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LbL assembly of albumin on nitric oxide-releasing silica nanoparticles using suramin, a polyanion drug, as an interlayer linker Hung-Chang Chou, Shih-Jiuan Chiu, and Teh-Min Hu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00534 • Publication Date (Web): 29 Jun 2015 Downloaded from http://pubs.acs.org on July 5, 2015
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LbL assembly of albumin on nitric oxide-releasing silica nanoparticles using suramin, a polyanion drug, as an interlayer linker Hung-Chang Chou,1 Shih-Jiuan Chiu2 and Teh-Min Hu1* 1
School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan, ROC;
2
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan,
ROC
*Corresponding author: Teh-Min Hu E-mail:
[email protected] School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan, ROC.
KEYWORDS: silica nanoparticles; nitric oxide; layer-by-layer assembly; albumin; protein corona; suramin
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ABSTRACT
Preformed protein corona of nanoparticles can be utilized as a promising formulation strategy for improving nano drug delivery. Nitric oxide (NO) is a labile molecule with extensive therapeutic implications. In this study, we test whether preformation of protein coatings can enhance the performance of NO-delivering nanoparticles. S-Nitroso (SNO) silica nanoparticles (SNO-SiNPs) were prepared using a nanoprecipitation method. For the first time, bovine serum albumin (BSA) was used to coat NO-releasing nanoparticles, facilitated by a polyanionic drug, suramin, under a layer-by-layer (LbL) scheme. Bare and coated nanoparticles were characterized by zetapotential, size, and spectroscopic measurements. We demonstrate that albumin/suramin-surface co-assembly has advantages in preventing particle aggregation during lyophilisation, controlling NO release and exerting an enhanced anticancer effect.
INTRODUCTION Recently, the nano-bio interactions (i.e. the interaction of nanoparticles (NPs) with biological components) have attracted much attention in the field of nanomedicine. It is now well recognized that the biological fates of therapeutic NPs are governed by their complex interaction with proteins and biological membranes. Several studies have shown that unprotected NPs can adsorb hundreds of different proteins in serum and cells to form “protein corona”.1-3 On the one hand, binding of opsonic proteins to NPs may facilitate blood clearance of these particles, thereby reducing their circulation time; on the other, cellular uptake and transport of NPs may be significantly enhanced or attenuated, depending on which specific proteins are adsorbed onto NPs.4 Thus, one aspect of nanoparticle engineering is to modify the particle surface to reduce undesirable particle-protein interactions.
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A promising strategy to counteract in vivo protein binding is to utilize protein binding per se, i.e. to pre-coat NPs with protective protein films such that the surface protein-binding sites of NPs are blocked. Indeed, it has been shown that pre-coating of liposomes or other NPs with albumin, a dysopsonic protein, exhibit reduced affinity of these NPs to serum proteins and cells.5, 6
Recently, further studies have demonstrated that preformed albumin coatings can increase the
blood circulation time, enhance the cellular uptake and modulate the toxicity of NPs.7-9 These findings pinpoint one of the utilities of employing protein coating to optimize nanoscale drug delivery systems. Besides, protein pre-coating of NPs may have additional advantages, which remain to be elucidated. First, by altering the physicochemical property of surfaces, the precoating strategy may circumvent the particle aggregation problem commonly encountered in formulating nanoformulations. Second, the surface protein film may serve as a barrier for protecting the payload inside NPs, and preferably, controlling its release. Thus, to fulfil the desired properties mentioned above, a versatile protein-coating method should be developed. This method should allow for the formation of stable protein films with tunable surface coverage. The layer-by-layer (LbL) technique is a simple and efficient way to form multilayer polymer films on various surfaces, based on repeat, alternate formation of distinct polymer layers via electrostatic or other supramolecular interactions.10-12 Although the method is viable for assembly of multilayer proteins on plane surfaces it is more challenging on particles, especially NPs.13 Moreover, for therapeutic NPs there is a need to search for the interlayer linking materials that is biocompatible. NO is a short-lived bioregulatory molecule. Various nano platforms have been proved successful in delivering NO.14-21 Remarkably, these NO-releasing NPs exhibit a wide range of potential therapeutic activities, including antitumor effect.22-25 In the present study, NO-releasing
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S-nitroso silica NPs (SNO-SiNPs) were prepared from a single silane source using a surfactantfree and energy-efficient approach.26 An LbL procedure is proposed to assemble bovine serum albumin (BSA) on SNO-SiNPs (Scheme 1). For the first time, suramin was used to function as an interlayer linker. Suramin is an old drug for treating human trypanosomiasis (sleeping sickness); its use in animals and human populations dates back to the early 1920s.27 After nearly 7 decades and in the early 1990s, basic and clinical studies demonstrate that suramin is a novel anticancer agent with multiple mechanisms of action.27, 28 One of the potential mechanisms has been associated with the ability of suramin to bind to multiple growth factors, including basic fibroblast growth factor (bFGF).27 The suramin binding would block the binding of growth factors to their receptors, thereby resulting in antiproliferative and anti-angiogenesis effects.29, 30 Moreover, the antiproliferative mechanism may be via disruption of cellular energy balance and cellular respiration.31 Recently, suramin has been shown to enhance the antitumor efficacy of traditional chemotherapeutic agents; notably, in such chemo-sensitization regimens, low nontoxic doses of suramin were used.32-35
Scheme 1. A schematic representation of the layer-by-layer procedure for the surface coating of S-nitroso silica nanoparticles (SNO-SiNP).
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Besides binding to multiple growth factors, suramin binds avidly to various other proteins, including albumin, with specific binding sites.36-38 Suramin has 6 negatively charged sulfonate groups, which is assumed to form salt bridges with the positively charged amino acid residues at low pH.36-38 In addition, the binding also involves hydrophobic interactions. Thus, we envision that suramin is a promising candidate for modulating protein coatings on therapeutic NPs, given its poly-anionic and protein-binding properties as well as established biocompatibility. Furthermore, we consider that the use of suramin as an LbL-coating material may offer additional advantages in cancer nanomedicine, given the atypical anticancer activities of suramin mentioned above. Here, we demonstrate how suramin can facilitate the assembly of albumin on nitric oxide (NO)-releasing silica NPs, thereby offering advantages of increasing formulation capability, controlling NO release and enhancing the anticancer activity. EXPERIMENTAL SECTION Chemicals 3-Mercaptopropyltrimethoxysilane (MPTMS), dimethyl sulfoxide (DMSO), albumin from bovine serum (BSA), suramin sodium salt, sodium hydroxide (NaOH), latex beads (polystyrene, 0.1 µm), citric acid, 2,3-diaminonaphthalene (DAN), sulfanilamide, N-(1naphthyl)ethylenediamine dihydrochloride, phosphoric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium nitrite (NaNO2), mercuric chloride, sodium chloride, potassium chloride, sodium phosphate (dibasic, Na2HPO4), potassium phosphate (monobasic, KH2PO4) were obtained from J.T.Baker (Phillipsburg, NJ, USA). Hydrochloric acid (HCl) was purchased from Merck (Darmstadt, Germany). Diethylenetriaminepentaacetic acid (DTPA) was obtained from TCI. (Tokyo, Japan). All chemicals and solvents were ACS reagent grade and used as
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received. Deionized water (18.2 MΩ·cm at 25 °C) was prepared using a Millipore Milli-Q gradient A-10 water-purification system (Bedford, MA, USA). Preparation of Bare and Coated S-Nitroso Silica Nanoparticles (SNO-SiNPs) Bare SNO-SiNPs were prepared using a nanoprecipitation-based procedure described previously.26 Briefly, a 1.5 mL aliquot of the organic phase (containing 200 mM MPTMS, 400 mM NaNO2, 0.5 M HCl in DMSO) preincubated for 24 h was injected immediately (~4s) with a 27-gauge syringe to 10 mL deionized water (the water phase). After aging for 1 h at room temperature (to avoid light), a total of 11 reaction mixtures were combined and centrifuged at 5500 rpm (Sorvall Super T21; DuPont Company, Wilmington, DE, USA) for 30 min at 4 °C to remove the supernatant. The remaining particle pellets were washed twice using 25 mL of icecold water. Eventually, the washed particles were redispersed in 2 mL deionized water (i.e. bare SNO-SiNPs; layer-0 particles). For the convenience of adjusting particle concentrations during protein coating, we determined particle concentrations using a turbidity measurement (Shimadzu UV-2450, Kyoto, Japan) and set optical density at 800 nm of 1.0 absorbance unit (OD800 nm = 1 a.u.) to be 1 particle concentration unit. The layer-by-layer (LbL) coating was initiated by adding 1 mL bare SNO-SiNPs (6 units) to 19 mL BSA solution (10 mg/mL) at pH 4. The mixture was incubated for 30 min at room temperature (in the dark) and then centrifuged at 5500 rpm (3615g) for 30 min at 4 °C (Sorvall Super T21). After removing the supernatant, the particle pellet was redispersed in 1 mL deionized water (i.e., B-SNO-SiNPs, layer-1 particles). Next, 1 mL B-SNOSiNPs and 1 mL of 20 mg/mL sodium suramin (pH adjusting to 4) were mixed and incubated under the same condition. Then, the mixture was centrifuged at 3600 rpm (1058g) for 30 min at 4 °C (Eppendorf Centrifuge 5402, Hamburg, Germany). After removing the supernatant, the particle pellet was redispersed in 1 mL deionized water (i.e. SB-SNO-SiNPs, layer-2 particles).
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Finally, repeat BSA coating was performed on layer-2 particles to form layer-3 particles (i.e. BSB-SNO-SiNPs), using the same coating condition as described in the initial step.. Determinations of Hydrodynamic Particle Sizes and Zeta Potential The hydrodynamic diameters of particles were determined by a dynamic-light-scattering (DLS) instrument (LB-500, Horiba instruments Inc., Irvine, CA, USA). Three repeated measurements were conducted for each sample. The surface charges of particles at different stages of coating were determined by a zeta-potential analyzer (ZetaPlus, Brookhaven Instruments Co., Holtsville, NY, USA). For each measurement, 10 zeta-potential readings were recorded and averaged. The data reported (sizes and zeta potential) are the mean ± SD of three independent experiments. Transmission Electron Microscopy (TEM) The TEM images were obtained from a Hitachi HT7700 120 kV high-contrast/highresolution digital TEM instrument operated at 75.0 kV, 8.0 µA. The samples were prepared on the previous day, stored at 4 °C. On the experimental day, one drop of a 20-fold diluted sample was placed on a carbon-Formvar-coated copper grid (300 mesh, type A; Electron Microscopy Sciences, Hatfield, PA, USA). After air-drying for 3 h, the TEM pictures were taken. Assay of SNO Concentrations The amounts of SNO groups in bare and coated SNO-SiNPs were determined using a modified Saville-Griess method.39 Briefly, mercuric chloride (50 mM) was added to 100 µL particle dispersions, followed by incubation at 60 °C for 15 min. After centrifugation (15996g for 10 min at 4 °C; Eppendorf Centrifuge 5402, Hamburg, Germany), 50 µL of the supernatant
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was taken and allowed to react with 950 µL of the Griess reagent for 15 min. The optical absorbance was measured at 540 nm and the nitrite concentration was determined using sodium nitrite standards (1-100 µM). Fluorescence Spectroscopy For the fluorescence quenching study of BSA, measurements were conducted for solutions containing BSA (1 mg/mL) and various concentrations of SNO-SiNPs (0.0025-2 units; 1 unit equivalent to O.D.800nm=1) at pH 4 in water. Two types of SNO-SiNPs were added to the solution: bare SNO-SiNPs (layer 0 particles) and suramin/BSA-treated SNO-SiNPs (layer 2 particles). The fluorescence spectra of BSA were acquired at an excitation wavelength of 280 nm and emission wavelengths of 310-500 nm (Infinite M200, Tecan Austria GmbH). For the fluorescence enhancing study of suramin, the first experiment was performed by adding increasing concentrations of the layer-1 BSA-coated SNO-SiNPs (0.1-3 units) to an aqueous suramin solution (10 mg/mL, pH 4). The second experiment was conducted by adding increasing concentrations of BSA (0.1-2 mg/mL) to an aqueous dispersion of suramin/BSA-coated SNOSiNPs (layer 2 particles). The fluorescence spectra of suramin were acquired at an excitation wavelength of 330 nm and emission wavelengths of 360-550 nm. Attenuated Total Reflection Fourier-Transform Infrared (ATR-FTIR) Spectroscopy The ATR-FTIR spectra were measured by a Nicolet iS5 Fourier-transform infrared spectrometer (Thermo Fisher Scientific Inc., Madison, WI, USA) with an iD3 ATR accessory (single-reflection Zinc Selenide 7 mm crystal; Fisher Scientific UK Ltd., Loughborough, UK). The nanoparticle suspension (10 µL) was placed above a ZnSe flat crystal plate. The background was corrected with deionized water. IR spectra were the results of 50 scans with a resolution of 4
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cm-1 in the spectral range of 4000-600 cm-1. Each IR spectrum was corrected for optical effects with the ATR correction algorithm in the OMINC software. Labeling of Coated Protein Fifty microliters of bare and coated SNO-SiNPs were added into 750 µL of PierceTM 660nm Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA). After 15 min, the solutions were centrifuged at 15996g for 10 min (Eppendorf Centrifuge 5402) to collect the pallets. Lyophilization Five hundred microliters of the nanoparticle suspension were filled in 5-ml glass vials. After freezing at -80 °C, the samples were placed into the drying chamber of a lyophilizer (FD5030, Panchum Scientific Corp., Kaohsiung, Taiwan), pre-cooled to -40 °C. Drying was performed at a pressure of 100 mTorr for 12 h. The freeze-dried samples were resuspended in 1 ml of deionized water for size evaluation. Kinetics of Nitric Oxide Release A nitric oxide-specific fluorophore, 2,3-diaminonaphthalene (DAN), was used to measure the kinetics of NO release.40 The phosphate-buffered saline (PBS) based release medium contains 0.225 mL of 20 mM DAN, 0.5 mL of 10 mM DTPA, and 8.275 mL of PBS. Freshly prepared bare and BSA-coated SNO-SiNPs (containing 100 µM SNO) were dispersed and diluted in the release medium and then incubated at 37 °C in the dark. At predetermined time intervals, 10 µL of sample was taken and added to 240 µL of 10 mM NaOH solution, followed by fluorometric measurements (excitation 375 nm and emission at 415 nm; Infinite M200, Tecan
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Austria GmbH). The concentration of the formed fluorescent product (2,3-napthotriazole, NAT) was determined using NAT standards (0.78-50 µM).41 Anticancer Cytotoxicity MCF-7 cells were seeded in triplicates at 5000 cells per well in 96-well plates and incubated in DMEM medium containing 10% FBS (culture medium). Following the overnight incubation, cancer cells were exposed to serial dilutions of bare and coated SNO-SiNPs in the culture medium for 72 h. Then, the medium was removed and replaced with MTT solution (Sigma-Aldrich, St. Louis, MO, USA) for 3 h at 37 °C. After removing the MTT solution, DMSO was added and the absorbance was measured at 540 nm using a microplate ELISA reader. Relative absorbance (% of negative control) was used to represent cell viability. Statistical Analysis Data are the mean ± SD of three independent experiments. Cell viability data were fitted according to a four-parameter logistic model (SigmaPlot 12.5). To compare bare and coated SNO-SiNPs, one-way analysis of variance (ANOVA) with a post-hoc test (Scheffe’s) was used (StatPlus 5.0). A value of P< 0.05 was considered significant and marked with a star symbol. RESULTS AND DISCUSSION In this study, we perform albumin assembly at pH near 4, at which BSA is positively charged (pI~4.7)42 and SNO-SiNPs are negatively charged (pI~1.5, Figure 1a). Before BSA binding, the surface charge of bare SNO-SiNPs (layer 0) is
