Hyaluronic Acid Nanovehicles with

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Magnetic Fullerene-DNA/Hyaluronic Acid Nanovehicles with Magnetism/Reduction Dual-Responsive Triggered Release Ling Wang, Yitong Wang, Jingcheng Hao, and Shuli Dong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01939 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Biomacromolecules

Magnetic

Fullerene-DNA/Hyaluronic

Nanovehicles

with

Acid

Magnetism/Reduction

Dual-Responsive Triggered Release Ling Wang, Yitong Wang, Jingcheng Hao, and Shuli Dong* Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special Aggregated Materials, Shandong University, Ministry of Education, Jinan 250100, P. R. China.

* Corresponding author E-mail: [email protected]. Tel.: +86-531-88363768 // Fax: +86-531-88564750 1

ACS Paragon Plus Environment

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ABSTRACT: We create the dual-responsive nanovehicle that can effectively combine and abundantly utilize magnetic and glutathione (GSH)-reductive triggers to control the drug delivery and achieve more intelligent and powerful targeting. In the nanovehicles, paramagnetic fullerene (C60@CTAF) was prepared via one-step modification of fullerene with magnetic surfactant CTAF by hydrophobic interaction for the first time. The perfect conjugation of C60 and CTAF increased the solubility or dispersity of fullerenes and qualify CTAF with more powerful assembly capability with DNA. DNA molecule in the nanovehicles acted as an electrostatic scaffold to load anticancer drug Dox as well as the important building block for assembly with C60@CTAF into C60@CTAF/DNA. The further combination of deshielding and targeting functions in reduction-responsive disulfide modified HA-SS-COOH coating on C60@CTAF/DNA complexes, could reduce the agglomeration and regulate the morphology of C60@CTAF/DNA complexes from irregular microstructures to more uniform ones. More importantly, the introduction of HA-SS-COOH provides a response to a simulating reductive extra-tumoral environment by efficient cleavage of disulfide linkages by GSH, and site-specific drug delivery to HepG2 cells. Amazingly, the final nanovehicles presented an increased magnetic susceptibility compared with paramagnetic CTAF, and they can “walked” under an applied magnetic field. Due to its facile fabrication, rapid responsiveness to extra tumoral environment, and its external automatic controllability by external magnet, the drug delivery nanovehicles constructed by magnetic fullerene-DNA/hyaluronic acid might be of great interest for making new functional nucleic-acid-based drug carriers. 2


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KEYWORDS: magnetic surfactant, paramagnetic fullerene, DNA, hyaluronic acid, drug delivery

3


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1. INTRODUCTION For cancers, the construction of versatile smart nanovehicles to efficiently improve the chemotherapy efficiency and avoid the severe side effects in healthy tissues, by means of effective targeting as well as controlled release of anticancer drugs, have attracted extensive attention.1 Nucleic acids have been acted as important building blocks for assembly materials due to their outstanding molecular self-recognition ability and unique structural motif.2 Although DNA molecule can act as an intercalating scaffold to load anticancer drug Dox,3 naked DNA cannot be ideal building blocks for drug delivery due to their poor intracellular uptake caused by the negative charge repulsion between DNA and cytomembrane.4 Complexation with cationic surfactants provides an effective method of facilitating intracellular delivery due to the efficient DNA condensation and relieving the negative charge of DNA backbone.5 As the concentration is above critical association concentration (cac), surfactant molecules would associate to form cationic micelles in the vicinity of DNA. These multivalent cations interact with DNA via electrostatic and hydrophobic interaction,5 making it possible to use nucleic acid-surfactant self-assembly materials for drug-delivery applications. Among various cationic surfactants, magnetic surfactant CTAF with magnetic counterion, [FeCl3Br]−, has a more effective capability to interact with DNA at high surfactant concentration.6 This kind of surfactants can be synthesized simply through coordinating cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) with FeCl3 in methanol.7,8 The magnetic counterions [FeCl3Br]− provide magnetic surfactants possibilities for controlling liquid surface property8 or producing magnetic emulsions9 without the help of magnetic nanoparticles. Meanwhile, the magnetic surfactants have advantages compared with magnetic nanoparticles including their facile fabrication, clean, good 4


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dispersibility, high sensitivity and inexhaustible stimulus.7,8 However, the applications of cationic surfactants in vivo have largely been limited by disadvantages including their inherent cytotoxicity. To solve the problem, fullerene was introduced as a scaffold to combination of more CTAF molecules by hydrophobic interactions, in order to enhance the electrostatic interaction between DNA and CTAF, and decrease the amount of magnetic surfactants. Compared with other scaffolds10, fullerene plays an important role in biological and medical applications as enzyme inhibitors, antiviral agents and photodynamic therapy agents.11 But the main challenge for fullerenes application is its highly hydrophobic property. Many efforts have been made to improve the solubility, dispersity and biocompatibility of fullerenes in water, through biomaterials12 or surfactant molecules13. The perfect combination of C60 and CTAF will bring an unexpected outcome for increasing the solubility or dispersity of fullerenes, as well as reducing CTAF concentration and improving the biocompatibility of magnetic surfactants. For another improvement of the biocompatibility of cationic surfactants, non-ionic hydrophilic PEG, negatively charged liposomes, polysaccharide-based hyaluronic acid (HA) and proteins were grafted or coated on the DNA complexes.14 The modifications shielded positive charge, reduced toxicity, restrain the undesirable interactions and aggregation, and enhance the systemic duration or even achieving tumor targeting. Among the shielding protections, HA has attracted more attention because of its excellent biocompatibility and biodegradability. As a natural linear polysaccharide, HA has vital roles in various biological functions including biomedical applications such as tissue engineering and drug delivery.15 Most importantly, HA has attracted extensive attention for site-specific drug delivery to tumor due to its high affinity toward CD44 receptor, which is overexpressed in 5


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various tumor cells.16 However, stable shielding including HA may lower the drug delivery efficiency due to the reduced cellular uptake and endosomal escape. The introduction of stimuli-responsive deshielding design could overcome this “shielding dilemma”. Furthermore, the differences of physicochemical microenvironment between tumor and normal tissues could be utilized to control the release of anticancer drugs. In contrast with normal tissues, the endosomes and lysosomes of tumor tissues are in a reductive state due to the high level of (glutathione) GSH or cysteine. The construction of nanovehicles with reduction-sensitive hyaluronic acid derivatives can effectively trigger the more intelligent controlled drug delivery and more effective targeting to tumor cells by utilizing subtle biochemical signals. In this study, we create a multiple responsive nanovehicle that can effectively combine and abundantly utilize magnetic and GSH-reductive triggers to control the drug delivery and achieve more intelligent and powerful targeting. The design of the smart nanovehicle is shown in Scheme 1. Specially, a protocol was developed for preparing magnetic fullerene (C60@CTAF) via one-step modification with paramagnetic surfactant CTAF. The perfect conjugation of C60 and CTAF could increase the solubility or dispersity of fullerenes and improve the whole positive charges, moreover, significantly increase the compaction capability of DNA than CTAF. DNA was used as an electrostatic scaffold to load an anticancer drug Dox, then DNA-Dox backbones assembling with the C60@CTAF to construct C60@CTAF/DNA complexes. The hydrophobic interaction between C60 and CTAF would not destroy the paramagnetic property of [FeCl3Br]−, and C60@CTAF presents a paramagnetic behavior similar to CTAF. Amazingly, after paramagnetic fullerene assembles with DNA backbones into C60@CTAF/DNA complexes, the complexes possess paramagnetic and weak superparamagnetic property. The strong hydrophobic 6


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interaction of magnetic [FeCl3Br]− with the alkyl chains of CTAF, making [FeCl3Br]− enter into the core of CTAF. The increased concentration of magnetic [FeCl3Br]− ions after C60@CTAF/DNA formation enrich the higher ferric content (a larger magnetic unit density), which led to a higher magnetic susceptibility value under an applied external magnetic field. The combination of deshielding and targeting functions in reduction-responsive

disulfide

modified

HA

(HA-SS-COOH)

coating

on

C60@CTAF/DNA complexes, would reduce the agglomeration and regulate the morphology of C60@CTAF/DNA complexes from irregular microstructures to more uniform ones. More importantly, the C60@CTAF/DNA/HA-SS-COOH vehicles would be stable in the circulation, with efficient shielding and specific interaction with the desired cells. Once entering the environments with lower redox potential in tumors, there occurs a direct cleavage of disulfide linkages of HA-SS-COOH, consequently, induces a GSH-reductive trigger for fast Dox release. We expected that the smart vehicle could achieve the goal of GSH reduction responsive controlled anticancer drug release as well as an exogenously remote magnetic controllability via disulfide bond modified hyaluronic acid and cationic surfactant with magnetic counterion, [FeCl3Br]−.

Scheme 1. Schematic representation of magnetic fullerene-DNA vehicles with reduction-sensitive hyaluronic acid derivatives. 7


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2. EXPERIMENTAL SECTION Chemicals and Materials. Hexadecyl trimethyl ammonium bromide (CTAB) and FeCl3·6H2O were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Fullerene C60 (99.9%) was provided by Suzhou Dade Carbon Nanotechnology Co., Ltd., China. Reduced glutathione (GSH), cysteamine hydrochloride (CSA·HCl, 99%), 1-ethyl-3-[3-(dime-thylamino)-propyl]

carbodiimide

(EDC)

and

N-hydroxy-

succinimide (NHS) were from J&K Scientific Ltd. Sodium hyaluronic acid (HA, the molecular weight of 370 kDa) was obtained from Freda Biochem Co., Ltd. (Shandong, China). Doxorubicin HCl (Dox, > 98%) was purchased from Dalian Meilun Biotech, China. Salmon testes double-strand DNA (dsDNA) sodium salt was purchased from Sigma-Aldrich (USA). Its molar weight was about 1000 bps, as determined by agarose gel electrophoresis (AGE), and its concentration was examined by considering DNA bases molar extinction coefficient to be 6600 mol−1 cm−1 at 260 nm. The absorbance ratio of DNA stock solution was 1.8 to 1.9 at 260 and 280 nm, suggesting no existence of protein. Thrice-distilled water was used to prepare each sample solution. UV–vis Spectrometry. UV spectra of DNA/surfactant mixture samples were examined by a U-4100UV/vis spectrometer, using 10 mm path length quartz cell at a wavelength range of 220-320 nm. Circular Dichroism (CD) Measurements. CD experiments were performed under a continuous flow of nitrogen using a Jasco-810 spectropolarimeter. Samples were located in 10 mm path length cells, and the scanning speed was controlled at 200 nm·min-1 with a wavelength range of 220-320 nm. Three scans were made and computer averaged for each sample. Zeta-Potential Measurements. The zeta potentials of the samples were 8


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measured using a Zeta PALS potential analyzer instrument (Brookhaven, USA), equipped with parallel-plate platinum black electrodes spaced 5 mm apart and a 10 mm path length rectangular organic glass cell. All samples were measured at a sinusoidal voltage of 80V with a frequency of 3 Hz. Each sample was measured ten times for the average value. 1H

NMR Measurements. All samples were dissolved in D2O (Aldrich product, ≥

99.9%). 1HNMR spectra were recorded on a Bruker Avance 400 spectrometer equipped with pulse-field gradient module (Z axis) using a 5 mm BBO probe at 400.13 MHz. SQUID Magnetometry. Dried samples were placed in sealed polypropylene tubes and mounted inside a plastic straw for measuring in a magnetometer with a superconducting quantum interference device (MPMSXL, Quantum Design, USA) and a reciprocating sample option (RSO). All the data were measured at 300 K. Scanning Electronic Microscopy (SEM). A JSM-6700 instrument (JEOL, Japan) was used to observe the surface morphology of the nanospheres. After sputter coating with gold, the samples were transferred onto the microscope stage and examined at ∼10 kV. Transmission Electronic Microscopy (TEM). A drop of dispersion liquid was dropped on TEM grid (copper grid, 3.02 mm, 200 mesh, and coated with Formvar film). After drying, TEM observations were performed with a JEOL’s JEM 100 cx TEM (Japan) at an accelerating voltage of 100 kV. Sample Preparation. The cationic surfactant, CTAF, was prepared as reported7,8 by mixing equal molar amount of CTAB and FeCl3·6H2O in methanol and stirring over night at room temperature and then dehydrated in vacuo at 80 °C overnight. The critical micelle concentration (cmc) as well as the dissociation constant of CTAF was 9


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0.42 mmol·L−1 and 0.68.6 Synthesis of C60@CTAF. Surfactants with fullerenes embedded were prepared by evaporation of fullerene and surfactant mixture in toluene solutions.17,18 Under dark conditions, 50 equiv. of CTAF was added to a purple toluene solution of 1 equiv. of C60 and stirred overnight at room temperature (yellow-brown solution) and then dehydrated in vacuo at 80 °C overnight. The C60@CTAF participating in technical characterization or DNA assembly progress was freshly prepared. Synthesis

of

Disulfide

Bond

Modified

HA

(HA-SS-COOH).

HA-SS-COOH was synthesized via two steps including condensation and oxidation. Thiolated hyaluronic acid derivative (HA-SH) was synthesized by the following process.19 Firstly, HA (400 mg) was dissolved thoroughly in 80 mL of deionized water. NHS (230 mg, 2 mmol) and EDC (383 mg, 2 mmol) was added into the mixture solution and kept reacting for 2 h at pH = 4.75 to activate the carboxylic group of HA. Subsequently, CSA·HCl (114 mg, 1 mmol) was added and stirred for 24 h under dark conditions, the solution was dialyzed (MWCO

3500 Da) exhaustively

against dilute HCl solution (pH 3.5) for 24 h and HCl solution (pH 3.5) with 100 mM NaCl for another 24 h. At last, the acidified solution was lyophilized to yield the HA-SH solid. The obtained HA-SH (200 mg, 0.5 mmol) in PBS buffer (50 mL) was reacted with 3-mercaptopropionic acid (100 equiv. to HA-SH, 5.31 g, 50 mmol) under O2. HA-SS-COOH was obtained by exhaustive dialysis (MWCO 3500 Da) and lyophilization. The chemical structures of HA and HA-SS-COOH were confirmed by 1

H NMR using D2O as a solvent. Assembly

of

C60@CTAF/DNA

Complexes

with

HA-SS-COOH.

C60@CTAF/DNA binary complexes were prepared by mixing DNA and fresh C60@CTAF solution and incubated at 25 °C for 30 min. HA-SS-COOH were then 10


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added to DNA complexes solution to fabricate ternary complexes incubating at 25 °C for 4 h. Cell Culture. The cytotoxicty study was conducted using MTT assay for HepG2 cell lines. Cells were incubated for 24 h at 37 °C under 5% CO2. The sample with different amount was added to the wells. 6-fold diluted MTT (0.15 mL/well) in PBS solution was added to each well and incubated at 37 °C for 4 h. The cell viability was colorimetrically detected by using a microplate reader (VERSA), which was used to measure the OD490 nm (absorbance value).

3. RESULTS AND DISCUSSION 3.1.

Synthesis

of

Magnetic

Fullerene-DNA

Vehicles

with

Reduction-Sensitive Hyaluronic Acid. 3.1.1.

Magnetic

Fullerene

and

Magnetic

Fullerene-DNA Vehicles.

Attempts have been developed to effectively improve the solubility and dispersity of extremely hydrophobic fullerenes in aqueous media. As reported, when surfactants applied above critical micelle concentration (cmc), they were available for C60 being encapsulated with CTAB.13 A longer aliphatic moiety in cationic surfactant facilitated encapsulation of C60 in the water and prevented aggregation,13 which indicates the stronger hydrophobic surfactants could effectively improve the solubility of fullerenes. For magnetic surfactant CTAF, the f-block coordinator [FeCl3Br]− have been proved to be stronger hydrophobic in our previous work6 and exhibit obvious hydrophobic interaction with the alkyl chain of cationic surfactants by Eastoe et al.7,8 The electrical conductivity measurements showing the cmc and the degree of dissociation ( for CTAF, CTAB and cetyltrimethylammonium chloride (CTAC), could provide a powerful evidence for the hydrophobicity of CTAF.6 The larger anions [FeCl3Br]compared with Br- and Cl-, should be less effective at screening cation-cation 11


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headgroup repulsions, thus increasing the cmc. However, the cmc of CTAF (0.42 mmol·L-1) is lower than CTAB (0.92 mmol·L-1) and CTAC (1.3 mmol·L-1). Besides, if the [FeCl3Br]- ions are not strong hydrophobic and cannot interact with the hydrophobic chains, they will bind around the polar groups of a micelle through the electrostatic interaction just like Br- and Cl-, the β of CTAF micelles (0.68) cannot be too much higher than that of CTAB (0.11) and CTAC (0.24). The specificity of magnetic [FeCl3Br]− make them be able to enter into the core of cationic CTAF micelles due to the hydrophobic interaction with the alkyl chains.6,7,8 Therefore, the existence of more hydrophobic coordinators [FeCl3Br]− enhance the conjugation of hydrophobic fullerenes with CTAF than CTAB. Normally, colloid particles with higher zeta potential values, have higher thermodynamic stability.20,21 The zeta potential measurement reflects the dispersity or stability of C60-surfactant at different stoichiometric ratios (r). CTAF could effectively disperse and encapsulate C60 at r ≤ 20:1000 (Table 1). Accordingly, the resulting CTAF modified C60 (C60@CTAF) at r = 20:1000 with a zeta potential of (85.5 ± 1.9) mV, and is selected as the model sample in the system. Table 1. Zeta potential (ξ) values of fresh C60@CTAF solution in different molar ratios at 298.0 ± 0.1 K. “p” indicated the C60@CTAF solution with visible black C60 clusters suspended in the solution. sample C60/CTAF C60/CTAF

ratio 5:1000 10:1000

ξ (mV) 52.5 ± 16.6 86.5 ± 1.1

C60/CTAF

15:1000

81.4 ± 5.2

C60/CTAF

20:1000

85.5 ± 1.9

C60/CTAF

40:1000

p

C60/CTAF

60:1000

p

C60/CTAF

80:1000

p

12


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Although DNA can work as an intercalating scaffold to load anticancer drug Dox,3 it cannot act as a perfect vehicle to delivery drugs. This is because DNA molecules (especially with high molecular weight) can hardly cross cell membranes independently due to its charge repulsion and high rigidity. Thus, a procedure that compact DNA molecule from an extended worm-like linear structure to a small-sized global state is necessary to solve this problem. As reported, multivalent cationic species with at least three positive charges can compact DNA by relieving the charge repulsion between the adjacent DNA backbones.22 Cationic surfactants can compact DNA by self-assembling into micelles in the vicinity of DNA backbones.23 Different from the reported work about the investigation of the interaction between DNA and CTAF,6 we put emphasis on the DNA compaction capability of C60@CTAF and magnetic controlled migration of DNA by applying low magnetic fields. To investigate potential applications of C60@CTAF in assembly and compacting DNA, we performed the measurements of UV-vis to obtain the basic understanding on the interaction behavior of DNA with different amounts of C60@CTAF or CTAF. Once DNA was compacted, due to the reduction in the charge repulsion between the adjacent phosphate groups, mixtures of DNA and cationic surfactants will aggregate and precipitate out from the solutions.24 Thus, we can use phase separation to indicate the DNA compaction. The phase diagram of DNA mixed with C60@CTAF and CTAF is shown in Figure 1a. One can see that 20 mol·L-1 CTAF is required to compact DNA efficiently, but for C60@CTAF, only 10 mol·L-1 is enough. In UV-vis spectra (Figure 1b), reduction in optical density at λmax = 260 nm (OD260) of 75 mol·L-1 DNA started at C60@CTAF = 10 mol·L-1, which is well in accord with the phase gram (Figure 1a). All these results demonstrated that compared to cationic CTAF, the complex C60@CTAF can significantly increase the compaction capability of DNA 13


Biomacromolecules

and the critical concentration for DNA compaction reduced significantly. The higher compaction efficiency contributes to the encapsulation of C60 by CTAF, which enables fullerene to act as scaffold loading CTAF with more positive charges (high zeta potential value). CTAF

b

a

Abs. (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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C60@CTAF

0.6

C60@CTAF -1

(molL ) 0 1 2 5 8 10 20 50

0.4

0.2

-1

CDNA = 0.075 mmolL

0.01

0.1

1

0.0

10

220

-1

240

logc ( mmolL )

260

280

300

320

Wavelength (nm)

Figure 1. (a) Phase behavior of 75 mol·L-1 DNA mixed with various amounts of C60@CTAF and CTAF, respectively. Open circles correspond to clear solutions, whereas filled circles correspond to turbid or macroscopically phase-separated samples. (b) UV-vis spectra show the presence of compaction behavior of DNA by C60@CTAF at different concentrations. 3.1.2. Targeted DNA Migration Induced by Magnetic Controllability for C60@CTAF/DNA Complex. As reported, CTAF is magneto-responsive surfactant because they contain high effective concentration of high-spin d5 FeIII centers in [FeCl3Br]−.7,8 The presence of counterions [FeCl3Br]− allows physico-chemical properties such as hydrophobicity, and electrical conductivity, to be controlled by an external magnetic field. SQUID magnetometry (Figure 2a) clearly shows the paramagnetic properties of CTAF as well as C60@CTAF, and their values for magnetic susceptibility are similar to those reported in the literature.7,8 The paramagnetic properties of C60@CTAF, indicate that the conjugation of C60 and CTAF cannot destroy the counterion [FeCl3Br]−. This study also reveals that the conjugation 14


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of C60 and CTAF appears to be a little increase in magnetic susceptibility. As discussed above, the magnetic [FeCl3Br]− could partly enter into the cationic micelles owing to the hydrophobic interaction with the alkyl chains.6,7,8 The enriched concentration of [FeCl3Br]− for the whole C60@CTAF maybe an important reason for enhancement in magnetic susceptibility. In order to investigate the controllability of the external magnet for DNA compaction by C60@CTAF, the UV-vis and CD measurements were performed. The experiments proceeded with a weak NdFeB magnet (0.25 T) positioned at the bottom of the solution composing 75 mol·L-1 DNA and 10 mol·L-1 C60@CTAF. It was found that the OD260 of DNA obviously decreased with the time exposed to the magnetic field (Figure 2b). Due to the test samples in Figure 2 coming from the upper solution of DNA solution, the decreased DNA concentration indicates an obvious migration of DNA towards the magnet at the bottom. After 96 h, about 60.3% DNA migrate towards the magnet to form aggregates or precipitates adjacent the magnet, and the raised baseline of absorption spectrum further confirms the aggregates in solution caused by C60@CTAF. But without the magnet (Figure 2c), the upper solution of DNA is just reduced by 39.9% with 10 mol·L-1 C60@CTAF, indicating the presence of magnet obviously enhancing the compaction efficiency of DNA. Furthermore, one can find in the absence of C60@CTAF, the OD260 of DNA (with or without external magnetic fields) are similar, illustrating that DNA cannot migrate by oneself without the help of C60@CTAF at magnetic field. The controllability of targeted DNA migration induced by the magnetic is also confirmed by CD measurements. In CD spectra, native ds-DNA has a positive peak at 275 nm by base stacking and a negative peak at 245 nm by helicity.25 Upon complexation with surfactants, CD peaks take a red-shift to longer wavelengths and decrease in intensity 15


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compared to ds-DNA in aqueous phase.26 Accordingly, the intensity of positive and negative bonds in CD spectra decreases with time, ascribed to a reduction in the concentration of DNA in the upper solution (Figure 2d). C60@CTAF with magnetic [FeCl3Br]− could be attracted by the magnetic field on the bottom, inducing an increased local concentration of C60@CTAF in the vicinity of magnet, which further results in the aggregates or precipitates of DNA adjacent the magnet. The generation of precipitates from DNA means a large reduction of DNA in the vicinity of magnet, and ultimately the DNA in the upper solution would migrate towards the magnet. The whole process is dynamic and continual, demonstrating a stepwise reduction in intensity of both bonds with time in CD spectra (Figure 2d). The main reason for the targeted DNA migration could be explained according to classical models describing colloidal particles and the work published by Eastoe et al.7,8,20 In the classical models,20 electric double layer of colloidal particles are divided into two regions, Stern layers and diffuse layers. In the Stern layer, counterions are strongly bound and move with the colloidal particles as a whole dynamic entity. In the second, counterions could move freely in the bulk and maintain dynamic equilibrium with those in the Stern layer.20 [FeCl3Br]- are hydrophobic and present strong hydrophobic interaction with the alkyl chains of cationic surfactants, and could enter into the core of C60@CTAF as a part in the Stern layers. Therefore, the magnetic [FeCl3Br]− in the Stern layer as well as those in the core of C60@CTAF micelles could be attracted as a whole dynamic entity of C60@CTAF by magnet. The published work by Eastoe and Tabor provide another mechanism for the magnetization of graphene oxide (GO) by magnetic ionic liquid surfactants or magnetic polyelectrolytes.27,28 They believed for magnetic materials with [FeCl4]-, the magnetic response of the co-flocculated network is caused by two processes. One is the adsorption of undissociated chains largely 16


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interacting with the GO sheets through polar interactions, which could direct interact with the magnetic field. The second is the interaction of dissociated, bound surfactant ions which form a diffuse layer in the proximity of the GO-surfactant interface. These dissociated species [FeCl4]- would still retain their counterions, and osmotically ‘‘drag” the co-flocculated network towards the magnet. According to the above theory, the dissociated counterion [FeCl3Br]- in diffuse layer might also osmotically ‘‘drag” the C60@CTAF/DNA towards the magnet. The above discussion provides a reasonable account for targeted DNA migration and contributes to a deeper understanding of the interaction between DNA and magnetic surfactants, which will expand the application of magnetic surfactant or magnetic polyelectrolytes.

1.0

CTAF C60@CTAF

a

0.6

0.5

Abs. (a. u.)

-1

Magnetic moment (emug )

0.8

0.0 -0.5

10 M

b

0h 24 h 48 h 72 h 96 h

0.4

0.2

-1.0 -60000 -40000 -20000

0

20000

40000

0.0 220

60000

240

260

Magnetic Field (Oe)

4

without C60@CTAF

c

DNA without magnet

CD (mdeg)

0.4

0.2

0.0 220

280

300

320

Wavelength (nm)

0.6

Abs. (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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0h 24 h 48 h 72 h 96 h

d

2

0

-2

240

260

280

300

220

320

Wavelength (nm)

240

260

280

300

320

Wavelength (nm)

Figure 2. (a) SQUID magnetometry of paramagnetic CTAF and C60@CTAF at 300 K. UV-vis (b) and CD (d) evidence of the reduction of DNA on the top of DNA/C60@CTAF solution, where DNA is 75 mol·L-1 and C60@CTAF 10 mol·L-1 vs time exposured to an external magnetic field (0.25 T). (c) UV-vis measurements of 17


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DNA after 96 h incubation show the importance of C60@CTAF and external magnetic fields for DNA migration: UV-vis of DNA with or without an external magnetic field (0.25T) and C60@CTAF/DNA complexes without an external magnetic field. 3.1.3. Synthesis of Reduction-Sensitive Hyaluronic Acid. HA is a natural acidic mucopolysaccharide with wide molecular, and the ones with low molecular weight was reported to induce particles with smaller size. 29 Therefore, we choose the low molecular weight HA (Mw = 370000 Da) for disulfide-linkage modification in the system. HA-SS-COOH was synthesized via two steps including condensation19 and oxidation (Figure 3a). HA-SH was synthesized by acting HA with CSA·HCl at pH = 4.75.19 To get HA-SS-COOH, HA-SH was reacted with 3-mercaptopropionic acid to recover a carboxyl group via a disulfide linkage. To avoid formation of HA-SH cross-linked dimer, 3-mercaptopropionic acid was used at 100-fold molar excess to the thiols group in HA. The 1H NMR spectrum (D2O) of HA-SS-COOH was shown in Figure 3b. Compared with the spectrum of HA, new resonances appeared at 2.57 and 2.87 ppm corresponding to protons of side chain (CH2CH2COOH).

a

18


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b

Figure 3. Synthesis and confirmation of disulfide bond modified HA: the synthetic route (a) and

1

H NMR spectrum (b) of reduction-sensitive hyaluronic acid

(HA-SS-COOH) in D2O.

3.1.4. Synthesis of Magnetic Fullerene-DNA with Reduction-Sensitive Hyaluronic Acid. In the nanovehicle, DNA molecule acted as an electrostatic building block for assembly with C60@CTAF into C60@CTAF/DNA. Due to the specific hydrophobicity of [FeCl3Br]−, magnetic CTAF possesses more effective DNA compaction capability because magnetic [FeCl3Br]− ions in the core of CTAF micelles make the polar groups of CTAF micelle much easier to contact with DNA back bones.6 Figure 4a demonstrates the morphology of C60@CTAF/DNA complexes in the solution of 75 mol·L-1 DNA and 100 mol·L-1 C60@CTAF. In this case, we could only obtain large scale irregular and dispersed microstructures mixed with some small spheres. The irregularity and large scale limit their drug delivery applications. The reason of irregular structure is that during the compaction process, positive 19


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C60@CTAF can significantly reduce the charge repulsion among adjacent DNA backbones through electrostatic and hydrophobic interactions,22 making an aggregation and association with DNA complexes.30 Meanwhile, C60@CTAF with multivalent charges could act as a bridge to link different DNA condensates.31 To solve such problems, protective polyanion-coating was developed to afford negatively charges for DNA complexes in lectures report,32 which eliminates the undesirable interactions. Therefore, we assemble HA-SS-COOH with C60@CTAF/DNA to reduce the agglomeration and to regulate the morphology of DNA complexes.

It turns out

that the addition of HA-SS-COOH could effectively adjust the morphology of C60@CTAF/DNA complexes to be almost spheres with diameters of around 400-500 nm

(as

shown

in

Figure

4,

SEM

(4b)

and

TEM

(4c)

images

of

C60@CTAF/DNA/HA-SS-COOH complexes). TEM images also demonstrated that C60@CTAF/DNA/HA-SS-COOH complex possessed a darker core area and a lighter shell area (the darker core area is C60@CTAF/DNA complex and the lighter shell area consists of HA-SS-COOH). Zeta potential measurements could provide better account for the formation of C60@CTAF/DNA/HASS-COOH complexes. As Figure 4d shown, the addition of negatively charged HA-SS-COOH to the DNA-C60@CTAF solutions (pre-incubated for 30 min) decreased the final zeta potential gradually from +9.93 mV to -9.91 mV (with 0.005 to 0.8 mol·L-1 HA-SS-COOH). These results indicate that HA-SS-COOH was deposited onto C60@CTAF/DNA complexes, leading to the formation of C60@CTAF/DNA/HA-SS-COOH ternary complexes via electrostatic interaction.

On

the

other

hand,

the

negative

20


repulsion

between

the

Page 21 of 37

C60@CTAF/DNA/HA-SS-COOH nanoparticles caused by the negative charges of HA-SS-COOH could diminish the undesirable aggregations of C60@CTAF/DNA complexes. Thus, shielding by grafting the HA-SS-COOH on the complex surface, could benefit for the intracellular uptake efficiency and enhancement of the duration. a

b

c

d10 Zeta Potential (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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5 0

0.8

0.5

0.3

0.1 0.08 0.05 0.03 0.01 0.008 0.005

-5 -10 -15

Figure 4. (a) SEM image of C60@CTAF/DNA complexes, SEM (b) and TEM (c) images of C60@CTAF/DNA/HA-SS-COOH complexes. (d) The zeta potentials () of DNA complexes (75 mol·L-1 DNA and 100 mol·L-1 C60@CTAF) conjugating with varied concentration of HA-SS-COOH.

3.2. Magnetic Properties of C60@CTAF/DNA/HA-SS-COOH Vehicle. The magnetic properties of C60@CTAF examined using SQUID in Figure 2a show a paramagnetic behavior. Amazingly, the complexes of C60@CTAF/DNA and C60@CTAF/DNA/HA-SS-COOH complexes both behave paramagnetic and weak superparamagnetic property and an increased magnetic susceptibility value (0.533 and 21


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Page 22 of 37

0.413 emu·g-1 respectively), indicating that they possess stronger magnetism at an applied

magnetic

field

(Figure

5).

Furthermore,

a

video

of

C60@CTAF/DNA/HA-SS-COOH complexes moved under the attraction of external magnet (1T) was supplied in Figure S1 to illustrate the magnetic response at room temperature in detail. The C60@CTAF/DNA/HA-SS-COOH complexes “walked” under the attraction of magnet directly. As described in part 3.1.2 about the classical models, electric double layer of colloidal particles is divided into two regions, the so-called Stern and diffuse layers.20 There exists a slipping plane that separates the two layers,20 and the location of the slipping plane is quite sensitive to polyelectrolytes. An interaction with oppositely charged polyelectrolytes would induce a displacement between the two layers, result in a release of counterions into diffuse layer. Theoretically, as C60@CTAF bind with DNA backbones, the magnetic [FeCl3Br]− in the diffuse layer would be released by the charge competition, but those in the Stern layer remain unchanged which could magnetize C60@CTAF/DNA complexes. However, our group and Eastoe et al. have proved the strong hydrophobic interaction of magnetic [FeCl3Br]− counterions with the alkyl chains of cationic surfactants, making [FeCl3Br]− in diffuse layer enter into the core of CTAF.6,7,8 The increased concentration of magnetic [FeCl3Br]− ions in the core of C60@CTAF from diffuse layers and those in the Stern layer synergetically increase counterion [FeCl3Br]− after complexation compared with C60@CTAF. The higher ferric content by [FeCl3Br]− enrichment had a larger magnetic unit density which led to a higher magnetic susceptibility value under an applied external magnetic field. Loading drugs 22


Page 23 of 37

via this magnetic vehicle could make the resulting biological entities be electively controlled by external magnetic force. 0.6

-1

Magnetic moment (emug )

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

Biomacromolecules

1 2

0.4 0.2 0.0 -0.2 -0.4 -0.6

-60000 -40000 -20000

0

20000 40000 60000

Magnetic Field (Oe)

Figure

5.

SQUID

magnetometry

of

C60@CTAF/DNA

(1)

and

C60@CTAF/DNA/HA-SS-COOH (2) complexes at 300 K.

3.3. Reduction-Responsive Properties of C60@CTAF/DNA/HA-SS-COOH Vehicles. It is reported that the total concentration of reducing agents GSH was at 2-20 mol·L-1 in extracellular fluids and normal tissues, but ranges from 2-20 mmol·L-1 under intracellular tumor conditions. Tumor tissues shows at least 100 to 1000 times higher concentrations of GSH levels compared to normal tissues.33 Therefore, glutathione could act as perfect and ubiquitous intracellular stimulus for rapid destabilization of nano-vehicles inside cells to accomplish efficient drug release. The reduction-sensitive shielding of DNA complexes are expected to be stable in the extracellular environment but detach timely under reducing conditions of cancers via disulfide cleavage. Taking concerns to the differences of GSH level between the normal and tumor tissues, in the present study, reduction-triggered deshielding of 23


Biomacromolecules

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

C60@CTAF/DNA/HA-SS-COOH complexes was characterized by zeta potential measurement and morphology observation in the presence of reductive reagent GSH (5 mmol·L-1) for 2 h in a large excess to the calculated amount of the disulfide bonds in the shielding. As predicted, the surface charge of DNA complexes (with 0.3 mol·L-1 HA-SS-COOH) changed from -6.20 ± 2.02 mV to -0.81 ± 0.16 mV after GSH treatment (Table 2). The DNA complexes with various reduction-sensitive shielding (0.1-0.8 mol·L-1 HA-SS-COOH) showed the similar increase trend in zeta potentials under reduction conditions (Table 2). This may be caused by the linkage between the carboxyl groups and the backbone of HA would be cleaved under 5 mmol·L-1 GSH conditions. Losing carboxyl group of HA-SS-COOH would reduce the negative charge of the shielding, increased positive zeta potential and consequently weaken the electrostatic interaction between HA-SS-COOH shielding and C60@CTAF/DNA. TEM images (Figure 4c) showed the intact global structure of C60@CTAF/DNA/HA-SS-COOH vehicle with a darker core area of C60@CTAF/DNA complex and a lighter shell area of HA-SS-COOH. The CD spectra of C60@CTAF/DNA/HA-SS-COOH vehicles (Figure S2) behave no opposite bands, indicating DNA molecules are completely compacted. However, after adding 5 mmol·L-1 GSH, a recovery of positive and negative bands (1) demonstrates the presence of free DNA (Figure S2). Under 5 mmol·L-1 GSH reductive conditions, one can find the destroyed structures of C60@CTAF/DNA/HA-SS-COOH complexes (Figure 6) and special substances with jujube-like morphology (Figure S3a). Figure S3b further proved the jujube-like materials were pure CTAF without other substances. 24


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It is sufficient to conclude that GSH (5 mmol•L-1) could destroy the structure of C60@CTAF/DNA/HA-SS-COOH complexes, which might induce a higher Dox release ratios. This result is similar to the reported conclusion, and addition of anions usually leads to the decomposition of DNA complexes by competitive dissociation of the DNA molecule from the polycation.34,35 Table 2. Zeta potential ξ of C60@CTAF/DNA/HA-SS-COOH complexes and those with 5 mmol·L-1 GSH treatment. Concentration

Zeta potential ξ (mV) of Zeta potential ξ (mV) of vehicles after 5 mmol·L-1

of HA-SS-COOH in vehicles -1

GSH treatment

vehicles (mol·L ) 0.1

-4.46 ± 1.32

-1.60 ± 0.18

0.3

-6.20 ± 2.02

-0.81 ± 0.16

0.5

-7.80 ± 2.05

-0.026 ± 0.36

0.8

-9.91 ± 2.10

-3.56 ± 2.11

Figure 6. TEM image of C60@CTAF/DNA/HA-SS-COOH complexes in the presence of reductive reagent GSH (5 mmol·L-1) for 2 h. 25


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Page 26 of 37

3.4. Drug Loading and Controlled Releasing Performance. Dox is the most utilized anticancer drug against a wide range of neoplasms, which can intercalate within the DNA strand due to the presence of flat aromatic rings in this molecule.3 Utilizing DNA as negatively charged scaffolds, Dox was initially mixed with dsDNA (stoichiometric ratio is 1: 5) in an aqueous solution to effectively entrap positively charged Dox on DNA backbones by the electrostatic interaction. By means of assembling the Dox-binding DNA with C60@CTAF, Dox can be equably incorporated into the C60@CTAF/DNA/HA-SS-COOH complexes with high efficiency. A significant increase in the zeta potential from +14.2 to +37.4 mV confirmed that the C60@CTAF/DNA/HA-SS-COOH complexes were filled with positively charged Dox (Figure S4). To mimic the intracellular release of tumor tissues and extracellular release, we investigated

the

Dox

release

behavior

from

the

Dox

loaded

C60@CTAF/DNA/HA-SS-COOH complexes for 12 h under different typical conditions (10 mol·L-1 GSH simulating extracellular release; 5 mmol·L-1 GSH mimicking intracellular release, respectively) in Figure 7a. Only 31%-40% Dox released from C60@CTAF/DNA coated with 0.005-0.8 mol·L-1 HA-SS-COOH, in the presence of 10 mol·L-1 GSH, while a much higher Dox release ratios of 92%-100% were obtained, in the mimetic microenvironments of tumor tissues. The nearly complete drug release is caused by the cleavage of the anionic shield of HA-SS-COOH as well as the de-compaction of C60@CTAF-DNA/HA-SS-COOH complexes in the presence of 5 mmol·L-1 GSH (Figure 6). Furthermore, Figure 7b 26


Page 27 of 37

definitely shows the rapid drug release when the vehicles were exposed to 5 mmol·L-1 GSH, and drug presented totally deliver into media after 120 min. It is particularly important, as a drug carrier, C60@CTAF/DNA/HA-SS-COOH should be engineered with high stability therefore achieving long circulation time and minimizing premature drug release. No noticeable release of drugs from vehicle in the extracellular conditions indicates excellent loading efficiency and stability in the mimetic microenvironments of normal issues. These results also clearly show that the rapid release is essentially caused by the intracellular of tumor tissues (5 mmol·L-1 GSH). 125

b

5 mM 10 M GSH

100

Dox Release (%)

a Dox Release (%)

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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75 50

100 80 60 40 20

5 mM 10 M GSH

25 0.8

0.5

0.3

0.1

0.08 0.05 0.03

0

0.01 0.008 0.005

Concentration of HA-SS-COOH (M)

0

20

40

60

80

100

120

Time (min)

Figure 7. The histogram of Dox release (a) and the time dependence of cumulative Dox release curves (b) from drug-loaded C60@CTAF/DNA/HA-SS-COOH complexes in the presence of 10 mol·L-1 or 5 mmol·L-1 GSH at 37 °C. 3.5. Cytotoxicity of C60@CTAF/DNA/HA-SS-COOH Vehicles for HepG2 Cells. The cytotoxicity of the C60@CTAF/DNA/HA-SS-COOH complexes was studied to examine their feasibility in bio-related applications. HA is a specific ligand for targeting to CD44-overexpressing cancer cells because it can specifically bind to 27


Biomacromolecules

CD44 receptor. Therefore, CD44 overexpressing human liver adenocarcinoma HepG2 cells were selected as model cancer cells for study. To evaluate the feasibility of the C60@CTAF/DNA/HA-SS-COOH complexes as a targeted drug-delivery platform, we performed the cytotoxicity test by means of a methyl thiazolyl tetrazolium (MTT) assay. As Figure 8 shows, after the cells were incubated with the sample for 24 h, the cytotoxicity was tested. Although the dose of C60@CTAF/DNA/HA-SS-COOH complexes up to 7.5 μg·mL−1, the cell viability still remained to be 83.37%. The result suggests that the vehicle itself has low cytotoxicity, confirming the good biocompatibility of this kind of vehicles. After the vehicles exposed to the microenvironments of tumor (high concentration of GSH), the responsive drug release behavior occurred by a cleavage of the anionic shield of HA-SS-COOH as well as the de-compaction of C60@CTAF-DNA/HA-SS-COOH complexes. Figure 8 exhibits dose-dependent cytotoxicity behavior of C60@CTAF/DNA/HA-SS-COOH complexes. An obviously high killing efficiency on HepG2 cells can be obtained 41.94% by adding 7.5 μg·mL−1 Dox-loaded vehicles.

100

Cell Viability (%)

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

Page 28 of 37

1 2

80

60

40 0.0

2.5

5.0

7.5 -1

Concentration (g·mL )

Figure 8. In vitro cytotoxicity of HepG2 cells after being incubated with different 28


Page 29 of 37

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amounts of C60@CTAF/DNA/HA-SS-COOH complexes for 24 h at 37 °C (1). Cytotoxicity assays of HepG2 cells treated with different amounts of Dox-loaded C60@CTAF/DNA/HA-SS-COOH complexes (2).

4. CONCLUSIONS In this work, a GSH and magnetic dual-responsive nano-vehicle composed of fullerene-DNA with magnetic counterion [FeCl3Br]− and reduction-sensitive hyaluronic acid has been designed. As a scaffold, fullerene was endowed with more positive charges by hydrophobic interaction with CTAF, which behaves a more powerful assembly capability with DNA. Shielding by grafting reduction-responsive HA-SS-COOH could regulate the morphology of C60@CTAF/DNA complexes into more similar spherical shape. More importantly, the finally designed nanovehicles presented an increased magnetic susceptibility compared to CTAF, furthermore, they can “walked” under an applied magnetic field. Complexation with HA-SS-COOH provides a response to a simulating reductive extra-tumoral environment by efficient cleavage of disulfide linkages of HA-SS-COOH by GSH, and site-specific drug delivery to HepG2 cells. Efficiently magnetic control over migration property was demonstrated for targeted drug delivery, and sensitively GSH-responsive drug release was proved for controlled cancer therapy. Owing to its facile fabrication, rapid responsiveness, and external automatic controllability, these nano-vehicles might be of great interest for making new functional nucleic-acid-based nanomachines for controlled and targeted drug delivery. ■ ASSOCIATED CONTENTs 29


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Supporting Information Additional figures. This information is available free in the charge via the internet at http://pubs.acs.org/.

Figure S1. Video of C60@CTAF/DNA/HA-SS-COOH complexes “walked” under an external magnetic field (1T). Figure S2. CD of C60@CTAF/DNA/HA-SS-COOH complexes in the presence and absence of GSH. Figure S3. TEM images of C60@CTAF/DNA/HA-SS-COOH complexes with GSH (a) and pure CTAF (b). Figure S4. The zeta potentials of C60@CTAF/DNA-Dox. ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +86-531-88363768. Fax: +86-531-88564464. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was financially supported by the NSFC (21420102006 & 21273136).

REFERENCES (1) Brambilla, D.; Luciani, P.; Leroux, J. C. Breakthrough discoveries in drug delivery technologies: the next 30 years. J. Control. Release 2014, 190, 9-14. (2) Wei, R.; Martin, T. G.; Rant, U.; Dietz, H. DNA origami gatekeepers for solid-state nanopores. Angew. Chem. 2012, 124, 4948-4951. (3) Zhu, G.; Zheng, J.; Song, E.; Tan, W. Self-assembled, aptamer-tethered DNA 30


Page 30 of 37

Page 31 of 37

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

Biomacromolecules

nanotrains for targeted transport of molecular drugs in cancer theranostics. PNAS 2013, 110, 7998-8003. (4) Zabner, J.; Fasbender, A. J.; Moninger, T.; Welsh, M. J. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 1995, 270, 18997-19007. (5) Grueso, E.; Cerrillos, C.; Hidalgo, J.; Cornejo, P. L. Compaction and decompaction of DNA induced by the cationic surfactant CTAB. Langmuir 2012, 28, 10968-10979. (6) Xu, L., Feng, L.; Hao, J.; Dong, S. Compaction and decompaction of DNA dominated by the competition between counterions and DNA associating with cationic aggregates. Colloid Surf. B 2015, 134, 105-112. (7) Brown, P.; Bushmelev, A.; Butts, C. P.; Eastoe, J. Properties of new magnetic surfactants. Langmuir 2013, 29, 3246-3251. (8) Brown, P.; Bushmelev, A.; Butts, C. P.; Schmidt, A. M. Magnetic control over liquid surface properties with responsive surfactants. Angew. Chem. 2012, 51, 2414-2416. (9) Brown, Butts, C. P.; Eastoe, J.; Trickett, K. Microemulsions as tunable nanomagnets. Soft Matter 2012, 8, 11609-11612. (10) Yan, X.; Blacklock, J.; Li, J.; Möhwald, H. One-pot synthesis of polypeptide-gold nanoconjugates for in vitro gene transfection. ACS Nano 2012, 6, 111-117. (11) Bakry, R.; Vallant, R. M.; Bonn, G. K. Medicinal applications of fullerenes. Int. J. Nanomed. 2007, 2, 639-649. 31


Biomacromolecules

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

Page 32 of 37

(12) Mba, M.; Jiménez, A. I.; Moretto, A. Templating the self-assembly of pristine carbon nanostructures in water. Chem. Eur. J. 2014, 20, 3888-3893. (13) Lee, J.; Kim, J. H. Effect of encapsulating agents on dispersion status and photochemical reactivity of C60 in the aqueous phase. Environ. Sci. Technol. 2008, 42, 1552-1557. (14) Ito, T.; Tanaka, N. I.; Niidome, T.; Koyama, Y.

Hyaluronic acid and its

derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation. J. Control. Release 2006, 112, 382-388. (15) Kogan, G.; Šoltés, L.; Stern, R. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol. Lett. 2007, 29, 17-25. (16) Zhong, Y.; Zhang, J.; Cheng, R.; Zhong, Z. Reversibly crosslinked hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug resistant CD44+ human breast tumor xenografts. J. Control. Release 2015, 205, 144-154. (17) Li, H.; Jia, X.; Li, Y.; Hao, J. A salt free zero-charged aqueous onion-phase enhances the stability of fullerene C60 in water. J. Phys. Chem. B 2006, 110, 68-74. (18) Li, H.; Hao, J. Phase behavior of salt-free catanionic surfactant aqueous solutions with fullerene C60 solubilized. J. Phys. Chem. B 2007, 111, 7719-7724. (19) Bian, S.; He, M.; Sui, J.; Cai, H.; Zhang, X. The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture. 32


Page 33 of 37

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

Biomacromolecules

Colloid Surf. B 2016, 140, 392-402. (20) Xu, L.; Chen, J.; Feng, L.; Dong, S.; Hao, J. Loading capacity and interaction of DNA binding on catanionic vesicles with different cationic surfactants. Soft Matter 2014, 10, 9143-9152. (21) Xu, L.; Feng, L.; Dong, R.; Hao, J.; Dong, S. Transfection efficiency of DNA enhanced by association with salt-free catanionic vesicles. Biomacromolecules 2013, 14, 2781-2789. (22) Bloomfield, V. A. DNA condensation by multivalent cations. Biopolymers 1997. (23) Anne-Laure, M.; Lee, C. T. Photoreverible DNA condensation using light-responsive surfactants. J. Am. Chem. Soc. 2006, 128, 6400-6408. (24) Carlstedt, J.; Lundberg, D.; Dias, R. S.; Lindman, B. Condensation and decondensation

of

DNA

by

cationic

surfactant,

spermine

or

cationic

surfactant-cyclodextrin mixtures: macroscopic phase behavior, aggregate properties, and dissolution mechanisms. Langmuir 2012, 28, 7976-7989. (25) Kypr, J.; Kejnovská, I.; Renčiuk, D. Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 2009. 37, 1713-1725. (26) Chen, W.; Gerasimov, J. Y.; Zhao, P.; Herrmann, A. High-density noncovalent functionalization of DNA by electrostatic interactions. J. Am. Chem. Soc. 2015, 137, 12884-12889. (27) McCoy, T. M.; Brown, P.; Eastoe, J.; Tabor, R. F. Noncovalent magnetic control and reversible recovery of grapheme oxide using iron oxide and magnetic surfactants. ACS Appl. Mater. Interfaces 2015, 7, 2124-2133. 33


Biomacromolecules

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(28) Hazell, G.; Navarro, M. H.; McCoy, T. M.; Tabor, R. F.; Eastoe, J. Responsive materials based on magnetic polyelectrolytes and grapheme oxide for water clean-up. J. Colloid Interface Sci. 2016, 464, 285-290. (29) Kim, J.; Park, K.; Hahn, S. K. Effect of hyaluronic acid molecular weight on the morphology of quantum dot-hyaluronic acid conjugates. Int. J. Biol. Macromol. 2008, 42, 41-45. (30) Cao, M.; Deng, M.; Wang, X.; Wang, Y. Decompaction of cationic gemini surfactant-induced DNA condensates by -cyclodextrin or anionic surfactant. J. Phys. Chem. B 2008, 112, 13648-13654. (31) Ran, S.Y.; Wang, Y.; Yang, G.; Zhang, L.

Morphology characterization and

single-molecule study of DNA-dodecyltrimethylammonium bromide complex. J. Phys. Chem. B 2011, 115, 4568-4575. (32) Trubetskoy, V. S.; Wong, S. C.; Wolff, J. A. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery. Gene Ther. 2003, 10, 261-271. (33) Cheng, R.; Feng, F.; Meng, F.; Zhong, Z. Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery. J. Control. Release 2011, 152, 2-12. (34) Iwaki, T.; Saito, T.; Yoshikawa, K. How are small ions involved in the compaction of DNA molecules? Colloid Surf. B 2007, 56, 126-133. (35) Koyama, Y.; Yamada, E.; Ito, T.; Yamaoko, T. Suger-containing polyanions as a self-assembled coating of plasmid/polycation complexes for receptor-mediated gene 34


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delivery. Macromol. Biosci. 2002, 2, 251-256.

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Hyaluronic Acid Nanovehicles with

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