Bioassemblies Fabricated by Coassembly of Protein Molecules and

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

Bio-Assemblies Fabricated by Co-Assembly of Protein Molecules and Mono-Tethered Single-Chain Polymeric Nanoparticles Qi Liu, Yuanyuan Ju, and Hanying Zhao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02895 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

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Bio-Assemblies Fabricated by Co-Assembly of Protein Molecules and Mono-Tethered Single-Chain Polymeric Nanoparticles

Qi Liu†,Yuanyuan Ju †, and Hanying Zhao*,†, ‡ †

Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University, Tianjin 300071, China

‡ Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China

ACS Paragon Plus Environment

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ABSTRACT: Molecular nanoparticles have been used as building blocks in the synthesis of functional materials. The grand challenges in the synthesis of the functional materials are precise control of the structures and functionalities of the materials by using nanoparticles with different architectures and properties. Mono-tethered single-chain polymeric nanoparticles (SCPN) are a type of nano-sized asymmetric particles formed by intramolecular crosslinking of linear diblock copolymer chains. Mono-tethered SCPNs can be used as elemental building blocks for the fabrication of well-defined advanced structures. In this research, synthesis of biohybrid materials based on co-assembly of bovine serum albumin (BSA) molecules and mono-tethered SCPNs is investigated. Due to the asymmetric structure of the SCPNs, positively charged SCPNs and negatively charged protein molecules co-assemble into biohybrid vesicles with SCPNs on the layers and protein molecules in the walls. The self-assembled structures were analyzed by using dynamic light scattering, transmission electron microscopy, cryo-transmission electron microscopy and atomic force microscopy. The average size of the biohybrid vesicles can be controlled by the molar ratio of SCPNs to BSA. The protein molecules in the biohybrid vesicles maintain most of the activities. This research paves a new way for the synthesis of functional biohybrid structures and the materials can be used as protein carriers.

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INTRODUCTION


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Molecular nanoparticles are a type of nanoparticles which are used as building blocks in the synthesis of advanced structures.1,2 In these years, the fabrication of materials with complex hierarchical structures by using molecular nanoparticles as elemental building blocks has aroused great interest. Self-assembly of molecular nanoparticles provides an efficient approach to the synthesis of materials with tunable structures, compositions and enhanced functionalities. In the past decade, fullerene, polyhedral oligomeric silsesquioxane and polyoxometalate have been used as building blocks in the synthesis of functional materials.2 Single-chain polymeric nanoparticles (SCPNs) are nanometer-sized particles prepared by intramolecular cross-linking of synthetic polymer chains.3-9 SCPNs have many interesting properties. For example, amphiphilic SCPNs are able to distribute at liquid-air inetrface reducing the surface tension of water, and self-assemble into delicate structures.10-15 A variety of methods, including covalent and noncovalent interactions, have been used to perform intramolecular folding/collapse, and SCPNs with different topological structures and chemical compositions have been synthesized.16-24 In oder to prepare functional materials, SCPNs with bio-functionalities have been synthesized.25-28 For example, Palmans and co-workers synthesized oxidase enzyme-mimic SCPNs with Ru(II)-based catalysts in the structures, and the authors used the SCPNs in the oxidation of alcohols.29 In addition, researches on the bioactivities and applications of the functional SCPNs in biological environments were performed.30 However, until now all the previous researches were focused on the bioactivities of individual SCPNs. In order to expand our knowledge about the molecular nanoparticles and find applications of SCPNs in the synthesis of new biohybrid materials with delicate structures, co-assembly of mono-tethered SCPNs and protein molecules is investigated in this research and active biohybrid assemblies are fabricated. The hybrid structures are endowed with SCPNs’ properties and protein functionalities, and can be used as a promising platform for a variety of applications, including protein and drug delievery.


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Scheme 1. Outlines for the synthesis of positively charged POEGMA-tethered single-chain polymeric nanoparticles (SCPNs), and selfassembly of SCNPs and bovine serum albumin (BSA) molecules.

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EXPERIMENTAL SECTION Materials. In order to remove the inhibitor, oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA, Mn =500 Da, 99%, Aldrich)

was purified by dissolving in dichloromethane and passing the monomer solution through a column with basic alumina. The synthesis and characterization of anthracene-9-carboxyl ethyl methacrylate (AnMA) can be found in our previous paper.31 To remove the inhibitor, 2(dimethylamino) ethyl methacrylate (DMAEMA) (Acros, 99 %) was purified with basic alumina column, and distilled under reduced pressure. The chain transfer agent (4-cyanopentanoic acid) dithiobenzoate (CPADB) was synthesized in this laboratory. 4,4’-Azobis (4cyanopentanoic acid) (ABCPA, Sigma Aldrich, 97%) was recrystallized from methanol, and after filtration it was dried in an oven under reduced pressure at room temperature. Iodomethane (Energy Chemical, 99%), bovine serum albumin (BSA, Genview, 96%), sodium tetraiodofluorescein (TIF, Sigma, 95%), fluorescein isothiocyanate (FITC, Alfa Aesar, 95%) were used as received.


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Reversible addition−fragmentation chain transfer (RAFT) polymerization of OEGMA. OEGMA (5.7 g, 12 mmol), ABCPA (8.0 mg, 0.029 mmol) and RAFT chain transfer agent CPADB (56 mg, 0.20 mmol) were dissolved in 4 mL of dry 1,4-dioxane in a 25 mL Schlenk flask. The solution was degassed by three freeze–pump–thaw cycles, and the RAFT polymerization was conducted at 60 °C for 10 h. The polymerization was stopped by quenching the Schlenk flask in liquid nitrogen. The pinky POEGMA was precipitated in cold diethyl ether. The yield was about 76%. The number-average molecular weight and molecular weight distribution of POEGMA were 20.7 K and 1.12, respectively. Synthesis of POEGMA46-b-(PDMAEMA182-co-PAnMA24). POEGMA (220 mg), ABCPA (0.40 mg, 0.0015 mmol), DMAEMA (471 mg, 3.00 mmol), and AnMA (100 mg, 0.300 mmol) were dissolved in 1.5 mL of dry 1,4-dioxane in a 10 mL Schlenk flask. The solution was degassed by three freeze–pump–thaw cycles, and the RAFT polymerization was conducted at 70 °C for 12 h. The polymerization was stopped by quenching the Schlenk flask in liquid nitrogen. The block copolymer was precipitated in hexane. The yield was about 60%. The numberaverage molecular weight and molecular weight distribution of the block copolymer were 66.3 K and 1.24, respectively. Preparation of positively charged mono-tethered SCPNs. POEGMA46-b-(PDMAEMA182-co-PAnMA24) (50 mg) was dissolved in 150 mL of dry DMF in a 250 mL quartz round bottom flask, and the solution was exposed to ultraviolet light by a lamp with a power of 26 W at a wavelength of 364 nm. The distance between the lamp and the quartz flask was about 15 cm. The intramolecular cross-linking reaction was conducted at 25 °C for 3 h. Iodomethane (12 μL, 0.19 mmol) was added to the solution and the quaternization reaction was conducted at 30 °C for 12 h in the dark. To remove DMF, the solution in a dialysis tubing (MWCO 7 kDa) was dialyzed against water for 24 h. Positively charged mono-tethered SCPNs were obtained after freeze-drying. Self-assembly of positively charged SCPNs and BSA. The self-assembly was performed by directly mixing of aqueous solutions of BSA and SCPNs at different molar ratios. A typical process was described as follows. Aqueous solution of BSA (2.5 mL) at a concentration of 1


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mg/mL was added into 1 mL of SCNPs solution at a rate of 3 mL/min. The solution was stirred (250 r/min) at 25 °C for 1 h. Self-assemblies with different molar ratios of SCPNs to BSA were prepared. Preparation of FITC-labeled BSA. FITC (9.03×10-4 mmol) dissolved in 10 mL PBS solution (50 mM, pH 8.0), was added into 10 mL of BSA aqueous solution (4.52×10-4 mmol ), and after that 30 mL PBS solution (50 mM, pH 8.0) was added into the above solution. The solution was stirred at 4 °C for 7 h in the dark. After the reaction, BSA solution was transferred to a dialysis tubing (MWCO 7 kDa) and dialyzed against water for 48 h to remove excess FITC. UV-vis was employed to monitor the dialysis process.

Preparation of TIF-labeled SCPNs. Aqueous solution of TIF (100 μL, 3.6×10-5 mmol) was added into 5 mL of aqueous solution of SCPNs (7.6×10-5 mmol). After stirring at 25 °C overnight, the mixture was transferred to a dialysis tubing (MWCO 7 kDa) and dialyzed against water for 48 h to remove excess TIF. UV-vis was employed to monitor the dialysis process. The dialysis was performed until no absorption of TIF was detected in water. Activity assay of BSA in the self-assembled structures. p-Nitrophenyl acetate (NPA) was selected as the substrate for the determination of esterase activity of BSA in the self-assembled structures. Briefly, 0.1 mL of aqueous solutions of self-assembled structures prepared at different molar ratios of SCPNs to BSA were mixed with 1.0 mL PBS solution of NPA (0.023 mg/mL). Esterase activity was monitored by measuring the absorbance of the 4-nitrophenolate anion at 400 nm. In order to compare the esterase activity, the concentration of BSA in the self-assembled structures is controlled to be equal to that of free BSA. Characterization. 1H NMR spectra of the POEGMA-tethered single-chain polymeric nanoparticles (SCPNs) and linear polymers were acquired on a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer by using CDCl3 as solvent. The apparent and absolute molecular weights of the SCPNs and precursors were determined on a size exclusion chromatograph (SEC), with a Hitachi L-2130 HPLC pump, three Shodex columns (5000−5K, 400−0.5K, and 5−0.15K molecular ranges), a refractive index detector (Hitachi L-2490), and


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Viscotek 270 dual detector (Malvern Instruments Ltd.). DMF containing 1 mg/mL LiBr was used as the eluent, and the flow rate was set at 1 mL/min. Poly(methyl methacrylate) (PMMA) standards were used for calibration. OMNISEC V4.7 software was used for the data acquisition and analysis. The steady state fluorescence spectra were acquired on a Shimadzu RF-5301PC fluorescence spectrophotometer, and a quartz cell of 1cm path length was used in the measurements. Transmission electron microscopy (TEM) images of the SCPNs and biohybrid vesicles were collected on a Tecnai G2 20 S-TWIN electron microscope. The TEM specimens were prepared by freeze-drying method. A carbon-coated copper grid was placed on a block of metal, precooled with liquid nitrogen, and the diluted aqueous solutions at a concentration of 0.1mg/mL was deposited on the copper grid and the liquid was frozen immediately. The frozen droplet was dried by lyophilization. The details for the preparation of the cryogenic Temperature-Transmission Electron Microscopy (cryo-TEM) specimens can be found in our previous paper.32 Imaging was performed on a TEM (FEI Tecnai G2 F20 S-Twin), which was operated at the working temperature below -175 °C. The AFM specimens were prepared by depositing aqueous solutions of the biohybrid vesicles at a concentration of 0.01mg/mL on mica discs, and observed on a Nanoscope IV atomic force microscope (Digital Instruments Inc.). The dynamic light scattering (DLS) measurements of the biohybrid vesicles were conducted on a Malvern Zetasizer Nano ZS, which is equipped with a 10 mW He–Ne laser with a wavelength of 632.8 nm. Zeta-potentials of the nanoparticles, protein molecules and hybrid vesicles were collected on a Malvern Zetasizer Nano S90. Static laser scattering (SLS) measurements of the hybrid vesicles were performed on a laser light scattering spectrometer (Brookhaven BI-200SM), which is equipped with a digital correlator (BI-9000AT) and a laser with a wavelength of 532 nm. The concentrations of the solutions in DLS and SLS measurements were 1 mg/mL. 3.

RESULTS AND DISCUSSIONS

As a proof of concept, positively charged mono-tethered SCPNs were synthesized and used as building blocks to make self-assembly with negatively charged BSA molecules (Scheme 1). The asymmetric SCPNs were prepared by intramolecular cross-linking of a linear diblock copolymer. Poly(oligo(ethylene glycol) monomethyl ether methacrylate)-block-poly(2-(dimethyl amino)ethyl methacrylate-co-(anthracene-9-


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carboxyl) ethyl methacrylate) (POEGMA-b-(PDMAEMA-co-PAnMA)) diblock copolymer was synthesized by two-step RAFT polymerization. Herein, OEGMA is a methacrylate-based macromonomer with nine pendant ethylene glycol units. It was demonstrated that POEGMA derivatives can significantly improve the efficiency of the formation of SCPNs.33,34 By using POEGMA as the macro-CTA, RAFT copolymerization of DMAEMA and AnMA leads to the synthesis of the diblock copolymer. Size exclusion chromatography (SEC) curves and 1H NMR spectra of POEGMA and POEGMA-b-(PDMAEMA-co-PAnMA) are shown in the Supporting Information, Figure S1 and S2. On the basis of 1H NMR results, the average repeating unit numbers of OEGMA, DMAEMA, and AnMA on a block copolymer chain were determined to be 46, 144, and 24, respectively. In this paper, the block copolymer is assigned as POEGMA46-b-(PDMAEMA144-co-PAnMA24). As shown in Scheme 1, anthracene photodimerization of AnMA units under UV irradiation in a dilute solution results in the formation of POEGMA-tethered SCPNs.35,36 The intramolecular anthracene photodimerization was monitored by recording the absorption of AnMA. The absorption spectra of POEGMA46-b-(PDMAEMA144-co-PAnMA24) under UV irradiation at different time intervals, as well as a standard curve of AnMA at different concentrations, are shown in the Supporting Information, Figure S3. Our calculation result indicated that 60% of AnMA units were involved in the formation of photodimers after 270 min of UV irradiation. 1H NMR spectrum of the POEGMA-tethered SCPNs is shown in the Supporting Information, Figure S4, where a peak at 5.32 ppm corresponding to the bridge-ring proton on the photodimer is observed, which demonstrates the anthracene photodimerization of AnMA units on the polymer chains.31 In the synthesis of SCPNs, intramolecular cross-linking results in reductions in hydrodynamic radius and the average apparent molecular weight due to the collapse of the linear polymer chains.37 SEC curves of the linear diblock copolymer before and after UV irradiation are shown in Figure 1a. Comparing to the linear precursor, an increase in the retention time after UV irradiation suggests a decrease in the hydrodynamic size of the block copolymer chains after intramolecular cross-linking. The absolute molecular weights (Mw), intrinsic viscosities and hydrodynamic radii of linear block copolymer and POEGMA-tethered SCPNs, as measured by SEC equipped with RI, RALS/LALS, and IV-DP detectors, were listed in the Supporting Information, Table S1. The SEC curves are shown in Figure S5, in the Supporting Information. The absolute molecular weights (Mw) of the linear block copolymer and SCNPs are both 82.4 kg/mol, demonstrating intramolecular cross-linking of the


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polymer chains under UV irradiation. It is also noted that in comparison to the linear precursor the average hydrodynamic radius and intrinsic viscosity of the SCPNs decrease as a consequence of the compact topological structure. The mono-tethered SCPNs were further characterized by TEM. Figure 1b shows a TEM image of SCPNs prepared by casting from DMF solution. To enhance the contrast between the particles and the background, the SCPNs were stained with OsO4 and PDMAEMA nano-sized domains in the particles were stained. In the TEM image, narrowly dispersed SCPNs are observed. The size analysis result in the inset of Figure 1b indicates that the average size of the SCPNs is around 10 nm.

Figure 1. (a) SEC traces of POEGMA46-b-(PDMAEMA144-co-PAnMA24) before (curve a) and after intramolecular anthracene photodimerization (curve b), (b) TEM image and size analysis of POEGMA-tethered SCPNs prepared by intramolecular anthracene photodimerization . In SEC measurements, DMF containing 1 mg/mL LiBr was used as the eluent and a refractive index detector was used.


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Figure 2. (a) TEM image and size analysis of positively charged SCPNs in aqueous solution, (b) size distribution and correlation function of positively charged SCPNs in aqueous solution measured by dynamic light scattering (DLS). The Z-average hydrodynamic diameter of the SCPNs is about 20 nm.

To prepare hydrophilic POEGMA-tethered SCPNs, PDMAEMA were quaternized with iodomethane and positively charged monotethered SCPNs were synthesized. In aqueous solution, the zeta potential of the SCPNs was measured to be 34 mV due to the positively charged quaternary ammonium salts on the surfaces. TEM image of the SCPNs prepared by casting from aqueous solution is shown in Figure 2a, where spherical core-shell structures with an average size of 17 nm are observed. Size distribution of the SCPNs is shown in the inset of Figure 2a. The SCPNs are composed of quaternized PDMAEMA (q-PDMAEMA) and hydrophobic anthracene groups/anthracene photodimers. In aqueous solution, SCPNs make nano-sized phase-separation, the hydrophilic q-PDMAEMA segments move to the surface and the hydrophobic components are left in the core part, resulting in the core-shell structures (Figure 2a). Because of


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the swelling of the q-PDMAEMA segments on the SCPNs, the average size of the POEGMA-tethered SCPNs in aqueous solution is bigger than the SCNPs in DMF. Dynamic light scattering (DLS) results of the mono-tethered SCNPs are shown in Figure 2b. The Z-average hydrodynamic diameter of the SCPNs is about 20 nm. A single relaxation processes can be observed in the correlation function of the mono-tethered SCPNs, indicating single species in aqueous solution (Figure 2b). BSA with an isoelectric point at pH 4.7 is negatively charged in neutral water, and was used as a model protein to make assembly with positively charged SCPNs. The self-assembly of positively charged POEGMA-tethered SCPNs and negatively charged BSA was performed by directly mixing of the two components in aqueous solution (Scheme 1). Fluorescence resonance energy transfer (FRET) method, which has a resolution as low as 1–10 nm, was used to demonstrate the co-assembly of the mono-tethered SCPNs and BSA. In FRET, the excitation energy absorbed by the donor is transferred to the acceptor through nonradiative dipole-dipole coupling, and the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between the the donor and the acceptor.38 The absorption of tetraiodofluorescein (TIF) overlaps the emission of fluorescein isothiocyanate (FITC),39 so the SCPNs were labeled with TIF (TIF-SCPNs) and BSA molecules were labeled with FITC (FITC-BSA). As shown in Figure 3a, under an excitation at 450 nm FITC-BSA shows strong emission spectrum at 519 nm and TIF-SCPNs present very weak fluorescence emission; however the co-assembled structures of TIF-SCPNs and FITC-BSA show a new strong emission at 553 nm, which demonstrates the energy transfer from FITC-BSA to TIF-SCPNs, and the co-assembly of the two building blocks.


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Figure 3. (a) Emission spectra of fluorescein isothiocyanate labeled BSA (FITC-BSA), tetraiodofluorescein (TIF) labeled SCPNs (TIF-SCPNs), and the assembled structures of FITC-BSA and TIF-SCPNs (TIF-SCPNs/FITC-BSA), all excited at 450 nm, (b) a TEM image and DLS curve of self-assemblies of SCPNs and BSA, (c) Berry plot of the self-assembled structures formed by BSA and SCPNs in aqueous solution, (d) atomic force microscopy (AFM) image and height profiles of hollow vesicles self-assembled by SCPNs, (e) A magnified TEM image of a typical vesicle and a carton picture showing the membrane structure of the vesicle, (f) a cryo-TEM image of assemblies formed by SCPNs and protein molecules. All the structures were assembled by SCPNs and BSA at a molar ratio of 2:1.

A TEM image and dynamic light scattering (DLS) curve of the co-assembled structures of SCPNs and BSA at a molar ratio of 2:1 are shown in Figure 3b. DLS result indicates that the hydrodynamic radii of the structures are in the range of 80 to 200 nm. TEM result demonstrates the SCPNs and BSA co-assemble into spherical structures. It is worthy to note that the spherical particles in the TEM image are almost “transparent”, which means in the TEM measurement electron beams are able to penetrate into the overlapped particles, and the particles may have hollow structures. Static light scattering measurements were employed to characterize the assemblies. The Berry plot is shown in Figure 3c, where the radius of gyration (Rg) was calculated to be around 152 nm. The ratio of the gyration radius to


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the hydrodynamic radius (Rg/Rh) was calculated to be 0.91, very close to 1.0, which implies that the mono-tethered SCPNs and BSA molecules self-assemble into hollow vesicles.40,41 An atomic force microscopy (AFM) image and height profiles of two typical particles are shown in Figure 3d. In the height profiles, the lateral dimensions are about 190, and 200 nm, respectively; however, the heights are only 19 nm, much smaller than the lateral sizes, which demonstrates the collapse of the hollow structures in the dry state. Figure 3e shows a magnified TEM image of a typical vesicle. The TEM specimen is not stained and iodine in the SCPNs enhances the contrast. As indicated by the dashed circle, the membrane of the vesicle is composed of two-layered SCPNs (dark phases) and protein layer (white phase). As illustrated by the carton picture in the insert of Figure 3e, the SCPNs located at the inner and outer surfaces of the membrane, and the protein molecules bind to the SCPNs through electrostatic interaction forming the inner wall. The assymetric structure of the POEGMA-tethered SCPNs makes it possible for the two building blocks to have the directional electrostatic interaction. Because of the steric hindrance of POEGMA chains, BSA molecules are only able to have electrostatic interaction with SCPNs on the opposite sides, which inhibits random coagulation of the two components. The co-assembly of SCPNs and protein molecules was further confirmed by cryo-transmission electron microscopy (cryoTEM). As indicated by an arrow in Figure 3f, the boundaries of the two overlapped particle can be observed, the transparency of the particles demonstrate the co-assembly of SCPNs and BSA molecules into hollow vesicles. More cryo-TEM images of the assemblies prepared at different molar ratios of SCPNs to BSA can be found in the supporting information, Figure S6.


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Figure 4. Dynamic light scattering results for the assembled structures prepared at different molar ratios of SCPNs to BSA: (a) correlation functions and (b) a plot of average hydrodynamic diameters versus molar ratio.

The sizes of the vesicles co-assembled by the mono-tethered SCPNs and BSA molecules are strongly dependent on the molar ratio of the two components. DLS results of the vesicles prepared at different molar ratios are presented in Figure 4a,b. Figure 4a compares the electric field time correlation functions g12(t) vs. time of the vesicles prepared at different molar ratios. DLS results indicate that the vesicles prepared at a molar ratio of 1:2 have the lowest decay rate, which means the vesicles have the largest sizes. A plot of the Zaverage size of the assembled structures vs. molar ratio of SCPNs to BSA is shown in Figure 4b. In the range of 4:1 to 1:2, the average size increases with the molar ratio; the average size is inversely proportional to the molar ratio in the range of 1:2 to 1:6. The largest vesicles are obtained at a molar ratio of 1:2. TEM results confirm this. Figure 5a-f show TEM images of self-assembled structures prepared at different molar ratios. As the molar ratio decreases, the average size of the vesicles increases and it reaches a maximum at 1:2. Vesicles with smaller sizes are obtained at high or low molar ratios.


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Figure 5. TEM images of the assembled structures prepared at different molar ratios: (a) 4:1, (b) 2:1, (c) 1:1, (d)1:2, (e) 1:4, (f) 1:6.

Figure 6. (a) Zeta potential values of SCPNs, BSA and vesicles prepared at different molar ratios of SCPNs to BSA, (b) esterase-like activities of BSA and hybrid vesicles prepared at a molar ratio of 2:1, and the hydrolysis of NPA in buffer solution. The concentration of NPS is 0.023 mg/mL and the esterase activity was monitored by measuring the absorbance of the 4-nitrophenolate anion at 400 nm.


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The driving force for the co-assembly is the electrostatic interaction between the positively charged POEGMA-tethered SCPNs and the negatively charged BSA molecules. The change of the surface charges on the vesicles with the molar ratio was monitored. Figure 6a shows zeta potential (ξ) values of SCNPs, BSA and the biohybrid vesicles assembled at different molar ratios. Vesicles prepared at low or high molar ratios are highly charged, but the vesicles fabricated at a molar ratio of 1:2 are almost neutral. Based on DLS, TEM and the zeta potential measurements, it can be concluded that the sizes of the vesicles are closely related to the surface charges, small vesicles are highly charged and large vesicles are lowly charged. The self-assembly of nanoparticles into colloidal superparticles is strongly dependent on the interparticle interactions, the attractive electrostatic interaction and the repulsive electrostatic interaction.39,42,43 In the vesicles fabricated at a molar ratio of 1:2, the attractive electrostatic interactions between SCPNs and BSA and the repulsive electrostatic interactions among SCPNs or BSA molecules keep balance, and the surface tension of the vesicle membrane is the lowest, so the vesicles with the largest size are obtained. In order to demonstrate this, a control experiment was performed. The colloidal solution of the structures prepared at at a molar ratio of 1:2 was stirred at high speed, and a TEM specimen was prepared by freeze-drying method. TEM result (Figure S7 in the Supporting Information) demonstrates that the spherical vesicles are deformed under high-speed stirring due to the low surface tension of the membranes, and distorted ellipsoids and cylinders are observed in the image. In contrast, the morphology of the vesicles prepared at 4:1 keeps unchanged. BSA displays esterase-like activity toward hydrolysis of NPA, and 4-nitrophenol is produced.44,45 Previous research demonstrated that morphology of the assemblies based on electrostatic interaction may be affected by the organic solvent in the solution.46 In order to avoid the possible effect of NPA on the morphology of the assemblies, the concentration of NPA is controlled at low level. The activities of native BSA and biohybrid vesicles were compared and the results are shown in Figure 6b. The activity of native BSA was set as 100% and the relative activity of BSA in the vesicles was calculated by measuring the absorbance of the 4-nitrophenolate anion at 400 nm. After


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2 h of hydrolysis, the esterase-like activity of BSA in the vesicles was measured to be about 84.4%, which means BSA molecules in the assemblies maintain most of the activities. 4.

CONCLUSIONS In conclusion, positively charged POEGMA-tethered SCPNs were prepared by intramolecular anthracene photodimerization and

quaternization of PDMAEMA. In aqueous solution, the SCPNs have core-shell structures with positive charges on the surfaces. Negatively charged BSA molecules can make co-assembly with the SCPNs into biohybrid vesicles. The average size of the vesicles is strongly dependent on the molar ratio of SCPNs to BSA. The largest vesicles can be prepared at a molar ratio of 1:2. Comparing to the native BSA, BSA molecules in the hybrid vesicles maintain 84.4% of the activity in the hydrolysis of NPA. The biohybrid vesicles can be used as an efficient platform for protein carriers.

ASSOCIATED CONTENT Supporting Information. Materials and characterization, 1H NMR spectra and SEC curves of SCPNs, block copolymer and its precursor, preparation of positively charged SCPNs and self-assembly with BSA, UV−vis spectrum of the absorptions of AnMA at different concentrations and block copolymer under UV irradiation, Cryo-TEM images of assemblies at different molar ratios, TEM image of assemblies at a molar ratio of 1:2 after stirring at high speed.

AUTHOR INFORMATION Corresponding Author


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* Hanying Zhao, Email: [email protected] ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (NSFC, 51673098 and 51473079), and technical support of Dr. Zhijie Zhang in Malvern Instruments Ltd was greatly appreciated. REFERENCES (1). Whitesides, G. M.; Grzybowski, B. Self-Assembly at All Scales. Science 2002, 295, 2418-2421. (2) Zhang, W.; Yu, X.; Wang, C. L.; Sun, H. J.; Hsieh, I. F.; Li, Y.; Dong, X. H.; Yue, K.; Horn, R. V.; Cheng, S. Z. D. Molecular Nanoparticles Are Unique Elements for Macromolecular Science: From “Nanoatoms” to Giant Molecules. Macromolecules 2014, 47, 12211239. (3) Hanlon, A. M.; Lyon, C. K.; Berda, E. B. What Is Next in Single-Chain Nanoparticles? Macromolecules 2016, 49, 2-14. (4) Gonzalez-Burgos, M.; Latorre-Sanchez, A.; Pomposo, J. A. Advances in Single Chain Technology. Chem. Soc. Rev. 2015, 44, 6122-6142. (5) Harth, E.; Van Horn, B.; Lee, V. Y.; Germack, D. S.; Gonzales, C. P.; Miller, R. D.; Hawker, C. J. A Facile Approach to Architecturally Defined Nanoparticles via Intramolecular Chain Collapse. J. Am. Chem. Soc. 2002, 124, 8653-8660. (6) Mecerreyes, D.; Lee, V.; Hawker, C. J.; Hedrick, J. L.; Wursch, A.; Volksen, W.; Magbitang, T.; Huang, E.; Miller, R. D. A Novel Approach to Functionalized Nanoparticles: Self-Crosslinking of Macromolecules in Ultradilute Solution. Adv. Mater. 2001, 13, 204−208. (7) Croce, T. A.; Hamilton, S. K.; Chen, M. L.; Muchalski, H.; Harth, E. Alternative o-Quinodimethane Cross-Linking Precursors for Intramolecular Chain Collapse Nanoparticles. Macromolecules 2007, 40, 6028-6031.


20

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(8) Cui, Z.; Cao, H.; Ding, Y.; Gao, P.; Lu, X.; Cai, Y. Compartmentalization of ABC Triblock Copolymer Single-Chain Nanoparticle via Coordination-Driven Orthogonal Self-Assembly Polym. Chem. 2017, 8, 3755-3763. (9) Perez-Baena, I.; Asenjo-Sanz, I.; Arbe, A.; Moreno, A. J.; Lo Verso, F.; Colmenero, J.; Pomposo, J. A. Efficient Route to Compact SingleChain Nanoparticles: Photoactivated Synthesis via Thiol–Yne Coupling Reaction. Macromolecules 2014, 47, 8270-8280. (10) Zhang, Y.; Zhao, H. Surface-Tunable Colloidal Particles Stabilized by Mono-Tethered Single-Chain Nanoparticles. Polymer 2015, 64, 277-284. (11) Zhou, F.; Xie, M.; Chen, D. Structure and Ultrasonic Sensitivity of the Superparticles Formed by Self-Assembly of Single Chain Janus Nanoparticles. Macromolecules 2014, 47, 365-372. (12) Wen, J.; Yuan, L.; Yang, Y.; Liu, L.; Zhao, H. Self-Assembly of Monotethered Single-Chain Nanoparticle Shape Amphiphiles. ACS Macro. Lett. 2013, 2, 100-106. (13) Li, W.; Kuo, C. H.; Kanyo, I.; Thanneeru, S.; He, J. Synthesis and Self-Assembly of Amphiphilic Hybrid Nano Building Blocks via SelfCollapse of Polymer Single Chains. Macromolecules 2014, 47, 5932-5941. (14) Wen, J.; Zhang, J.; Zhang, Y.; Yang, Y.; Zhao, H. Controlled Self-Assembly of Amphiphilic Monotailed Single-Chain Nanoparticles. Polym. Chem. 2014, 5, 4032–4038. (15) Zhang, Y.; Zhao, H. Surfactant Behavior of Amphiphilic Polymer-Tethered Nanoparticles. Langmuir 2016, 32, 3567-3579. (16) Mavila, S.; Eivgi, O.; Berkovich, I.; Lemcoff, N. G. Intramolecular Cross-Linking Methodologies for the Synthesis of Polymer Nanoparticles. Chem. Rev. 2016, 116, 878-961.


21

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Page 22 of 26

(17) Perez-Baena, I.; Barroso-Bujans, F.; Gasser, U.; Arbe, A.; Moreno, A. J.; Colmenero, J.; Pomposo, J. A. Endowing Single-Chain Polymer Nanoparticles with Enzyme-Mimetic Activity. ACS Macro. Lett. 2013, 2, 775-779. (18) Lyon, C. K.; Prasher, A.; Hanlon, A. M.; Tuten, B. T.; Tooley, C. A.; Frank, P. G.; Berda, E. B. A brief User's Guide to Single-Chain Nanoparticles. Polym. Chem. 2015, 6, 181-197. (19) Hanlon, A. M.; Chen, R.; Rodriguez, K. J.; Willis, C.; Dickinson, J. G.; Cashman, M.; Berda, E. B. Scalable Synthesis of Single-Chain Nanoparticles under Mild Conditions. Macromolecules 2017, 50, 2996–3003. (20) Knöfel, N. D.; Rothfuss, H.; Willenbacher, J.; Barner-kowollik, C.; Roesky, P. W. Platinum (II)-Crosslinked Single-Chain Nanoparticles: An Approach towards Recyclable Homogeneous Catalysts. Angew. Chem. Int. Ed. 2017, 56, 4950–4954. (21) Huerta, E.; van Genabeek, B.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A. A Modular Approach to Introduce Function into SingleChain Polymeric Nanoparticles. Macromol. Rapid Commun. 2014, 35, 1320-1325. (22) Zhang, S.; Pang, X.; Guo, D.; Zheng, B.; Cui, S.; Ma, H. Single-Chain Polymers Achieved from Radical Polymerization under SingleInitiator Conditions. Langmuir 2012, 28, 14954−14959. (23) Liang, J.; Struckhoff, J. J.; Hamilton, P. D.; Ravi, N. Preparation and Characterization of Biomimetic β‑Lens Crystallins Using SingleChain Polymeric Nanoparticles. Langmuir 2017, 33, 7660−7668. (24) Njikang, G.; Liu, G.; Hong, L. Chiral Imprinting of Diblock Copolymer Single-Chain Particles. Langmuir 2011, 27, 7176–7184. (25) Latorre-Sánchez, A.; Pomposo, J. A. Recent Bioinspired Applications of Single-Chain Nanoparticles. Polym. Int. 2016, 65, 855-860.


22

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(26) Bai, Y.; Feng, X.; Xing, H.; Xu, Y.; Kim, B. K.; Baig, N.; Zhou, T.; Gerwirth, A. A.; Lu, Y.; Oldfield, E.; Zimmerman, S. C. J. Am. Chem. Soc. 2016, 138, 11077-11080. (27) Terashima, T.; Mes, T.; De Greef, T. F. A.; Gillissen, M. A. J.; Besenius, P.; Palmans, A. R. A.; Meijer, E. W. Single-Chain Folding of Polymers for Catalytic Systems in Water. J. Am. Chem. Soc. 2011, 133, 4742-4745. (28) Chan, D.; Yu, A. C.; Appel, E. A. Single-Chain Polymeric Nanocarriers: A Platform for Determining Structure–Function Correlations in the Delivery of Molecular Cargo. Biomacromolecules 2017, 18, 1434-1439. (29) Artar, M.; Souren, E. R. J.; Terashima, T.; Meijer, E. W.; Palmans, A. R. A. Single Chain Polymeric Nanoparticles as Selective Hydrophobic Reaction Spaces in Water. ACS Macro. Lett. 2015, 4, 1099-1103. (30) Liu, Y.; Pujals, S.; Stals, P. J. M.; Paulöhr, T.; Presolski, S. I.; Meijer, E. W.; Albertazzi, L.; Palmans, A. R. A. Catalytically Active SingleChain Polymeric Nanoparticles: Exploring Their Functions in Complex Biological Media. J. Am. Chem. Soc. 2018, 140, 3423–3433. (31) Ji, X.; Zhang, Y.; Zhao, H. Amphiphilic Janus Twin Single-Chain Nanoparticles. Chem. Eur. J. 2018, 24, 3005- 3012. (32) Tao, Y.; Ma, X.; Cai, Y.; Liu, L.; Zhao, H. Coassembly of Lysozyme and Amphiphilic Biomolecules Driven by Unimer−Aggregate Equilibrium. J. Phys. Chem. B 2018, 122, 3900-3907. (33) Terashima, T.; Sugita, T.; Fukae, K.; Sawamoto, M. Synthesis and Single-Chain Folding of Amphiphilic Random Copolymers in Water. Macromolecules 2014, 47, 589−600. (34) Wong, E. H. H.; Lam, S. J.; Nam, E.; Qiao, G. G. Biocompatible Single-Chain Polymeric Nanoparticles via Organo-Catalyzed RingOpening Polymerization. Acs Macro. Lett. 2014, 3, 524−528.


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Page 24 of 26

(35) Frank, P. G.; Tuten, B. T.; Prasher, A.; Chao, D.; Berda, E. B. Intra-Chain Photodimerization of Pendant Anthracene Units as an Efficient Route to Single-Chain Nanoparticle Fabrication. Macromol. Rapid Commun. 2014, 35, 249–253. (36) Tian, J.; Liu, G.; Guan, C.; Zhao, H. Amphiphilic Gold Nanoparticles Formed at a Liquid–Liquid Interface and Fabrication of Hybrid Nanocapsules Based on Interfacial UV Photodimerization. Polym. Chem. 2013, 4, 1913–1920. (37) Pomposo, J. A.; Perez-Baena, I.; Buruaga, L.; Alegría, A.; Moreno, A. J.; Colmenero, J. On the Apparent SEC Molecular Weight and Polydispersity Reduction Upon Intramolecular Collapse of Polydisperse Chains to Unimolecular Nanoparticles. Macromolecules 2011, 44, 8644−8649. (38) Förster, T. 10th Spiers Memorial Lecture. Transfer Mechanisms of Electronic Excitation. Discuss. Faraday Soc. 1959, 27, 7–17. (39) Fan, W.; Liu, L. Zhao, H. Co-Assembly of Patchy Polymeric Micelles and Protein Molecules. Angew. Chem. Int. Ed. 2017, 56, 8844 – 8848. (40) Xie, D.; Jiang, M.; Zhang, G.; Chen, D. Hydrogen-Bonded Dendronized Polymers and Their Self-Assembly in Solution. Chem. Eur. J. 2007, 13, 3346–3353. (41) Khougaz, K.; Astafieva, I.; Eisenberg, A. Micellization in Block Polyelectrolyte Solutions. 3. Static Light Scattering Characterization. Macromolecules 1995, 28, 7135–7147. (42) Wang, T.; LaMontagne, D.; Lynch, J.; Zhuang, J.; Cao, Y. C. Colloidal Superparticles from Nanoparticle Assembly. Chem. Soc. Rev. 2013, 42, 2804–2823. (43) Maye, M. M.; Luo, J.; Lim, I. S.; Han, L.; Kariuki, N. N.; Rabinovich, D.; Liu, T.; Zhong, C. Size-Controlled Assembly of Gold Nanoparticles Induced by a Tridentate Thioether Ligand. J. Am. Chem. Soc. 2003, 125, 9906–9907.


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(44) Means, G. E.; Bender, M. L. Acetylation of Human Serum Albumin by p-Nitrophenyl Acetate. Biochemistry 1975, 14, 4989–4994. (45) Yang, X.; Chen, D.; Zhao, H. Silica Particles with Immobilized Protein Molecules and Polymer Brushes. Acta Biomater 2016, 29, 446– 454. (46) Ding, Y.; Cai, M.; Cui, Z.; Huang, L.; Wang, L.; Lu, X.; Cai, Y. Synthesis of Low-Dimensional Polyion Complex Nanomaterials via Polymerization-Induced Electrostatic Self-Assembly. Angew. Chem. Int. Ed. 2018, 57, 1053 –1056.

For TOC Only

Bio-Assemblies Fabricated by Co-Assembly of Protein Molecules and Mono-Tethered Single-Chain Polymeric Nanoparticles Qi Liu, Yuanyuan Ju and Hanying Zhao


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Bioassemblies Fabricated by Coassembly of Protein Molecules and

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