Filamentous Viruses Grafted with Thermoresponsive Block Polymers

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

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Filamentous Viruses Grafted with Thermoresponsive Block Polymers: Liquid Crystal Behaviors of a Rodlike Colloidal Model with “True” Attractive Interactions Shuaiyu Liu,† Chunxiong Zheng,† Zihan Ye,† Baptiste Blanc,‡ Xueli Zhi,† Linqi Shi,† and Zhenkun Zhang*,† †

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Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 300071 Tianjin, China ‡ Department of Physics, Brandeis University, Waltham, Massachusetts 02453, United States S Supporting Information *

ABSTRACT: Understanding how attractive interactions among rigid polymers or rodlike particles influence their liquid crystal (LC) phase behavior is of fundamental and practical importance. This question has not been fully answered yet, mainly due to the shortage of model systems with “true” pairwise attractions on a single particle level while with excellent colloidal stability. Herein, we report on a well-defined rodlike system that fulfills such criteria, through covalently grafting the free end of the thermoresponsive PNIPAM block of poly(ethylene glycol)-block-poly(N-isopropylacrylamide) (PEG-b-PNIPAM) onto the classic rodlike model systemthe fd or M13 viruswhich is the hallmark in understanding the LC behaviors of rigid polymer or rodlike particles. Increasing temperature induces dehydration and collapse of the PNIPAM chains onto the virus surface and therefore introduces attractions among the viruses, while the outer hydrophilic PEG block offers steric stabilization to prevent interparticle aggregation, gelation, or other dynamically arrested states. The influence of the temperature, and consequently of the attraction strength between rodlike particles, on the LC phase behaviors of hard rodlike particles was thoroughly investigated via a forced phase separation assisted by a low-speed centrifuge, leading to an apparent phase diagram in the space of attractive strength and isotropic−nematic coexisting LC phases. At T ≪ LCST, the block polymers are in the fully hydrophilic state and the rodlike system behaves as hard rods. Its LC behaviors can be quantitatively described by the flexibility-corrected Onsager’s hard rod theory. Although the forced phase separation is not truly in phase equilibrium, increasing temperature to induce the collapse of the PNIPAM blocks does lead to theoretically predicted widening of the isotropic−nematic coexisting concentrations with increasing temperature. Fitting our experimental data with advanced theories reveals several physical parameters that probably characterize the LC phase of rigid polymers or rod systems with attractive interactions in general.

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materials.6,15,20 For instance, there have been great efforts to disperse carbon nanotubes into the LC state to transform such precious nanomaterials into macroscopic foams or fibers with controlled internal structures.15 In addition, intensive attention has recently been paid to direct drying of the LC phase of the CNCs into films, which can be further used as one-dimensional photonic materials and templates for inorganic materials with chiral internal nanopores.6,22−24 Similarly, dispersing rigid conjugated polymers into the LC state is a promising means to process them into photovoltaic materials.12 Furthermore, LC behaviors of amyloid fibrils from abnormal aggregation of certain proteins have been extensively investigated recently due to their relevance to many neurodegenerative disorders.21,25−27

INTRODUCTION Rigid polymers in solvents or rodlike particles in the suspension state can form lyotropic liquid crystals (LLCs) above certain concentrations.1−10 Early observations of such phenomena dated back to many decades ago, followed by theoretical predictions and numerous experimental works on various kinds of rigid polymers or rodlike particles. The recent renaissance of interest in lyotropic LCs of such polymers or particles has been stimulated by successful preparation of a wide spectrum of rigid polymers or anisotropic nanocolloids, such as cylindrical polymer brushes,11 conjugated polymers,12 semiconductor or metallic nanorods,13,14 carbon nanotubes,3,15,16 cellulose nanocrystals (CNC),6 rodlike DNA origami,17 nanofilaments from supramolecular assembly,18−21 etc. The LC states of such novel polymers or rodlike particles offer an excellent means to facilitate their self-assembly into higher level structures and to process them into functional © XXXX American Chemical Society

Received: March 30, 2018 Revised: September 22, 2018

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DOI: 10.1021/acs.macromol.8b00674 Macromolecules XXXX, XXX, XXX−XXX

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polymer−rod segregation leads to other exotic superstructures like colloidal membranes, smectic stacks, etc.52−54 Therefore, model systems with tunable pairwise attractions on the single particle level that avoid gelation or the phase segregations in the case of depletion induced attractions has yet to be developed. In the current work, through combining filamentous viruses with thermoresponsive block polymers, we aim to prepare a well-defined rodlike system that has “true” pairwise attractions on the single particle level while can be colloidally stabilized (Scheme 1). Because of their precious size monodispersity, well-defined surface properties, and large aspect ratio that fulfills the criterion of Onsager’s theory, rodlike viruses such as the fd, M13, and their mutants have been extensively used to test Onsager’s theory in the past decades.1,31,32,55 These viruses mainly consist of proteins that have a refractive index similar to water. Therefore, there are negligible van der Waals attractions among viruses in aqueous suspensions. By treating the electrostatic double layer (EDL) of the virus with an effective diameter, or grafting with hydrophilic polymers, the fd or M13 virus can be essentially regarded as hard rods that interact with each other via excluded volume of Onsager’s style.32,56,57 After taking into account of semiflexibility of the viruses, excellent consistency has been found between the experimental LC phase behaviors of the viruses and Onsager’s hard rod theory.32 Therefore, it is safe to say that the rodlike fd or M13 virus represents the best rodlike model that can be used to test many fundamental problems of LC phase of rod particles.31 Inspired by such unique properties of these rodlike viruses and their performance in the fundamental research of the LC, we shall here introduce tunable attractions between these rods by grafting a thermosensitive block polymer to the virus surface. The molecularly well-defined block copolymer poly(ethylene glycol)-block-poly(N-isopropylacrylamide) (PEG-b-PNIPAM) is designed, and the free end of the thermoresponsive PNIPAM block was chemically tethered onto the virus surface (Scheme 1). Increasing temperature to the lower critical solution temperature (LCST) induces dehydration and collapse of the PNIPAM block onto the virus surface. Therefore, attraction among the viruses is introduced by hydrophobic interactions of the dehydrated PNIPAM block, while the PEG block offers steric stabilization to prevent interparticle aggregation. The strength of attractive interactions can be tuned by varying either the temperature around the LCST of PNIPAM or the molecular weight of the PNIPAM blocks. Under the condition where the rodlike virus backbone can be treated as the hard rod, the liquid phase behavior of rodlike particles with attractions can be investigated for the first time.

From a fundamental research point of view, Onsager and Flory have derived seminal theories for rodlike colloidal particles and rigid polymers, respectively.28,29 Especially, based on hard rod models that only interact with each other via excluded volumes, Onsager’s theory can quantitatively predict the isotropic−nematic LC phase transition of rodlike particles and have been confirmed with simulations and experimental model systems.30−32 However, either Onsager’s or Flory’s theory focuses on idealized systems with highly simplified interparticle interactions that barely exist in reality. Complicated interactions between real particles or rigid polymers are always haunting for discrepancies between theoretical predictions and experimental observations.33,34 Especially, attractive interactions of various natures often dominate interactions of the aforementioned carbon nanotubes, rigid conjugated polymers, amyloid fibrils, and so on.25,34−36 Although unique material properties are observed on a single particle level of these precious particles, strong interparticle attractions lead to agglomeration or gelation with increasing particle concentrations. Such behaviors severely hinder the LC-based processing of them into macroscopic materials and delay their practical applications. Interparticle attraction is also believed to stand behind the concentration-dependent competition of in vivo gelation and LC formation of amyloid fibrils of proteins, which is elusive to be understood at the moment and might relate to the origin of neurodegenerative disorder diseases.25 Therefore, understanding the LC behavior of rodlike particles or rigid polymers with attractive interparticle interactions based on well-defined model systems is not only fundamentally important but also has very important practical relevance. Inspired by the abnormal LC behaviors of rodlike particles with attractive interactions, various kinds of theories dealing with such situations have appeared and been tested with simulations, leading to a consensus that the isotropic (I)−nematic (N) coexisting concentrations in the phase diagram will widen with increasing attractive strength: i.e., the number density of the rod in the isotropic liquid phase (pi) will decrease while that of the nematic LC phase (pn) will increase.37−41 Compared to such theoretical progress, experimental investigations of real systems with attractive interactions have lagged far behind, mainly due to the shortage of well-defined system with controllable attractive interactions. Early efforts have been dedicated to some systems like poly(γ-benzyl L-glutamate) (PBLG), rodlike mineral particles, and so on, which have attractive interactions of unknown origins.42−44 However, the attraction in these cases is often uncontrollable and leads to gelation or other dynamically arrested states that defies systematic and unambiguous investigation of the equilibrium phase separation. Grafting rodlike systems with only thermoresponsive polymers to introduce attractions have encountered the same situations as the systems often form reversible gels upon heating to induce attractions.35,45−48 Instead of direct pairwise interparticle attractions, another kind of effective attraction can be realized by depletion effects.49−51 Through adding nonadsorptive polymers to suspensions of rodlike particles, Dogic and Fraden have observed widening of the I−N transition, together with abnormal phase behaviors and large discrepancy when comparing with theoretical predictions.50 This is due to strong partitioning of the added polymer (the depletant) into the two I−N phase, and therefore the rods in each phase experience different effective attractions. In addition, the strength of attraction can only be tuned by increasing the polymer concentration to certain ranges, above which strong

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EXPERIMENTAL SECTION

Materials. Methoxy poly(ethylene glycol) (PEG-OH) with a molecular weight of 5K and 2K were supplied by Sigma-Aldrich. The salts such as Trima and sodium phosphate for preparing the buffer solution were obtained from Dingguo Biotechnology Co. LTD (Beijing, China). Other chemicals such as N-isopropylacrylamide (NIPAM), 2,2′-azobisisobutyronitrile (AIBN), N-acryloxysuccinimide, dimethylphenylphosphine (DMPP), and n-hexylamine were obtained from J&K Scientific (Beijing, China) and used without further purification. Solvents were supplied by local suppliers. The standard drying procedure of dioxane, THF, and diethyl ether was further performed to make sure the solvents are anhydrous. Ultrapure water from a Milli-Q UltraPure system (18.2 mΩ·cm) (Millipore) was always used. The fd or M13 virus was grown and purified following the standard biochemical protocol using the ER2738 strain of E. coli as the host bacteria. B


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Scheme 1. Schematic Illustration of the Synthesis Procedure of Poly(ethylene glycol)-block-poly(N-isopropylacrylamide) (PEG-b-PNIPAM) with an N-Hydroxysuccinimide (NHS) Ester Group at the End of the PNIPAM Block (A) and Grafting of the Block Polymers onto the Surface of the Virus (B)

Synthesis of PEG-b-PNIPAM End-Functionalized with N-Hydroxysuccinimide (NHS) Ester. We followed the general procedure of refs 58 and 59 to prepare the PEG-b-PNIPAM via RAFT polymerization. This procedure started with the synthesis of the macro chain transfer agent (CTA)-PEG end-functionalized with a dithiobenzoyl group (PEG-CTA, see the Supporting Information for the detailed procedure). With PEG-CTA, the typical RAFT procedure for synthesis of PEG5k-b-PNIPAM3k was the following: PEG5k-CTA (0.50 g, 0.1 mmol), NIPAM (0.30 g, 2.60 mmol), and AIBN (5.00 mg, 0.03 mmol) as the initiator were charged into a Schlenk flask that was connected to a vacuum system. Anhydrous dioxane was used as the solvent for RAFT polymerization. The solution was degassed by three freezing−thaw cycles and finally sealed under nitrogen. The polymerization was performed at 65 °C under constant stirring for 24 h. The reaction mixture was poured into diethyl ether, and the precipitates were collected by vacuum filtration. The PEG5k-b-PNIPAM3k was collected as pink solids after drying in a vacuum oven at 40 °C. The molar ratio of PEG-CTA, NIPAM, and AIBN for synthesis of PEG5kb-PNIPAM8k, PEG2k-b-PNIPAM3k, and PEG2k-b-PNIPAM8k was 1:71:0.3, 1:26:0.3, and 1:71:0.3, respectively. Aminolysis was used to transfer the dithiobenzoate group of the afore-synthesized PEG-b-PNIPAM into the −SH group.60,61 For this, PEG-b-PNIPAM and anhydrous THF were charged under nitrogen into a Schlenk flask with a magnetic stir bar. DMPP and n-hexylamine were then added under nitrogen. The molar ratio of PEG-b-PNIPAM, DMPP, and hexylamine was 1:1.2:10. The mixture was degassed by three freeze−thaw cycles and finally sealed under nitrogen. The mixture was brought to 25 °C and kept at this temperature for 2 h. The polymer was poured into diethyl ether, and the precipitate was collected by filtration and dried under vacuum at 30 °C overnight. The thiol-terminated PEG-b-PNIPAM (PEG-b-PNIPAM-SH) was obtained as a white powder. Finally, the thiol−ene Michael addition reaction of PEG-bPNIPAM-SH with N-acryloxysuccinimide was performed to functionalize the free end of PNIPAM with NHS group (PEG-b-PNIPAMNHS). PEG-b-PNIPAM-SH and N-acryloxysuccinimide with a molar ratio of 1:10 was dissolved in anhydrous THF in a Schlenk flask. Under nitrogen atmosphere, triethylamine was added. The mixture was degassed by three freeze−thaw cycles and finally sealed under nitrogen. The reaction was performed at 25 °C for 24 h under

constant stirring. The product was collected by precipitation in diethyl ether, vacuum filtration, and drying in a vacuum oven. The NHS endfunctionalized PEG-b-PNIPAM was stored under −20 °C to prevent hydrolysis of the NHS group. All the polymers from each step were characterized by 1H NMR spectroscopy and GPC (Figures S1−S6 in the Supporting Information). The thermoresponsive behavior of PEG-b-PNIPAM was investigated by light scattering.62 All the results are listed in the Supporting Information. Grafting the fd Virus with PEG-b-PNIPAM. The fd virus was dispersed in phosphate buffer (100 mM, pH 8.0) to form suspensions with a concentration of 2 mg mL−1 and cooled in a water−ice bath (with a typical temperature around 4 °C). PEG-b-PNIPAM-NHS (500 mg) was dissolved in 200 μL of anhydrous DMF, and the resulted solution was added dropwise to 5 mL of the virus suspension under stirring. The mixture was further stirred in the water−ice bath for 1 h and then stored at room temperature overnight. To remove the excess polymer, the polymer grafted virus was spun down by an ultracentrifuge at RCF = 260000g for about 6 h and then dispersed in a phosphate buffer. Such a procedure was repeated at least three times. Finally, the polymer-grafted virus in phosphate buffer was dialyzed against phosphate buffer at 4 °C for 96 h, with the dialysis buffer changed every 12 h. After this extensive purification, PEG-bPNIPAM grafted viruses were transferred into Tris-HCl buffer (ionic strength = 110 mM, pH 8.2) by dialysis. SDS-PAGE of the polymer grafted virus was used to qualitatively confirm the successful grafting (see the Supporting Information). The number of polymers grafted onto each virus was estimated by measuring the difference of the specific refractive index increment of the pristine and polymer grafted viruses, as reported in our previous work.63 Temperature-Dependent Solution Behaviors of PEG-b-PNIPAM Grafted Rodlike Viruses. The main goal is to check whether the suspension of PEG-b-PNIPAM grafted rodlike viruses is in the sol or gel state upon increasing temperature. A system consisting of a refrigerated water bath (Haake A28) and circulator (Haake SC 100) (Thermo Fisher, USA) with a temperature accuracy of ±0.02 °C and the capability of fast heating and cooling was always used for the temperature control. Suspensions of viruses or polymer grafted viruses were put into thin-wall cylindrical glass vials of 1 mL volume. The glass vials were equilibrated at each temperature at least 60 min before any C


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Cryo-Transmission Electron Microscopy (TEM) of PolymerGrafted Virus in the Sol or Gel State. The following procedure was used to prepare the virus sample for the cryo-TEM to avoid influence of flow induced ordering of the virus inside the sample. Virus suspensions was prepared as 3 mg mL−1 in phosphate buffer (pH = 8.0 100 mM), 15 μL of which was taken and deposited onto a lacey copper grid. The excess solution was blotted with a filter paper. The grid with a layer of the virus suspension was placed inside a climate chamber at room temperature for 10 min. The evaporation of the solvent was prevented by keeping the relative humidity close to saturation (100%). The temperature of the climate chamber was then increased to 40 °C for 2 min. After this, the grid was plunged into a bath of liquid ethane cooled with liquid nitrogen. With a Cryoholder 626 (Gatan), samples were kept at a temperature of −170 °C and were observed with a JEOL 2200FS electron microscope at a nominal magnification of 40000 under low-dose conditions. The TEM operates at 200 kV and is equipped with an omega energy filter. Instruments. The molecular weight of polymers was determined on a Waters 1525/2414 gel permeation chromatograph (GPC), and polystyrene was used as the standard and THF as the mobile phase. 1 H NMR spectra was recorded on an AVANCE III 400 MHz spectrometer (Bruker). The concentration of the virus was determined with a UV−vis 2550 UV−vis spectrometer (Shimadzu). Centrifugation and ultracentrifugation were respectively performed on an Allegra X15R benchtop centrifuge and Optima L-90K ultracentrifuge (Beckman Coulter, USA). The atomic force microscopy (AFM) images were obtained with an etched silicon tip (RTESP, Bruker; spring constant k = 40 N/m; frequency f 0 = 300 kHz) in the tapping mode on a NanoScopeR IIIa (Veeco) AFM system. Image processing was performed with NanoScope 5.31r1 software. The liquid crystal behavior of the virus suspension was observed with an Olympus BX53P polarized optical microscope (POM). LC MS was performed with an Agilent 6520 Q-TOF LC/MS instrument. Main Conclusions of Theories about Liquid Crystal Behaviors of Rodlike Particles. We summarize some basic facts about the well-established I−N LC phase equilibrium of rodlike particles, which is characterized by the coexistence of an isotropic liquid phase (I) with an anisotropic nematic LC phase (N).32 The volume fractions (ϕ) of rods in each phase (ϕI and ϕN) are different. The classic Onsager’s hard rod theory in the limit of infinitely long rigid rods predicts the following relations for rodlike hard particles: ϕI = 3.3 (Deff/L) and ϕN = 4.2 (Deff/L), where Deff and L are the effective diameter and contour length of the particles, respectively. The fd or M13 virus is a semiflexible rod with a persistence length (P) of 2.2 μm. Khokhlov and Semenov extended Onsager’s theory to include semiflexible rods with arbitrary values of P (KB theory).65 Later on, Chen performed an accurate numerical calculation of the KB theory.66 For the fd virus with a contour length (L) of 880 nm and flexibility of L/P ∼ 0.36, Fraden and Grelet derived and experimentally confirmed the following quantitative equations for the volume fraction or the concentration in mg mL−1 of the rod at the I−N LC transition based on Chen’s calculation of the KB theory:32,56

observation. The birefringence of the samples was checked between a homemade cross-polarizer with a plane white light source. Gelation was assessed by the inverted tube method. For this, 100 μL of the virus suspension was placed into the capped thin-wall cylindrical glass vials with an inner diameter of 5 mm, and the vials was inverted upside down at each temperature. A complete gelation is achieved if the suspension stops flow and keeps its shape for more than 30 min. Temperature-Dependent Liquid Crystal Behaviors of PEG-bPNIPAM Grafted Rodlike Viruses. The lowest virus concentration (C*), at which the nematic LC phase becomes unstable and the suspensions start to turn into the isotropic liquid phase, was determined by diluting a concentrated virus suspension. For this, each kind of the polymergrafted viruses was suspended in Tris-HCl buffer at room temperature to form suspensions with a virus concenration of 30 mg mL−1. At such concentration and room temperature, all of the suspensions were in a fully developed nematic LC phase. The suspension was diluted with Tris buffer that was at the same temperature as the virus suspension. Each time, 5 μL of buffer was added, and the suspension was vortexed thoroughly and then incubated under the water bath with preset temperature for at least 30 min. When approaching the critical point, the suspensions was also checked by a polarized optical microscope with a 50× objective to make sure there is no any nematic LC droplet that could not be discerned by the naked eye. The virus concentration was then determined as the C*. Temperature-Dependent Isotropic Liquid (I)−Nematic (N) LC Phase Equilibrium of PEG-b-PNIPAM Grafted Rodlike Viruses. Here only the system of virus grafted with PEG5k-b-PNIPAM3k (fd-N3E5) was investigated. The I−N LC phase equilibrium state is usually achieved by phase separation of a suspension into two coexisting LC phases: one is the isotropic liquid phase that appears as dark between the crossed polarizers, and the other is the colorful nematic LC phase. Autonomous phase separation by long-term static storing of a suspension of fd-N3E5 with a concentration that close to the I−N transition was not successful. Besides, long-term storing leads to bacterial growth and solvent evaporation, especially at high temperature, which can influence the LC phase behavior. To avoid such influences, the following procedure was used to speed up the I−N phase separation. Suspensions of fd-N3E5 with a concentration of 30 mg mL−1 were diluted with buffer at the same temperature to a point that many tiny nematic droplets were dispersed in a bulk suspension. A low-speed centrifuge (RCF = 100g) was applied for few minutes to spin down the nematic droplets into a continuous phase.64 The system was remixed by vortex to redisperse the continuous nematic phase into even smaller droplets to speed up the exchange of rodlike particles between the tiny nematic droplets and the bulk isotropic phase. After storing for at least 36 h, the system was subjected to the same lowspeed centrifuge. The final system was further incubated at each temperature for 3 h, during which the virus concentration of each phase was measured. This procedure was continued for several weeks until that the variation of the virus concentration in each phase is within experimental error. Light Scattering (LS) Characterization. Polymer grafted viruses were suspended in 100 mM phosphate buffer to form suspensions of 0.005 mg mL−1. Dusts were removed by filtering the suspension through a 0.45 μm Millipore syringe filter into precleaned dust free vials for LS. Centrifuging at a speed of 1000g for 3 h was performed to further exclude dusts. The LS measurements was performed using an ALV/CGS-3 goniometer (ALV-Alangen, Germany) equipped with an ALV/LSE-5003 multiple-τ digital correlator and a JDS-Uniphase solid-state He−Ne laser with an output power of ca. 22 mW and an operating wavelength of 632.8 nm. The LS measurements were performed at a scattering angle of 20°. The temperature was controlled by the above Haake A28/SC 100 system with a temperature accuracy of ±0.02 °C, and the actual temperature of the indexmatching solvent inside the sample chamber was monitored by an ALV/Pt-100 probe. The relative scattering intensity (Ir) in the temperature range 5−55 °C was measured as the intensity minus the solvent scattering. The samples were equilibrated at each temperature for 30 min before LS measurements.

ϕI−N = 4.8Deff /L

ϕI−N =

C* =

C*NA π LDeff 2 Mw 4

19.2M w 1 NA πL2 Deff

(1)

(2)

(3)

where ϕI−N and C* are the volume fraction and concentration in mg mL−1 of the virus particle at the I−N LC phase transition, respectively; L and Mw are the contour length and molecular weight of the fd virus, respectively; NA is Avogadro’s constant. Hereafter, we refer to the above conclusions as the flexibility-corrected hard−hard rod theory. The fd or M13 virus is highly charged and can be treated as semiflexible rods with an effective diameter (Deff) that is determined by the electrostatic double layer (EDL).1,32 The EDL and therefore D


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Macromolecules Deff can be regulated by the ionic strength (I). For polymer grafted viruses, the EDL layer becomes confined under the tethered polymer layer above certain I so that the intervirus interactions are purely determined by the grafted polymer layer.56,57 In such cases, the effective diameter (Deff) of polymer grafted fd virus can be estimated by the bare diameter of 6.6 nm of the fd virus plus 4 times the radius of gyration, Rg, of the polymer, as discussed in the recent work.56

The main structure of the fd virus is a rodlike protein capsid consisting of around 2700 major coat proteins (P8) that assemble in a helical way around the encapsulated DNA (Scheme 1B).69 The solvent-exposed part of the coat protein has two amino groups and five carboxyl groups that endow the whole virus with vast possibilities for chemical modifications.70−72 The grafting of the above tailor-designed block copolymers to the virus surface was performed following previous procedures,63 resulting in four kinds of polymer grafted viruses with the end of PNIPAM tethering to the virus surface. The molar ratio of block polymers to the surface amine groups of the virus was high to achieve the maximum grafting density that is normally low due to the steric effect inherent to the “grafting to” procedure.63 The successful grafting of the block polymers to the virus was confirmed by SDS-PAGE analysis on the disassembled virus (Figure 1A). There are two bands

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RESULTS AND DISCUSSION Synthesis of Semitelechelic Block Polymers and Their Grafting to Rodlike Viruses. The PEG-b-PNIPAM block copolymers were synthesized by RAFT polymerization.58,59,61 For this, methoxypoly(ethylene glycol)s (mPEGs) with a wellcontrolled molecular weight (Mw) were first end-functionalized with a thiocarbonylthio moiety and then used as the macrochain-transfer agent for RAFT polymerization of NIPAM (Scheme 1A). This technique enables to control the length (or Mw) of the PEG and PNIPAM block, i.e., the strength of steric repulsion and attraction, respectively. The thiocarbonylthio moiety at the end of the PNIPAM block was converted into the −SH group, which was further coupled with N-acryloxysuccinimide via the efficient thiol−ene click chemistry.60,61 In this way, PEG-b-PNIPAM block copolymers with an N-hydroxylsuccinimide ester (NHS) groups coupled to the end of the PNIPAM block were successfully prepared (Table 1). The NHS Table 1. Physiochemical Parameters of the EndFunctionalized Block Polymers block polymer

Mn (theory)a

Mn (NMR)b

Mn (GPC)c

PDId

TLCST

N per viruse

N3E2 N8E2 N3E5 N8E5

5000 10000 8000 13000

5503 11040 8616 13886

5441 9554 8689 13700

1.11 1.20 1.16 1.16

39 33 40 39

∼390 ∼290 ∼300 ∼260

a

Expected number-average molecular weight (Mn) from polymerization stoichiometry. bMn calculated by H1 NMR data. cMn by GPC in THF. dPolydispersity index (PDI) by GPC in THF. eNumber of the grafted polymer per virus, as estimated by the refractive index difference of the pristine virus and that grafted with block polymers.

Figure 1. Characterization of PEG-b-PNIPAM grafted fd viruses. (A) SDS-PAGE analysis of the major coat protein (p8) of the virus with or without polymer grafting. Lanes a and b are the marker proteins and the pristine virus, respectively. From lane c to f is the virus grafted with PEG2k-b-PNIPAM3k, PEG5k-b-PNIPAM3k, PEG2k-b-PNIPAM8k, and PEG5k-b-PNIPAM8k, respectively. (B) Schematic illustration of the major coat protein (P8) with or without grafted polymers. (C) AFM of the fd virus grafted with PEG3k-b-PNIPAM5k. (D) Contour length distribution based on statistics of around 100 particles presented in AFM images.

end group offers the possibility to graft such block copolymers to the virus surface or other proteins via the reaction of the surface amino groups with the NHS groups.47,63 The spacer between the NHS group and the PNIPAM backbone is critical to decrease steric effects inherent in the “grafting to” method to create polymer grafted protein complex.63 Herein, four kinds of PEG-b-PNIPAM-NHS were synthesized and fully characterized by NMR and GPC (Table 1 and Figures S1−S6). Hereafter, we refer to the polymer as NmEn, with N and E standing respectively for the PNIPAM and PEG block, while m and n represent the Mw of each block in units of kilodaltons. The polymerization degree index (PDI) of each block copolymer is less than 1.2, indicating that all of the polymers have a narrow Mw distribution. As expected, the LCST of the block copolymers as determined by light scattering (Figure S7) is slightly higher than that of pure PNIPAM due to increasing hydrophilicity introduced by the PEG block.61,67 Micellation behavior of the block polymers was probed by dynamic light scattering (DLS) (Figure S8). Upon heating, the PNIPAM blocks collapse and aggregate, leading to micelles with a collapsed PNIPAM core and a PEG shell.62,68 All these results confirm the well-defined thermoresponsive behavior of the as-prepared block polymers.

appearing in cases of polymer grafted viruses that correspond respectively to the pure coat protein and those grafted with polymers (Figure 1B). The number of polymers per virus was estimated by the difference in the refractive index of the intact virus, polymer grafted viruses, and the block polymers (see the Supporting Information).56,63 More than 200 polymers per virus were achieved, and the exact number depends on the molecular weight of the polymers (Table 1). Such grafting density is enough to fully cover the virus surface, and the intervirus interaction is only dictated by the grafting polymer layer above a certain ionic strength, as confirmed by previous works.63 The copolymer grafted virus was investigated with AFM, which reveals the rodlike shape of the polymer−virus conjugates with excellent monodispersity (Figure 1C,D).63 Therefore, grafting well-controlled polymers obtained by living E


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and cooling was systematically investigated, and qualitative results are presented in Figure 2. First of all, when heated above the respective LCST of each block copolymer, the suspension of fd-NmEn will either be in the free-flowing sol state or turn into hydrogels, critically depending on the molecular architecture of the grafted block copolymers (i.e., the ratio of PEG to PNIPAM). Among the four kinds of block copolymer grafted viruses, fd-N8E2 formed transparent self-supporting hydrogels at T > LCST while those grafted with other three kinds of block copolymers are still in the free-flowing sol state. Cryo-TEM analysis of such sol state at T > LCST reveals discrete single viruses, suggesting that the particles are well-dispersed and no intervirus aggregation or bundling exists (Figure 3A,C). In contrast, in the gel state of

polymerization techniques to highly monodisperse viruses is an exquisite and robust way of creating monodisperse rodlike model systems with tunable properties. Hereafter, we refer to the polymer grafted viruses as fd-NmEn, with N and E standing respectively for the PNIPAM and PEG block, while m and n represent the Mw of each block in the units of kilodaltons. To our best knowledge, this is probably the first example of virus-based bioconjugates grafted with a thermosensitive block polymer. Temperature-Dependent Solution Behaviors of the Block Copolymer Grafted Viruses. Previous works have indicated that nano/colloidal particles or proteins grafted with only PNIPAM will aggregate or form gels upon heating above the LCST of PNIPAM due to the hydrophobic attractions introduced by the dehydrated and collapsed PNIPAM.45,47,73,74 For the current PEG-b-PNIPAM grafted fd viruses, the PNIPAM block is expected to collapse onto the virus surface at T > LCST and might form interconnected hydrophobic patches wrapping the thin virus backbone of a diameter of only 6.6 nm (Figure 2A). In this way, the PEG-b-PNIPAM grafted

Figure 3. Internal structure of virus suspensions in the sol and gel state as revealed by cryo-TEM (A, B) and aggregation behavior of polymer grafted viruses in highly dilute suspensions as revealed by light scattering (C, D). Viruses in (A) and (C) are the fd virus grafted PEG5k-b-PNIPAM3k (fd-N3E5) while (B) and (D) fd-N8E2. Red arrows in (B) highlight bundles of viruses. The virus concentration of the samples for cryo-TEM and LS is 3 and 0.005 mg mL−1, respectively.

Figure 2. Temperature-dependent solution behaviors of block copolymer grafted rodlike viruses. (A) Schematic illustration of the conformational change of the PEG-b-PNIPAM chains grafted on the virus surface upon varying temperature. The gray cylinder and the red and blue curve lines represents the virus, PNIPAM, and PEG, respectively. Deff refers to the effective diameter as determined by the grafted polymers. (B) Visual inspection of the suspensions of PEG-bPNIPAM grafted viruses upon heating and cooling. I and N refer to the isotropic liquid and nematic LC phase, respectively. The accompanying cartoon represents possible arrangement of the polymer grafted virus in suspension. The PEG-b-PNIPAM grafted viruses were suspended in Trs-HCl buffer with an ionic strength of 110 mM and pH 8.2.

fd-N8E2, cryo-TEM reveals bundles that consist of several viruses, clearly due to intervirus aggregation along their long axis. Light scattering of a highly diluted fd-N8E2 suspension was also performed at a scattering angle of 20° to detect any aggregation of the polymer grafted virus upon heating (Figure 3C,D). No pronounced change occurs to the relative scattering intensity from the fd-N3E5 suspension upon heating and cooling. In contrast, the scattering intensity of the fd-N8E2 suspension increases significantly when approaching the LCST of the grafted polymers (Figure 3D), suggesting that large particles of several viruses form. Hysteresis that is typical of PNIPAM-based materials was also observed upon cooling. The normalized autocorrelation functions (ACF) of fd-N3E5 from dynamic light scattering (DLS) at T below and above LCST can overlapped with each other, showing an exponential decay consistent with a single population of particles (Figure S9A,B). In the case of fd-N8E2, a rising shoulder at longer relaxation times was found in the ACF at T > LCST (Figure S9C), which can be attributed to aggregates larger than single virus.

virus transforms into a structure that consists of a thin filamentous virus core, a collapsed and dehydrated PNIPAM intermediate layer, and a PEG shell (Figure 2A).75 The collapsed PNIPAM layer is expected to introduce interparticle attractions while the outermost shell of PEG can offer steric repulsions.76 The two interactions are competitive with each other and might lead to rich solution behaviors. To test this, the solution behavior of PEG-b-PNIPAM grafted fd viruses upon heating F


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Table 2. List of the Critical Concentrations (C*) at Which the Transition of the Nematic LC Phase to Isotropic Liquid Phase Occurs, Effective Diameter (Deff) of PEG-b-PNIPAM Grafted Rodlike Viruses, and Radius of Gyration (⟨Rg⟩) of PEG-bPNIPAM at Various Temperatures fd-N3E2

fd-N8E2

fd-N3E5

fd-N8E5

T (°C)

C* (mg mL−1)

Deff (nm)

Rg (nm)

C* (mg mL−1)

Deff (nm)

Rg (nm)

C* (mg mL−1)

Deff (nm)

Rg (nm)

C* (mg mL−1)

Deff (nm)

Rg (nm)

4 15 22 33 40 50

10.10 10.50 11.02 14.64 15.32 15.87

21.29 20.87 19.51 12.25 11.70 13.55

3.67 3.57 3.23 2.02 1.86 1.73

7.59 7.87 9.93 gel gel gel

28.33 27.32 21.65

5.43 5.18 3.76

8.89 9.12 10.09 12.46 13.28 14.73

24.18 23.45 21.31 17.26 16.19 15.66

4.39 4.21 3.68 2.66 2.40 2.26

6.37 7.26 8.12 10.90 12.19 13.15

33.75 32.28 26.48 19.72 17.64 16.35

6.79 6.42 4.97 3.28 2.76 2.44

The gelation of the fd-N8E2 suspension at T > LCST is a confirmative evidence of the existing attraction among the PEG-b-PNIPAM grafted viruses.47,77−79 For fd-N8E2, the PNIPAM block with a Mw of 8K is much longer than the PEG block with a Mw of 2K. The collapsed PNIPAM may form large interconnected hydrophobic patches around the filamentous viruses,75,79 giving rise to strong interparticle attractions. The short-range steric repulsion offered by the PEG2k chains is not strong enough to prevent viruses from sticking to each other during random Brownian collision, and the whole system turns into the gel state.67,80 Such behavior is consistent with theoretical predictions.79 Because of the reversible transition of PNIPAM between the hydrophobic and hydrophilic state, the sol−gel transition of fd-N8E2 is reversible and can be manipulated by heating and cooling (Figure 3D). In the case of the three other kinds of viruses, the PEG block can offer steric repulsions strong enough to counteract the attraction of the collapsed PNIPAM block and stabilize the whole system from forming intervirus aggregations at T > LCST.67 Viruses mainly consist of proteins with a refractive index close to that of aqueous buffers, and van der Waals attractions between proteins in aqueous system are weak. If there is any enhanced attraction in the case of polymer grafted viruses, it should be due to the grafted polymers. The attraction is believed to stem from hydrophobic interactions induced by the collapsed PNIPAM layer that wraps the virus74,81 Therefore, by controlling the molecular architecture of the grafted block copolymers, we succeed in preparing model systems that have pronounced interparticle attractions while are colloidally stabilized by steric repulsions. It is noted here that qualitative investigation revealed another interesting phenomenon. Upon increasing temperature, some polymer grafted virus suspensions that are originally in the nematic LC phase as indicated by the weak birefringence between the crossed polarizer turned into the isotropic phase without any birefringence. Virus suspensions with such behavior have a virus concentration that is very close to the critical concentration at which the isotropic−nematic LC phase transition occurs. This transition takes place around a critical temperature (Tc) in the range 15−20 °C, which is much lower than the LCST of PNIPAM. This nematic-toisotropic transition at temperatures much less than LCST is relative to the decreasing effective diameter (Deff) of the polymer grafted virus induced by the conformational change of PEG-b-PNIPAM, which we will quantitatively discussed in the next section. Temperature-Dependent Liquid Crystal Behavior. As stated in the Introduction, the filamentous M13, fd viruses, and their mutants have played a pivotal role in the fundamental

investigations and testing of various kinds of theories of the liquid crystal behavior of rodlike particles or rigid polymers.1,31,55 These viruses are highly charged and can be treated as hard rods with an effective diameter (Deff) that is determined by the electrostatic double layer (EDL).1,32 In the current work, we focus on the high ionic strength region (I = 110 mM), where the EDL is compressed to the virus surface so that the intervirus interactions are purely determined by the polymer layer.56,57 In this way, we can diminish the influence of the electrostatic repulsion and single out the short-range steric interaction and tunable attractions introduced by the PEG and collapsed PNIPAM blocks, respectively. It is noted here that the pH value of the Tris-HCl buffer we used varies with temperature, which will influence the surface charge density of the virus and eventually the range of the EDL.82 Such an influence can be neglected at I = 110 mM where the EDL is confined near the virus surface, and the intervirus interactions are dominantly determined by the grafted polymers. Quantitative equations as summarized in eqs 1−3 have been derived and confirmed in the cases of the bare or PEG grafted fd viruses.56,57 First of all, the nematic LC behavior of the current polymer grafted viruses was qualitatively investigated at various temperatures that range from 4 °C to T > LCST. Starting from a suspension of polymer grafted viruses that was in the fully developed nematic LC phase as characterized by strong birefringence under crossed polarizers, the lowest critical concentration (C*) at which the nematic LC phase can exist was determined by diluting the suspension with buffer at the same temperature to the point where the birefringence just disappears (Table 2). C* at each temperature was used to estimate the effective diameter (Deff) of the polymer grafted rodlike viruses based on eq 3 (Table 2), which are derived from Onsager’s theory of hard rods after taking into account semiflexibility of the rodlike virus.56 Deff is equal to the diameter of the bare virus plus 4 times the radius of gyration Rg of the block polymers, and therefore Rg was also derived.56 All of these quantities are listed in Table 2. During calculation with eqs 1−3, we used the same persistence length of 2.2 μm as the bare fd virus although polymer grafting and temperature might have influence on the flexibility of the virus. Previous works have demonstrated that polymer grafted fd viruses have the same flexibility as the bare one as long as the grafting density in the range of few hundreds of polymeric chains per virus.56,63 Fraden and co-worker have found that the persistence length only varies between 2.1 and 2.3 μm in the temperature range 5−65 °C.64 Therefore, we neglect any potential variation of the persistence length due to polymer grafting and temperature variation. G


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Macromolecules At T < LCST, all polymer grafted viruses can form the nematic LC phase above C*, which is much lower than that of the bare virus at the same ionic strength (I = 110 mM, pH = 8.2) (Table 2 and Figure 4A).57 Such phenomena can be

such behavior is elusive but might be due to the fact that the neighboring PEG block changes the water structure surrounding the PNIPAM blocks, which facilitates dehydration of the PNIPAM blocks. The effective diameter (Deff) of the polymer grafted rodlike viruses at each temperature also derived from the critical concentration C* by eq 3 (Table 2).56 As will be discussed further, this estimation is not accurate since our system have interparticle attractions at T > LCST but do qualitatively show the tendency of the size change of the polymer grafted viruses upon varying temperature. As expected, Deff decreases upon increasing temperature, which should be due to the conformational change of the PNIPAM block, since that the contour diameter of virus is temperature-independent and the PEG block is hydrophilic in the investigated temperature range. The degree of the change of Deff is closely correlated to the length of the PNIPAM block, and a change of Deff of 9 and 16 nm was observed between the lowest and highest temperature for fd-N3E5 and fd-N8E5, respectively. The radius of gyration Rg of the block polymers grafted on the virus surface was also calculated and is listed in Table 2 based on the fact that Deff is equal to the diameter of the bare virus plus 4 times Rg.56 At T < LCST, Rg has a value in the range 3−7 nm, which is comparable to the value of 5 nm for a single and free PEG-bPNIPAM as determined by small-angle neutron scattering.85 At T > LCST, Rg decreases to values of 1−3 nm, which is even smaller than Rg of pure PEG5k (∼3.3 nm). As will be further discussed later, such values are physically impossible and indicate the failure of the semiflexibility corrected Onsager’s hard rod theory at T > LCST, where interparticle attractions start to play a role. Temperature-Dependent Isotropic−Nematic LC Phase Equilibrium. The above results confirm that the collapsed PNIPAM block of the PEG-b-PNIPAM grafted viruses can induce attractions between the rods and the attraction can be regulated by the temperature. From now on, we shall investigate the temperature dependent isotropic (I)−nematic (N) LC phase equilibrium. The flexibility corrected hard-rod theory is used as the reference, for which some basic facts derived from theory are summarized in the Experimental Section. With the well-defined system of fd-N3E5, we determined the apparent I−N LC phase coexistence at several temperatures in the range 4−50 °C by a forced phase separation procedure. Ideally, true phase separation and equilibrium should be achieved by Brownian diffusion, which will take more than several months in the case of such long rods as the fd or M13 virus. During such a long time window, bacteria growth and solvent evaporation,1,32 especially at high temperature, might severely influence the phase separation and the accurate determination of the virus concentration in each phase. To speed up the phase separation, a suspension of pure fd or M13 viruses deep in the nematic LC phase is normally prepared first and diluted to the state at which tiny nematic droplets are dispersed in an isotropic background.1,32 The nematic droplets automatically settle down due to their higher density and coalesce into a homogeneous nematic phases at the time scale of days or weeks. However, in the cases of polymer grafted viruses, such automatically settling down of the nematic droplets is extremely slow for unknown reasons as tested on the time scale of one year, and a I−N LC phase diagram has never been reported with other polymers such as PEG grafted fd or M13 viruses. Therefore, the following method, hereafter referred as forced phase separation, was used to establish the

Figure 4. Critical concentration (C*) of the isotropic to nematic transition of PEG-b-PNIPAM grafted rodlike viruses at several typical temperatures (A). (B) Temperature-dependent effective diameter (Deff) derived from Onsager’s hard rod theory after taking into account the semiflexibility of the rodlike virus.

understood with the relation between the critical concentration (C*) and the effective diameter of the rods (Deff), C* ∝ 1/Deff, as derived from Onsager’s hard rod theory (eq 3). Polymer grafting will increase the effective diameter (Deff) of the rods and therefore shifts the apparent critical concentration of I−N LC phase transition to a lower value.57 At T > LCST, except for fd-N8E2 that forms gels as discussed above, the other three kinds of polymer grafted viruses can still form free-flowing nematic LC phases above C*. This is a clear confirmation that the rodlike particles are in the individual particle state despite the intervirus attractions introduced by the collapsed PNIPAM blocks. In the whole temperature range investigated herein, C* increases with increasing temperature. For instance, in the case of fd-N3E2, there is around 5 g mL−1 increase of C* from 4 °C, where the PNIPAM block should be in the fully hydrated coil state, to T = 40 °C where the PNIPAM block is fully dehydrated and collapsed. Moreover, we noted that the increase of C* already starts at T < LCST, where the PNIPAM is normally considered as hydrophilic. There exits another critical temperature (Tc) that is well below the LCST of the PNIPAM block. At T < Tc, C* only slightly increases with increasing temperature and pronounced increase occurs from Tc and LCST. Tc is found around 15−20 °C and does not show significant variation with the exact molecular structure of the grafted PEG-b-PNIPAM. This critical phenomenon is due to the unique behaviors of PNIPAM-b-PEG as confirmed by many previous works.83 Although the coil to globule transition of pure PNIPAM occurs at ca. 32 °C, the dehydration of the PNIPAM block in PNIPAM-b-PEG has been observed to start at 17−20 °C, at which a PNIPAM-rich phase will form surrounded by the hydrophilic PEG.83,84 The exact reason for H


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at T > LCST. It is noted here that the I−N coexisting phase behavior of the fd or M13 virus is only slightly dependent on the temperature.64 Although what is presented in Figure 5 is not a true phase diagram obtained by thermodynamic phase separation, some interesting features are revealed by this apparent I−N phase behavior. First of all, upon increasing the temperature, the CI shifts to lower concentrations while CN to higher ones; i.e., the width of the biphase area increases with temperature. The degree of shifting of CN to high concentrations is much higher than that of CI. Such classic widening behaviors of the I−N coexisting concentrations at each temperature qualitatively agree with the prediction of many theories dealing with rods with attractions.28,40,50 The enhanced widening of the I−N coexisting concentrations upon increasing temperature suggests that the strength of interparticle attraction increases with increasing temperature. Therefore, we realize a system with pairwise interparticle attractions on a single particle level, the strength of which can be conveniently regulated in a wide range of temperatures. In addition, while most pronounced widening of the two phase areas occurs when temperature approaches LCST, slight widening can already be observed at T < LCST. Such widening is consistent with the temperature dependent collapsing and dehydration of the PNIPAM block of PEG-b-PNIPAM as discussed above, which starts around 17−20 °C and then becomes fully dehydrated and collapsed around the LCST. The temperature-dependent widening of the coexisting biphase area can be quantified by the coexisting I−N width, ΔCN−I = (CN − CI)/CI (Figure 5B). ΔCN−I has the value of ca. 0.14 at 4 °C, which is consistent with the theoretical prediction of Onsager’s theory after taking into account the flexibility of the virus.56 This is therefore another confirmative indication that the block polymer is fully hydrophilic under the condition of T = 4 °C and I = 110 mM and that the PEG-b-PNIPAM grafted virus behaviors like hard rods. ΔCN−I increases slightly with increasing temperature at T < LCST, and a sharp jump occurs at the LCST and then levels off at a value of ca. 2.7 above the LCST, after which PNIPAM becomes completely dehydrated and the strength of interparticle attraction becomes constant. It is noted here that the effective diameter (Deff) of PEG-bPNIPAM grafted viruses will also decrease in our system due to collapsing of the PNIPAM blocks as discussed above (Figure 2A). Because the concentrations at each phase, CI and CN, are proportional to 1/Deff based on Onsager’s hard rod theory (eq 3), variation of Deff will lead to the shifting of the phase diagram. In another words, the temperature-dependent widening of the I−N coexisting concentrations might be the synergic effects of the decreasing Deff and the increasing strength of intervirus attractions. We plot the I−N coexisting concentrations for a system with no attractive interactions but with varying Deff (dashed lines in Figure 5A). There is no widening if only the decreasing effective diameter plays the sole role. Therefore, the pronounced widening at each specific temperature should be due to interparticle attractions. It is noted here that data presented in Figure 5A can be considered only as the apparent I−N LC phase equilibrium at best, since it is not based on thermodynamic phase separation by the translational diffusion of the particles. Monitoring of older samples in the forced phase separation state that have been stored at some temperatures for more than one year indicates that the virus concentrations of each phase shift to higher

apparent I−N LC phase behaviors. At each temperature, a suspension of the polymer grafted rodlike virus deep in the nematic LC phase was diluted with buffer at the same temperature until droplets of nematic domains were dispersed in the background of the isotropic liquid phase. After being further incubated at such stage at least for 36 h, low-speed centrifugation was applied to spin down the nematic droplets and force them to coalesce with each other, leading to two phases that consist of an upper dark isotropic phase and a lower birefringent nematic LC phase (inset of Figure 5A). The virus concentrations

Figure 5. Temperature-dependent apparent isotropic liquid (I)−nematic (N) LC phase behaviors of PEG-b-PNIPAM grafted rodlike viruses via a forced phase separation assisted by low-speed centrifuge. The polymer grafted virus under study is the fd virus grafted with PEG5k-b-PNIPAM3k (fd-N3E5) suspended in Tris-HCl buffer with an ionic strength of 110 mM and pH of 8.2. (A) Phase diagram of the polymer grafted viruses in the plane of temperature and concentrations. The solid squares and circles are the virus concentration in the equilibrium isotropic and nematic LC phase, respectively. The dashed lines are the predicted phase diagram of hard rod with decreasing diameter based on Onsager’s hard rod theory. The open cycles are the data of fd-N3E5 in Figure 4A. Insets: photo of a suspension with coexisting I and N LC phases. (B) Coexisting I−N width, ΔCN−I = (CN − CI)/CI, versus temperature. The dashed line corresponds to ΔCN−I = 0.14, which is derived from the theoretical prediction of Onsager’s hard rod theory after taking into account the flexibility of the virus.

of each phase (CI and CN for the isotropic and nematic LC phase, respectively) were then determined. This procedure was continued for several weeks until the virus concentrations of each phase are close to constant values within experimental error. The final values were used to construct the apparent phase diagram as shown in Figure 5A. The possibility to separate the two phases at temperature well above the LCST of the PNIPAM block further confirms that the grafted viruses are in the suspended sol state consisting of individual rods with Brownian motion, although interparticle attractions are expected I


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molar mass of the virus are well-defined. All of these simplify the data fitting. In general, there is good fitting between the theory and our data except for those at the lowest temperature, i.e., 4 °C (Figure 6A). From this fitting, together with the fact that the

values (Figure S10). Such shifting probably reflects the evolving phase separation due to the translational diffusion of the rods but also might be due to centrifuge forces, bacteria growth, solvent evaporation, etc.1,32 The general widening of the I−N coexisting concentration in Figure S10 has a trend similar to that of Figure 5A. Therefore, the apparent I−N phase behaviors of Figure 5A might reflect some core features of the true phase equilibrium. Comparison with Recent Theoretical Predictions. As stated in the Introduction, there are many theoretical predictions about the liquid crystal behavior of rodlike particles or rigid polymers with attractive interactions, a few of which can accommodate certain experimental data with limited success.34,39−41 The challenge in theoretical dealing of such cases is the detailed and accurate definition of the attractive potentials that is often dependent on the orientation of the particle.41,86 Flory’s classic lattice theory of rigid polymers can account for interpolymer attractions using the parameter of the Flory−Huggins parameter (χ) between the LC mesogens and the solvent, which has been later extended to rigid polymers grafted with side polymers that are highly related to our system.28,38 However, the lattice model involves many stringent assumptions in the treatment of a system of ordered rigid rods, and such theory can only qualitatively compare with experimental results. In the case of colloidal rods with effective attractions realized by the depletion effect,49 detailed theoretical phase diagrams have been constructed but pronounced discrepancy was found when comparing with experimental data.50 Among theories based on the more accurate Onsager’s excluded volume theory by introducing interparticle attractions of various natures, we noticed recent theoretical works from Jackson’s group in their efforts to account for the complicated LC behavior of the rigid polypeptide−PBLG in DMF, which has a temperature-dependent intermolecular attractive interaction.37 On the basis of a coarse-grained model consisting of a hard spherocylinder decorated with an effective attractive square-well (SW) potential that acts at the center of mass of the particle, the authors have developed van der Waals−Onsager algebraic equations of state capable of describing the isotropic liquid and nematic LC phases of attractive rodlike particles. To compare with the experimental I−N data of PBLG in DMF, two temperature-dependent parameters can be played: the first one is the particle volume (Vm) of the spherorods, and the second one is the depth of the enveloping isotropic SW attraction potential (ε0). Quantitative comparison with the experimental data of the I−N transition of PBLG in DMF was achieved by treating the PBLG molecule as a spherocylinder that can change its shape (specifically the length of PBLG) and the attraction potential. In many aspects, our system is comparable to this theoretical model of PBLG in DMF. Our filamentous virus grafted with polymers can be treated as hard rod systems.56 The attraction between rods is induced by the dehydrated PNIPAM while the system is colloidally stabilized by PEG that offers steric repulsion. The effective diameter of our current system and therefore the Vm of the rods change with temperature. The strength of attractions is also temperature-dependent. We therefore used their model to fit our data of Figure 5A (the details of fitting are presented in the Supporting Information). One advantage of our current system is that the mass concentration and therefore the number density (ρ) of the rod can be accurately determined by UV spectroscopy. In addition, other parameters such as length and

Figure 6. Comparison of experimental results and theoretical prediction. (A) Fitting of the results listed in Figure 5A with the theoretical works from Jackson’s group. (B) Temperature-dependent effective diameter (Deff) of the polymer grafted viruses. Cycles are Deff that are derived from the fitting in (A) while squares are Deff of fd-N3E5 in Table 2 based on the flexibility-corrected Onsager’s hard rod theory. The dashed line is the effective diameter of the rodlike fd or M13 virus grafted with PEG5k. (C) Isotropic square-well depth of the effective attractive square-well (SW) potential at various temperatures; the line is a guide for the eye.

contour length of the virus does not change with temperature, the effective diameter (hereafter referred as DAtt eff ) at each temperature can be extracted from Vm and has a value of 15−20 nm in the whole temperature range (Figure 6B). Values of the effective diameter based on the flexibility-corrected Onsager’s hard rod theory (DHard eff ) are also listed in Figure 6B (the Deff column of fd-N3E5 in Table 2, see the section “Temperature-Dependent Liquid Crystal Behavior”). At the Hard lowest temperature 4 °C, DAtt eff has a value of 15 nm while Deff is ca. 24 nm. It is well-established that the same virus grafted with only PEG with an Mw of 5K (PEG5k) has a Deff of ca. 19 nm.57 Accordingly, the minimum value of the effective diameter of PEG5k-b-PNIPAM3k grafted virus should be at least 19 nm as determined by the outer PEG5k block. Therefore, the theoretical works by Jackson’s group underestimate the value of the effective diameter at 4 °C, where attractions between PEG5k-b-PNIPAM3k grafted viruses barely exist. We also recall here that the coexisting I−N width, ΔCI−N, of PEG5k-b-PNIPAM3k grafted virus has a value of 0.14 at 4 °C, which is exactly the prediction based on the semiflexible hard rods.56 Therefore, the PEG5k-b-PNIPAM3k grafted virus can be treated as a hard rod, and the flexibility-corrected Onsager’s hard rod theory describes the system very well, as already discussed above.66 DHard eff gives a more rational estimation of the effective diameter. The DHard of the PEG5k-b-PNIPAM3k eff grafted virus is larger than that of the one grafted with only PEG5k, which is due to the additional PNIPAM block. Because DHard is equal to the diameter of the bare virus plus eff 4 times the radius of gyration (Rg) of the block polymers, this J


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results of our system of attractive rods, methods to achieve true phase separation and more advanced theories would still be required.

gives aRg of the block copolymer of 4.5 nm, which is comparable to the value in the literature.85 With increasing temperature, the DAtt eff derived from the above theoretical fitting slightly increases at first and then a sharp jump occurs when T approaches the LCST of the grafted block copolymer. At T > TLCST, DAtt eff eventually levels off and has the value of ca. 20 nm, which is close to the 19 nm of the same virus grafted with only PEG5k.57 Such a value seems reasonable if considering the fact that the virus is enveloped by the fully collapsed PNIPAM layer at T > LCST, onto which is attached with stretched PEG5k chains (right inset of Figure 6B). In contrast, DHard from the flexibility-corrected Onsager’s hard eff rod theory (eq 3) is much more sensitive to the temperature change and sharply decreases with increasing temperature even at T < LCST. DHard eventually decreases to 16 nm at T > eff LCST. Such a value is physically impossible since the minimum Deff of the PEG5k-b-PNIPAM3k grafted virus should be at least 19 nm. Such a discrepancy suggests that the flexibilitycorrected Onsager’s hard rod theory (eq 3) is not appropriate for estimating the effective diameter as long as there is interparticle attractions, especially at T > LCST. Fitting with the current model also gives the square-well depth (ε0) of the attraction potential that is listed in Figure 6C as ε0/kbT. This first indicates that weak attractive interactions already exist at T < LCST. This is consistent with our previous discussions that slight dehydration of PNIPAM in the PNIPAM-b-PEG blocks already starts at T < LCST although sharp transition often occurs at the LCST. As expected, the strength of attractive interactions ε0 increases with increasing temperature and eventually saturates at 3.5 × 10−3kbT at T > LCST. We noticed that Zaccone and co-workers recently quantified the attraction potential of a spherical core−shell model system consisting of a polystyrene nanoparticle as the core and a cross-linked PNIPAM thin layer as the shell.74 The authors assumed that the range of attractive interaction (δ) is ca. 10 nm, which is the typical range of the attractive interaction between two organic hydrophobic surfaces. They found that the depth of the effective attraction well Vmin is 12kbT at T > LCST. This value is much higher than our estimation. However, this discrepancy is probably due to the fact that the attraction potential in the current Jackson’s theory is averaged over all the rotational configuration of the rod, leading to the interaction potential with a range of L + D and a uniform value ε0.37 As discussed in the Supporting Information, if assuming that the similar attractive potential used by Zaccone et al. is acting around our polymer grafted virus at T > LCST, one actually derives ε = 3.2 × 10−3kbT, which is in remarkable agreement with the one obtained by our fitting. Based on the above discussion, the flexibility-corrected Onsager’s hard rod theory can account for the current system at the temperature range that is much less than LCST, especially at 4 °C, where attraction is very weak or negligible. The fitting of our data to the theoretical works by Jackson’s group based on two adjustable parameters gives a rational estimation of the effective diameter at T > LCST, where clear inter-rod attractions are introduced by the fully dehydrated PNIPAM blocks while aggregation is prevented by the PEG chains. Although a very good fitting between theory and data in Figure 5 was found with only two adjustable parameters, the current model does not consider neither the flexibility of the virus nor the specific nature of the interactions.86 In addition, the apparent phase behaviors in Figure 5 were not obtained by true thermodynamic phase separation. To fully understand the

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CONCLUSIONS In summary, several kinds of well-defined PNIPAM-b-PEG were prepared via RAFT polymerization, and a NHS group was introduced to the end of the PNIPAM block. Such polymers were grafted onto the surface of well-defined rodlike fd virus via the end of the PNIPAM chain, leading to block polymer−virus bioconjugates. Increasing temperature induces the dehydration and collapse of the PNIPAM blocks and therefore introduces interparticle interaction among the particles while the PEG chains offer steric repulsion to prevent aggregation, gelation, or other dynamically arrested states. In this way, a rodlike system with “true” pairwise attractions on a single particle level has been realized. The strength of the attractive interaction can be controlled by the temperature or by the molecular structure of the thermosensitive block polymer. With these systems, the temperature-dependent apparent liquid crystal (LC) phase behaviors of the polymer grafted virus were fully investigated via a forced phase separation assisted by low-speed centrifuge. At T < LCST, especially, at 4 °C, where the block polymers are in the fully hydrophilic state, the rodlike system behaves as hard rods, and its LC behaviors can be quantitatively described by the flexibilitycorrected Onsager’s hard rod theory. Increasing temperature to induce the collapse of the PNIPAM block leads to widening of the isotropic−nematic LC phase coexistent concentrations at each temperature, which qualitatively agrees with the predictions of many theories dealing with rods with attractive interactions. The fitting of our data to the theoretical works by Jackson’s group based on two adjustable parameters give a rational estimation of the effective diameter of polymer grafted virus at T > LCST, where clear inter-rod attraction are introduced by the fully dehydrated PNIPAM blocks. The current work will encourage more theoretical works to gain accurate mapping of the LC phase behavior of rigid polymers or rods with attractive interactions, which will definitely facilitate the LC-based processing of the above-mentioned technologically important polymers or colloidal particles.

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ASSOCIATED CONTENT

S Supporting Information *

. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b00674.

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Experimental details of synthesis of the macro-chaintransfer agent PEG-DTB, NMR and GPC of the block polymers, other characterizations of the polymer grafted viruses, fitting of experimental data with theory and supplementary figures (DOCX)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Z.Z.). ORCID

Linqi Shi: 0000-0002-9534-795X Zhenkun Zhang: 0000-0002-3480-2381 Author Contributions

S.L., C.Z., and Z.Y. contributed equally to this work. K


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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21774064, 21274067, 21620102005, 91527306, and 51390483), the Fundamental Research Funds for the Central Universities, Natural Science Foundation of Tianjin, China (No. 17JCYBJC16900), and PCSIRT (IRT1257). We acknowledge support from the NSF MRSEC DMR-1420382. Z.Z thanks Prof. Zvonimir Dogic for his generous support during Z.Z’s one year stay at Brandeis University as a visiting scholar and also for his critical reading of the manuscript. Prof. Seth Fraden from Brandeis University is acknowledged for his stimulating discussion and reading of the manuscript. The authors acknowledge Prof. Chi Wu for his insightful comments about the light scattering.

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Filamentous Viruses Grafted with Thermoresponsive Block Polymers

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