Cary-100 Bio; Varian Biotech, US) with a Peltier thermostatted temperature controller. The measurement was performed as a function of temperature from 20 C to 65 C at a heating rate of 1 C/minute. Copolymer solution in THF at 0.01 w/v% was slowly added into 10 mM phosphate buffer at pH 7, which was composed of 0.1547 w/v % of sodium phosphate dibasic heptahydrate and 0.0584 w/v % of sodium phosphate monobasic monohydrate to give a total concentration of 0.004 w/v% in order to characterize self-assembled micellar structures. As THF solvent was evaporated from the polymer solutions, the amphiphilic copolymers were self-


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assembled into micellar structures. The aqueous copolymer solutions were sonicated at room temperature for two minutes to prepare homogeneous micelle structures. The hydrodynamic diameter of self-assembled nanostructures was measured at different temperatures, and their zetapotential values were analyzed using dynamic light scattering (DLS) (Zeta-sizer Nano ZS90; Malvern Instruments, Malvern, UK). Preparation of oppositely charged ANPs using EHD co-jetting Each solution of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) was prepared to produce ANPs with oppositely charged and stimuli-responsive compartments via EHD co-jetting. Fluorescein isothiocyanate isomer 1 (FITC) and Nile red were used as fluorescent dyes to observe the interface between compartments using confocal laser scanning microscopy (CLSM; Leica TCS SP, Leica, Germany). FITC was chemically conjugated with poly(NIPAM-co-SA-coAAm) via a reaction between amine groups of poly(NIPAM-co-SA-co-AAm) and isothiocyanate groups of FITC. Poly(NIPAM-co-SA-co-AAm) was dissolved in 100 mM sodium bicarbonate buffer at pH 8.2 to give a total concentration of 6.0 mg/ml; this amine-free buffer was used to prevent formation of unstable intermediates. The amines of poly(NIPAM-co-SA-co-AAm) were reacted with a 15- to 20-fold molar excess of FITC in DMSO at 1.0 mg/ml for two hours at room temperature under dark conditions, followed by addition of 5.0 M NaCl solution to aggregate FITC-conjugated poly(NIPAM-co-SA-co-AAm), dissolution of the copolymer in deionized water, then dialysis in a large amount of deionized water for two days to remove unreacted reagents. Purified FITC-conjugated poly(NIPAM-co-SA-co-AAm) was freeze-dried and stored in a desiccator. Prior to ANP preparation, EHD jetting of poly(NIPAM-co-SA-co-AAm) and poly(NIPAM-co-SA-co-AAc) was conducted separately to obtain isotropic nanoparticles (INPs)


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as controls. The colloidal self-assembly patterns of the positively charged poly(NIPAM-co-SAco-AAm) INPs and negatively charged poly(NIPAM-co-SA-co-AAc) INPs were analyzed as controls under aqueous conditions. To obtain ANPs with poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) compartments, each polymer was dissolved at a final concentration of 24 w/v% in a mixture of DMF and chloroform at a 7:3 volume ratio. Nile red was added into the jetting solution of poly(NIPAM-co-SA-co-AAc) at 0.01 w/v%, and FITC-labelled poly(NIPAM-co-SA-co-AAm) was added into poly(NIPAM-co-SA-co-AAm) solution at 1.0 w/v% to observe each compartment using CLSM. Each polymer solution was loaded into a 1.0 mL syringe (BD, Franklin Lakes, NJ, USA) and the syringe was connected to a dual-channel needle (FibriJet® SA-3610; Micromedics, Inc, St Paul, MN, USA) with side-by-side geometry. The two syringes were fixed with a syringe pump (KD Scientific, Inc, Holliston, MA, USA) to control their flow rates, and a power supply (Nano NC, Seoul, Korea) was used to apply voltage between dual needles and an aluminum foil with a thickness of 0.018 mm (Fisherbrand; Thermo Fisher Scientific, Waltham, MA, USA) as a collecting substrate. The cathode was connected to the dual needle and the anode was connected to the aluminum foil, with a distance between the dual needle and collecting substrate of 15-20 cm. High voltage was applied in the range of 10-15 kV and the flow rate of the two syringes was 0.20 - 0.25 ml/hour. A high-resolution digital camera (D-90; Nikon Corporation, Tokyo, Japan) was used to visualize the biphasic Taylor cone, jet stream, and jet break-up during EHD co-jetting. Characterization of ANPs and their colloidal self-assembly The ANPs and their colloidal self-assembly were characterized in dry and swollen states by CLSM, with He/Ne and argon lasers (Leica TCS SP) in fluorescence and bright field modes.


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Physically crosslinked INPs and ANPs were suspended in deionized water and PBS at pH 7.4 and imaged to characterize colloidal stability, swelling properties, and degree of colloidal selfassembly (ANPs only). The He/Ne- and argon lasers were used for excitation of FITC and Nile red at 488 nm and 543 nm, respectively. The emission spectral range was controlled to minimize overlap of dye emission signals. The wavelength ranges of FITC and Nile red for spectral emission were adjusted to 500–535 nm and 592–647 nm, respectively. ANPs were characterized using DLS in deionized water and PBS at pH 7.4 at 25 C and 37 C to assess colloidal self-assembly and stimuli-responsiveness. Scanning electron microscopy (SEM, VEGA-SB3; TESCAN, Czech Republic) with a focused beam at 0.5-30 kV was used to characterize an average diameter, size distribution, and surface morphology of INPs and ANPs in the dry state. For SEM, the nanoparticles were coated with a thin, conductive platinum layer using a K575X Turbo Sputter Coater (Emitech Ltd, Ashford, UK).

Results and Discussion A schematic of ANP preparation by EHD co-jetting of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) is shown in Figure 1(a). A polymer concentration of 24 w/v% in jetting solvent (DMF and chloroform at a volume ratio of 7:3) was used for both copolymers to produce a viscosity that maintained a clear interface between jetting solutions within a Taylor cone, and a spherical nanostructure shape. Nile red and FITC dyes were used separately to observe the interface between jetting solutions and to determine the degree of anisotropic compartmentalization. One jetting solution contained 24 w/v% poly(NIPAM-co-SA-co-AAc) and 0.01 wt% Nile red, the other contained 24 w/v% poly(NIPAM-co-SA-co-AAm) and 1 wt% FITC-


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conjugated poly(NIPAM-co-SA-co-AAm). Nile red was used as a hydrophobic dye for encapsulation within the poly(NIPAM-co-SA-co-AAc) compartment; FITC was chemically conjugated with poly(NIPAM-co-SA-co-AAm) because of its hydrophilicity and fast release from ANPs under aqueous conditions. Each polymer solution was loaded into an individual syringe connected to a dual channel needle with side-by-side geometry. The two syringes were fixed with a syringe pump and their flow rates were simultaneously controlled at 0.20-0.25 ml/hour. When high voltage (10-15 kV) was applied between a dual channel needle and an aluminum foil as a collecting substrate, a thin jet stream was formed at the vertex of the stable biphasic Taylor cone when the electrostatic force overcame the surface tension of the two jetting solutions. Once the DMF/chloroform solvent mixture in the electrohydrodynamically atomized droplets evaporated, ANPs with two discrete compartments were formed, as confirmed by fluorescence imaging. Figure 1(b) shows schematics and fluorescence images of the ANPs and their supracolloidal selfassembly. The ANPs and self-assembled nanostructures were stabilized under aqueous conditions by physical crosslinking of SA moieties in both copolymer compartments via hydrophobic interactions. As temperature increased from 25 C to 37 C, ANPs in PBS collapsed due to the thermally responsive poly(NIPAM)-based copolymers in both compartments. In contrast, when ANPs were added to deionized water rather than PBS, self-assembled supracolloidal nanostructures were formed due to electrostatic interactions between positively and negatively charged compartments. The degree of self-assembly and thermal responsiveness of the ANPs were largely controlled by the charge density of each compartment and solution ionic strength. The syntheses of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) for the preparation of stimuli-responsive, physically crosslinkable, and charged ternary copolymers are shown in Figures 2(a) and (b). The two copolymers were synthesized via free radical 13 ACS Paragon Plus Environment

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polymerization of NIPAM, SA, and AAc or AAm monomers in ethanol at 83 C, with two different feed molar ratios (96.5:3.0:0.5 and 96:3:1) to produce different charge densities. SA monomers were used to produce physically crosslinked ANPs via hydrophobic interactions between stearyl chains, while NIPAM monomers were used for thermal responsiveness. AAc and AAm were used to introduce opposite charges in each compartment. Figure 2(c) and (d) show 1H NMR spectra of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) in DMSO-d6, respectively. A singlet peak at 3.9 ppm indicates the C-2 proton of the isopropyl group on the NIPAM, and a triplet peak at 0.9 ppm reveals the presence of the proton of the terminal methyl group on the SA moiety. The 1H NMR peak of the carboxylic group from the AAc moieties was at 12.0 ppm, and that of proton adjacent to the amine group from the AAm moieties was at 3.0 ppm. Table 1 shows the apparent molar ratio of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm), determined by calculating the area under each 1H NMR peak (Figures 2(c) and (d)). The apparent molar ratio of SA moieties was greater than the feed molar ratio of the SA, indicating that the SA monomers were incorporated into polymer chains with higher reactivity. In addition, the molecular weight and polydispersity index (PDI) of the copolymers were obtained by GPC using THF as mobile phase. GPC traces of (a) poly(NIPAM-co-SA-co-AAc) and (b) poly(NIPAM-co-SA-coAAm) are shown in Figure S1. When the monomer feed molar ratio of NIPAM, SA, and AAc or AAm was 96:3:1, poly(NIPAM-co-SA-co-AAc) had a weight-average molecular weight of 43,800 g/mol with a PDI of 1.64, while poly(NIPAM-co-SA-co-AAm) had a weight-average molecular weight of 26,800 g/mol with a PDI of 1.54. When the feed molar ratio was 96.5:3.0:0.5, poly(NIPAM-co-SA-co-AAc) had a weight-average molecular weight of 46,000 g/mol with a PDI of 1.71, while poly(NIPAM-co-SA-co-AAm) had a weight-average molecular weight of 29,200 g/mol with a PDI of 1.64. Although these copolymer sets were synthesized under identical 14 ACS Paragon Plus Environment

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conditions (monomer concentration, monomer-to-initiator ratio, solvent, and polymerization time), the molecular weight of poly(NIPAM-co-SA-co-AAc) was higher than that of poly(NIPAM-co-SA-co-AAm), most likely due to different polymerization kinetics. The unique thermal transition behavior of the copolymers, characterized by UV absorbance as a function of temperature, is shown in Figure 2(e). Poly(NIPAM)-based random or block copolymers have been shown to be temperature-responsive, exhibiting lower critical solution temperature (LCST) behavior.47 Generally, poly(NIPAM)s are soluble below the LCST due to hydrogen bonding between amide groups of NIPAM and water molecules, and are insoluble above the LCST due to collapse of poly(NIPAM) chains via hydrophobic interactions between isopropyl groups, resulting in a sharp transition of UV absorbance at the LCST. The phase transition of the two copolymers was measured in 3 mM phosphate buffer at pH 5.6 as a function of temperature (Figure 2(e)). Buffer composition and solution conditions were optimized to compare LCST values under identical conditions, because the copolymers have different solubility depending on ionic strength and pH. The LCST, as known as the inverse transition temperature (Tt), was defined as the temperature at which the value of the first derivative of UV absorbance as a function of temperature was maximal. Incorporation of hydrophobic SA moieties into poly(NIPAM) caused a decrease in LCST, while hydrophilic AAc and AAm moieties caused an increase in LCST. Copolymers made from 0.1 w/v% poly(NIPAM-co-SA-co-AAc) or poly(NIPAM-co-SA-coAAm) at a 96:3:1 feed molar ratio in phosphate buffer at pH 5.6 showed a thermally-triggered phase transition at 40.6 C and 40.5 C, respectively (Table 2). Copolymers formed using a 96.5:3.0:0.5 feed molar ratio exhibited their LCST behavior at 38.9 C and 31.5 C – lower than at a 96:3:1 feed molar ratio. This was likely due to lower charge densities and a greater number of SA moieties (Table 1). 15 ACS Paragon Plus Environment

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When the ternary copolymers were dissolved in deionized water at 0.1 w/v%, they formed selfassembled micellar structures due to their amphiphilic characteristics. The self-assembled micelles exhibited a negative -potential for the copolymer containing AAc, and a positive -potential for the polymer containing AAm (Figure 2(f)). Apparent pKa was defined as the pH value at which the concentration of protonated carboxyl groups of poly(NIPAM-co-SA-co-AAc) or protonated amine groups of poly(NIPAM-co-SA-co-AAm) was identical to that of deprotonated groups. Apparent pKa values of poly(NIPAM-co-SA-co-AAc) at 96:3:1 and 96.5:3.0:0.5 feed molar ratios were 3.94 and 4.07, respectively. The AAc-containing copolymer was negatively charged because AAc moieties become deprotonated with increased pH. Poly(NIPAM-co-SA-co-AAm) at 96:3:1 and 96.5:3.0:0.5 feed molar ratios had apparent pKa values of 10.56 and 10.52, due to the AAm moieties. This copolymer becomes protonated with decreased pH, showing a positive -potential value. As the pKa values are affected by the content of charged groups and hydrophobic moieties of the copolymers, the pKa values of copolymers with different feed molar ratios varied.48 Poly(NIPAM-co-SA-co-AAc) micelles showed similar -potential values despite the increase in feed molar ratio of AAc moieties, while poly(NIPAM-co-SA-co-AAm) micelles showed an increase in -potential value with an increased feed molar ratio of AAm (Figure 2(f) and Table 2). The lack of a change in -potential for poly(NIPAM-co-SA-co-AAc) micelles might be attributed to a similar surface charge density on micelles despite the increased apparent molar ratio of AAc (from 1.5 to 2.1).49 However, -potential values of poly(NIPAM-co-SA-co-AAm) micelles increased as the apparent molar ratio of AAm increased from 0.7% to 1.1%. The hydrodynamic radii of self-assembled micelles of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) under aqueous conditions at 25 C (below the LCST) and 50 C (above the LCST) are shown in Figure 2(g-j). Each copolymer solution in THF at 0.01 w/v% was 16 ACS Paragon Plus Environment

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added into 10 mM phosphate buffer at pH 7 in a dropwise manner to give a final concentration of 0.004 w/v%, and self-assembled micelles were formed at 25 C following slow evaporation of THF. Both copolymers have amphiphilic properties, leading to core-shell micellar structures with a hydrophobic core of SA moieties and a hydrophilic shell of NIPAM and AAc or AAm moieties. Their hydrodynamic radii changed in response to temperature due to poly(NIPAM). The average hydrodynamic radius of poly(NIPAM-co-SA-co-AAc) at a 96.5:3.0:0.5 feed molar ratio in phosphate buffer at pH 7 was 67.5 ± 2.6 nm at 25 C and 56.0 ± 2.4 nm at 50 C (Figure 2(g)). The average hydrodynamic radius of poly(NIPAM-co-SA-co-AAm) at a 96.5:3.0:0.5 feed molar ratio decreased from 118.0 ± 1.3 nm to 99.0 ± 1.8 nm with the increase in temperature under identical conditions (Figure 2(h)). As temperature increased, the average micellar size of both copolymers decreased due to collapse of poly(NIPAM) chains above their LCST. Likewise, the average hydrodynamic radius of poly(NIPAM-co-SA-co-AAc) at a 96:3:1 feed molar ratio in phosphate buffer with pH 7 was 88.0 ± 3.2 nm at 25 C and 62.0 ± 1.3 nm at 50 C (Figure 2(i)). The average hydrodynamic radius of poly(NIPAM-co-SA-co-AAm) at a 96:3:1 feed molar ratio decreased from 148.6 ± 1.3 nm to 118.5 ± 4.5 nm with the increase of temperature. (Figure 2(j)). Greater micelle sizes were produced by a feed molar ratio of 96:3:1 than 96.5:3.0:0.5 likely due to copolymer chain extension resulting from repulsive forces between charged AAc and AAm groups. INPs and ANPs were prepared via electrohydrodynamic (EHD) jetting and co-jetting. SEM images of INPs of (a) poly(NIPAM-co-SA-co-AAc), (b) poly(NIPAM-co-SA-co-AAm) at a 96:3:1 feed molar ratio, and (c) ANPs in a dried state for analysis of surface morphology and size distribution are shown in Figure 3. ANPs were spherical, and no significant beads-on-a-string structures were observed for either INPs or ANPs. These results indicate that EHD jetting and co17 ACS Paragon Plus Environment

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jetting conditions were appropriate for synthesis of discrete INPs and ANPs. Approximately 70 nanoparticles were selected randomly from SEM images to determine average diameter and size distribution. Average diameters for poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-coAAm) INPs were 585 ± 161 nm and 404 ± 228 nm, respectively; ANPs had an average diameter of 617 ± 150 nm. Similar average diameters and size distributions were observed for INPs and ANPs formed from the two copolymers (Figure 3(d)). CLSM, bright-field, and merged images of the INPs in dry and swollen states are shown in Figure 4. The INPs were collected on a slide glass located on an aluminum foil as a collecting substrate, and suspended in deionized water. Approximately 50 INPs were selected for analysis of size and size distribution. Poly(NIPAM-co-SA-co-AAc) INPs at a 96:3:1 feed molar ratio in (a) dry state and (c) deionized water had average diameters of 444 ± 201 nm and 2.2 ± 0.45 m, respectively, while poly(NIPAM-co-SA-co-AAm) INPs at a 96:3:1 at feed molar ratio had average diameters of 416 ± 222 nm and 1.40 ± 0.28 m (b) dry and (d) in deionized water, respectively (Table 3). These INPs were stabilized under aqueous conditions by physical crosslinking of SA moieties, and formed colloids due to interactions between carboxylic or amine groups on their surfaces. The size distributions of INPs in dry and swollen states are shown in Figures 8 (a) and (b). The swelling ratio of poly(NIPAM-co-SA-co-AAc) INPs in deionized water was 136.1, while that of poly(NIPAM-co-SA-co-AAm) INPs under identical conditions was 38.4. This large difference in swelling ratio is potentially due to a different degree of ionization of charged groups of each copolymer in deionized water, and is in agreement with -potential values in Table 2. The swelling ratios of INPs in deionized water were lower at a feed molar ratio of 96.5:3.0:0.5 than 96:3:1 because of the decreased charge density. In PBS, INP swelling ratios were reduced because of shielding of charge repulsion by the salt solution. 18 ACS Paragon Plus Environment

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CLSM images of ANPs with poly(NIPAM-co-SA-co-AAc) and Nile red in one compartment, and poly(NIPAM-co-SA-co-AAm) and FITC-conjugated poly(NIPAM-co-SA-co-AAm) in the other, are shown in Figures 5 and 6. The ANPs were collected on a slide glass and suspended in deionized water and PBS separately, followed by CLSM imaging. A distinct interface between ANP compartments was observed, with distinct fluorescence signals. CLSM images of ANPs in a dry sate clearly show an anisotropic distribution of copolymers, without diffusion between compartments during EHD co-jetting. Average ANP sizes were 0.92 ± 0.39 m and 0.99 ± 0.37 m for the two feed molar ratios. Figures 5(b-d) show that the ANPs made using a 96.5:3.0:0.5 feed molar ratio formed self-assembled supracolloidal nanostructures with 2.73 ± 0.58 multimers in deionized water. In contrast, at a 96:3:1 feed molar ratio, self-assembled supracolloidal nanostructures were formed, with 6.21 ± 0.55 multimers on average (Figures 6(b-d)). This reflects a higher degree of clustering between ANPs made using a 96:3:1 feed molar ratio than using a 96.5:3.0:0.5 feed molar ratio, due to different compartment charge densities. The average multimerization of the ANPs in deionized water was determined by analysis of ~50 particles from CLSM images (Figure 8(d)). In addition, as shown in Figure 5 (b3-d3), the negatively charged poly(NIPAM-co-SA-co-AAc) compartments of the ANPs were generally interfaced with the positively charged poly(NIPAM-co-SA-co-AAm) compartment of the others in deionized water, representing that there was directionality in the ANP multimers. Furthermore, the average percentage of the ANPs with oppositely charged compartments adjacent to each other was determined by analysis of approximately 50 ANPs of the multimers from the CLSM images. The percentage of those ANPs in the total number of ANPs with a 96.5:3.0:0.5 feed molar ratio was 51.8% while that of ANPs at a 96:3:1 feed molar ratio was 67.9 %. It suggests that charge density of the copolymers of the ANPs also affected the degree of directionality in the ANP multimers. 19 ACS Paragon Plus Environment

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On the other hand, no defined shapes of the different ANP multimers were observed. It was reported that the charged Janus spheres assembled into energetically stable structures or less symmetric clusters with different shapes depending on the particle number.19 Self-assembled micelles and INPs of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SAco-AAm) exhibited negative and positive zeta-potential values in deionized water, respectively (Tables 2 and 3). Poly(NIPAM-co-SA-co-AAc) micelles showed similar -potential values despite the increase of apparent molar ratio of AAc moieties, due to a high degree of ionization of carboxyl groups. In contrast, poly(NIPAM-co-SA-co-AAm) micelles showed an increase in -potential with an increase in apparent molar ratio of AAm. The difference in -potential values of poly(NIPAMco-SA-co-AAm) caused by the different apparent molar ratios resulted in different degrees of clustering in deionized water. Therefore, the apparent molar ratios of AAc and AAm in the copolymers determined the surface charge densities of the oppositely charged compartments, which in turn controlled colloidal self-assembly. In general, the overall shape of the self-assembled supracolloidal nanostructures differed from the well-ordered structures of individual ANPs and INPs, which tend to have specific geometrical shapes, as reported before.50 It was reported that colloidal micron-sized particles whose surface charges on two hemispheres were uniformly balanced exhibited a net charge of zero, and significant chaining of the particles was observed due to dipole-dipole interactions in aqueous solution. In contrast, in our study, ANP -potentials at 96.5:3.0:0.5 and 96:3:1 feed molar ratios in deionized water were -15.58±0.40 mV and 13.06±0.65 mV. The charge imbalance of each ANP compartment is potentially due to a different degree of ionization of charged groups in the two compartments. In addition to particle net charge, it was reported that head-to-tail assembly of positively- and negatively charged surfaces of poly(styrene)-based, micron-sized Janus particles led to self-assembly into chains, while nano20 ACS Paragon Plus Environment

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sized particles formed a ring structures, suggesting that the size of Janus particles influenced definite geometrical shapes of clusters.51-52 In contrast, as mentioned, no defined shapes of the different ANP multimers were observed, potentially due to both of the size polydispersity and the relative charge imbalance at two compartments of ANPs. CLSM images of ANPs with 96:3:1 and 96.5:3:0.5 feed molar ratios in PBS at 25 C and 37 C were obtained to examine the effect of ionic strength and temperature on colloidal self-assembly and hydrodynamic diameter (Figures 7 and S2). Surface charges on each compartment were lower in PBS than in deionized water due to shielding of charge at higher ionic strength. The -potential values of the ANPs with 96.5:3.0:0.5 and 96:3:1 feed molar ratios in PBS were -4.50 ± 1.84 mV and -6.10 ± 2.16 mV, respectively. Consequently, the electrostatic interaction between compartments was greatly reduced, and no colloidal self-assembly occurred. In addition, thermally responsive ANPs with 96.5:3.0:0.5 and 96:3:1 feed molar ratios collapsed in PBS as the temperature increased from 25 C to 37 C, and their average diameter decreased from 2.03 ± 0.44 μm to 1.37 ± 0.40 μm or 1.73 ± 0.40 μm to 1.26 ± 0.44 μm, respectively (Figures 7 and S2). However, no significant effect of temperature on the hydrodynamic diameter of both ANPs in deionized water was observed because the transition temperatures of the ternary copolymers within the ANPs were >37 C. The swelling and collapse ratio of ANPs with a 96:3:1 feed molar ratio in PBS were 8.54 and 0.31, respectively, while those of ANPs with a 96.5:3.0:0.5 feed molar ratio were 6.72 and 0.39. The swelling ratio was defined as the volume ratio of swollen ANPs to dry ANPs; the collapsed ratio was calculated by dividing the particle volume in a collapsed state with that in a swollen state. Figures 8(c) and S2 show the size distributions of ANPs of copolymers with 96:3:1 and 96.5:3.0:0.5 feed molar ratios in dry and swollen states in an aqueous environment at


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different temperatures. ANP size decreased significantly due to the collapse of poly(NIPAM) chains within each compartment at 37 C. The degree of self-assembly of ANPs due to electrostatic interactions between the oppositely charged compartments was largely controlled by the charge density of the copolymers and the solution ionic strength. The supracolloidal nanostructures showed intrinsic properties from the ANPs as well as properties from the self-assembled structures. These results suggest that the ANPs may be useful as drug delivery carriers, colloidal crystals for switchable displays, and building blocks for ion-sensitive gels, depending on the functionality and stimuli-responsiveness of each ANP compartment. The two compartments enable them to incorporate two different drugs, potentially with temporally decoupled release of the two drugs in a controlled manner via stimuli responsiveness. As colloidal crystals for an electronic paper, the oppositely charged ANPs may show field-directed colloidal assembly patterning, such that the negatively charged compartments are directed to positive electrodes in response to an electric field. Thus, these ANPs could be useful as advanced materials for electronic displays due to the controllable charged density at each compartment. In addition, the ANPs could form viscous gels at high particle concentration depending on ionic strength. Different types of ion-sensitive gels could be created by controlling the degree of self-assembly of ANPs, which would be useful for sensing the environment.

Conclusions In summary, we presented colloidal self-assembly of oppositely charged, stimuli-responsive ANPs depending on charge density at each ANP compartment and solution ionic strength. Two different

thermally

responsive

poly(N-isopropylacrylamide)-based

ternary

copolymers,

poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm), were synthesized via free 22 ACS Paragon Plus Environment

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radical polymerization, and the ANPs with oppositely charged compartments were prepared by EHD co-jetting of the two copolymers. The ANPs were physically crosslinked via hydrophobic interactions among SA moieties of both copolymers. ANPs with a 96.5:3.0:0.5 monomer feed molar ratio formed clusters of 2.73 ± 0.58 multimers in deionized water; ANPs with a 96:3:1 feed molar ratio formed clusters of 6.21 ± 0.55 multimers. In PBS, no clustering occurred due to screening of the charged groups. The ANPs were collapsed in PBS at 37 C due to the thermalresponsiveness of the poly(NIPAM)-based ternary copolymers. In conclusion, this new class of ANPs with oppositely charged and thermally responsive compartments opens new routes toward self-assembled supracolloidal nanostructures, with potential biomedical and industrial applications as drug delivery carriers, colloidal crystals, and ion-sensitive gels.

Disclosure The authors have no conflicts of interest to declare.

Supporting Information Included with three pages and two figures. Acknowledgements This work was supported by the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Korea (2015R1D1A1A01058026, 2018R1A6A1A03024231), the Ministry of Science and ICT, Korea (2018R1A2B6006411), and the Agency for Defense Development through Chemical and Biological Research Center, Korea. 23 ACS Paragon Plus Environment

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Figure Captions Figure 1. (a) A Schematic for preparation of ANPs composed of negatively charged poly(NIPAMco-SA-co-AAc)

and

positively

charged

poly(NIPAM-co-SA-co-AAm)

via

electrohydrodynamic(EHD) co-jetting with side-by-side needle geometry. One polymer solution contained poly(NIPAM-co-SA-co-AAc) and the other contained poly(NIPAM-co-SA-co-AAm). (b) Schematics and fluorescence images of ANPs and their colloidal self-assembly via electrostatic interactions. The degree of self-assembly was controlled by compartment charge density and solution ionic strength. ANPs collapsed in PBS at high temperature due to the thermallyresponsive poly(NIPAM) copolymers in both compartments. The scale bar in the fluorescence images is 4 m. Figure 2. Synthesis of (a) poly(NIPAM-co-SA-co-AAc) and (b) poly(NIPAM-co-SA-co-AAm) at feed molar ratios of NIPAM, SA, and AAc or AAm of 96.5:3.0:0.5 and 96:3:1 via free radical polymerization, using AIBN as initiator. The 1H NMR spectra of (c) poly(NIPAM-co-SA-co-AAc) and (d) poly(NIPAM-co-SA-co-AAm) with 96:3:1 feed molar ratio of NIPAM, SA, and AAc or AAm monomers were obtained in DMSO-d6 at 400 MHz. The chemical shift of the C-2 proton of the isopropyl group on the NIPAM was 3.9 ppm and that of the proton of terminal methyl group on the SA moiety was 0.9 ppm. The chemical shift of the carboxylic group from the AAc moieties was 12.0 ppm, and that of proton adjacent to the amine group from the AAm moieties was 3.0 ppm. (e) Phase transition behavior of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-coAAm) with different feed molar ratios in 3 mM phosphate buffer at pH 5.6, as characterized by UV absorbance as a function of temperature. (f) -potential values of self-assembled micelles of the ternary copolymers in deionized water. Hydrodynamic radii of (g, i) poly(NIPAM-co-SA-coAAc) and (h, j) poly(NIPAM-co-SA-co-AAm) with (g, h) 96.5:3.0:0.5 and (i, j) 96:3:1 feed molar 25 ACS Paragon Plus Environment

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ratio under aqueous environment at 25 C (below the LCST) and 50 C (above the LCST). Both copolymers had amphiphilic properties, leading to core-shell micellar structures composed of the hydrophobic core of the SA moieties and the hydrophilic functional shell of NIPAM and AAc or AAm moieties. Figure 3. SEM images of INPs of (a) poly(NIPAM-co-SA-co-AAc), (b) poly(NIPAM-co-SA-coAAm), and (c) ANPs at a 96:3:1 feed molar ratio for analysis of surface morphology and size distribution in a dry state. Scale bars are 5 m in (a-c) and 1 m in inset images. (d) Size distributions of INPs and ANPs at a 96:3:1 feed molar ratio. Approximately 70 nanoparticles from SEM images were selected randomly to analyze average diameter and size distribution. Figure 4. (a1-d1) CLSM, (a2-d2) bright-field, (a3-d3) merged images of poly(NIPAM-co-SA-coAAc) INPs and poly(NIPAM-co-SA-co-AAm) INPs, in a dry state (a, b) and in a swollen state in deionized water (c, d). Fluorescence images of poly(NIPAM-co-SA-co-AAc) INPs at a 96:3:1 feed molar ratio (a) dry and (c) in deionized water are shown using the Nile red channel, while images of poly(NIPAM-co-SA-co-AAm) INPs (b) dry and (d) in deionized water are shown using the fluorescein channel. INPs were stabilized by physically crosslinking via interactions between the alkyl chains of the SA moiety, and were in a colloidal state due to interactions between charged groups on the INP surfaces. The scale bar is 4 m. Figure 5. CLSM images of ANPs with a 96.5:3.0:0.5 feed molar ratio, where the two compartments had poly(NIPAM-co-SA-co-AAc) with Nile red and poly(NIPAM-co-SA-coAAm) with FITC-conjugated poly(NIPAM-co-SA-co-AAm), in (a) dry state and (b-d) deionized water as swollen state. The ANPs spontaneously formed self-assembled supracolloidal nanostructures with 2.73 ± 0.58 multimers on average. Poly(NIPAM-co-SA-co-AAc) and 26 ACS Paragon Plus Environment

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poly(NIPAM-co-SA-co-AAm) compartments are shown using fluorescent signals of (a1-d1) Nile red and (a2-d2) fluorescein, respectively. The two oppositely charged compartments are shown with a merged channel in (a3)-(d3). The scale bars are (a, c) 4 m, (b) 8 m and (d) 2 m. Figure 6. CLSM images of ANPs with a 96:3:1 feed molar ratio in (a) dry state and (b-d) deionized water as swollen state. ANPs formed self-assembled supracolloidal nanostructures with 6.21 ± 0.55 multimers, a higher degree of clustering than at a 96.5:3.0:0.5 feed molar ratio (b-d). The apparent molar ratio of AAc and AAm of poly(NIPAM)-based ternary copolymers determined the different compartment charge densities and controlled the degree of clustering in deionized water. Both copolymer compartments are shown using the fluorescent signals of (a1-d1) Nile red and (a2-d2) fluorescein. The two compartments are shown using a merged channel in (a3-d3). Scale bars are (a, c) 4 m, (b) 8 m and (d) 2 m. Figure 7. CLSM images of ANPs with a 96:3:1 feed molar ratio in PBS, showing the effect of solution ionic strength and temperature on colloidal self-assembly and the hydrodynamic diameter. No colloidal self-assembly was observed in PBS due to the interference of electrostatic interactions in the salt solution. Furthermore, the thermally responsive ANPs collapsed in PBS as temperature increased from (a) 25 C to (b) 37 C, as indicated by a reduced average diameter. Both copolymer compartments are shown with fluorescent signals of (a1-b1) Nile red and (a2-b2) fluorescein. The two oppositely charged compartments are shown with a merged channel in (a3-b3). Scale bars are 4 m in all images. Figure 8. Size distributions of INPs of (a) poly(NIPAM-co-SA-co-AAc) and (b) poly(NIPAMco-SA-co-AAm) with 96:3:1 feed molar ratios in a dry state and in deionized water as a swollen state, and (c) the ANPs with a 96:3:1 feed molar ratio of NIPAM, SA, and AAc or AAm in a dry 27 ACS Paragon Plus Environment

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state and in PBS as a swollen state, at 25 C and 37 C. The sizes of INPs and ANPs of the copolymers were largely increased in the swollen state compared to the dry state. The size of ANPs was greatly reduced at 37 C due to collapsed poly(NIPAM) chains in each compartment. (d) Multimerization degree of ANPs in deionized water.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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Figure 8.


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Table 1. Feed and 1apparent molar ratio, 2number-average molecular weight (Mn), 3weightaverage molecular weight (Mw), and 4polydispersity index (PDI) of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm). Apparent molar ratio was determined by 1H NMR spectra in DMSO-d6. Values of Mn, Mw and PDI were obtained by GPC analysis using THF as the mobile phase.


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Table 2. 1LCST of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) in 3 mM phosphate buffer at pH 5.6. 2 The -potential of self-assembled micelles of poly(NIPAM-co-SAco-AAc) and poly(NIPAM-co-SA-co-AAm) in deionized water. To characterize the -potential of the micelles, the ternary copolymers were dissolved in deionized water at 0.1 w/v%. 3The average hydrodynamic radius of self-assembled micelles was measured after 0.01 w/v% copolymer solutions dissolved in 1 ml THF were slowly added into 25 ml of deionized water to give a final concentration of 0.004 w/v%. The measurements were performed with five different batches (N=5). 4The size reduction ratio was defined as the radius at 25 C divided by the radius at 50 C.


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Table 3. The -potential value and average diameter of INPs of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SA-co-AAm) in deionized water. 1The INPs were dissolved in deionized water at a final concentration of 0.1 w/v% to characterize -potential value. 2The average diameters of INPs were determined by analysis of 50 randomly selected INPs from fluorescence images. Measurements were performed with five different batches (N=5). 3The swelling ratio was defined as the volume ratio of swollen to dry INPs.


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TABLE OF CONTENTS (TOC) GRAPHIC

Oppositely charged, thermally-responsive anisotropic nanoparticles (ANPs) self-assembled into supracolloidal nanostructures via electrostatic interactions, exhibiting both temperature-responsive 43 ACS Paragon Plus Environment

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properties of the ANPs and collective properties from self-assembly.


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Figure 1. (a) A Schematic for preparation of ANPs composed of negatively charged poly(NIPAM-co-SA-coAAc) and positively charged poly(NIPAM-co-SA-co-AAm) via electrohydrodynamic(EHD) co-jetting with sideby-side needle geometry. One polymer solution contained poly(NIPAM-co-SA-co-AAc) and the other contained poly(NIPAM-co-SA-co-AAm). (b) Schematics and fluorescence images of ANPs and their colloidal self-assembly via electrostatic interactions. The degree of self-assembly was controlled by compartment charge density and solution ionic strength. ANPs collapsed in PBS at high temperature due to the thermallyresponsive poly(NIPAM) copolymers in both compartments. The scale bar in the fluorescence images is 4 μm. 316x419mm (150 x 150 DPI)

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Figure 2. Synthesis of (a) poly(NIPAM-co-SA-co-AAc) and (b) poly(NIPAM-co-SA-co-AAm) at feed molar ratios of NIPAM, SA, and AAc or AAm of 96.5:3.0:0.5 and 96:3:1 via free radical polymerization, using AIBN as initiator. The 1H NMR spectra of (c) poly(NIPAM-co-SA-co-AAc) and (d) poly(NIPAM-co-SA-co-AAm) with 96:3:1 feed molar ratio of NIPAM, SA, and AAc or AAm monomers were obtained in DMSO-d6 at 400 MHz. The chemical shift of the C-2 proton of the isopropyl group on the NIPAM was 3.9 ppm and that of the proton of terminal methyl group on the SA moiety was 0.9 ppm. The chemical shift of the carboxylic group from the AAc moieties was 12.0 ppm, and that of proton adjacent to the amine group from the AAm moieties was 3.0 ppm. (e) Phase transition behavior of poly(NIPAM-co-SA-co-AAc) and poly(NIPAM-co-SAco-AAm) with different feed molar ratios in 3 mM phosphate buffer at pH 5.6, as characterized by UV absorbance as a function of temperature. (f) ς-potential values of self-assembled micelles of the ternary copolymers in deionized water. Hydrodynamic radii of (g, i) poly(NIPAM-co-SA-co-AAc) and (h, j) poly(NIPAM-co-SA-co-AAm) with (g, h) 96.5:3.0:0.5 and (i, j) 96:3:1 feed molar ratio under aqueous environment at 25 °C (below the LCST) and 50 °C (above the LCST). Both copolymers had amphiphilic properties, leading to core-shell micellar structures composed of the hydrophobic core of the SA moieties


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and the hydrophilic functional shell of NIPAM and AAc or AAm moieties. 400x604mm (150 x 150 DPI)


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Figure 3. SEM images of INPs of (a) poly(NIPAM-co-SA-co-AAc), (b) poly(NIPAM-co-SA-co-AAm), and (c) ANPs at a 96:3:1 feed molar ratio for analysis of surface morphology and size distribution in a dry state. Scale bars are 5 μm in (a-c) and 1 μm in inset images. (d) Size distributions of INPs and ANPs at a 96:3:1 feed molar ratio. Approximately 70 nanoparticles from SEM images were selected randomly to analyze average diameter and size distribution. 415x281mm (150 x 150 DPI)


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Figure 4. (a1-d1) CLSM, (a2-d2) bright-field, (a3-d3) merged images of poly(NIPAM-co-SA-co-AAc) INPs and poly(NIPAM-co-SA-co-AAm) INPs, in a dry state (a, b) and in a swollen state in deionized water (c, d). Fluorescence images of poly(NIPAM-co-SA-co-AAc) INPs at a 96:3:1 feed molar ratio (a) dry and (c) in deionized water are shown using the Nile red channel, while images of poly(NIPAM-co-SA-co-AAm) INPs (b) dry and (d) in deionized water are shown using the fluorescein channel. INPs were stabilized by physically crosslinking via interactions between the alkyl chains of the SA moiety, and were in a colloidal state due to interactions between charged groups on the INP surfaces. The scale bar is 4 μm. 244x184mm (150 x 150 DPI)


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Figure 5. CLSM images of ANPs with a 96.5:3.0:0.5 feed molar ratio, where the two compartments had poly(NIPAM-co-SA-co-AAc) with Nile red and poly(NIPAM-co-SA-co-AAm) with FITC-conjugated poly(NIPAMco-SA-co-AAm), in (a) dry state and (b-d) deionized water as swollen state. The ANPs spontaneously formed self-assembled supracolloidal nanostructures with 2.73 ± 0.58 multimers on average. Poly(NIPAM-co-SA-coAAc) and poly(NIPAM-co-SA-co-AAm) compartments are shown using fluorescent signals of (a1-d1) Nile red and (a2-d2) fluorescein, respectively. The two oppositely charged compartments are shown with a merged channel in (a3)-(d3). The scale bars are (a, c) 4 μm, (b) 8 μm and (d) 2 μm. 338x448mm (150 x 150 DPI)


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Figure 6. CLSM images of ANPs with a 96:3:1 feed molar ratio in (a) dry state and (b-d) deionized water as swollen state. ANPs formed self-assembled supracolloidal nanostructures with 6.21 ± 0.55 multimers, a higher degree of clustering than at a 96.5:3.0:0.5 feed molar ratio (b-d). The apparent molar ratio of AAc and AAm of poly(NIPAM)-based ternary copolymers determined the different compartment charge densities, and controlled the degree of clustering in deionized water. Both copolymer compartments are shown using the fluorescent signals of (a1-d1) Nile red and (a2-d2) fluorescein. The two compartments are shown using a merged channel in (a3-d3). Scale bars are (a, c) 4 μm, (b) 8 μm and (d) 2 μm. 339x446mm (150 x 150 DPI)


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Figure 7. CLSM images of ANPs with a 96:3:1 feed molar ratio in PBS, showing the effect of solution ionic strength and temperature on colloidal self-assembly and the hydrodynamic diameter. No colloidal selfassembly was observed in PBS due to the interference of electrostatic interactions in the salt solution. Furthermore, the thermally responsive ANPs collapsed in PBS as temperature increased from (a) 25 C to (b) 37 C, as indicated by a reduced average diameter. Both copolymer compartments are shown with fluorescent signals of (a1-b1) Nile red and (a2-b2) fluorescein. The two oppositely charged compartments are shown with a merged channel in (a3-b3). Scale bars are 4 μm in all images. 337x224mm (150 x 150 DPI)


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Figure 8. Size distributions of INPs of (a) poly(NIPAM-co-SA-co-AAc) and (b) poly(NIPAM-co-SA-co-AAm) with 96:3:1 feed molar ratios in a dry state and in deionized water as a swollen state, and (c) the ANPs with a 96:3:1 feed molar ratio of NIPAM, SA, and AAc or AAm in a dry state and in PBS as a swollen state, at 25 °C and 37 °C. The sizes of INPs and ANPs of the copolymers were largely increased in the swollen state compared to the dry state. The size of ANPs was greatly reduced at 37 °C due to collapsed poly(NIPAM) chains in each compartment. (d) Multimerization degree of ANPs in deionized water. 409x308mm (150 x 150 DPI)


Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles for

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