How Does Hydrogen Bonding Influence Morphology?

Article pubs.acs.org/Macromolecules

Probing into Homopolymer Self-Assembly: How Does Hydrogen Bonding Influence Morphology? Yunqing Zhu, Lin Liu, and Jianzhong Du* School of Materials Science and Engineering, Tongji University, 4800 Caoan Road, Shanghai, 201804, China S Supporting Information *

ABSTRACT: Self-assembly of amphiphilic homopolymers composed of both hydrophilic and hydrophobic components in each repeating unit is burgeoning in recent years due to their facile synthesis compared to block copolymers. However, ordered homopolymer nanostructures are very limited, and solid TEM evidence for the formation of vesicles and other complex morphologies is necessary to address the mechanistic insights of homopolymer self-assembly. Presented in this article are the studies on the morphological transition, the structure analysis, and the formation mechanism of homopolymer self-assembly. First, a series of amphiphilic homopolymers, poly(2-hydroxy-3-phenoxypropyl acrylate) (PHPPA) with various molecular weights (MWs) have been designed and synthesized by the reversible addition−fragmentation chain transfer (RAFT) process. Second, upon simply changing the homopolymer’s chain length or cosolvents during self-assembly, a wide range of new homopolymer-based nanostructures can be obtained, such as large compound micelles (LCMs), simple vesicles, large compound vesicles (LCVs), and hydrated large compound micelles (HLCMs) as a result of different intensity of inter/ intra-polymer hydrogen bonding in the homopolymer self-assemblies. Moreover, micrometer-sized branched cylinders are formed by premixing PHPPA36 and PHPPA103 homopolymers, which is not observed by self-assembly of PHPPA36 and PHPPA103 individually. Third, we claim that the structures of homopolymer self-assemblies are much different from their block copolymer analogues due to homopolymer’s fuzzy hydrophobic and hydrophilic domains compared to block copolymer’s distinct ones. We confirm that the structure of micelle core or vesicle membrane (alike to each other in nature) consists of both hydrophilic and hydrophobic moieties, which is different from block copolymer micelles or vesicles with hydrophobic cores or membranes. Also, a dye encapsulation experiment is employed to identify and distinguish a new nanostructure, HLCMs, from LCMs. Our study has provided a new perspective on homopolymer self-assembly. mimicking the origin of life.21−23 However, the main problem to mimic life by amphiphilic block copolymers is their poor similarity to DNA, RNA, polypeptide or protein due to the strictly distinguishable hydrophobic and hydrophilic segments. For example, in a natural system such as heme, the boundary between the hydrophobic and hydrophilic domains are not so clear. Very recently, homopolymer self-assembly has been investigated aiming to simplify the synthetic route of polymers.24,25 However, comparing with numerous studies on block copolymer self-assembly, study on homopolymer selfassembly is really limited. Pioneering studies have been conducted by Thayumanavan et al., focusing on the selfassembly behavior of amphiphilic homopolymer.26−28 For instance, a type of homopolymer was prepared and was able to form micelle-like structure due to its inherent amphiphilic molecular structure which possesses a relatively long hydro-

1. INTRODUCTION Life itself is notoriously complex and even the present-day simplest bacterium has hundreds of genes and thousands of proteins. Compared to a covalent bond, each hydrogen bond is extremely weak. However, millions of H-bonds together represent an extremely strong force that keeps two DNA strands together. Other polar groups of the base rings can form external hydrogen bonds with surrounding water that give the molecule extra stability. More importantly, interpolymer hydrogen bonding plays a key role in inducing DNA assembly, determining protein structure and packing chitosan molecules.1,2 Therefore, the investigation of hydrogen bonding controlled self-assembly is of great significance.3−12 Recently, amphiphilic block copolymers have been selfassembled into a range of nanostructures such as micelles, cylinders, vesicles, large compound micelles, jellyfish, and worm-like structures, and other morphologies.13−16 The enhanced stabilities and lower critical aggregation concentrations (CACs) of polymers than liposomes17 make the polymeric self-assembly a promising approach to a range of fields from drug delivery to nanosensors,18−20 and eventually © 2012 American Chemical Society

Received: October 18, 2012 Revised: November 23, 2012 Published: December 13, 2012 194

dx.doi.org/10.1021/ma302176a | Macromolecules 2013, 46, 194−203

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Scheme 1. Preparation of PHPPA Homopolymers Which Can Be Self-Assembled into a Range of Nanostructures Induced by Inter/Intra-Polymer Hydrogen Bondinga

a Key: aLarge compound micelles (LCMs), composing of hydrophilic surface (blue) and hydrophobic body (yellow). bVesicles with a membrane, consisting of both hydrophilic (blue) and hydrophobic (yellow) moieties in the membrane. clarge compound vesicles (LCVs): the yellow cores inside LCVs are hydrophobic zones with highly intensive inter/intra-polymer H-bonding. dhydrated large compound micelles (HLCMs) with large amount of unbonded −OHs or −COOHs inside their body. ecross-section model of branched cylinders with −OHs or −COOHs covered on their surface which only emerges in homopolymer binary system.

2. EXPERIMENTAL SECTION

phobic side chain and short hydrophilic carboxylic acid group in every repeating unit.26 Furthermore, a range of amphiphilic homopolymers with molecular structures similar to the former one can self-assemble into hollow aggregates (high hydrophobic content) and spherical micelles (low hydrophobic content) in water.28 Du et al. discovered that a tiny amount of hydrophobic end groups in very hydrophilic homopolymers are the main driving force for the formation homopolymer micelles and complex micelles.29,30 The transition between different morphologies is a very important issue in self-assembly. For example, vesicles are considered as one of important prerequisites for the origin of life due to their unique membrane compartment that surrounds all present-day cells and acts as boundaries.31 Micelles without the membrane compartment are found to be the material source for the division of membrane compartment to form next generation vesicles.32,33 To date, there are only very limited reports on the hydrogen bonding controlled homopolymer self-assembly. For example, Jiang et al. developed noncovalently connected micelles (NCCMs) based on interpolymer hydrogen bonding between two homopolymers with respective proton-donor and protonacceptorgroups.34,35 However, hydrogen bonding controlled transition of various nanostructures such as LCVs, LCMs, HLCMs, cylinders, and vesicles based solely on one kind of homopolymer has not been reported yet. Herein, we designed a new homopolymer, poly(2-hydroxy-3phenoxypropyl acrylate) (PHPPA), which has fuzzy boundaries between hydrophobic and hydrophilic domains. Polar hydroxyl group has been introduced into the repeating unit to afford both inter- and intrapolymer hydrogen bonds, mimicking the self-assembly masterpiece of nature, DNA, and controlling the morphology of PHPPA self-assembly (see Scheme 1).

Materials. 2-Hydroxy-3-phenoxypropyl acrylate (HPPA) and 2bromo-2-methyl propionic acid was purchased from Aladdin Chemistry, Co. The HPPA monomer was passed through an alumina B column to remove the inhibitor before use. Carbon disulfide, 1dodecanethiol, 2, 2′-azobis(2-methylpropionitrile) (AIBN), dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), dialysis tubing (3.5 or 8−14 kDa molecular weight cutoff), dimethylformamide (DMF), tetrahydrofuran (THF), and other solvents were purchased from Sinopharm Chemical Reagent CO., Ltd. (SCRC, Shanghai, China) and used as received. AIBN was recrystallized from MeOH and stored at −25 °C before use. Characterization. GPC. The homopolymer molecular weights and polydispersities were characterized using a THF GPC conducted by a Waters Breeze 1525GPC analysis system with two PL mix-D columns with HPLC grade THF as the eluent at a flow rate of 1.0 mL/min at 35 °C. The homopolymers were dissolved in THF and filtered prior to analysis. 1 H NMR. 1H NMR Spectra were recorded using a Bruker AV 400 MHz spectrometer at room temperature with CDCl3 as solvent. DLS. The dynamic light scattering (DLS) was used to determine the hydrodynamic radius (Rh) and polydispersity of self-assemblies formed from homopolymers in aqueous solution. The field correlation function, g(1)(t), is calculated from the measured intensity auto correlation function, g(2)(t) through the Siegert equation (eq 1).

g(2)(τ − 1) = β|g(1)(τ )|

(1)

In a polydispersed spheres solution, the g(1)(t) (first-order electric field correlation function) is related to G(Γ) (the line width distribution function) by36

g(1)(t ) =

∫0

∞

G(Γ)e−Γt dΓ

(2)

where t refers to the delay time. Both the cumulant analysis and the CONTIN analysis are employed as data processing methods. The cumulant analysis (eq 3) can be applied to describe logarithm of g(1)(t) as a series expansion of time (t). ⟨Γ⟩, the first cumulant, is the decay rate of the process which yields the z-averaged diffusion coefficient and 195


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15 min. CS2 was then injected into the mixture and followed by an immediate yellow turning of the solution. After stirring for another 15 min, 2-bromo-2-methylpropionic acid (3.71 g, 21.8 mmol) was added and the reaction mixture was kept stirring for 20 h at room temperature. The solvent was removed by rotary evaporation at 35 °C and the orange residue was extracted into CH2Cl2 (3× 200 mL) from 1.0 M HCl (200 mL). Afterward, the organic phase was washed with water (2× 200 mL) and brine (200 mL) successively and dried over anhydrous MgSO4 overnight. The solvent was then evaporated under reduced pressure at 35 °C and the residue was then recrystallized from n-Hexane to yield a bright yellow crystal. Yield: ∼76%. 1H NMR spectrum is shown in Figure S1, Supporting Information. Synthesis of Poly(2-hydroxy-3-phenoxypropyl acrylate) (PHPPA) by RAFT Process. In a typical polymerization of PHPPA, a flask of 25 mL with a rubber plug and a magnetic stirrer bar was loaded with AIBN radical initiator (1.80 mg, 1.10 × 10−2 mmol), DDMAT CTA (20.0 mg, 5.49× 10−2 mmol), HPPA (1.22 g, 5.49 mmol), and DMF (1.96 mL). Before immerged in an oil bath setting at 70 °C, this mixture was flushed with Ar for 15 min to be deoxygenated. The relative molar ratio of [HPPA]/[DDMAT]/[AIBN] was 100:1:0.2. Twenty h later, the reaction was terminated by cooling to room temperature. The bright yellow polymerization solution was then diluted with DCM (50 mL) and washed with deionized water (3× 50 mL) for wiping out DMF. The organic phase was then dried over anhydrous MgSO4 for 2 h and vacuum evaporated in a rotary evaporator at 35 °C, after which a bright yellow solid appeared. 1H NMR spectrum is shown in Figure 1. The conversion of HPPA monomer is shown in Table 1.

the second cumulant (μ2) is correlated to the second moment of the distribution of relaxation times by eq 4.

In[g(1)(t )] = − Γ t +

μ2 =

∫0

∞

⎛ μ2 ⎞ 2 ⎛ μ3 ⎞ 3 ⎜ ⎟t − ⎜ ⎟t + ... ⎝2⎠ ⎝6⎠

G(Γ)(Γ − Γ )2 dΓ

(3) (4)

where G(Γ) is a line width distribution function and Γ is given by Γ = Dq2

(5)

where D is the translation diffusion coefficient, and q [=(4πn/ λ0) sin(θ/2)] is the scattering vector, θ is the scattering angle, n is the refractive index of the solvent, and λ0 is the wavelength of the incident light. ⟨Γ⟩ and μ2 can be acquired directly by the cumulant methods and meanwhile the hydrodynamic radius distribution M (Rh) can be obtained by the CONTIN program in the correlator. Then with the assist of the Laplace inversion, the line width distribution function G(Γ) can be figured out based on the measured g(1)(t). For a diffusive relaxation, Γ can be interrelated to D (the translational diffusion coefficient) by eq 5. On the basis of the mathematical relation mentioned above, G(Γ) can be converted to M (Rh) according to eq 6, namely the Stokes−Einstein equation.

Rh

=

kBT 6πηD

(6)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. The hydrodynamic diameters of different concentrations of aqueous homopolymer vesicle solutions were characterized by a Zetasizer Nano series instrument (Malvern Instruments ZS 90). The scattering angle was fixed at 90°. Data processing was carried out using cumulant analysis of the experimental correlation function and analyzed using Stokes−Einstein equation to calculate the hydrodynamic diameters of homopolymer aggregates. All the aqueous aggregates solutions were analyzed using disposable cuvettes. FT-IR. FT-IR spectra were recorded using an EQUINOXSS/ HYPERION 2000 (Bruker Co., Ltd., Germany) in ATR mode. SLS. The statistic light scattering (SLS) was used to determine the radius of gyration (Rg) and was conducted using ALV/5000E laser light scattering (LLS). The data were analyzed using the Zimm plot method on ALV software to determine Rg. The UV−Vis Absorption Spectra. The UV−vis absorption spectra of aqueous homopolymer aggregates were acquired using a U-3010 spectrophotometer (HITACHI) to monitor the changing in concentration of homopolymer aggregates. TEM. All the self-assembly solutions were diluted at ambient temperature. Copper grids were surface-coated to form a thin layer of amorphous carbon. Each sample (3 μL) was then dropped onto the carbon-coated grid and dried at ambient environment. To stain those aggregates, phosphotungstic acid (PTA; 2 w/w %) solution (10 μL) was dropped onto a hydrophobic film (Parafilm), then those sampleloaded grids were laid upside down on the top of the PTA solution droplet and soaked for 2 min. After that a filter paper was used to carefully blot the excess PTA solution. The grids were dried under ambient environment overnight. Imaging was performed on a JEOL JEM-2100F instrument at 200 kV equipped with a Gatan 894 Ultrascan 1k CCD camera. SEM. SEM was utilized to observe the surface morphologies of branched cylinders. To obtain SEM images, a drop of solution was spread on a silica wafer and left until dryness. It was coated with platinum and viewed by a FEI Quanta 200 FEG electron microscopy operated at 15 kV. The images were recorded by a digital camera. Synthesis of Chain Transfer Agent (CTA). 2-(Dodecylthiocarbonothioylthio)-2-methylpropanoic acid (DDMAT) was prepared by adding 1-dodecanethiol (5.00 g, 24.2 mmol) to a suspension of K3PO4 (5.14 g, 24.2 mmol) in acetone (125 mL) and kept stirring for

Figure 1. Typical 1H NMR spectrum of PHPPA71 homopolymer in CDCl3.

Table 1. Homopolymers by RAFT Process polymer

DPa

Mn,GPC (Da)b

Mw/ Mnb

Mn,NMRc (Da)

convnd (%)

PHPPA36 PHPPA71 PHPPA103

50 100 150

11k 18k 30k

1.15 1.29 1.46

9k 16k 23k

72 71 69

a

Theoretical degree of polymerization assuming 100% monomer conversion. bMn and Mw/Mn were determined by GPC in THF. c Calculated Mn by 1H NMR, corresponding to the real composition in the first column. dConversion of HPPA monomer.

Synthesis of Ethyl-Based PHPPA (Et-PHPPA). A flask with a stirring bar was charged with PHPPA71 (250 mg, 1.56× 10−2 mmol), ethanol (2.90 mg, 6.25× 10−2 mmol), DMAP (1.00 mg, 7.80× 10−3 mmol), DCC (6.50 mg, 3.13× 10−2 mmol), and anhydrous THF (2 mL). After stirred at room temperature for 24 h, the mixture was transferred into a dialysis tube (3.5 kDa molecular weight cutoff) and dialyzed against deionized water for 48 h. The obtained self-assembly solution was lyophilized to give a yellow sticky solid. Yield: 82%. 196


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Table 2. Self-Assembly of PHPPAx Homopolymers sample

homopolymera

cosolventb

pH

diameter (nm)c

ζ potential (mV)

membrane thickness (nm)d

morphologye

1 2 3 4 5 6 7

PHPPA71 PHPPA36 PHPPA71 PHPPA103 PHPPA71 PHPPA36/103f Et-PHPPA71g

THF/water DMF/water DMF/water DMF/water DMF/water DMF/water DMF/water

7.0 7.0 7.0 7.0 11.0 7.0 7.0

617 432 580 746 421 − 567

−20.9 −27.7 −26.3 −24.5 −33.6 − −5.1

52 − 68 66 − − −

vesicles and LCMs LCVs vesicles vesicles HLCMs branched cylinders −

a Subscripts of PHPPAx refer to the degree of polymerization by 1H NMR. bInitial concentration (Cini) of PHPPAx (where x = 36, 71, and 103) in organic solvent is 1.0 mg/mL and the volume ratio of cosolvent to water is 1/2, v/v. cZ-Averaged hydrodynamic diameter obtained right after dialysis by DLS. dMembrane thickness cannot be acquired in the case of LCVs and LCMs. eMorphology by TEM. fHomopolymer binary system containing both PHPPA36 and PHPPA103 (1/1, w/w). gEthyl-based PHPPA71.

Figure 2. (A) DLS study of PHPPA71 homopolymer vesicle prepared in the mixture solvent of THF/water (1/2, v/v) at a Cini of 1.0 mg/mL, 25 °C and pH 7.0 (7 days after dialysis). (B) TEM image and illustration diagram for the structure of the homopolymer vesicle in Sample 1. (C) Electron transmittance chart related to the red scan-line in (B), and the membrane thickness is ∼60 nm. (D) Illustration diagram of homopolymer chains with (a) low, (b) medium, and (c) high hydrophobicity in different regions of the vesicle membrane and the inter/intra-polymer hydrogen bonding formed between homopolymers. Self-Assembly of PHPPA Homopolymer. Homopolymer vesicles and other nanostructures were generally prepared according to the following protocol. As the poor solvent, 4.0 mL of deionized water or 4.0 mL of water at pH 11.0 was added dropwise to 2.0 mL of homopolymer solution in DMF or THF over a period of 2 h under vigorous stirring. Right after that, 14 mL of excess poor solvent was poured into the solution for “quenching” the structures of those selfassemblies and the residual DMF or THF was removed by dialyzing against deionized water. The detailed information is listed in Table 2. Water-Soluble Dye Encapsulation Efficiency of Different Homopolymer Self-Assemblies. In a typical experiment, 3.0 mL of PHPPA solution (in THF, at the concentration of 1.0 mg/mL) was mixed with 1.0 mL of predissolved Rhodamine B (RB) solution (in THF, at the concentration of 0.5 mg/mL). Then, 8.0 mL of DI water was added dropwise into the mixed solution over a period of 2 h under vigorous stirring. Thereafter, the obtained RB-loaded self-assembly solution and the free dye were transferred into a dialysis tube (cutoff Mn = 8000− 14000 Da) and immersed into 500 mL of DI water, which was refreshed every half hour to remove the free RB. The whole dialysis process lasted 7 h. Dye encapsulations were characterized by measuring the UV absorbance at 554 nm.

Figure 3. Relationship between Dh of sample 1 and time. Sample 1 was kept still during the experiment and supernatant was extracted to monitor the change in Dh. Over the first 7 days, Dh decreased rapidly from 617 to 291 nm. Finally the Dh reached a steady state at about 180 nm.

3. RESULTS AND DISCUSSION 3.1. Design and Synthesis of Amphiphilic Homopolymers. PHPPAx homopolymers (where x = 36, 71, 103, determined by 1H NMR, see Table 1) were synthesized via RAFT polymerization. The typical 1H NMR spectra of DDMAT (RAFT reagent) and PHPPA homopolymer are shown in Figure S1 (Supporting Information) and Figure 1, respectively. The calculation of the degree of polymerization (DP) is discussed in Supporting Information (Table S1) as well. Both 1H NMR and GPC confirmed the successful synthesis of homopolymers.

3.2. Morphology Studies of Homopolymer SelfAssembly. PHPPA71 homopolymer forms vesicles in the mixture solvent of THF/water (sample 1 in Table 2), as confirmed by TEM in Figure 2B. The hydrodynamic diameter (Dh) of PHPPA71 vesicles determined by dynamic light scattering (DLS) is 617 nm right after dialysis. However, as shown in Figure 2A and Figure 3, after being kept still for 7 days, the Dh decreases to 291 nm accompanied by some microscopic precipitates. Thus, we monitored the change in Dh 197


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indicating a vesicular structure. This is consistent with TEM analysis of vesicles (Figure 2B). However, the membrane thickness of those PHPPA71 homopolymer vesicles varies from 8 to 200 nm, with some coexistent LCMs (Figure S3, Supporting Information).36,37 It was reported that a broad molecular weight distribution favors vesicle formation with uniform vesicle membrane.38,39 Also, the membrane thickness of vesicles can be considered as a power law with the hydrophobic proportion.40−42 Most recently, an morphology, which is nearly identical to Figure S3C (Supporting Information), was reported by Shunmugam et al.43 They proposed that the structure of their vesicle membrane is the same as traditional block copolymer vesicle. However, we think the homopolymer vesicle membrane is probably too thick to be a bilayer or interdigitated structure. Therefore, we propose that the vesicle membrane consists of PHPPA chains with different amphiphilicity, as illustrated in Figure 2D, which is quite different from the traditional block copolymer vesicle membrane with bilayer or interdigitated hydrophobic segments. Outside homopolymer vesicle membrane, H-bonds form between −OHs and surrounding water molecules, while the hydrophilicity decreases gradually inside homopolymer vesicle membrane due to the formation of inter/ intra-polymer H-bonds (Figure 2D), causing a variation in the hydrophobic proportion and finally the membrane thickness discrepancy. Considering that THF molecules have relatively weak hindrance against H-bond formation,44 excessively formed inter/intra-polymer H-bonds might be responsible for the formation of some coexistent LCMs. Information about the inter/intra- polymer H-bonding interaction was provided by FT-IR spectra (Figure 4). Two significant changes can be seen in the absorbance peak of −OHs in PHPPA71 spectra. Comparing the spectra of PHPPA71 solution and lyophilized PHPPA71 self-assembly, the peaks at about 3451 and 1732 cm−1 are shifted to lower wavenumbers of 3380 and 1728 cm−1, respectively, along with the expanding and strengthening of −OH absorbance. The forming of strong inter/intra-polymer H-bonding in aggregation phase might contribute to the above obvious shifting in the PHPPA71 spectra.10

Figure 4. FT-IR spectra of PHPPA71. (A) PHPPA71 solution in CHCl3 at a concentration of 150 mg/mL. (B) lyophilized PHPPA71 selfassembly. The peak shift from 3451 to 3379 cm−1 indicates the forming of strong hydrogen bonding between −OHs, and the changing of peak at 1732 to 1728 cm−1 is resulted from the hydrogen bonding formed between −OH and CO.

with time and found that the Dh of Sample 1 decreased with time (see Figure 3) and finally reached a steady state at around 180 nm. In fact, the hydrophilic coronas in homopolymer nanostructures are not as many as block copolymer ones to maintain their suspension in water. Therefore, the decrease in particle size is caused by sedimentation of large particles. Owing to this phenomenon, Dh in this paper was obtained right after dialysis except for those with special notes. To further verify the vesicle structure in sample 1 (36 days after dialysis), the radius of gyration (Rg) was measured by static light scattering (SLS). Usually, the Rg/Rh value can predict the particle morphology. For example, a solid sphere has an Rg/Rh of 0.774, while a thin-layer hollow sphere of 1.00.37 By simultaneously monitoring the Rg and Rh values on the same light scattering apparatus (ALV 5000), an Rg of 97 nm (Figure S2, Supporting Information) and an Rh of 87 nm were recorded. Therefore, the Rg/Rh value of sample 1 is 1.10,

Figure 5. (A) TEM image and (B) illustration diagram of PHPPA71 HLCMs (sample 5, self-assembled in the mixture solvent of DMF/water at pH 11.0). Those dark dots appearing inside the HLCMs with similar size are dehydrated cores (different from the PTA residue on the surface of HLCMs), which are represented by yellow spheres in the illustration diagram. 198


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Figure 6. (A) Calibration curves of Rhodamine B in DI water. (B) UV absorbance of Rhodamine B loaded self-assembly solution of sample 1 (selfassemble in the mixture solvent of THF/water at pH 7.0) and sample 5 (self-assembled in the mixture solvent of DMF/water at pH 11.0).

dots have much low contrast compared with the PTA staining agent residue (black dots with sharp contrast) around each HLCMs. The HLCMs’ structure is further studied by Ellman’s assay. It is well-known trithiocarbonate groups in the homopolymer (from DDMAT RAFT agent) might be degraded at high pH to form thiols, which will increase the hydrophilicity of homopolymer.53 Thus, Ellman’s assay was utilized to examine the presence of thiols in this HLCMs solution. However, UV− vis analysis showed no absorbance at 412 nm, which means no thiols were formed during the self-assembly. This confirmed that the trithiocarbonate groups and the hydrophobic dodecyl group forming the dehydrated core of the small micelles inside the HLCMs (as shown in Figure 5), which have little possibility to access with water (see Figure 5).53,54 3.4. Thermal Effect on Morphology. Since H-bonding is sensitive to thermal effect, sample 1 (self-assembled in THF/ water at pH 7.0) and sample 5 (self-assembled in DMF/water at pH 11.0; 7 months after dialysis) based on PHPPA71 homopolymer were stirred at 60 and 4 °C to monitor the changing in morphology. Over 48 h, Dh of sample 1 at 60 °C decreased from 555.4 (obtained just before heating) to 438.3 nm accompanied by a visual transition from white turbid to bluish solution. However, sample 5 at 60 °C barely showed a size change, which we believe after being self-assembled at the basic condition, there was no more inter/intra-polymer hydrogen bonds can be further cleaved. In contrast, when stirred at pH 7.0 and 4 °C, sample 1 did not show any size change. However, at the same condition, sample 5 experienced a steady decline in Dh from 174.7 to 116.6 nm. This observation is consistent with their structure of LCMs and HLCMs and evidently supports our assumption of inter/intrapolymer H-bonds effect. 3.5. Effect of Chain Length. A series of PHPPAx (x = 36, 71, and 103) with different molecular weights were selfassembled in the mixture of DMF and water (Table 2, samples 2−4) to reveal the relationship between chain length and morphology. DLS studies reveal that the Dh grows with the increasing of molecular weight (MW) of PHPPAx (Figure S4, Supporting Information) (The raw correlation data and the cumulant fit curves are shown in Figure S5, Supporting Information). According to TEM observations in Figure 7, the MW has great impact on the morphology of PHPPAx

Table 3. Calculated Dye-Encapsulation Efficiency of Sample 1 and Sample 5 sample

self-assembly condition

1

THF/water (1/2, v/v) at pH 7.0 DMF/water (1/2, v/v) at pH 11.0

5

morphology

dye encapsulation efficiency (wt %)

LCMs

10.6

HLCMs

16.2

3.3. Structure of Hydrated Large Compound Micelles (HLCMs). Similar PHPPA71 homopolymer structure to LCM was found in Sample 5 (Table 2), which was self-assembled in the mixture solvent of DMF/water (1/2, v/v) at pH 11.0. However, as shown in Figure 5, it showed a much complex morphology than LCMs in Sample 1. Actually those particles are hydrated large compound micelles (HLCMs). To distinguish the difference between LCMs and HLCMs, cryoTEM was initially considered. However, it is found that even by cryo-TEM, many artifacts might be introduced during the sample preparing process,45−50 and may not be solid evidence as well. Additionally, Libera and co-workers found that there was a thickness effect, which confined the characterization of hydrated soft material by cryo-TEM (Sample thickness should be below 180 nm).51 Thus, in the case of HLCMs, due to its hydrated structure and relatively large diameter (421 nm), it is not appropriate for cryo-TEM characterization, so as the watercontaining vesicles and LCVs with large diameter. Therefore, we first designed an indirect experiment to verify the difference of the internal structure between LCMs and HLCMs by comparing the dye encapsulation efficiencies of both kinds of particles (Figure 6). It was found that the dye encapsulation efficiencies of sample 1 and sample 5 are 10.6 and 16.2 wt % (Table 3), respectively. Considering that the dye is watersoluble Rhodamine B, we believe that the higher dye encapsulation efficiency of sample 5 is due to its hydrated structure as HLCMs. Normally, at higher pH, the inter/intrapolymer H-bonds will be cleaved.52 As a result, more −OHs are stretched into water rather than being trapped in H-bonds. Therefore, though HLCMs afford a structure similar to LCMs, HLCM has a much looser internal body containing large amount of water. Besides, conventional TEM revealed that those dark dots inside every HLCMs in Figure 5 might be dehydrated homopolymer cores. This is because those dark 199


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Figure 7. TEM images of homopolymer self-assemblies (prepared in the mixture solvent of DMF/water at pH 7.0) stained by phosphotungstic acid (PTA): (A) PHPPA36, LCVs, (B) PHPPA71, vesicles, (C) PHPPA103, vesicles, (D) IFFT TEM image of a representative PHPPA36 LCV, and (E and F) close-up TEM images of PHPPA71 and PHPPA103 vesicles, respectively. The mean vesicle sizes in parts B and C are 580 and 432 nm, respectively. (G) The corresponding schematic representation of homopolymer vesicle, which has a membrane consisting of both hydrophilic (blue) and hydrophobic (yellow) components.

homopolymer self-assemblies. For example, PHPPA36 forms large compound vesicles (LCVs) (sample 2), whereas PHPPA71 and PHPPA103 form vesicles (samples 3 and 4). Since MW itself cannot change the volume ratio of hydrophilic/hydrophobic moieties in PHPPA, the morphology transformation should be caused by inter/intra-polymer H-bonds. Compared with PHPPA71 and PHPPA103, PHPPA36 has much better chain mobility, especially in the case of polymers with strong Hbonding interaction,55 which is one of the critical factors in determining the self-assembly morphology.56 Upon addition of water, PHPPA36 forms large amount of inter/intra-polymer Hbonds by adjusting conformation, leading to the formation of LCVs (Figure 7A and inverse fast Fourier transform (IFFT) image in Figure 7D, and more TEM images in Figure S6, Supporting Information). Given that the MWs of PHPPA71 and PHPPA103 are nearly doubled/tripled compared with PHPPA36, their chains are too frozen to change the conformation upon the addition of water.57,58 Consequently, less inter/intrapolymer H-bonds form during the self-assembling process

Figure 8. Colloid stability measurement of sample 3 (A) and sample 7 (B) in aqueous solutions.

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Figure 9. TEM images of PHPPA36/PHPPA103 binary system: (A) branched cylinders, (B) close-up image of branched cylinders, where those black dots appearing inside the cylinder are residual phosphotungstic acid (PTA) stains. SEM images of PHPPA36/PHPPA103 binary system: (C) low magnification and (D) high magnification.

and relatively low hydrophobicity induces the formation of vesicles (Figure 7, parts B and C). Unfortunately, these selfassemblies are too big to be characterized by SLS to calculate Rg/Rh. Similar to sample 1, after being kept still for about a month, there was slight microscopic precipitates in sample 3. Those precipitates were carefully extracted and easily redispersed in water by shaking. DLS and TEM were employed to manifest the morphology of the precipitates, as shown in Figure S7, Supporting Information. The Dh of the precipitates is 628 nm, which is bigger than 580 nm of sample 3, indicating that the decrease in Dh is caused by the precipitation of bigger particles rather than the dimensional shrinking of particles. TEM image in Figure S7 (Supporting Information) shows that the precipitates have the same structure of vesicle as the aggregates remaining in water. Thus, we are convinced that the formation of precipitate is not the consequence of instability of PHPPA self-assembly, yet aroused by its huge size. 3.6. ζ Potential Analysis and Its Effect on Colloid Stability. Different from amphiphilic block copolymer selfassembly, there are no significant hydrophilic coronas to colloidally stabilize homopolymer self-assemblies. Therefore, surface charge is the key factor keeping homopolymer selfassembly steady. ζ potential studies shown in Table 2 reveal that all of the homopolymer self-assemblies are negatively charged, as a result of carboxyl end group on their surface. We also found that by varying cosolvents and pH values during selfassembly process, the ζ potentials of homopolymer selfassemblies can be tuned. For example, Sample 1 (selfassembled in THF/water at pH 7.0) has a ζ potential lower than that of sample 3 (self-assembled in DMF/water at pH 7.0), −20.9 mV and −26.3 mV, respectively, which means more −COOHs were trapped in H-bonds in sample 1 due to

stronger inter/intra-polymer H-bonding. For sample 5, carboxyl formed carboxylate in the presence of NaOH during the selfassembly (preventing −COOHs from forming H-bonds), which endows sample 5 with the highest ζ potential of −33.6 mV among those samples. To evaluate the effect of −COOH on self-assembly’s ζ potential, Et-PHPPA71 was synthesized and self-assembled (Sample 7) at the same circumstance as sample 3. As we expected, ζ potential of sample 7 dropped significantly to merely −5.1 mV. Consequently, sample 7 had poorer colloid stability. As can be seen from Figure 8, after being monitored for 8 days the transmittance of sample 3 only increased by 6.73%, while sample 7 experienced a tremendous rise by 34.38%. Therefore, it is obvious that −COOH is critical for maintaining colloid stability of homopolymer self-assembly. 3.7. Homopolymer Binary System. Block copolymer binary system (self-assembly using a premixed blend of two different polymers) has raised great attention for the past decade.59−61 Contrary to low MW amphiphiles, the component exchange between polymeric amphiphiles is relatively slow as a result of the low critical aggregate concentration (CAC),62,63 which allows the premixed binary polymer mixture to form selfassemblies with unified dominant morphology instead of dual structures. In other words, binary system self-assembly undergoes a disequilibrium state as a consequence of nonergodicity (i.e., locally isolated).64 To investigate the binary system of homopolymer, a mixture of PHPPA36/PHPPA103 (1/ 1, w/w) was self-assembled (Table 2, Sample 6). Figure 9 displays representative TEM and SEM images of self-assemblies emerged in this binary system (More TEM images are shown in Figure S8, Supporting Information). Even though PHPPA36 and PHPPA103 form LCVs and vesicles, respectively (Figure 7), branched cylinders emerge in sample 6. On the basis of 201


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Education (20110072110048), the Bayer Science & Education Foundation, Bayer-Tongji Eco-Construction & Material Academy, the startup package (0500144022), and the open fund for characterization (0002011002) of Tongji University.

Eisenberg and co-workers’ research, the decline in hydrophobicity enables amphiphilic copolymers to exhibit transitions from vesicles, to cylinders (a decrease in interfacial curvature).65,66 Interfered by PHPPA103, PHPPA36 fails to pack into LCVs with high density of inter/intra-polymer Hbonds, inducing decline in hydrophobicity. Therefore, branched cylinders appear. SEM images of sample 6 clearly show that these branched cylinders are composed of spherical micelles.

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4. CONCLUSIONS In summary, we have successfully designed an amphiphilic PHPPA homopolymer with −COOH as the end group to enhance the colloidal stability. TEM observation indicates that PHPPA homopolymer is able to self-assemble into vesicles as well as other distinct colloidal structures, such as HLCMs, LCVs, LCMs, and even branched cylinders in binary system, which is closely depending on its molecular weight and the selfassembly conditions such as solvent properties and solution pH. The inter/intra-polymer hydrogen bonding effect plays a key role in the formation of various nanostructures by homopolymer self-assembly. Additionally, a new membrane structure of homopolymer vesicles is proposed which is quite different from that of block copolymer vesicles (bilayer or interdigitated). Admittedly, the lack of big hydrophilic coronas decreases the colloidal stability of homopolymer self-assembly, but the introduction of −COOH as the end group increases the negative surface charge (verified by ζ potential) and prevents the aggregation of self-assembled nanoparticles. Overall, homopolymer has the same ability to form a range of nanostructures with either thermodynamic or kinetic stability as block copolymer, yet only one-step of polymerization is required in preparation of homopolymer, which opens up a much convenient and effective way to prepare vesicles and other structures for potential applications in drug delivery, biosensor, nanoreactor, and enzyme-catalyzed reaction.

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ASSOCIATED CONTENT

S Supporting Information *

Characterizations of PHPPA homopolymer and self-assembled nanostructures by 1H NMR, TEM, DLS and SLS. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Fax: +86 (021) 69584723. Telephone: +86 (021) 69580239. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.D. is supported by the National Natural Science Foundation of China (21074095 and 21174107), Pujiang project of Shanghai science and technology commission (10PJ1409900), SRF for ROCS of SEM, the program for professor of special appointment (Eastern Scholar) at Shanghai institutions of higher learning, Shanghai 1000 Plan, the CXY project and the key project of innovative scientific research of Shanghai education commission (11CXY16 and 11ZZ31), Fok Ying Tong Education Foundation (132018), the fundamental research funds for the central universities (0500219141), new century excellent talents in universities of Ministry of Education (NCET-10-0627), Ph.D. program foundation of Ministry of 202


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