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Cite This: Langmuir XXXX, XXX, XXX−XXX
Multilayer Assembly of Tannic Acid and an Amphiphilic Copolymer Poloxamer 188 on Planar Substrates toward Multifunctional Surfaces with Discrete Microdome-Shaped Features Lianghong Peng,†,‡ Fang Cheng,†,§ Yanan Zheng,†,‡ Zengqian Shi,∥ and Wei He*,†,‡
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State Key Laboratory of Fine Chemicals, ‡Department of Polymer Science and Engineering, and §School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian, Liaoning 116023, China ∥ Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore S Supporting Information *
ABSTRACT: Tannic acid (TA) is a natural polyphenol compound with a broad spectrum of biological activities, the most notable of which being antioxidation. Poloxamer 188 (P188), a synthetic triblock copolymer of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide), is amphiphilic in nature and best known for its ability to seal structurally damaged cellular membranes. The integration of both substances onto planar substrates could bring a new option for multifunctional coatings that are advantageous for implantable biomedical devices. Here, we demonstrate the feasibility of multilayer assembly of TA/P188 toward such a coating based on hydrogen bonding between phenolic hydroxyls of TA and ether groups of P188, and the unique surface feature it generates. The interactions between these two compounds were studied both in solution and in substrate-supported layer-by-layer assembly. The multilayer assembly process exhibits an exponential growth pattern as characterized by UV−vis spectrophotometry and quartz crystal microbalance with dissipation. Morphologically unique, microdome-shaped surface features emerge and evolve with the number of layers assembled. Such features bring a reservoir function to this coating, as demonstrated by the loading of hydrophobic nile red dye. Furthermore, the presence of TA in the multilayers was revealed by silver nitrate staining, and its antioxidation activity was demonstrated through a 2,2-diphenyl-1-picryl-hydrazyl free-radical scavenging assay.
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INTRODUCTION Tannic acid (TA) is a naturally occurring polyphenol compound. Structurally, TA features a glucose core esterified with five digallic acid units (Scheme 1a). Each digallic acid unit carries five phenolic hydroxyl groups, rendering a total of 25 −OH groups per TA molecule. TA has been of great interest for a wide variety of applications, ranging from healthcare, food industry, nanotechnology to consumer products.1−5 Among these applications, the biomedical relevance of TA can be attributed largely to its attractive biological properties, for example, antimicrobial,6 antioxidant,7 anticarcinogenic,8 and antimutagenic.9 To apply TA for biomedical usages, different approaches have been explored, one of which being in the form of layer-by-layer (LbL) assembly. Its participation in LbL process driven by electrostatic interaction, hydrogen bonding, or coordination interaction has been enabled by the unique structure of TA. Being a weak polyacid (pKa ≈ 8.5), TA has acted as a polyanion component to build LbL films or capsules with polycations, such as poly(dimethyldiallylamide),10 poly(allylamine hydrochloride),7,10 quarternized poly(N-vinylpyridine),7,11 and chitosan,12 as well as with zwitterionic material poly(sulfobetaine methacrylate).13 In LbL, based on hydrogen © XXXX American Chemical Society
bonding interactions, the phenolic hydroxyls of TA function as hydrogen bond donors and can be paired with various neutral polymers to form multilayer assembly, including poly(Nvinlypyrrolidone),11,14−16 poly(N-vinyl caprolactam),11,14 poly(N-isopropylacrylamide),11,14 poly(2-oxazoline)s,17 and derivatives of poly(N-vinylamide),18 poly(ethylene glycol),11,16,19 and poly(triethylene glycol methyl acrylate-co-tocopheryl acrylate).20 To achieve coordination-driven LbL assembly of TA, metal-chelating capability of polyphenols has been capitalized, with most studies focusing on pairing of TA and iron(III) ions.21 Poloxamers, better known under the commercial trade name of Pluronic, are a family of synthetic triblock copolymers of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO-b-PPO-b-PEO). These polymers are amphiphilic in nature because of the presence of both hydrophilic PEO and hydrophobic PPO components and have been extensively studied as nonionic surfactants for decades. Applications of Received: June 12, 2018 Revised: August 10, 2018
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DOI: 10.1021/acs.langmuir.8b01982 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir Scheme 1. Chemical Structures of TA (a) and P188 (b)
Reagents Co., Ltd. (Tianjin, China). Hydrogen peroxide was obtained from Guangfu Fine Chemical Research Institute (Tianjin, China). 2,2Diphenyl-1-picryl-hydrazyl (DPPH) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nile red was obtained from Solarbio (Beijing, China). All reagents were used as received. Ultrapure water with a resistivity of 18.2 MΩ cm was used for all aqueous solution preparation and rinsing procedures. Substrates for the LbL assembly included silicon wafers (1 × 1 cm) and quartz plates (d = 24 mm). Prior to LbL deposition, substrates were prepared using the protocol reported in our previous study.34 Briefly, substrates were cleaned by treatment with a boiling piranha solution made up by mixing concentrated sulfuric acid with 30% hydrogen peroxide at a volume ratio of 3:1 (Caution: piranha solutions should be handled with extreme care because of their violent reaction with organics). The cleaned substrates were then primed with a PEI layer by incubation in PEI solution (1.5 mg/mL, pH 7) for 30 min, followed by rinsing with water for 5 min. Preparation of TA/P188 Mixtures. TA solution and P188 solution were prepared separately by dissolving a given amount of each material in ultrapure water. The solution pH was adjusted to desired values using 1 M HCl or NaOH. The TA/P188 mixtures were prepared by direct mixing of the TA and P188 stock aqueous solutions at ratios corresponding to different mass fractions of TA in the final mixtures. The mixtures were subjected to turbidity studies using UV−vis spectroscopy and size measurements by dynamic light scattering (DLS). Preparation of TA/P188 Multilayers. In a typical deposition process, the PEI primed substrate was sequentially immersed in solutions of TA (0.5 mg/mL, pH 3) and P188 (0.5 mg/mL, pH 3) for 10 min at room temperature, respectively. A rinsing step with HCl solution of pH 3 was included in between each incubation phase to remove excess materials from the surface. This cycle was repeated until the desired number of TA/P188 bilayers was reached. The resulting LbL construct was termed as (TA/P188)n, where n represents the number of bilayers. All assemblies were performed on silicon substrates, except for the UV−vis study where quartz plates were used. Characterizations. UV−vis spectra were obtained on a U-3900 spectrophotometer (Hitachi, Japan). For turbidity study of the TA/ P188 mixtures, absorbance at 450 nm was recorded where TA and P188 solutions have no absorption bands. Investigations of the TA/ P188 multilayer assembly process in real time were carried out using a Q-Sense E1 QCM-D system (Biolin Scientific, Sweden). The assembly was performed on silica-coated 5 MHz AT-cut quartz crystals (Q-Sense). The QCM-D instrument was operated with a flow cell attached to a peristaltic pump to achieve a flow rate of 50 μL/min. A cleaned crystal was mounted in the flow cell and equilibrated until stable flat baselines of frequency (F) and energy dissipation factor (D) were obtained at 25 °C. After being primed with a PEI layer, TA solution (0.5 mg/mL, pH 3) was pumped over the crystal. When a
poloxamers have been diverse, covering petroleum industry, cosmetics, bioprocessing, agricultural processes, pharmaceutics, biomedicine, nanoscience, and more.22−27 Depending on their molecular compositions, the properties of poloxamers can vary greatly. For poloxamer 188 (P188, Scheme 1b), which is composed of a PPO block of about 1800 g/mol and 80% weight content of PEO, its most notable biological function seals structurally damaged cellular membranes.28 For example, studies have shown that the mechanical membrane injuryinduced neuronal death could be mitigated by P188 through its action of membrane integrity restoration and preventing further disruption of cytoskeleton.29,30 The role of P188 in improving the survival of non-neuronal cells, such as fibroblasts and skeletal muscle cells against severe membrane damaging injury has also been demonstrated.31,32 In this paper, we report the LbL assembly of TA with P188 on planar substrates. The motivation is to explore the feasibility of a bioactive coating that could integrate the antioxidative attribute of TA with membrane sealing capability of P188. Such a multifunctional coating could find applications for implantable biomedical devices, addressing adverse biological responses33 related to mechanical insertion injury and foreign body reaction induced by these implants. It is rationalized that multilayers of TA/P188 could be achieved based on the formation of hydrogen bonds between phenolic hydroxyls of TA and ether groups of P188. The interactions between these two compounds were studied both in solution and in substrate-supported LbL assembly. The multilayer assembly process was investigated using UV−vis spectrophotometry and quartz crystal microbalance with dissipation (QCM-D). The assembled multilayers were characterized for surface chemistry and morphology. Interestingly, unique microdome-shaped surface features were observed. Such features bring an additional reservoir function to this coating, as demonstrated by the loading of hydrophobic nile red dye. Furthermore, the presence of TA in the multilayers was revealed by silver nitrate staining as well as free-radical scavenging assay.
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MATERIALS AND METHODS
Materials. Branched polyethyleneimine (PEI, Mw = 1800 g/mol) and TA were obtained from Alfa Aesar (Ward Hill, MA, USA). P188 was purchased from Adamas (Shanghai, China). Hydrochloric acid and sulfuric acid were obtained from Tianjin Chemical Reagent No. 3 Plant (Tianjin, China). Sodium hydroxide was from Tianli Chemical B
DOI: 10.1021/acs.langmuir.8b01982 Langmuir XXXX, XXX, XXX−XXX
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Langmuir
Figure 1. Turbidity measurements and the corresponding photographs of various TA/P188 mixtures (pH = 3). (a) TA and P188 mixed in equal mass ratio with original concentrations of each component ranging from 0.01 to 0.5 mg/mL. (b) Samples prepared by mixing TA and P188 solutions at different mass fractions of TA.
Figure 2. Hydrodynamic diameters obtained from DLS measurements of TA/P188 mixtures (pH = 3) prepared by mixing TA and P188 either (a) in equal mass ratio with varying original concentrations or (b) at different mass fractions of TA. plateau was reached for both F and D, the solution was switched to a pH 3 rinsing buffer. After the signals were stabilized, P188 solution (0.5 mg/mL, pH 3) was introduced until F and D reached another plateau. Afterward, another rinsing step followed. These cycles repeated until the desired number of TA/P188 bilayers was achieved on the crystal. Throughout the entire process, changes in frequency and dissipation factor were monitored continuously. Surface chemistry of the assembled TA/P188 multilayers was characterized by acquiring infrared spectra in the attenuated total reflection (ATR) mode. A Thermo Fisher 6700 Fourier transform infrared (FTIR) spectrometer (Thermo Fisher, USA) equipped with a mercury cadmium telluride detector was used. Each spectrum was the average of 16 scans between 650 and 4000 cm−1 at a resolution of 4 cm−1. Optical microscopy and fluorescent microscopy were carried out using a BX53 upright fluorescent microscope (Olympus, Japan). An Asylum Research Cypher S atomic force microscope (AFM, Oxford Instruments, USA) was used to characterize the morphology of the hydrated multilayers. Tapping-mode imaging was carried out using Olympus AC240TSA-R3 cantilevers with a nominal spring constant and resonance frequency of 2 N/m and 70 kHz, respectively. Silver Nitrate Assay. To visually locate TA in the multilayer construct, a silver nitrate assay was adapted from the literature.35,36 Briefly, (TA/P188)8-coated silicon samples were incubated overnight in 25 mM AgNO3 aqueous solution under protection from light. After rinsing with water to remove excess AgNO3, the samples were examined in water microscopically. Nile Red Loading. (TA/P188)8-coated silicon samples were incubated in nile red solution (0.1 μg/mL in ultrapure water) at room temperature for 12 h under orbital shaking. Afterward, the samples were rinsed with ultrapure water four times, followed by fluorescent imaging in water. Free-Radical Scavenging Assay. The antioxidation potential of TA/P188 multilayers was evaluated using a DPPH free radical scavenging assay.20 Briefly, DPPH working solution was prepared by mixing 1 mL of ultrapure water with freshly prepared DPPH stock solution (3 mL, 0.025 mg/mL in methanol). The silicon wafers assembled with (TA/P188)n multilayers (n = 0, 4, 8, and 12) were
immersed in the DPPH working solution for 30 min at room temperature. UV−vis spectra of the solutions were then collected to track the change in the characteristic absorbance peak of DPPH radicals at 515 nm.
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RESULTS AND DISCUSSION Complexation of TA with P188 in Aqueous Solution. It is well-known that the study of mixtures of complementary building pairs in solution can provide insights on parameters favorable for their multilayer assemblies on surfaces.37,38 Therefore, we first investigated the formation of waterinsoluble complexes between TA and P188. The mixed solutions were characterized for cloudiness using direct visual inspection and the turbidimetric method. At a mass ratio of 1:1, a transparent solution was observed when the mixture was prepared with 0.01 mg/mL TA and 0.01 mg/mL P188 (Figure 1a). However, at higher concentrations, cloudy mixtures were obtained with a noticeable increase in turbidity as the concentrations of the parent TA and P188 solutions increased up to 0.5 mg/mL (Figure 1a). The effect of mass fraction of TA in the TA/P188 mixtures on complexation was also studied with a constant final concentration of P188 being 0.025 mg/ mL in the mixture. As shown in Figure 1b, mixtures prepared with TA mass fraction less than 0.5 were optically transparent, whereas those at 0.5 or more appeared as milky dispersions. The complexation behavior of TA with P188 was further characterized by DLS. Interestingly, despite the mixture of 0.01 mg/mL TA and 0.01 mg/mL P188 solution being transparent, the DLS measurement of the mixture showed the presence of particles with average hydrodynamic size of 132 nm (Figure 2a), suggesting the formation of TA/P188 complexes. As the concentrations of TA and P188 increased, the resulting particles became larger, reaching 500 nm for the mixture of C
DOI: 10.1021/acs.langmuir.8b01982 Langmuir XXXX, XXX, XXX−XXX
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Figure 3. (a) UV−vis absorption spectra of LbL assembly of up to eight bilayers of TA/P188. (b) Absorbance at 222 nm as a function of number of assembled bilayers.
Figure 4. In situ characterization of alternating deposition of TA and P188 at a concentration of 0.5 mg/mL on QCM crystals. (a) Time evolution of the QCM frequency during the assembly process. (b) Frequency change and (c) adsorbed mass per unit area plotted against the number of layers during the deposition of TA and P188. (d) Corresponding dissipation response as a function of time.
was formed (Figure S1). Considering the pKa of TA being ∼8.5, this transition at pH 9 could be attributed to the dissociation of TA/P188 complex because of TA deprotonation. A similar effect of pH on complex stability was reported for mixtures of TA and poly(N-vinlypyrrolidone), where optically clear solution was obtained only after a specific critical ionization of TA was achieved.11 LbL Assembly of TA with P188. With information gained from the studies of TA/P188 complexation in solution, we then carried out LbL assembly onto solid substrates using acidic (pH = 3) solutions of TA (0.5 mg/mL) and P188 (0.5 mg/mL). The assembly process was monitored by UV−vis spectrophotometry of the multilayers on quartz substrates. As shown in Figure 3a, two absorbance peaks at 222 and 278 nm were observed, and their intensity increased with the number of bilayers. Both peaks are characteristics of the TA component, suggesting the successful incorporation of TA into the assembled multilayers. A plot of the absorbance at 222 nm against the number of bilayers revealed that the growth of the TA/P188 multilayers was close to an exponential growth pattern (Figure 3b). The trend holds when the peak at 278 nm was plotted against the number of bilayers (Figure S2). This result is consistent with the previous studies of LbL assembly
0.25 mg/mL TA and 0.25 mg/mL P188 (Figure 2a). For the sample made with 0.5 mg/mL TA and 0.5 mg/mL P188, it was too concentrated to allow direct DLS measurements. Shown in Figure 2b were the hydrodynamic diameters of various TA/ P188 complexes prepared with different mass fractions of TA. The size of the complexes first increased when the mass fraction of TA changed from 0.1 to 0.5 and then decreased when TA content in the mixture was further increased up to 0.9 (Figure 2b). It is worth noting that, although the mixture prepared from TA mass fraction of 0.9 displayed much smaller particles than those of 0.5, it appeared more opaque (Figure 1b). The results suggest that factors other than particle size may have influenced the cloudiness of the mixture, such as refractive index of the TA/P188 complexes. It should be pointed out that the concentrations of P188 used here were much lower than its critical micellization concentration (i.e., ∼4 mg/mL),39 minimizing the formation of P188 micelles and their interference with interpretation of the DLS results. The above studies were carried out at pH 3, a condition where TA molecules are protonated to present phenolic hydroxyl groups available for hydrogen bonding. When exposed to increasing pH values, the TA/P188 mixture was cloudy except at pH 9, where completely transparent solution D
DOI: 10.1021/acs.langmuir.8b01982 Langmuir XXXX, XXX, XXX−XXX
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Figure 5. (a) FTIR spectra of TA, P188, and an assembled (TA/P188)12 film. (b) Curve fitting of the −C−O−C− region of a (TA/P188)12 film.
Figure 6. (a) UV−vis absorption spectra and (b) QCM frequency change obtained from the studies of (TA/P188)8 stability under various pH conditions. The arrowheads indicate the introduction of solutions of different pH into the QCM flow cell.
Throughout the assembling process, the change in dissipation was relatively small (
