Engineering Multifunctional Films Based on Metal-Phenolic Networks

Article pubs.acs.org/journal/abseba

Engineering Multifunctional Films Based on Metal-Phenolic Networks for Rational pH-Responsive Delivery and Cell Imaging Hongshan Liang,†,§ Jing Li,†,§ Yun He,†,§ Wei Xu,†,§ Shilin Liu,†,§ Yan Li,†,§ Yijie Chen,†,§ and Bin Li*,†,‡,§ †

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Hubei Collaborative Innovation Centre for Industrial Fermentation, Hubei University of Technology, Wuhan 430068, China § Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China ‡

ABSTRACT: Here, we reported a designable pH-responsive system on the basis of coordination bonded metal-tannic acid (TA) networks on zein/quaternized chitosan (HTCC) nanoparticles (NPs). Zein/ HTCC NPs has been developed as a promising delivery system for natural bioactives in our lab previously. The coordination bonds between either the “NH2-metal” or the “metal-TA” are sensitive to pH variations that would release encapsulated drugs under designated pH conditions. Metal-TA film-coated zein/HTCC NPs had a spherical structure with particle size of 110−120 nm. Doxorubicin (DOX) was used as a model anticancer drug for release study and cell viability. The in vitro release profile of DOX loaded metal-TA films coated zein/ HTCC NPs (DOX-zein/HTCC-TA/metal NPs) showed a significant pH-responsiveness when changing the amount or the type of metal ions. In in vitro cell assays, the blank zein/HTCC-TA/metal NPs showed low cytotoxicity, but the DOX-loaded NPs exhibited a high cytotoxic activity against HepG2. To impart enhanced imaging properties of metal-TA films, we used EuIII to chelate with ligand named 2-thenoyltrifluoroacetone (TTA) to intense fluorescence intensities of EuIII-TA films so as to develop pH-responsive zein/HTCC-TA/metal NPs for anticancer drug delivery as well as cell imaging. KEYWORDS: coordination bonding, pH-responsive, drug delivery, nanoparticles, cell imaging alkaline or acidic blocks.18−20 The formation and breakage of metal ion−ligand coordination bonds are response to pH changes due to the reason that metal ions and protons, all regarded as Lewis acids, could compete to combine with the ligand, a kind of Lewis base.15,21 So, intensive research has been carried out on the assembly of metal−ligand coordination materials. Tannic acid (TA), which contains galloyl groups, could interact with a variety of materials through hydrogen bonding, electrostatic interactions or hydrophobic interactions.22,23 In addition, TA has been well recognized as a promising material to self-assembly of coordination bonding architecture on NPs due to strong metal chelation ability and significant binding affinity to materials surfaces.24−26 Previous reports have shown that TA could coordinate with a range of metal ions for the assembly of films and capsules.27−30 Zein, an alcohol-soluble corn storage protein, has been widely utilized to fabricate self-assembly as potential biomaterial for colloidal delivery systems.31 Such biopolymeric colloidal particles has been regarded as a good candidate in food and pharmaceutical applications for controlled and enhanced

1. INTRODUCTION Stimuli-responsive drug delivery systems aim to readily modulate the take-up or release of guest molecules with high specificity to targeted cells in a controlled manner.1,2 Selfassembled biodegradable nanoparticles (NPs), on the basis of natural polymers, have attracted considerable attention in recent years, which provide a variety of promising applications in drug delivery systems.3,4 Among them, protein-based systems are regarded as an appealing class for drug and gene delivery because of the enhanced absorbability and low toxicity in the degradation of end products.5,6 Increasing evidence has shown that NPs with particle size from 100 to 200 nm favors retention in tumors for the enhance permeability and retention (EPR) effect.7 Additionally, smart systems can targeted release drugs in response to external stimuli, typically including temperature,8,9 pH,1,10,11 light irradiation,12,13 ionic strength.14 Of these stimuli responsive systems, the pH-sensitive systems are of vast interest due to the acidic extracellular pH environment of most cancer tissues. Therefore, a pH-sensitive transporter can selectively deliver drugs in the tumor tissues while maintaining a low level under physiological conditions, to reduce the side effects of drugs.15 Common routes for pH-sensitive systems involve pHsensitive bonds16,17 or protonation of copolymers based on © XXXX American Chemical Society

Received: August 25, 2015 Accepted: January 13, 2016

A

DOI: 10.1021/acsbiomaterials.5b00363 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

briefly vortexed. Immediately following this, 100 μL of fresh metal solution (72 mM of FeCl3, AlCl3 or EuCl3 solutions) was added and the dispersion was vortexed. The final concentration of DOX was 100 μg/mL. The absorbance of DOX at 480 nm was measured with UV/ vis spectrophotometer (UV-1100, MAPADA). Being centrifuged at 4000 × g for 30 min, the free DOX was obtained by calculating the DOX content in the filtrate receiver. The encapsulation efficiency was defined as the drug content that was entrapped into NPs or NPs-TA/ metal and calculated as follows

delivery of heparin,32 gitoxin,33 fish oil,34 curcumin,35 etc. The complex NPs prepared with the self-assembly of zein and quaternized chitosan (HTCC) has been regarded as a promising delivery system for curcumin in our lab previously.36 This hydrophilic polysaccharide (HTCC) was used as a hydrophobic/hydrophilic balance to avoid the rapid take-up of zein NPs by macrophage protein.37,38 Moreover, the NH2 in HTCC could also form coordination bonding with metal ions, which are sensitive to external pH variations.1 Herein, we represented a pH-responsive delivery system, tailored by the formation and breakage of metal ion-ligand coordination bonds, which are response to pH changes. Because of the diverse chelation ability of phenolic materials, TA was coordinated to FeIII, AlIII, or EuIII to generate robust metal-TA films on zein/HTCC NPs. To impart enhanced imaging properties of metal-TA films, we used EuIII to chelate with ligand named 2-thenoyltrifluoroacetone (TTA) to intense fluorescence intensities of EuIII-TA films.27 In this work, a kind of coordination bonding based pH-responsive delivery system on the basis of zein/HTCC NPs was developed for anticancer drug delivery as well as cell imaging.

EE (%) =

total DOX − free DOX 100% total DOX

2.5. In Vitro Release Study. In vitro release study was conducted using dialysis method under sink conditions. Briefly, an aliquot of drug loaded DOX-zein/HTCC NPs (DOX-NPs) or DOX-zein/HTCCTA/metal NPs (DOX-NPs-TA/metal) was placed in a 3500 MWCO dialysis bag, then immersed in 50 mL 0.01 M PBS (pH 7.4, 6.2, 5.0, or 4.0), followed by gently shaken at 100 rpm in a water bath (37.0 ± 0.5 °C). At appropriate intervals, a 1 mL dissolution sample was collected and the concentration of DOX was measured. 2.6. Cell Culture. HepG2 cells were cultivated in DMEM, supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin at 37 °C with 5% CO2. 2.7. Cytotoxicity Assay. The in vitro cytotoxicity of free DOX and DOX loaded NPs was evaluated by MTT assay. Briefly, 1 × 104 cell/well HepG2 cells were seeded in 96-well plates and incubated for 24 h attachment. The medium was then replaced by NPs (either blank or 1 to 4 μg/mL) with further incubation. After 24 h, 200 μL of MTT solution (0.5 mg/mL) was added to each well, and the cells were incubated for another 4 h. The medium was removed carefully and the resulting formazan crystals were dissolved by adding 150 μL of DMSO. Then, the absorbance values (A) at 490 nm were measured by using a microplate reader (Genios, Tecan, Mannedorf, Switzerland). Cell viability (%) was expressed by the following equation

2. MATERIALS AND METHODS 2.1. Materials. Zein (Z0001) was purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). CS was purchased from Yuhan Ocean Biochemical Co., Ltd. (Zhejiang Yuhuan, China), with deacetylation degree of 91%, and molecular weight (Mw) of 8,000− 15,000. HTCC was synthesized using previous method with the quaternization degree of 62%.39 Tannic acid was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Iron(III) chloride hexahydrate (FeCl3·6H2O), aluminum(III) chloride hexahydrate (AlCl3.6H2O), europium(III) chloride hexahydrate (EuCl3·6H2O), and 3-(N-morpholino)-propanesulfonic acid (MOPS) were purchased from the Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX), 2-thenoyltrifluoroacetone, phosphate buffer solution (PBS), and MTT [3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] were purchased from SigmaAldrich (St. Louis, MO, USA). Dubelcco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin-EDTAn and penicillinstreptomycin mixtures were from GibcoBRL (Carlsbad, CA, USA). Other chemicals used were of analytical grade. 2.2. Preparation of Metal-TA-Coated Zein/HTCC NPs. All solutions were freshly prepared for immediate use. The standard preparation process was described as follows: Zein (10 mg/mL) was dissolved in 75% alcohol-aqueous solution. HTCC was dissolved in MOPS buffer (10 mM, pH 7.4). Then, 0.5 mL of zein solution was immediately poured into 9.5 mL of HTCC solution with the weight ratio of 1:1. Next, 50 μL of TA solution (24 mM) was dropwise added and the dispersion was briefly vortexed. Immediately following this, 100 μL of fresh metal solution (72 mM of FeCl3, AlCl3 or EuCl3 solutions) was added and the dispersion was vortexed. The obtained product was then purified by dialysis (MWCO 15000) for 36 h and next freeze-dried for 48 h. 2.3. Characterization of Metal-TA-Coated Zein/HTCC NPs. The particle size and zeta potential was measured by a dynamic light scattering instrument (Nano-ZS90, Malvern, U.K.). Morphological structures of nanoparticles were observed via Transmission electron microscope (TEM) (JEOL, JEM-2100F, Japan). The chemical structure of samples was monitored by FTIR (Jasco Inc., Easton, MO). The samples were first lyophilized and then prepared as KBr pellets. XPS was operated on an axis ultra DLD apparatus (Kratos, U.K.). 2.4. Loading of DOX into NPs. For DOX loading, the stock solution of 4 mg/mL DOX was first mixed with zein solution for 60 min. Then the above solution was added to HTCC solution dissolved in MOPS buffer (10 mM, pH 7.4) with magnetic stirring. Next, 50 μL of TA solution (24 mM) was dropwise added and the dispersion was

cell viability (%) =

Abs490 nm of treated group 100% Abs490 nm of control group

The toxicities of the DOX-loaded NPs were also expressed as values of the half maximal inhibitory concentration (IC50). 2.8. Intracellular Uptake Study. HepG2 cells were cultured in a 35 mm Petri dish at a seeding density of 1 × 105 cells/well and cultured at 37 °C with 5% CO2 for 24 h. Then the cells were treated with DOX-loaded NPs and incubated for various times (2, 4, 6, or 12 h). After removing the NPs and washing the wells three times with cold PBS, the cells were stained with DAPI for 30 min. Then the cells were observed under a confocal laser scanning microscopy (CLSM) (Zeiss LSM 710, Germany). 2.9. Engineering EuIII -TTA-TA Fluorescent Films on Zein/ HTCC NPs for Cell Imaging. To impart enhanced imaging properties of metal-TA films, EuIII was used to chelate with ligand named 2-thenoyltrifluoroacetone (TTA) to intense fluorescence intensities of EuIII-TA films.27 The standard preparation process of zein/HTCC-TA/TTA-EuIII fluorescent NPs was similar to the protocol described in 2.2 except the addition of TTA. Briefly, after preparation of zein/HTCC NPs, the next addition order was 50 μL TA solution (24 mM), different volume (10,20,30,40,50,60,70, 80, and 90 μL) of fresh EuCl3 solution (72 mM) and then different volume of TTA (72 mM, ethanol). The EuIII: TTA concentrations were kept at a 1:2 ratio. The excess TA and metal ions were ultrafiltered through 10 kDa MWCO Amicon filter ultracentrifuging at 4000 × g for 30 min. The fluorescence spectral measurements were performed at 20 °C on a F-4600 (Hitachi, Japan). Zein/HTCC-TA/TTA-EuIII NPs solutions were excited at 360 nm. Morphological structures of nanoparticles were observed via Transmission electron microscope (TEM) (JEOL, JEM-2100F, Japan). In intracellular uptake study, DOX-loaded NPs were replaced by zein/HTCC-TA/TTA-EuIII NPs. The incubation time was 4, 12, or 24 h, respectively. Confocal laser scanning microscopy (CLSM) (Zeiss LSM 710, Germany) was used to collect the fluorescence images. B


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Scheme 1. Illustration of the Synthesis and Structures of DOX-Loaded Zein/HTCC NPs Coated by Metal-TA Films and the Proposed Model for pH-Dependent Drug Release in Intracellular Delivery Processing of DOX-Loaded Zein/HTCC-TA/Metal NPs in Tumor Cells

Figure 1. TEM image and size distribution of (a, b) zein/HTCC NPs, (c, d) zein/HTCC-TA/FeIII NPs, (e, f) zein/HTCC-TA/AlIII NPs, and (g, h) zein/HTCC-TA/EuIII NPs at a zein: HTCC ratio of 1:1 w/w with the initially used metal ion concentration of 72 mM. 2.10. Statistics Analysis. All of the data were expressed as the mean ± standard error. ANOVA analysis and Student’s t test were performed to compare the significant difference. The value of p < 0.05 was considered to be significant.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Metal-TACoated Zein/HTCC NPs. Because of the metal chelation of TA, it can act as a polydentate ligand for metal ion coordination.40 The materials surface binding affinity of TA makes metal-TA films compactly coat on NPs.41 In addition, NH2 in zein/HTCC NPs could form coordination bonding with metal ions. Scheme 1 showed the facile preparation process of functional metal-phenolic networks based on coordination bonding on zein/HTCC NPs. Figure 1 showed the TEM images and typical size distribution profiles of zein/HTCC NPs (Figure 1a, b), zein/ HTCC-TA/FeIII (Figure 1c, d), zein/HTCC-TA/AlIII (Figure 1e and 1f) and zein/HTCC-TA/EuIII (Figure 1g, h). As can be seen, TEM images showed that metal-TA-coated NPs shared features of a spherical shape and more homogeneous distribution compared with zein/HTCC NPs. FTIR was used to characterize the intermolecular interactions of NPs. The

Figure 2. Fourier transform infrared spectroscopy (FTIR) spectra of different samples. Zein, zein powder, TA, tannic acid powder, HTCC, HTCC powder, NPs, zein/HTCC NPs, NPs-TA/FeIII, zein/HTCCTA/FeIII NPs, NPs-TA/AlIII, zein/HTCC-TA/AlIII NPs, NPs-TA/ EuIII, zein/HTCC-TA/EuIII NPs. The NPs were prepared at zein: HTCC ratio of 1:1 w/w with the initially used metal ion concentration of 72 mM.

C


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Figure 3. XPS survey spectra of (a) zein/HTCC NPs, (b) zein/HTCC-TA/FeIII NPs, (c) zein/HTCC-TA/AlIII NPs, and (d) zein/HTCC-TA/EuIII NPs. The NPs were prepared at zein: HTCC ratio of 1:1 w/w with the initially used metal ion concentration of 72 mM.

Table 1. Element Composition and Content on the Surface of Zein/HTCC NPs, Zein/HTCC-TA/FeIII NPs, Zein/ HTCC-TA/AlIII NPs, and Zein/HTCC-TA/EuIII NPs sample zein/HTCC zein/HTCCTA/FeIII zein/HTCCTA/AlIII zein/HTCCTA/EuIII

C

O

N

S

Fe

76.74 68.04

18.74 23.68

3.82 5.05

0.36 1.82

1.41

68.76

23.60

5.87

0.79

68.67

24.77

5.27

0.97

Al

Eu

0.98 0.32

Table 2. Characterization of DOX-Loaded NPs (pH 7.4) (Results are Displayed As Mean ± Standard Deviation (n = 3)) sample

size (nm)

PDI

zeta potential (mV)

DOX-NPs DOX-NPsTA/FeIII DOX-NPsTA/AlIII DOX-NPsTA/EuIII

106.9 ± 1.9 145.9 ± 3.3

0.17 ± 0.02 0.18 ± 0.01

27.1 ± 1.7 33.0 ± 2.3

66.3 ± 2.9 83.1 ± 3.5

139.4 ± 2.8

0.16 ± 0.01

28.3 ± 1.6

80.7 ± 2.6

135.9 ± 1.7

0.20 ± 0.02

31.1 ± 3.3

78.4 ± 3.2

encapsulation efficiency (%)

Figure 4. TEM image of DOX-loaded (a) zein/HTCC NPs, (b) zein/ HTCC-TA/FeIII NPs, (c) zein/HTCC-TA/AlIII NPs, and (d) zein/ HTCC-TA/EuIII NPs. The NPs were prepared at a zein: HTCC ratio of 1:1 w/w with the initially used metal ion concentration of 72 mM.

coated NPs (NPs-TA/metal), the band of amide II group shifted from 1540 cm−1 in zein and 1567 cm−1 in HTCC to 1544 cm−1 in NPs, indicating that the electrostatic interaction was an intermolecular force between zein and HTCC. Due to the acidic nature of the galloyl groups in TA, there existed electrostatic interaction between TA and zein/HTCC NPs. Three common bands at 1716, 1616, and 1538 cm−1 of TA were coordinated to carbonyl CO vibration of the TA ester bond, CC stretching vibrations of aromatic ring and carbon chain, respectively.43 The vibration peak at 1204 cm−1 in TA could be assigned to HO-C in hydroxyl group.43 Seen from the FTIR spectrum of NPs-TA/FeIII, NPs-TA/AlIII, and NPs-TA/ EuIII, the decrease in intensity of the HO-C stretching confirmed the coordination bonds between phenolic groups

a DOX-NPs, DOX-NPs-TA/FeIII, DOX-NPs-TA/AlIII, and DOX-NPsTA/EuIII represented DOX-loaded zein/HTCC NPs, zein/HTCCTA/FeIII NPs, zein/HTCC-TA/AlIII NPs, and zein/HTCC-TA/EuIII NPs. The NPs were prepared at a zein: HTCC ratio of 1:1 w/w with an initially used metal ion concentration of 72 mM.

representative spectra of zein, HTCC, TA, zein/HTCC NPs, and metal-TA-coated NPs were shown in Figure 2. In the infrared spectra, vibration peaks of 1500−1700 cm −1 correspond to amide I and amide II bonds.42 Comparing the spectra of zein and HTCC with zein/HTCC NPs or metal-TAD


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Figure 5. In vitro release profiles of DOX from (a) DOX-NPs, (b) DOX-NPs-TA/Fe1III, (c) DOX-NPs-TA/Fe2III, (d) DOX-NPs-TA/Fe3III, (e) DOX-NPs-TA/Fe4III, (f) DOX-NPs-TA/Fe5III, (g) DOX-NPs-TA/AlIII, and (h) DOX-NPs-TA/EuIII in PBS under different pH conditions. DOXNPs represented DOX-loaded zein/HTCC NPs. DOX-NPs-TA/Fe1III, DOX-NPs-TA/Fe2III, DOX-NPs-TA/Fe3III, DOX-NPs-TA/Fe4III, and DOXNPs-TA/Fe5III represented DOX-loaded zein/HTCC-TA/FeIII NPs with initially used FeIII concentration of 36, 54, 72, 90, and 108 mM, respectively. DOX-NPs-TA/AlIII or DOX-NPs-TA/EuIII represented DOX loaded zein/HTCC-TA/AlIII NPs or zein/HTCC-TA/EuIII NPs with AlCl3 or EuCl3 initially used concentration of 72 mM. All the NPs were prepared at zein: HTCC ratio of 1:1 w/w.

Figure 6. In vitro cytotoxicity of the (a) blank NPs, (b) DOX-loaded NPs against HepG2 cells incubated for 24 h. In all panels, the indicated concentrations are DOX doses. It should be noted that for evaluating DOX-loaded NPs, equal concentrations of blank NPs were employed to eliminate the effect of vehicles in MTT assay. Data displayed as mean ± SD (n = 6). * p < 0.05, **p < 0.01, versus the DOX-NPs group.

or O−CO group.28,44 Fe 2p signal, Al 2p signal, and Eu 2p signal was clearly observed in the survey scan spectrum of NPs -TA/FeIII (Figure 3b), NPs-TA/AlIII (Figure 3c), and NPs-TA/ EuIII (Figure 3d), respectively. However, the survey scan spectrum of zein, HTCC and TA all contains C and O, which could not confirm the deposition of TA on the surface of NPs. We then further measured the C/O ratio of the samples (Table 1). As is well-known, TA was rich in oxygen. Tracing the

of TA and metal ions when compared with the spectrum of TA.27 XPS was employed to identify the surface elements present on the samples. Figure 3a displayed the spectrum of zein/ HTCC NPs, in which C 1s, O 1s, N 1s, and S 2p core levels existed obviously. Three peak components of C 1s core-level photoelectron spectrum located at 284.2, 285.5, and 287.3 eV (Figure 3a), which are coordinated to C−C, C−O, and CO E


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The in vitro release profile of DOX from DOX-loaded NPs was monitored under different pH conditions (pH 7.4, 6.2, 5.0 or 4.0) that simulated normal physiological environment, in flammatory microenvironment, endosomal compartment, and lysosomal compartment, respectively.45 Figure 5a showed that about 70% of DOX was released at pH 7.4 from zein/HTCC NPs into bulk within 5 h. At pH 4, the cumulative release of DOX was about 87%. Zein/HTCC NPs itself also showed pHresponsive release of DOX because hydrophobic interactions and electrostatic repulsions between the NPs and DOX were decreased in acidic environment.46−48 In comparison, the kinetic release profile of DOX from FeIII-TA coated NPs was found to be concentration dependent as a function of FeIII concentration. At lower FeIII concentration (36 mM), NPsDOX-TA/FeIII1 showed a relative slower release profile in the release media and about 55% of the DOX was released from NPs after 5 h incubation at pH 7.4 while approximately 80% DOX was released within 5 h at pH 4 (Figure 5b). Increasing the FeIII concentration to 54 mM, the release of the DOX was even lower (about 42%) at pH 7.4 within 5 h (Figure 5c). Further increase the concentration of FeIII, the release amount was lower than 40%, after incubation of 24 h from NPs-DOXTA/FeIII3 (Figure 5d). However, the NPs showed a more rapid release profile at pH 6.2, 5, and 4 in comparison to that at pH 7.4 (Figure 5d), indicating the highly pH-responsive release properties. As FeIII concentration increased to 90 mM or 108 mM, no dramatic changes about release profile were observed (Figure 5e, f) compared with NPs-DOX-TA/FeIII3 at FeIII concentration of 72 mM. When AlIII or EuIII was chosen as the coordinating center, at AlIII or EuIII concentration of 72 mM, it witnessed a faster release of DOX than FeIII at the same concentration (Figure 5g and 5h). When at lower metal concentration, the coordination binding positions of TA or NH2- were unsaturated and the metal− ligand films were thin, whereas increasing the amount of FeIII resulted in thick metal−ligand films.40,41 The metal−ligand films had controllable pH-dependent degradability, which led to an association of DOX release profile with the film degradation kinetics.49−52 Due to the different pH conditions between extracellular fluids and tumor site and/or inside tumor cells, zein/HTCC NPs was a favorable deliver system for triggered drug release when combined with metal-phenolic networks. 3.3. Cytotoxicity Assay. The in vitro cytotoxicity of DOXloaded zein/HTCC NPs and metal-TA coated NPs was evaluated by MTT assay. In the investigated range of concentrations, the cytotoxic effect of DOX-loaded zein/ HTCC NPs and metal-TA coated NPs on the cell was dosedependent. Although DOX-loaded metal-TA coated NP showed decreased toxicity compared with DOX-loaded zein/ HTCC NPs, they still demonstrated significant antitumor activity (Figure 6b). The antitumor activities were also evaluated using the values of the half maximal inhibitory concentration (IC50). For HepG2 cells, the IC50 of DOX-NPs was 1.17 ± 0.02 μg/mL, whereas those for DOX-NPs-TA/FeIII, DOX-NPs-TA/AlIII and DOX-NPs-TA/EuIII were 1.75 ± 0.24 μg/mL, 1.28 ± 0.11 μg/mL and 1.29 ± 0.17 μg/mL respectively (Table 3). Seen from the release profiles, DOXNPs experienced faster release of DOX compared with those coated NPs at pH 7.4 or 6.2 which led to more free DOX enter into the cells. Free DOX can freely diffuse through the plasma membrane and enter the active site.53 However, the DOXloaded metal-TA coated NPs traversed the plasma membrane

Table 3. IC50 Values for DOX-Loaded NPs and DOX against HepG-2 Cellsa samples

cell

DOX-NPs DOX-NPs-TA/FeIII DOX-NPs-TA/AlIII DOX-NPs-TA/EuIII

HepG2 HepG2 HepG2 HepG2

IC50 (μg/mL) 1.17 1.75 1.28 1.29

± ± ± ±

0.02 0.24** 0.11 0.17

a

DOX-NPs, DOX-NPs-TA/FeIII, DOX-NPs-TA/AlIII, and DOX-NPsTA/EuIII represented DOX-loaded zein/HTCC NPs, zein/HTCCTA/FeIII NPs, zein/HTCC-TA/AlIII NPs, and zein/HTCC-TA/EuIII NPs. Data displayed as mean ± SD (n = 3). **p < 0.01, versus DOXNPs group.

Figure 7. CLSM images of intracellular uptake of (a) DOX-NPs, (b) DOX-NPs-TA/FeIII NPs, (c) DOX-NPs-TA/AlIII NPs, and (d) DOXNPs-TA/EuIII NPs by HepG2 cells. Cells were counter-stained with DAPI for nuclei. The scale bars represent 20 μm.

amount of C 1s and O 1s, we got that the C/O ratio of NPs, NPs-TA/FeIII, NPs-TA/AlIII, and NPs-TA/EuIII was 4.09, 2.87, 2.91, and 2.77 respectively, once again conforming the successful coating of metal-TA films on zein/HTCC NPs. 3.2. Drug Encapsulation and Release Profiles. As illustrated in Scheme 1, a facile process was utilized to produce DOX-loaded NPs. Because zein/HTCC NPs were promising drug deliver systems for hydrophobic molecules,36 DOX could be loaded through interaction with zein by hydrophobic interactions. The particle size of DOX-encapsulated zein/HTCC NPs was 106.9 nm with ideal PDI and the encapsulation efficiency reached 66.3%. After coated by FeIII-TA, AlIII-TA, or EuIII-TA films, the particle size increased slightly, reaching 145.9, 139.4, and 135.9 nm, respectively, with relatively small PDI and the EE was greatly improved to 83.1, 80.7, and 78.4% (Table 2). In Figure 4, TEM micrograph displayed that the incorporation of DOX in metal-TA-coated NPs (Figure 4b−d) showed clearer spherical shape and more homogeneous distribution than in zein/HTCC NPs (Figure 4a). F


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Figure 8. Engineering multifunctional EuIII-TTA-TA films for imaging. (a) Normalized fluorescence spectra of suspensions of EuIII-TTA-TA films coated zein/HTCC NPs excited at 360 nm. (b) pPhotographs of the corresponding suspensions, excited at 365 nm. (c) TEM image and (d) size distribution of Eu3III-TTA-TA films coated zein/HTCC NPs. (e) CLSM images of intracellular uptake of Eu3III-TTA-TA films coated zein/HTCC NPs by HepG2 cells. Cells were counter-stained with DAPI for nuclei. The scale bars represent 20 μm. NPs-TA/EuIII represented EuIII-TA coated zein/HTCC NPs. NPs-TA-TTA-Eu1III, NPs-TA-TTA-Eu2III, NPs-TA-TTA-Eu3III, NPs-TA-TTA-Eu4III NPs-TA-TTA-Eu5III, NPs-TA-TTA-Eu6III, NPs-TA-TTA-Eu7III, NPs-TA-TTA-Eu8III, and NPs-TA-TTA-Eu9III represented EuIII-TTA-TA-coated zein/HTCC NPs with EuCl3 solution volume of 10, 20, 30, 40, 50, 60, 70, 80, and 90 μL with the initially used concentration of 72 mM, respectively. The EuIII: TTA concentrations were kept at a 1:2 ratio.

increase in the amount of EuIII-TTA, the fluorescence intensity of the EuIII-TTA-TA coated NPs solution enhanced. However, if further increase the concentration of EuIII-TTA, the detected fluorescence intensity decreased, which might be due to fluorescence inner filter effect.60 Figure 8b showed photographs of the EuIII-TTA-TA films suspensions and excited at 365 nm. We additionally examined the TEM images and typical size distribution profiles of EuIII-TTA-TA coated zein/HTCC NPs. Compared with EuIII-TA films without TTA, the addition of TTA did not cause morphological changes and the NPs shared features of a spherical shape. Also, Figure 8d showed a typical size distribution profile of the NPs. To evaluate the fluorescence property, we extended the investigation of EuIII-TTA-TA-coated zein/HTCC NPs for cell imaging. HepG2 cells were used as the model cells to evaluate the intracellular uptake of EuIII-TTA-TA-coated NPs and the endocytosis of EuIII-TTA-TA-coated NPs was observed by CLSM. A few scattered fluorescence dots could be observed at 4 h, whereas dramatically enhanced fluorescence appearing after 12 and 24 h of incubation. Figure8e showed that the red fluorescence was mainly localized in a perinuclear region after incubated for 24 h, which indicated that the released EuIII-TTA complex preferentially targeted nucleoli, similarly to previous studies.61,62 The good fluorescence property of the NPs make it highly promising for use in biomedical imaging.

through endocytosis, which was much slower than passive diffusion, leading to higher IC50.54 More importantly, compared with the blank NPs, the metalTA coated NPs did not show significant cytotoxicity (Figure 6a), which made this system reliable as safe nanoplatforms for drug delivery. 3.4. Intracellular Uptake Study. To evaluate the intracellular uptake of DOX-loaded NPs in HepG2 cells, we observed the endocytosis of DOX-loaded NPs under CLSM. As shown in Figure 7a, only 2 h of incubation, the red fluorescence was appeared in the cytoplasm and nuclei for DOX-zein/ HTCC NPs, indicating fast internalization of DOX released from zein/HTCC NPs. The intensity of red fluorescence increased when extending the incubation time. For FeIII-TA coated DOX-zein/HTCC NPs, minor red fluorescence could be observed at 2 h, indicating limited DOX released (Figure 7b), while the AlIII-TA or EuIII-TA coated DOX-zein/HTCC NPs witnessed faster internalization of DOX inside the cells (Figure 7c, d). After 6 h of incubation, the fluorescence was disseminated in the whole cytoplasm for AlIII-TA or EuIII-TA coated DOX-zein/HTCC NPs. However, there were still fewer DOX released for DOX-NPs-TA/FeIII, consistent to drug release results. Seen from the cellular uptake results, for DOXNPs-TA/metal, they showed that the NPs would internalize into cells and partially release drug in lysosomes from cleavage of coordination bond. 3.5. Engineering EuIII -TTA-TA Fluorescent Films on Zein/HTCC NPs for Cell Imaging. Organic Eu-based materials display high luminescence emission efficiency making them ideal for many imaging applications.55−57 TTA always used as a ligand, serve to facilitate intramolecular energy transfer from ligands to rare-earth ions and excite state stimulating metal centered luminescence.58,59 In this paper, we reported that the EuIII-TTA-TA films were utilized to impart imaging property of zein/HTCC NPs. Figure 8a showed the fluorescence spectra of the films. The observed red fluorescence in EuIII-TTA-TA films is mainly due to the 5D0 → 7F2 transition around 613 nm.27 As can be seen clearly, with the

4. CONCLUSION We report a simple and rapid strategy to prepare pH-responsive zein/HTCC-TA/metal NPs by constructing the coordination bonding. The resultant DOX-loaded zein/HTCC-TA/metal NPs were approximately 145.9 nm, 139.4 and 135.9 nm in diameter for DOX-zein/HTCC-TA/FeIII NPs, DOX-zein/ HTCC-TA/AlIII NPs and DOX-zein/HTCC-TA/EuIII NPs, respectively, with a spherical shape and high encapsulation efficiency. The controlled release of anticancer drugs were tailored by pH values as well as the concentrations and types of the metal ions. In vitro cell assays, the blank zein/HTCC-TA/ metal NPs showed low cytotoxicity, but the DOX-loaded NPs G


Article


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exhibited a high cytotoxic activity against HepG2 cells. To impart enhanced imaging properties of metal-TA films, we used EuIII to chelate with ligand named 2-thenoyltrifluoroacetone (TTA) to intense fluorescence intensities of EuIII-TA films.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-27-63730040. Fax: +86-27-87282966. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful the financial support from the National Natural Science Foundation of China (31371841) and acknowledge the support from colleagues of Key Laboratory of Environment in colleagues of Key Laboratory of Environment.

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REFERENCES

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Engineering Multifunctional Films Based on Metal-Phenolic Networks

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