Aggregation Behavior and Antioxidant Properties of Amphiphilic

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

Aggregation Behavior and Antioxidant Properties of Amphiphilic Fullerene C60 Derivatives Co-Functionalized with Cationic and Nonionic Hydrophilic Groups Mengjun Chen, Shengju Zhou, Luxuan Guo, Lin Wang, Fuxin Yao, Yuanyuan Hu, Hongguang Li, and Jingcheng Hao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03681 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 4, 2019

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Aggregation Behavior and Antioxidant Properties of Amphiphilic Fullerene C60 Derivatives Co-Functionalized with Cationic and Nonionic Hydrophilic Groups Mengjun Chen,1 Shengju Zhou,2 Luxuan Guo,1 Lin Wang,3 Fuxin Yao,1 Yuanyuan Hu,1 Hongguang Li,1* Jingcheng Hao1* 1

Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special Aggregated

Materials, Shandong University, Ministry of education, Jinan, 250100, China 2

School of Chemistry and Chemical Engineering Shandong University of Technology, Jinan, 250014,

Zibo, 255049, China. 3

Analytical Center of Qilu Normal University, Jinan, 250100 ,China.

Corresponding author: [email protected]

Phone: 0931-4968829

Fax: 0931-4968163

[email protected]

Phone: 0531-88366074 Fax: 0531-88364750

KEYWORDS: fullerene, amphiphilic, aggregation, scavenge, radical

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ABSTRACT Amphiphilic derivatives of fullerene C60 are attractive from viewpoints of supramolecular chemistry and biomedicine. The establishment of relationships among the molecular structure, aggregation behavior and properties such as scavenging radicals of the amphiphilic C60 derivatives is key to push these carbon nanomaterials to real applications. In this work, six monosubstituted C60 derivatives were synthesized by one-step quaternization of their neutral precursors which bear Percec monodendrons terminated with oligo(poly(ethylene oxide)) (o-PEO) chain(s). The main difference among the C60 derivatives lies in the number and substituted position of the o-PEO chain(s) within the Percec monodendron. Derivative with a 4-substitution of the o-PEO chain still showed limited solubility in water. Other derivatives possessing two or three o-PEO chains exhibited much improved solubilities and rich aggregation behavior in water. It was found that the formation of aggregates is regulated both by the number and the substituted pattern of the o-PEO chains. Typical morphologies include nanosheets, nanowires, vesicles, nanotubes and nanorods. Although the structures of the C60 derivatives are different from those of traditional surfactants, their aggregation behavior can be also well explained by applying the theory of critical packing parameter. Interestingly, the capabilities of the C60 derivatives to scavenge the hydroxyl radicals (OH ) followed the same order of their solubility in water, where the compound bearing three o-PEO chains with a 2,3,4- substitution got the champion quenching efficiency of

97.79%

at

a

concentration

of

0.15


mg mL-1

( 0.11

mmol L-1).

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INTRODUCTION Reactive oxygen species (ROS) refer to a general term for lively substances consisting of oxygen. Major ROS include hydroxyl radical (OH -), superoxide radical anion (O2•-), singlet oxygen (1O2) and peroxides (H2O2).1 To maintain a healthy body, the oxidation and anti-oxidation should be balanced. Although our system contains some endogenous antioxidant defense systems, they are incomplete to entirely prevent the occurrence of the accumulation of ROS. This unbalance will cause oxidative stress, which is one of the major reasons in cellular damage and aging.2,3 To develop exogenous antioxidants which can prevent such physiological disorder is always desired. Fullerenes are a class of cage molecules composed entirely of carbon. Among them, C60, which has a regular icosahedral structure with 20 six-membered rings and 12 five-membered ones, is the most abundant member. Since its discovery in 19854 and gram-scale production in 1990,5 C60 has received great attention and its rich physicochemical properties have led to a variety of applications including superconductors,6,7 field-effect transistors,8-10 heterojunction organic solar cells11,12 and artificial photosynthetic systems.13,14 The chemically attachment of various hydrophilic groups to the intrinsically hydrophobic C60 sphere and noncovalent complexation of pristine C60 with hosts produced water soluble C60, which amicably invited C60 to the fields of biology and life science.15-18 Early discoveries included the cleavage19 and condensation20 of DNA, and inhibition of HIV-1 protease.21 Later, new biological activities have been identified, including anti-bacterial activity,22 anti-cancer effect,23,24 and immunological properties.25 As one of the most important properties, the ability of C60 to scavenge radicals was first reported in 1991.26 After that, the type of radicals which can react with C60 was quickly expanded from the initially-discovered benzyl and methyl radicals to others.27 This feature of C60 has soon been applied to water soluble C60 to prevent oxidative and aging associated disorders in different pre-clinical in vitro and in vivo models.28 For chemically functionalized C60 derivative, the radical scavenging capability is heavily influenced by the means of chemical modification, including the type of the reaction adopted


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of the o-PEO chains is fixed, the compounds show very similar structure with the only difference on the substituted position of the o-PEO chain(s) within the Percec monodendron. Thus, they seem to be ideal compounds for deeper studies on their solution properties and biological activities. Unfortunately, they are insoluble or only slightly soluble in water, indicating that the o-PEO chain(s) alone can not balance with the highly hydrophobic C60 sphere. To solve this problem, in this work we transferred these neutral C60 derivatives to ionic ones (Scheme 1, 2a-f) through a facile one-step quaternization. After a thorough investigation on the aggregation behavior of the highly water soluble compounds bearing two and three o-PEO chains (2b-f), their capabilities to scavenge OH -, which is considered to be the most reactive component among all ROSs, were evaluated by electron spin resonance (ESR) measurements. Different annihilation effects were observed with the highest quenching efficiency up to 97.79% for 2e at a concentration of 0.15 mg·mL-1 ( 0.1 mmol L-1). Experimental Section Chemicals and Materials Iodomethane (CH3I,

99.5%) was bought from Sun Chemical Technology (Shanghai) Co., Ltd.

Tetrabutylammonium hexafluorophosphate (TBAPF6) was purchased from Sigma-Aldrich. Dichloromethane used in the electrochemical experiments is of spectral purity and was obtained from Sigma-Aldrich. Toluene and methanol used in silica gel column chromatography are of analytical grade and were obtained from local suppliers. All the chemicals were used without further purification unless other stated. High-purity water was obtained from a water purification system (Ulupure Instrument Co. Ltd.). Details for the syntheses and characterizations of the neutral C60 derivatives 1a-f can be found elsewhere.44 Synthesis The ionic fullerene derivatives 2a-f were facilely synthesized through one-step quaternization of the pyrrolidine moieties in 1a-f by using CH3I as both the solvent and the reactant, as illustrated in Scheme 1. CH3I is in large excess to ensure the complete dissolution and efficient conversion of 1a-f. In a


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typical synthesis, 5 mL CH3I with pre-dissolved 1a (0.2 g) was injected into a 15 mL two-neck flask which was performed three times vacuum-argon cycles in advance. The mixture was heated to reflux under stirring and allowed to react for 5 days. After that, CH3I was removed under reduced pressure and the crude product was purified by silica gel column chromatography using toluene/methanol mixture (3:1, v/v) as the eluent. The effluent containing the target product was collected and most of the organic solvent was removed under reduced pressure. After further dried in an vacuum oven at 40 C (12-24 h), 2a was obtained as a brown solid (0.1961 g, yield: 86%). MALDI-TOF MS: calculated (without I-): 1032.06, Found: 1032.06. 2b-2f were obtained by the same procedures. 2b (0.2017 g, yield: 90%), MALDI-TOF-MS: calculated (without I-): 1193.23, Found: 1193.05. 2c (0.1972 g, yield: 88%), MALDI-TOF-MS: calculated (without I-): 1193.23, Found: 1194.12. 2d (0.1482 g, yield: 67%), MALDI-TOF-MS: calculated (without I-): 1355.42, Found: 1355.24. 2e (quantitative, silica gel column chromatography is not necessary), MALDI-TOF-MS: calculated (without I-): 1355.42, Found: 1354.93. 2f (quantitative, silica gel column chromatography is not necessary), MALDI-TOF-MS: calculated (without I-): 1355.42, Found: 1354.92. Measurements of the Radical Scavenging Capabilities The radical scavenging experiment was done with the help of electron spin resonance measurements (ESR) on a JEOL JES-FA200 EER spectrometer. H2O2 was chosen as the OH - generator and dimethyl pyridine N-oxide (DMPO) was used as the trapping agent for OH -. Other factors, including the volume of the sample to be tested, the amount of H2O2 added and the amount of DMPO, are fixed. To evaluate the quenching ability of 2a-f toward OH -, H2O was selected as the control. All data were collected in the dark with a cumulative time of 1 min after 4 min of ultraviolet light irradiation. The quenching efficiency (

e)

was defined as follows, e

= (c0 – cf) / c0

where c0 is the initial concentration of OH - while cf is that of the remaining OH - after quenching. ACS Paragon Plus Environment

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Characterizations MALDI-TOF MS spectra were recorded on an AXIMA Confidence TM mass spectrometer (Shimadzu, Japan) with gentisic acid as the matrix. Thermogravimetric analysis (TGA) measurements were carried out on DSC 822e (Piscataway, NJ) under nitrogen with a scanning speed of 10 °C/min. FTIR spectra were obtained on a VERTEX-70/70v FTIR spectrometer (Bruker Optics, Germany). UV-vis spectra were recorded using a HITACHIU-4100 spectrophotometer (Hitachi, Japan) with a scanning speed of 600 nm/min. Cyclic voltammetry (CV) measurements were carried out on a CHI 600E electrochemical analyzer with a glassy carbon electrode as the working electrode and a Pt plate as the quasi-reference electrode. Dichlorobenzene and TBAPF6 were selected as the solvent and supporting electrolyte, respectively. The ferrocene/ferrocenium (Fc/Fc+) redox couple was used as internal reference for the potential calibration. Zeta potential and the size distribution of the aggregates were recorded on the Malvern Zetasizer ZS with a DTS1070 folded capillary cell. Differential scanning calorimetry (DSC) measurements were performed on DSC8500 (PerkinElmer, USA). Samples were measured in aluminum pans under nitrogen flow. An empty aluminum pan was used as the reference. The samples were heated at 10 °C·min-1. For transmission electron microscopy (TEM) observations, 5 O& of the specimen was placed on a copper grid, and the excess solution was wicked away with a piece of filter paper. The copper grid was then dried with an infrared lamp and observed on a JEOL JEM-1400 (Japan) at an accelerating voltage of 120 kV. For cryo-TEM observations, the sample ( 4 O&A was dropped onto a micro grid under high humidity (>80%). Excess sample was removed with two pieces of filter paper, leaving a thin film sprawling on the micro grid, which was plunged into liquid ethane pre-liquefied with liquid nitrogen. The vitrified sample was transferred into a sample holder (Gatan 626) and observed on a JEOL JEM-1400 TEM (120 kV) at ~ -174 °C. The images were recorded on a Gatan multiscan CCD. In freeze-fracture transmission electron microscopy (FF-TEM) observations, a small amount of sample ( 4 L) was dropped on the specimen carrier. The sample was frozen by plunging into liquid propane


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cooled by liquid nitrogen. Fracturing and replication were carried out using freeze-fracture apparatus (EM BAF 060, Leica, Germany) at a temperature of -160 C. Pt/C was deposited at an angle of 45 to shadow the fracture surface and C was deposited at an angle of 90 to consolidate the fracture surface. The replicas were transferred onto a copper grid and then checked using a JEOL JEM-1400 TEM (Japan) at an accelerating voltage of 120 kV. The optimized structures of 2a-f were obtained by Materials Studio 8.0 in vacuum. All the experiments were carried out at room temperature unless other stated. Results and Discussion Solubility and Physicochemical Properties of 2a-f Figure 1 gives photos of the saturated aqueous solutions of 2a-f. For comparison, those of 1a-f are also shown. For 2a which has only one o-PEO chain, there was no distinguishable change of the solubility in water before and after the quaternization. For other five compounds with two (2b, 2c) or three (2d-f) o-PEO chains, significant increase of the solubility in water has been observed after the quaternization. The highest concentration achieved was determined to be 0.5 mmol·L-1 for 2b, 0.2 mmol·L-1 for 2c, 1.0 mmol·L-1 for 2d, 2.0 mmol·L-1 for 2e and 1.0 mmol·L-1 for 2f, respectively. Thus, the solubility of 2a-f follows an order of 2a < 2c < 2b < 2d

2f < 2e.

1a

1b

1c

1d

1e

1f

2a

2b

2c

2d

2e

2f

Figure 1. Photos of saturated aqueous solutions of 1a-f and 2a-f, each of which is equilibrated with trace amount of solid at the bottom. The increase of the water solubility of the C60 derivatives after quaternization is unambiguously ascribed to the introduction of the ionic moiety, i.e., the quaternary ammonium cation on the pyrrolidine ring. However, it should be noted that the synergistic effect of the Percec dendron cannot be neglected


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as the ionic C60 derivative lacking such lateral functional group showed quite limited solubility in any single solvent including water.20 This conclusion gains further proof from the fact that the compounds with three o-PEO chains (2d-f) have higher solubilities in water compared to those with two o-PEO chains (2b, 2c). When the number of the o-PEO chains is the same, the water solubility of different compound could be influenced by the substituted pattern of the o-PEO chain. For compounds with two o-PEO chains, 2,4-substituted pattern (2b) creates a better solubility while for those with three o-PEO chains, 2,3,4-substituted pattern (2e) shows the best performance. This observation is consistent with the phenomena observed for other Percec type dendrons where a small change in the substitution position would induce a big change of the properties.45 Besides the water solubility, other physicochemical properties have also been changed by the quaternization. From FTIR spectra (Figure 2a, b), it can be seen that compared to 1a-f, the vibration peaks for the asymmetric and symmetric methylene stretching bands at 2930 cm-1 and 2850 cm-1 and those for methyl stretching bands at 2960 cm-1 become more obvious and sharper after quaternization. This observation indicated that the o-PEG chain(s) in 2a-f are in a more ordered state than those in 1a-f, which can be ascribed to the increased inter-compound interaction induced by the introduction of the additional electrostatic repulsion among the quaternary ammonium cations on the pyrrolidine rings. This conclusion gained further proof from the different appearance between the neutral C60 derivatives and the ionic ones. For example, while 1f behaves as a highly viscous liquid with a Tg at -13 oC, 2f exists as a solid at room temperature and exhibits no phase transition or glass transition point within the investigated temperature range (-40 oC to 100 oC). From cyclic voltammograms (Figure 2c, Table 1), the average potential (E1/2) of the first couple of redox peaks for 2a-f was determined to be -1.188 0.059 V, which is less negative compared to that of 1a-f (-1.226

0.034 V).44 In addition, while only

three couples of the redox peaks were observed for 1a-f, up to four couples have been detected for 2a-f. These observations indicated that 2a-f are easier to accept electrons than 1a-f due to the introduction of ionic headgroups, which is consistent with the observations on other fulleropyrrolidiums such as those


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bearing alkyl chains.46

(a)

1a

(b)

2a

1b

1d 1e 1f

(c)

2f

2b

Transmittance / a.u.

1c

Transmittance / a.u.

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

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2e

2c 2d

30 A

2d

2e

2c 2f

2b 2a

3000

2900

2800

Wavenumber / cm

-1

2700

3000

2900

2800

Wavelength / cm

2700

-1

0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -3.0 + E (vs Fc/Fc ) / V

Figure 2. (a) FTIR spectra of 1a-f at 3050-2700 cm-1. (b) FTIR spectra of 2a-f at 3050-2700 cm-1. (c) Cyclic voltammograms of 2a-f in dichlorobenzene (1.0 mmol·L-1) using ferrocene (Fc) as a reference and 50 mmol·L-1 TBAPF6 as supporting electrolyte. The scan rate is 0.1 V·s-1. Table 1. Summary of the physicochemical properties of 2a-f Solubility in water E1/2 for the first Type of aggregates / mmol·L-1 reduction / Va 2a / -1.194 (-1.283) / 2c 0.2 -1.179 (-1.221) Nanosheets 2b 0.5 -1.160 (-1.241) Nanosheets, vesicles Vesicles, nanotubes, 2d 1.0 -1.300 (-1.225) nanorods 2f 1.0 -1.132 (-1.179) Nanowires 2e 2.0 -1.16 (-1.208) Nanowires a Values in the brackets are from corresponding neutral precursors. b Values obtained at a concentration of 0.15 mg mL-1

Quenching of OH / %b / 53.70 82.35 89.97 86.58 97.79

It is known that the basicity of the nitrogen on the fulleropyrrolidine ring is quite low due to the presence of the C60 moiety which is highly electron deficient.47,48 Thus, decomposition could occur for fulleropyrrolidiums especially upon heating. Typical reactions include the back reaction from the ionic compound to the neutral one, as demonstrated for C60 derivatives bearing long alkyl chains.46 TGA analysis (Figure S1) on 2a-f showed that the decomposition temperatures are in the range of 130-160 C, which are lower compared to the neutral molecules whose decomposition temperatures are over 200 ACS Paragon Plus Environment

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C (1d, 1f) or 300 C (1a-c, 1e). Even though, we noticed that the thermal stability of 2a-f at moderate temperatures is acceptable. At a temperature of 100 C, the weight loss is below 2 % (inset of Figure S1). When stored at room temperature, 2a-f can be stable up to at least four weeks without detectable decomposition as monitored by thin layer chromatography and MALDI-TOL-MS. UV-vis Absorption Having a -conjugated, highly-curved structure, C60 exhibits rich absorptions in UV and visible regions.49 Upon chemical functionalization, the carbon-carbon double bond (C=C) will be destroyed, which can significantly alter the shape of the spectra. Besides chemical functionalization, the UV-vis spectrum of C60 is also sensitive to the microenvironment where the C60 moieties are located. Specifically for the monosubstituted C60 derivatives, the peak at 429 nm is sharp when dissolved in a good solvent, which becomes broad or totally disappears upon formation of aggregates.50 This feature makes UV-vis a powerful technique to facilely detect the state of a given C60 derivative in water, i.e., individuals or aggregates. The absorptions of 2b-d in a wavelength range of 230-600 nm are given in Figure 3a. Two absorption bands around 257 nm and 322 nm were observed. Compared to the spectra of pristine C6049 and highly water soluble C60 monoadduct,50 the absorptions of 2b-d have greatly broadened, which is indicative for the existence of inter-sphere interaction. This conclusion gained further proof from the total disappearance of the peak at 429 nm (Figure 3b). Absorptions at other concentrations were also checked. It was found that the peak at 429 nm did not come down to a concentration of 1 µmol L-1 (Figure S2), indicating that aggregates formed already in the highly dilute solutions. From these observations, it can be concluded that even though the compound was co-functionalized with the o-PEO chains and the cationic moiety, aggregation cannot be fully suppressed due to the presence of the large, highly hydrophobic C60 sphere. The aqueous solutions of 2b-f are transparent to the naked eyes. Consistent with the visual inspection, statistics and fittings on the absorption at 500 nm gave straight lines for all the five compounds, preventing obvious light scattering effect. The slope of the line, which is determined by the Moore


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extinction coefficient of each compound, differs from each other. This could be partially ascribed to the different substitution pattern of the attached Percec monodendron, which can influence the electronic structure of the phenyl as well as the interaction between the phenyl and the pyrrolidine ring. Another factor came from the different aggregation behavior of 2b-f in water, which formed aggregates with varying morphologies and sizes in water, as will be discussed in detail in the following section.

0.12 0.06

2d

0.18

1.2

0.5

(b)

2b 2c 2d 2e 2f

0.4 0.3 0.2

1.0

0.1 0.0

0

50

100 150 200

c / mol L

2b

-1

Absorbance

0.24 2f

Absorbance

0.30

Absorbance at 500 nm

0.36

(a)

2e

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

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0.6

2c

0.00 250

0.8

300

350

400

450

500

550

600

0.4 400

Wavelength / nm

410

420

430

440

450

Wavelength / nm

Figure 3. (a) UV-vis absorption of 2b-f at a concentration of 10 mol·L-1. Inset is variation of the absorbance at 500 nm as a function of the concentration of 2b-f. The straight lines are guides for the eyes. (b) UV-vis absorption in the range of 400-450 nm at a concentration of 200 mol·L-1 for 2b-f. Aggregation Behavior in Water Considering the important role of the aggregate formation of the C60 derivative played in determining its biological activity, examination of the aggregation behavior of the water soluble C60 derivative is a prerequisite before any of its biological tests is made. From a viewpoint of colloid and interface science, investigation of the aggregation behavior of water soluble C60 derivatives44,51-64 is quite interesting, as in this new class of amphiphiles, the hydrophobic part is a rigid carbon sphere instead of the flexible alkyl chain(s). For traditional surfactant, the morphology of the aggregate formed in aqueous solutions can be well predicted by the critical packing parameter p,65 which can be simplified to the ratio of the cross sectional area of the hydrophobic part to that of the hydrophilic part. When p > 1, reverse structures are preferred where the hydrophilic parts form the inner phase and the hydrophobic parts stay outside. At p ACS Paragon Plus Environment

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1, the compound looks like a cylinder and tends to self-associate into lamellar structures. At 1/2 < p < 1, the compound looks like a truncated cone which leads to the formation of unilamellar and multilamellar vesicles. Further decrease of p facilitates the formation of micelles with various morphologies including rodlike, threadlike or wormlike micelles (1/3 < p < 1/2) and globular ones (p 1/3). For 2a-f, following the order of the solubility in water (2a < 2c < 2b < 2d

2f < 2e), a continuous

decrease of p following can be deduced. In the following, we will give a detailed survey of the aggregation behavior of 2a-f in water following the same order.

Figure 4. Characterizations of the aggregates formed by 2c (a-c) and 2b (d-h) in water. (a) TEM image of the saturated solution of 2c ( 200 mol L-1). (b) TEM image of 100 mol L-1 2c. The magnification is the same with that of image a. (c) Size distribution of the aggregates formed by 2c at different concentrations. (d) TEM image of 100 mol L-1 2b. (e) Cryo-TEM image of 100 mol L-1 2b. (f) Size distribution of the aggregates formed by 2b at different concentrations. (g) FF-TEM image of 200 mol L-1 2b. (h) TEM image of 200 mol L-1 2b. The magnification of images g and h is the same with that of image e. The poor water solubility of 2a makes the study of its aggregation behavior tedious. We then focused our attention on the aggregation behavior of 2b-f, which were probed by a combination of imaging techniques including transmission electron microscopy (TEM) observations, cryo-TEM observations ACS Paragon Plus Environment

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and freeze fracture TEM (FF-TEM) observations. Besides, the size distribution of the aggregates has also been probed by dynamic light scattering (DLS). For 2c which is only slightly soluble in water, TEM observations on the saturated solution ( 200 mol L-1) showed the presence of nanosheets with sizes of 100-300 nm (Figure 4a). No aggregates with other shapes have been detected. When the concentration decreased to 100 mol L-1, the number density of the nanosheets also decreased, but their sizes remained unchanged (Figure 4b, 4c). For 2b, globular and thin nanosheets with sizes of 100-400 nm were observed at a concentration of 100 mol L-1 (Figure 4d). Besides these nanosheets, smaller aggregates of 30-75 nm were also detected by cryo-TEM (Figure 4e). The contrast in the central part of the aggregate is lower, indicating that they are vesicles. Judging from the result of size distribution (Figure 4f), the dominated type of aggregates are vesicles and the nanosheets, which have much larger sizes, are the minority. When the concentration increased to 200 mol L-1, vesicles were observed exclusively as confirmed by FF-TEM observations (Figure 4g). Clearly, an aggregate transition from nanosheets to vesicles was induced by the increase of concentration. In addition, unlike the vesicles found in the sample of 100 mol L-1 which existed individually, those formed at 200 mol L-1 tend to adhere each other. This phenomenon became more obvious in the saturated solution ( 500 mol L-1), as seen from a typical FF-TEM image shown in Figure S3. The adhesion of the vesicles accounted for the concentration-induced increase of the average size, as seen from Figure 4f. As the vesicles formed by 2b have thick walls and the interaction among C60 spheres is relatively strong,61 they are robust and available under common TEM without significant shrinkage (Figure 4h). From Figure 4e and 4h, it can be seen that the vesicles are non-spherical with rough surfaces. Vesicles with irregular shapes, termed faceted vesicles, have already been observed in systems containing cationic/anionic surfactant mixtures,66-68 and theoretical description of the formation mechanism has appeared.69 As the C60 sphere has a relatively big size with high rigidity, it has a high tendency to self-associate and crystalize in aqueous solutions. The experimental evidence obtained in current study indicated that amphiphilic C60 derivatives could be a new class of candidates for the ACS Paragon Plus Environment

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fabrication of faceted vesicles.

Figure 5. Characterizations of the aggregates formed by 2d-f in water. (a,b) Cryo-TEM images of 100 mol L-1 2d. (c) Size distribution of the aggregates formed by 2d at different concentrations. (d) Cryo-TEM image of 100 mol L-1 2f. (e) Cryo-TEM image of 1.0 mmol L-1 2e. (f) Cryo-TEM image of 2.0 mmol L-1 2e. The magnification of images e and f is the same with that of image d. (g) Size distribution of the aggregates formed by 2f and 2e at different concentrations. The dashed line is a guide for the eyes. Morphologies of the aggregates formed by the compounds with three o-PEO chains are summarized in Figure 5. Image a gives a typical cryo-TEM image for 2d at 100 mol L-1, where polymorphism was confirmed. The big vesicles have sizes around 100 nm and the smaller ones are only 10-50 nm. Image b gives a cryo-TEM image with higher magnification to highlight the structural features of the small vesicles. Unlike the vesicles with thick walls formed by 2b, the vesicles formed by 2d have thin walls which enclose a large inner compartment. This feature made them unstable during drying and attempts to capture them by TEM failed. Besides vesicles, aggregates with other morphologies were also observed. The nanotubes (Figure 5a, indicated by the arrow heads, Figure S4) are straight with lengths ranging from 80 to 120 nm and diameters around 15 nm. Besides vesicles and tubes, the presence of nanorods was also confirmed (Figure 5a, indicated by the arrow, Figure S5). Analysis on the size ACS Paragon Plus Environment

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distribution of the aggregates at different concentrations indicated that the curves superposed totally (Figure 5c), indicating that the variation of concentration mainly influenced the number density of the aggregates instead of their sizes. For 2f and 2e, formation of nanowires was confirmed by cryo-TEM observations as shown in Figure 5d-f. The nanowires formed by 2f have diameters of 3-4 nm and lengths up to several hundred nanometers (Figure 5d). They showed some flexibility and could bend at certain points. The nanowires formed by 2e, with diameters below 3 nm (Figure 5e, 5f), are thinner compared to those formed by 2f. The formation of the aggregates with a higher curvature for 2e indicated that the cross-sectional area of its hydrophilic part is larger, which resulted in a smaller p. Analysis on the size distribution of the nanowires at different concentrations (Figure 5g) showed that the nanowires formed by 2e are smaller with a narrower size distribution than those formed by 2f at the same concentration. It was also found that the sizes of the nanowires decreased with increasing concentration for both 2e and 2f. These observations are fully consistent with the results from imaging studies. To get a deeper understanding of the aggregation behavior of 2a-f in water, the configuration of each C60 derivative was optimized in vacuum by the software of Materials Studio 8.0. The geometry of each compound was described within the theory of p. The results, together with illustrations of the aggregates formed by 2a-f, are summarized in Scheme 2. While the sectional area of the hydrophobic part of 2a-f is basically determined by the C60 moiety and is nearly unchanged, that of the hydrophilic part varies significantly with the variation of the o-PEO chain(s) and their substituted positions within the Percec monodendrons. As a result, p changes accordingly from 2a to 2f, leading to the variation of the solubility and aggregation behavior. A comparison between the optimized configurations of 2b and 2c showed that the 2,4-substituted pattern (2b) creates a larger cross sectional area of the hydrophilic part and a smaller p than the 3,5-substituted pattern (2c) does, which is consistent with experimental observations where the aggregates formed by 2b (mainly vesicles) have higher curvatures compared to those formed by 2c (exclusively nanosheets). For compounds with three o-PEO chains, the optimized


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configurations showed that the 2,3,4-substituted pattern (2e) creates the largest cross sectional area of the hydrophilic part and the smallest p, which is also consistent with the experimental observations where 2e formed very thin nanowires with high curvature. Thus, it can be concluded that although the structures of 2a-f are much more complicated than those of traditional surfactants, their aggregation behavior in water can be largely interpreted by the theory of p. As depicted in Scheme 2, from 2c to 2e the aggregates changed gradually from nanosheets (corresponding to lamellae in the case of traditional surfactants) to vesicles and further to nanotubes, nanorods (corresponding to the rodlike micelles in the case of traditional surfactants) and nanowires (corresponding to threadlike or wormlike micelles in the case of traditional surfactants) caused by the continuously increase of the cross sectional area of the hydrophilic part.

Scheme 2. Structures of 2a-f optimized in vacuum and illustration of their aggregation behavior in water governed by the critical packing parameter p. Green balls: The C60 moieties. Blue tails: The o-PEO chains. The descriptions of the aggregates within the brackets denote the common names


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adopted in the case of traditional surfactants. It should be noted that in Scheme 2, the optimization was performed on individual compound. As most C60 derivatives have more than one o-PEO chains attached to the C60 sphere through a rotatable phenyl ring, their spatial arrangement within the aggregates should be taken into consideration, which may result in deviation of p from the value deduced for the individual compound. For example, from Scheme 2 it seems that the two o-PEO chains in 2b, which are highly extended, create a cross sectional area of the hydrophilic part larger than that of 2d-f. However, as the number density of the o-PEO chains in 2b is lower which means that they have a larger potential to be compressed, the real value of the cross sectional area of the hydrophilic part for 2b could be smaller when forming aggregates. This high degree of freedom of the molecular arrangement caused by the relatively complicated structures of the C60 derivative could also lead to the appearance of more than one metastable conformations, which may account for the polymorphism observed in the aggregation behavior of certain compound, such as 2d. This feature of the C60 derivative is different from traditional surfactants where the degree of freedom of the molecular arrangement within the aggregates is much lower due to the simpler structure of both the hydrophilic and hydrophic parts. 75

c / mol L

-1

100 200

60

/ mV

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

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45

30

15

0 2c

2b

2d

2f

2e

Figure 6. Zeta potential of the aggregates formed by 2b-f at different concentrations. The dashed line is a guide for the eyes denoting the value of 30 mV. All the aqueous solutions of 2b-f are quite stable, with no detectable changes upon storage. Zeta ACS Paragon Plus Environment

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potential ( ) measurements, which were carried out at concentrations of 100 and 200 mol L-1, indicated that all the aggregates are positively-charged with values well above 30 mV (Figure 6). These highly charged surfaces, together with the steric hindrance created by the o-POE chains, account for the high stability for the aggregates formed by 2b-f. Radical Scavenging Capabilities To date, evaluations of the radical scavenging capabilities of water soluble C60 derivatives are restricted to fullerenols,34,36-43 carboxylated fullerenes30-35 and a fulleropeptide.70 These studies suffered from the ill-defined molecular structures of the C60 derivatives, which made it difficult to establish the structure-property relationship. On the other hand, some water soluble C60 derivatives with defined structures have been synthesized and their aggregation behavior in water has been investigated.52,53,55-64 Unfortunately, further evaluations on their radical scavenging capabilities are absent. Having made clear the aggregation behavior of 2b-f in water, we transfer to the evaluations on their capabilities to scavenge OH by ESR (see experiments section for details). At a concentration of 0.15 mg mL-1, effective quenching of OH was observed (Figure 7a, b). Interestingly, the capabilities of the compounds to scavenge OH , characterized by water, i.e., 2c < 2b < 2d

2f < 2e. The highest

e,

e

followed the same order of their solubilities in

reached 97.79 % for 2e. Note that at a concentration

of 0.15 mg mL-1, the compounds with two o-PEO chains (2b, 2c) have a slightly higher molar concentration ( 125.7 mol L-1) than those with three o-PEO chains (2d-f, 110.7 mol L-1). However, they showed lower capabilities to scavenge OH , indicating that the number of o-PEO chains is a principal factor that influences

e.

For compounds with two o-PEO chains, it was found that the

compound with a 2,4-substituted pattern (2b) is superior to that with a 3,5-substituted pattern (2c). For compounds with three o-PEO chains, the compound 2,3,4-substituted pattern (2e) is advantageous over those with 3,4,5- and 2,4,6-substituted patterns (2d, 2f). These observations are fully consistent with the results from solubility tests and aggregation behavior studies. For water soluble C60 derivative, the C60 sphere is responsible for the scavenging of OH . Upon the formation of aggregates, the C60 spheres ACS Paragon Plus Environment

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gather together to form hydrophobic microdomains, leading to a reduction of the effective surface area available for the quenching of OH . For the C60 derivative with a higher water solubility, a smaller p will be created which leads to the formation of aggregates with higher curvatures. At the interface between the hydrophobic microdomains and the hydrophilic outerlayer, a higher curvature would enlarge the exposed surface area of the C60 spheres for the scavenging of OH , accounting for the observed sequence of radical scavenging capabilities of 2b-f. 2d 2f 2e

H 2O 2

(a)

2c 2b

318

320

322

324

326

(b)

322.4

328

80

0.05 mg mL

-1

-1

0.10 mg mL -1 0.15 mg mL

plates vesicles

nanowires

60 40 20 0

2c

2b

2d

2f

2e

322.8

323.0

(d) 100 Quenching Efficiency / %

(c) 100

322.6

mT

mT Relative peak area / %

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80

60

2d 2f 2e

40

2c 2b 20 0.04

0.08

0.12

Concentration / mg mL

-1

0.16

Figure 7. (a) ESR spectra of 250 mol·L-1 H2O2 aqueous solution at the presence of 0.15 mg mL-1 2b-f. Each solution contains 50 µmol·L-1 DMPO as the probe. (b) Magnified spectra in the range of 322.4-323.0 mT (the peak indicated by the ellipse in a). (c) Relative peak area at varying concentrations of 2b-f. The type of aggregates for each compound is also indicated. For 2b and 2d which showed polymorphic aggregation, only the dominant aggregates (i.e., vesicles) are indicated for clarity. The area of the peak obtained at the absence of the C60 derivative is designated as 100%. (d) Statistics of the quenching efficiency of OH - caused by 2b-f at different concentrations. The lines are guides for the ACS Paragon Plus Environment

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eyes. Investigations were also carried out on other two series of samples with concentrations of 0.10 and 0.05 mg mL-1. The results are summarized in Figure 7c and 7d. For 2e and 2f,

e

only decreased

slightly when the concentration was decreased to 0.10 mg mL-1, which showed a sudden drop if the concentration was further decreased to 0.05 mg mL-1. For 2b-d, a dose-dependent effect was observed where

e

decreased continuously with decreasing concentration. When the concentrations of 2b-f were

changed, the ratio of the number of individuals to that of the aggregates might change. In addition, the size of the aggregates may be also different as noticed for the nanowires formed by 2b, 2e and 2f. These combined effects accounted for the nonlinear relationship between

e and

the concentration of most C60

derivatives, especially for 2f and 2e. A deeper understanding toward the radical scavenging capabilities as well as other biological activities of 2b-f would rely on further experimental studies on their photophysical properties. The kinetics of the radical scavenging reaction is also yet to be characterized. Even though, current study proved the importance of molecular design on the regulation of the aggregation behavior and biological activities of water soluble C60 derivatives where a subtle change in the molecular structure would induce big differences. The relationship among the structure, aggregation behavior and radical scavenging capabilities of 2b-f could act as a guide for the design of new water soluble C60 derivatives with more interesting aggregation behavior and improved biological activities in future. This is especially important considering that although the aggregation behavior and the radical scavenging capabilities of water soluble C60 derivatives have been reported separately, the combined analysis as presented here is quite rare. Conclusions In summary, we have synthesized a series of water soluble C60 derivatives bearing a quaternary ammonium cation and o-PEO chains (2a-f). Change of the number and substituted position of the o-PEO chains induced variations of their solubility in water with an order of 2a < 2c < 2b < 2d ACS Paragon Plus Environment

2f

Aggregation Behavior and Antioxidant Properties of Amphiphilic

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