Article pubs.acs.org/Langmuir
Interaction of Hyaluronan with Cationic Nanoparticles Fouzia Bano,† Mónica Carril,†,§ Paolo Di Gianvincenzo,†,‡ and Ralf P. Richter*,†,∥,⊥,# †
CIC biomaGUNE, Paseo Miramon 182, 20009 Donostia - San Sebastian, Spain CIBER-BNN, Paseo Miramon 182, 20009 Donostia - San Sebastian, Spain § Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain ∥ Université Grenoble Alpes, Grenoble 38041 Cedex 9, France ⊥ CNRS, DCM, BP 53, Grenoble 38041 Cedex 9, France # Max-Planck-Institute for Intelligent Systems, Heisenbergstrasse 3, 70569 Stuttgart, Germany ‡
S Supporting Information *
ABSTRACT: The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies−compact nanoscopic spheres and extended microscopic fibers−upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.
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INTRODUCTION
surrounding binding partners is believed to be functionally important.2,8 Its biocompatibility, physicochemical properties, and biological effects make HA also attractive for applications in biomaterials11−13 and bionanotechnology.14,15 Applications as drug delivery carriers are particularly interesting,16−19 because HA is biodegradable and can target cell surface receptors such as CD44 (that is overexpressed in selected cell types including tumor cells) while simultaneously carrying therapeutic molecules. The preparation of HA-containing nanosized carriers has been achieved, for example, through cross-linking of chemically modified HA20,21 or complexation of HA with other polymers.22−24 The latter strategy has been mainly used for embedding drugs by hydrophobic interactions upon complex formation. Complexes with covalently linked inorganic nanoparticles (NPs) have also been reported.25,26 From a physicochemical perspective, HA has several salient features. First, HA presents one carboxylate group per disaccharide, or per nm along the chain contour,27 and thus is highly charged around physiological pH.28 Second, HA is
The polysaccharide hyaluronan (HA) is ubiquitous in the extracellular space of vertebrates, and is widely used for medical, pharmaceutical, and cosmetic applications. It is a structurally simple polymer, composed of linearly repeating disaccharide units of glucuronic acid and N-acetylglucosamine that are linked via alternating β-1,4 and β-1,3 glycosidic bonds. The number of disaccharide units per chain can extend to as many as 10 000, which corresponds to a contour length of as high as 10 μm. HA-rich matrices are of prime importance in a wide range of physiological and pathological processes including immune regulation,1 inflammation,2 fertilization,3 tumor development,4 osteoarthritis, and atherosclerosis.5 Apart from being bioactive itself, HA is now also recognized as a “pericellular cue”;4 that is, it serves as a versatile scaffold within which other molecules are organized and regulated. A variety of proteins and proteoglycans, called hyaladherins, can bind to HA. They confer different conformational states to HA,6 mediate its cross-linking and/or attachment to the cell surface,7,8 and thereby engender selfassembly into large and typically strongly hydrated supramolecular architectures.9,10 Due to its large size, HA chains can interact with many binding partners simultaneously, and the dynamic remodeling of HA-rich matrices as a function of the © 2015 American Chemical Society
Received: April 24, 2015 Revised: July 3, 2015 Published: July 5, 2015 8411
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how imaging by transmission electron microscopy (TEM) and atomic force microscopy (AFM) can be combined to investigate the morphology of the complexes that HA free in the solution phase forms with TMA-NPs. Measurements with free HA provide insight into the complexes that are formed with individual HA molecules and are of particular relevance in the context of nanomaterials. We show that the multivalent interactions between HA and cationic NPs give rise to a variety of complexes with distinct morphologies, and that the ratio of the ionizable groups on HA and NPs is an important determinant of the type of morphology formed.
intrinsically hydrophilic, meaning that the polymer remains soluble in aqueous even when the charges are screened.29−31 Third, HA is semiflexible in the sense that its persistence length (a measure for the chain stiffness) is larger than the size of the chemical monomer (a disaccharide; 1 nm in length) from which the HA chain is made. This property arises from hydrogen bonds between adjacent monosaccharides that stiffen the polymer chain locally. 27 Specifically, the intrinsic persistence length of HA has been found to lie between 4 and 8 nm,31−35 and is comparable to the dimensions of many globular proteins. In contrast, most other known hydrophilic, linear polymers are flexible, i.e. their persistence lengths are comparable to the size of the chemical monomer from which they are made and thus much smaller than that of HA. Thanks to the local stiffening, HA chains tend to occupy a large aqueous volume,29,36 and thus effectively hydrate, even though their capacity to associate water in the form of hydration shells is not better than, for example, that of other hydrophilic saccharides such as dextran.37 Thus, the combination of local chain stiffness with a high charge content and an intrinsic hydrophilicity make HA perhaps unique among natural and synthetic simple polymer strands.31 The polyanionic nature of HA enables complexation of unmodified HA with polycationic molecules or nanomaterials. This has been studied38 and exploited for materials design, for example, in the assembly of polyelectrolyte multilayers,17,39−41 and to provide enhanced functionality to cationic NPs.42−44 Depending on the size and nature of the NPs and HA, complexes of various sizes were observed when mixing HA with NPs in solution. For example, the reversible formation of large aggregates of plasmonically active gold NPs (46 nm diameter) capped with cetyltrimethylammonium bromide in the presence of HA was employed to design a simple colorimetric assay to detect hyaluronidase (a bladder cancer biomarker) in urine.42 In another example, cationic iron oxide NPs smaller than 40 nm formed clusters of 80−160 nm in diameter with HA, and were successfully tested as potential carriers for peptides into cells using HA as a targeting molecule.43 Bhang et al. used complexes of HA with positively charged quantum dots (6 nm) to selectively target cells for in vivo imaging: the cluster size in this case could be tuned between 50 and 120 nm by adjusting the mixing ratio of quantum dots and HA, and precipitation occurred at elevated quantum dot loads.44 The physicochemical parameters that determine the size and morphology of HA/NP complexes have to our knowledge not been investigated. For the rational design of HA-containing materials toward biomaterials and bionanotechnological applications, but also in view of HA’s biological function as a pericellular cue, it is desirable to better understand how the physicochemical properties of HA determine the “packaging” of this large macromolecule by (typically much smaller) binding partners. In the present study, we focus on the interaction with spherical nanoparticles presenting a high density of trimethylammonium on their surface (TMA-NPs). These cationic nanoparticles are chosen as a simplified yet well-defined model for other charged nanomaterials, with a size comparable to the persistence length of HA and the dimension of many globular proteins. On the one hand, we exploit quartz crystal microbalance (QCM-D) to study the effect of cationic nanoparticles on films of HA that are end-grafted to a surface and form a so-called polyelectrolyte brush. These measurements inform about large scale effects that are of relevance for hydrated HA films and other HA-based matrices such as hydrogels. On the other hand, we demonstrate
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EXPERIMENTAL SECTION
Materials. A “working buffer” solution of 150 mM NaCl, 10 mM HEPES, and 2 mM CaCl2 at pH 7.4 was prepared in ultrapure water (resistivity 18.2 MΩ/cm). Lyophilized streptavidin (SAv; Sigma) was dissolved in ultrapure water at a stock concentration of 1 mg/mL, stored at −20 °C, and used within 1 week after thawing. Lyophilized HA, biotinylated at its reducing end and with a well-defined molecular mass of 58 ± 3 kDa (Select-HA B50; b-HA) as well as nonbiotinylated HA of 0.45 ± 0.03 MDa (Select-HA 500), 1.16 ± 0.06 MDa (SelectHA 1000) and 2.38 ± 0.13 MDa (Select-HA 2500) were purchased from Hyalose (Oklahoma City, OK). For reconstitution, HA was dissolved in ultrapure water at a stock concentration of 1 mg/mL and gently shaken overnight, stored at −20 °C, and used within 1 week after thawing. Small unilamellar vesicles containing dioleoylphosphatidylethanolamine-cap-biotin and dioleoylphosphatidylcholine (both from Avanti Polar Lipids, Alabaster, AL) at a molar ratio of 1:19 (bSUVs) were prepared by sonication as described in detail previously,45 stored at 4 °C, and used within 2 months. Preparation of HA Brushes. HA brushes were prepared, as described in detail previously,46 and demonstrated in Figure 2A. Briefly, supported lipid bilayers (SLBs) were formed from b-SUVs by the method of vesicle spreading on silica-coated QCM-D sensors,47 and coated with a dense monolayer of the biotin-binding protein streptavidin (SAv). b-HA was then grafted through biotin at the reducing end to the SAv surface. Synthesis of Cationic NPs. Trimethylammonium-coated gold NPs (TMA-NPs) were prepared by ligand exchange on octanethiolcoated gold NPs in dichloromethane following a procedure described by Rotello et al.48 The precursor octanethiol-coated gold NPs were prepared as described before (Supporting Information Figure S1).49 Trimethylammonium-terminated thiolated ligand was prepared from commercially available starting materials in two reaction steps following a reported procedure.48 After dialysis and lyophilization, TMA-NPs were suspended in ultrapure water (1 mg/mL), stored at 4 °C and used within 3 months. TMA-NP size and stability were characterized by transmission electron microscopy (TEM), and surface charge by ζ-potential measurements (Zetasizer Nano ZS; Malvern Instruments Ltd., Malvern, U.K.) in aqueous solution. Quartz Crystal Microbalance with Dissipation Monitoring. QCM-D measures the changes in resonance frequency, Δf, and dissipation, ΔD, of a sensor crystal upon molecular binding events on its surface. The QCM-D response is sensitive to the mass (including hydrodynamically coupled water) and the mechanical properties of the surface-bound layer. Measurements were performed with a Q-Sense E4 system equipped with Flow Modules (Biolin Scientific, Västra Frölunda, Sweden) on silica-coated QCM-D sensors (QSX303; Biolin Scientific) at a working temperature of 23 °C. The system was operated in flow mode with flow rates typically between 5 and 20 μL/ min. Δf and ΔD were measured at six overtones (n = 3, 5, ..., 13), corresponding to resonance frequencies of f n ≈ 15, 25, ..., 65 MHz; changes in dissipation and normalized frequency, Δf = Δf n/n, of the seventh overtone (n = 7) are presented. Any other overtone would have provided comparable information. Before use, the QCM-D sensors were immersed in an aqueous solution of 2% sodium dodecyl sulfate for 30 min, rinsed abundantly with ultrapure water, blow-dried with N2, and treated with UV/ozone (Bioforce Nanoscience, Ames, 8412
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Figure 1. Characterization of TMA-NPs. (A) Scheme of a TMA-NP. The gold core and a few pendant chains are shown (not to scale). (B) Transmission electron micrograph visualizing the gold cores as black dots; the inset shows a histogram of the gold core size with a Gaussian fit giving 2.3 ± 0.7 nm (mean ± SD). (C) Atomic force micrograph visualizing TMA-NPs on mica as protrusions of a few nm in height. The four horizontal lines indicate the location of height profiles, shown in the inset in matching colors.
Figure 2. Interaction of TMA-NPs with HA brushes. (A) QCM-D responses (frequency shifts, Δf, and dissipation shifts, ΔD) for the assembly of an HA brush (schematically shown in inset) and the interaction of TMA-NPs with the HA brush. Start and duration of incubation steps with different samples are indicated by arrows (numbers in brackets indicate final concentrations in μg/mL in working buffer); during all other times, the surface was exposed to plain working buffer. Changes in Δf and ΔD between 180 and 190 min do not only reflect changes on the surface but also result from a change in the viscosity and density of the surrounding solution50 owing to the presence of an elevated NaCl concentration. The curves in gray correspond to the binding of TMA-NPs to SAv in the absence of HA. (B) Parametric plots of ΔD/−Δf (a measure for film softness) vs −Δf (a measure for the amount of material in the film) for the formation of the HA film and the binding of TMA-NPs as shown in A. Three phases can be discerned: during the formation of the HA film (phase I) the softness remains essentially constant (phase I; plateau in ΔD/−Δf), addition of the TMA-NPs first induces a drastic rigidification (phase II; strong decrease in ΔD/−Δf) followed by a slight softening of the film (phase III, slight increase in ΔD/−Δf). The duration of these phases is also indicated in (A). (C) Scheme of morphological changes in the HA brush upon TMA-NP binding suggested based on QCM-D data. IA) for 30 min. For sufficiently rigid films, that is, when ΔD/−Δf ≪ 0.4 × 10−6/Hz,50 the areal mass density can be estimated with the Sauerbrey equation, that is, Δm = −CΔf, where C = 18.06 ng/cm2/Hz is the mass sensitivity constant for a sensor with a fundamental resonance frequency of 4.95 MHz. Transmission Electron Microscopy. TEM measurements were performed on a JEOL JEM 2100F microscope. A volume of 2 μL of aqueous solutions of TMA-NPs, or HA/TMA-NP complexes prepared in desired ratios, was placed on an ultrathin carbon film supported by a copper grid (Ted Pella, Inc., Redding, CA). Prior to use, the carbon surface was hydrophilized by plasma treatment. Samples were allowed to dry overnight under ambient conditions. TEM images were
analyzed with ImageJ software; histograms of NP sizes were generated with Origin software. Atomic Force Microscopy. Samples for AFM measurements were prepared from the same solutions as for TEM. TMA-NPs or HA/ TMA-NP complexes were used at the same concentrations as for TEM, except for molar mixing ratios of 1:100 and 1:1000, which were diluted 6-fold in ultrapure water. 7 μL of solutions were deposited on freshly cleaved mica substrates (V-1 grade mica sheet; SPI, West Chester, PA), and dried under ambient conditions for at least 24 h. AFM images were recorded with a Nanowizard II (JPK Instruments, Berlin, Germany). Intermittent contact mode images of various sizes (1−20 μm) were obtained under ambient conditions at scan rates 8413
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estimate the grafting density for the present HA film (Δf = −22 Hz) to be 1.0 pmol/cm2. This corresponds to a root-meansquare (rms) distance between anchor points of 13 nm. We also know from previous characterization of these HA brushes with reflection interference contrast microscopy that their thickness is around 100 nm.46,52 To a first approximation, the mean mesh size of this solvated HA film is expected to have the same magnitude as the rms anchor distance.53 The TMA-NPs are smaller and should thus readily penetrate into the brush. TMA-NPs induced a strong and continuous QCM-D response on HA brushes (Δf = −275 Hz), without any tendency of equilibration within 1 h of incubation (Figure 2A, colored curves, 110−170 min). As a control, we also tested the binding of TMA-NPs on SAv-coated SLBs (Figure 2A, gray curves, 110−170 min). The response in this case was pronounced (Δf = −90 Hz), although much smaller than in the presence of the HA film and rapidly saturating (i.e., within 10 min). The saturable binding kinetics and the magnitude of the QCM-D responses on the SAv-covered SLB are in reasonable agreement with expectations for the formation of a dense TMA-NP monolayer. For a close-packed monolayer of 4.7 nm diameter particles with a gold core of 2.3 nm, a frequency shift around −60 Hz would be expected using the Sauerbrey equation when accounting for the density of gold and the organic coating, and the contribution of interstitial solvent to the frequency response.50 The slightly stronger response observed here may be due to the presence of a subpopulation of larger particles that would make an overproportional contribution to the frequency shift.54 No further response was observed upon rinsing in working buffer, indicating that TMA-NPs bind strongly to SAv. We propose that attractive electrostatic interactions between SAv, which is negatively charged at pH 7.4,55 and the TMA-NPs are at least in part responsible for this effect. The much larger response on HA brushes suggests that TMA-NPs bind in large amounts, corresponding to multiple layers. Upon rinsing in buffer (Figure 2A; colored curves, 170− 180 min), the frequency increased (and the dissipation decreased), indicating release of some TMA-NPs. The QCMD response upon rinsing was relatively slow, however, and TMA-NPs could not be eluted in 1 M NaCl (Figure 2A; colored curves, 180−190 min), indicating that the TMA-NPs are rather strongly bound to the HA film. Detailed inspection of the dissipation response upon TMANP binding revealed a nonmonotonous response, indicating that the interaction proceeds in several phases that are distinct in how binding affects the mechanical properties of the composite HA/TMA-NP film. This can be appreciated in a parametric plot (Figure 2B), in which the ratio ΔD/-Δf for the HA film formation and TMA-NP binding is plotted as a function of −Δf. To a first approximation, the parameter ΔD/ −Δf provides a measure for the softness of the film,50 while −Δf is a measure for the amount of material in the film. One can see that ΔD/−Δf remains approximately constant and is relatively high (0.4 × 10−6/Hz) throughout the formation of the HA film itself (phase I). Exposure of TMA-NPs then first leads to a drastic decrease in ΔD/−Δf, indicating pronounced rigidification of the film (phase II), followed by a moderate increase, indicating some softening (phase III). Toward the end of phase II and throughout phase III, ΔD/ −Δf is much smaller than the reference value 0.4 × 10−6/Hz (Figure 2B), implying that the so-called Sauerbrey equation provides a good estimate of the film’s areal mass density.50
between 0.6 and 1.0 Hz with silicon cantilevers with 40 N/m nominal spring constant and tips with 8 nm nominal radius (TESP; Bruker AFM Probes). Images were second-order flattened to account for sample tilt, and further analyzed by data processing software (JPK Instruments or open source Gwyddion).
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RESULTS AND DISCUSSION Preparation and Characterization of TMA-NPs. Watersoluble cationic NPs (Figure 1A) were prepared by ligand exchange on octanethiol-coated gold NPs with a trimethylammonium thiol. TEM characterization (Figure 1B) revealed the particles to be stable over several months and the gold core to have a diameter of 2.3 ± 0.7 nm (Figure 1B, inset). Electrophoretic light scattering of 0.5 μM TMA-NPs in water at 25 °C revealed a positive ζ-potential (6.6 ± 0.3 mV), as expected. The TMA-NPs could be deposited well-dispersed on mica and repeatedly imaged by AFM without apparent perturbation (Figure 1C). AFM height profiles revealed a diameter of the dry particles of 4.7 ± 1.9 nm (95 particles were analyzed) (Figure 1C, inset), consistent with the size of the gold core plus an organic coating of 1 to 2 nm thickness. Considering the contour length of the TMA ligand (2.4 nm) the coating thickness appears reasonable, i.e. the ligands are unlikely to be completely stretched, at least in the dry state. The apparent width of the NPs in the AFM images is much larger (a few 10 nm), most likely due to tip convolution effects. Characterization of TMA-NP Interaction with HA Brushes by QCM-D. To study how cationic NPs interact with HA, we used HA brushes (Figure 2A, inset) as a welldefined model system of HA matrices. HA brushes were prepared by grafting of HA polymers with a biotin at the reducing end (b-HA) to a streptavidin (SAv)-coated biotinylated supported lipid bilayer (SLB). The molecular mass of bHA was chosen as 58 kDa. Based on the length (1.0 nm) and molecular mass (0.4 kDa) of an HA disaccharide,27 this corresponds to a contour length of 145 nm. The use of such a relatively short polymer in QCM-D measurements is advantageous, because the thickness of the HA brush remains confined to within the penetration depth of the acoustic shear wave used to sense the film properties, thus facilitating data analysis. At the same time, the HA brush thickness remains sufficiently large to study effects at a scale that goes well beyond the size of individual TMA-NPs. The buildup of the HA brushes, and the effect of TMA-NPs on the HA brushes was monitored by QCM-D (Figure 2B). The two-phase response in frequency and dissipation (Figure 2A, 10−20 min) upon incubation of a silica surface with small unilamellar vesicles containing 5% biotinylated lipids (b-SUVs), and the final shifts of Δf = −26 ± 1 Hz and ΔD < 0.5 × 10−6, are characteristic for the formation of an SLB of good quality.47 The subsequent responses for streptavidin (SAv), which was first incubated at 1 μg/mL for 10 min and then at 10 μg/mL for another 10 min (Figure 2B, 30− 50 min) were consistent with the formation of a dense protein monolayer.51 A strongly hydrated and soft HA brush formed through the grafting of b-HA via the reducing-end biotin (Figure 2B, colored curves, 68−98 min).46 Here, the softness of the HA film is evidenced by the strong increase in dissipation. It is not possible to quantify the grafting density of HA from the QCM-D response alone.50 In earlier work,52 however, we had coupled QCM-D with spectroscopic ellipsometry, an optical mass-sensitive method, to calibrate the QCM-D frequency shift upon end-grafting of 58 kDa HA against the HA surface density (see Figure S3C in ref 52). Based on these data, we can 8414
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Langmuir From Δf = −140 Hz measured at the minimum in ΔD/−Δf, we can thus estimate an adsorbed amount of 2.5 μg/cm2, which includes the solvent in the film. If the film had the density of water, ρ = 1.0 g/cm3, this would correspond to a film thickness of 25 nm. In reality, the density is higher due to the presence of the gold NPs, and the film thickness therefore even lower. Compared to the virgin HA brush (∼100 nm), the thickness is thus reduced by at least 4-fold, indicating that the HA brush compacts upon binding of TMA-NPs. A plausible explanation (Figure 2C) for the observed responses is that the NPs induce HA cross-linking through the interaction of the many positive charges on the NP with the negatively charged HA, accompanied by film rigidification and collapse (phase II), as long as the NP load is sufficiently low. As the NP load increases, the electrostatic repulsion between NPs becomes dominant, leading to the disruption of cross-links, softening and likely also swelling (phase III). Taken together, the QCM-D data revealed a strong binding of TMA-NPs to HA and nontrivial effects on the morphology and mechanical properties of HA films. Characterization of HA/TMA-NP Interaction in Solution Phase by TEM and AFM. We next asked if similar effects would also occur upon interaction of HA with TMANPs in the solution phase. For this purpose, the morphology of HA/TMA-NP complexes formed in solution was characterized by TEM and AFM. Samples consisted of aqueous solutions of mixtures of TMA-NP and HA (1.16 MDa) which were allowed to interact for at least 2 days without shaking or stirring. To 50 nM aqueous solutions of TMA-NPs, HA was added in proportions ranging from 50 to 0.05 nM to obtain solutions with molar HA/TMA-NP ratios of 1:1, 1:10, 1:100, and 1:1000. Based on the molecular mass of HA, we expect each HA chain to contain 2900 carboxyl groups. In comparison, gold NPs of 2 nm diameter are expected to accommodate around 200 ligands of TMA according to the calculations described by Murray.56 Hence, the number of charges per HA chain exceeds the charges on a single NP by far, and the ratios of ionizable groups for these HA/TMA-NP mixtures are around 1:0.07, 1:0.7, 1:7, and 1:70, respectively. The solutions were deposited on a copper-grid-supported ultrathin carbon film for TEM imaging (Figure 3), and in parallel on mica surfaces for AFM imaging (vide infra). The organization of TMA-NPs in the TEM images varied drastically as a function of the HA/TMA-NP mixing ratio. This provides evidence that TMA-NPs and HA form complexes of distinct morphologies depending on the mixing ratio, even if the HA molecules are not directly visible in the TEM images due to lack of contrast in the unstained sample. For the 1:1 molar mixing ratio (Figure 3A), particle clusters of heterogeneous shape and typically a few 10 nm in extension were observed. The 1:10 ratio (Figure 3B) provided clusters with a consistent circular morphology yet similar extension. At a ratio of 1:100 (Figure 3C), much larger and elongated aggregates formed, with a few 10 nm in width and several 100 nm and more in length. For the 1:1000 ratio (Figure 3D), the distribution of TMA-NPs was fairly even, not unlike the pure TMA-NP solution (Figure 1B), i.e. isolated clusters could not be clearly identified. The occurrence of stable clusters with well-defined shape at a molar HA/TMA-NP ratio of 1:10 was remarkable and merited further analysis. The smallest circular clusters measured around 25 nm in diameter, although bigger clusters were also found (Figure 4). Among 165 systematically analyzed clusters, 65%
Figure 3. Transmission electron micrographs of unstained HA(1.16 MDa)/TMA-NP complexes at molar mixing ratios of 1:1 (A), 1:10 (B), 1:100 (C), and 1:1000 (D), corresponding to ratios of ionizable groups of 1:0.07, 1:0.7, 1:7, and 1:70, respectively. Single TMA-NPs are visible as black dots. HA is not visible due to lack of contrast.
had a diameter between 25 and 75 nm. Clusters smaller than 25 nm were not found. None of the clusters was larger than 205 nm, indicating that smaller clusters form preferentially, and that they are temporally stable. The individual TMA-NPs within the clusters could readily be discerned on the TEM images, irrespective of cluster size, suggesting that the particles retain some distance to each other and that they are arranged in a plane along the carbon support. Most likely, the planar arrangement does not represent the native morphology of the HA/TMA-NP complex, but the consequence of the spreading of a soft, spherical complex into two dimensions upon adhesion to the surface and/or subsequent drying. A statistical analysis of the number NNP of TMA-NPs per cluster as a function of cluster diameter d reveals a power-law dependence d = 11.0 nm × NNP0.44 (Figure 4C). The power is close to 0.5, indicating that the average distance between neighboring NPs in the planar cluster is independent of cluster size, and we can estimate an rms distance of 10 nm. The number of TMA-NPs in the smallest analyzed clusters was 11.9 ± 1.4, where the averaging was performed over the 10% of analyzed clusters that featured the smallest NNP. This is close to the stoichiometry of 10 TMA-NPs per HA chain in the mix, suggesting that these clusters contain a single HA chain. Interestingly, the ratio of ionizable groups on HA and TMANPs in this mixture is 1:0.7. The cationic NPs in the smallest clusters would thus just about balance the negative charges of an HA chain. We hypothesized that it is this balance that leads to the formation of dense and stable complexes. To test this, we prepared HA/TMA-NP mixtures using HA polymers of different sizes (0.45, 1.16, and 2.38 MDa) in which the ratio of ionizable groups was maintained at 1:0.7 and the NP concentration was unchanged. As expected, stable complexes were found for all HA sizes (Figure 5A). Importantly, the number of TMA-NPs in the smallest observable clusters was 8415
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Figure 4. Statistical analysis of clusters obtained at 1:10 HA/TMA-NP ratios. (A) Examples of aggregates of different sizes imaged by TEM. (B) Histogram of aggregate diameters (79 micrographs containing a total of 165 aggregates were systematically analyzed). (C) Relation between the aggregate diameter d and the number NNP of TMA-NPs per aggregate (log−log scale). The line is the best fit with a power law, i.e., d = 11.0 nm × NNP0.44.
cases, and that isolated HA chains are not sufficiently well attached to the mica support to allow their imaging with the applied AFM conditions. The arrangement of the TMA-NPs, the larger area imaged and the reduced surface coverage compared to TEM, however, revealed a clearer picture of the complexes obtained at 1:100 (Figure 6E,F) and 1:1000 (Figure 6G,H) mixing ratios. It is notable that a few compact complexes (Figure 6E, blue arrow) coexist with large, extended suprastructures (Figure 6E, yellow arrow). The extended suprastructures, discerned by TEM only for 1:100 ratios (Figure 3C), apparently persist for the 1:1000 ratio. The AFM images suggest that TMA-NPs arrange with HA into extended linear fibers. This can be appreciated particularly well in Figure 7, which presents images of a larger surface area. The length of the fibers can exceed the contour length of individual HA chains (2.9 μm) by far, indicating that they comprise several concatenated and perhaps also bundled HA chains. It is also notable that the fibers can branch (Figure 7D, white arrows). It has previously been reported that the deposition and drying process can significantly affect the morphology of HA polymers (see ref 57 and references therein), and one may ask if the extended superstructures seen by TEM (Figure 3C) and AFM (Figures 6E−H and 7) adequately reflect the morphologies of the TMA-NP/HA complexes in the solution phase. Inspection of several distinct locations on the carboncoated grids (by TEM) and on the mica substrates (by AFM) revealed these superstructures to be homogeneously distributed across the surface. This indicates that they were stably immobilized and not displaced by the receding edge of the drying water droplet. It is very likely that the stable attachment is aided by electrostatic interactions of the negatively charged surfaces with residual charges on the TMA-NPs, because TMANPs bind stably (Figure 1B,C) whereas HA does not under the employed incubation conditions.57 This suggests that the linear
found to scale with HA molecular mass (Figure 5B). The slope was 10.8 ± 0.2 NPs per MDa of HA, corresponding to a charge ratio of 1:0.9 which is close to the overall charge ratio of the mix. This provides a strong indication that the smallest clusters contain a single HA chain, irrespective of the HA size. It also demonstrates how the size of HA/TMA-NP complexes can be tuned simply by adjusting the HA molecular mass. HA(1.16 MDa)/TMA-NP complexes deposited on mica were also systemically characterized by AFM, in parallel to TEM. AFM (Figure 6) and TEM (Figure 3) data correlate well in the sense that the same types of aggregates are observed as a function of the mixing ratio. For example, AFM imaging of the HA/TMA-NP complexes at a molar mixing ratio of 1:1 (Figure 6A,B) revealed compact complexes (Figure 6A, white arrows), as well as apparently isolated TMA-NPs. Moreover, at a 1:10 ratio (Figure 6C,D), compact circular morphologies of various diameters dominated, and large, elongated aggregates are again observed for the 1:100 ratio. The surface topography of all aggregates was irregular. Globular protrusions could frequently be discerned within them, and their height of a few nanometers (Figure S2) indicates that these represent individual NPs (cf. Figure 1C). Apparently, the TMA-NPs in the HA/TMA-NP complexes arrange into planar monolayers on mica as well as on the carbon-coated TEM grids. The AFM images though provided additional information about the morphology of the HA/TMA-NP complexes. Detailed inspection of Figure 6D reveals a corona of 0.6 nm thickness surrounding the compact TMA-NP clusters. This corona was reproducibly observed for the 1:10 mixing ratio. Its height and extension suggest that it represents an essentially compact monolayer of HA57 where the AFM resolution limits preclude identification of individual chains. The presence of HA could not be directly revealed for the other mixing ratios. This suggests that HA does not arrange into a dense layer in these 8416
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Figure 7. Large AFM scans of HA(1.16 MDa)/TMA-NP complexes on mica surface for molar mixing ratios of 1:100 (A-B) and 1:1000 (C,D). Panels (B) and (D) show magnified images of the regions surrounded by black dashed squares in (A) and (C), respectively. White arrows in (D) indicate branch points in the extended suprastructures.
extension of the fibers over tens of micrometers is not an artifact of the sample preparation procedure. We can, however, not exclude that hydration-induced bundling of HA might occur at lengths similar to or smaller than the distance between TMA-NPs, and the fine structure of the fibers revealed by AFM at a length scale of 10 nm should thus be interpreted with caution. Comparing the data by QCM-D on TMA-NP binding to HA brushes, and the data by TEM and AFM on HA/TMA-NP complexes formed in solution reveals striking similarities. Small amounts of TMA-NP have a strong effect and induce HA to
Figure 5. Smallest clusters obtained as a function of HA size. HA of different molecular masses were mixed with TMA-NPs; the ratio of ionizable groups on HA and TMA-NP was fixed to 1:0.7. (A) Examples of smallest clusters imaged by TEM, with HA size as indicated. (B) Number of TMA-NPs per cluster, NNP, for the smallest observable clusters as a function of HA molecular mass. To obtain the data points, between 65 and 165 clusters were systematically analyzed for the different HA molecular masses; mean and standard deviations were taken over the 10% of the clusters with the smallest NNP. The dotted line is a linear fit through the origin and gives a slope of 10.8 ± 0.2 NPs per MDa of HA, corresponding to a ratio of ionizable groups on HA and TMA-NPs of 1:0.9.
Figure 6. AFM topographic images of HA(1.16 MDa)/TMA-NP complexes deposited on mica. The molar mixing ratios were (A,B) 1:1, (C,D) 1:10, (E,F) 1:100, and (G,H) 1:1000. Panels (B), (D), (F), and (H) show magnifications of the regions surrounded by black dashed squares in (A), (C), (E), and (G), respectively, to reveal more details about the complexes. White arrows in (A) indicate compact complexes. The height profile along the black line in (D) is shown as an inset, and reveals a corona of 0.6 nm thickness and several 10 nm width around the NP assembly (delimited by pink arrows on the right of the complex). Blue and a yellow arrows in (E) indicate a compact complex and an extended suprastructure, respectively. 8417
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of HA chains, both in solution and when assembled into a brush on surfaces. Smaller amounts of NPs (i.e., below ratios leading to a balance of ionizable groups on HA and NPs) lead to the collapse of HA brushes and the formation of compact aggregates in solution. When ionizable groups are balanced, well-defined spherical complexes form that are soft, solvated and stable. Larger amounts of NPs produce a softening of HA brushes and lead to the formation of extended fiberlike complexes with HA in solution. The study thus provided insight into the reorganization of HA matrices by multiply charged NPs, and routes for the preparation of well-defined HA complexes, with potential applications in materials science and nanomedicine.
organize in a nonlinear way, leading to the collapse of HA brushes (observed by QCM-D, Figure 2) and to the formation of compact aggregates in solution (observed by TEM, Figure 3A,B, and by AFM, Figure 6A−D). Large amounts of TMANPs with respect to HA, on the other hand, result in elongated morphologies in solution (observed by TEM, Figure 3C, and by AFM, Figures 6E−H and 7), consistent with the partial softening of the HA brushes observed by QCM-D in the presence of excess TMA-NP (Figure 2). We tentatively explain the morphological diversity by the potential for multivalent interactions between TMA-NPs and HA and the polyelectrolyte nature of the interaction partners. HA polymers alone adopt an extended conformation due to the repulsion of charges along the chain and a relatively high intrinsic chain stiffness.31 TMA-NPs can engage in multiple simultaneous interactions with HA, intra- and/or interchain, and thus act as cross-linkers when HA is in excess. This and the accompanying entropically favorable release of counterions (or effectively, the reduced electrostatic repulsion) in the complex promote the gradual compaction of HA chains as the relative amount of TMA-NP increases, until the amounts of ionizable groups on HA and TMA-NPs are approximately balanced. Under this condition, the most compact (spherical) complexes form. Excess of TMA-NPs over HA (in terms of ionizable groups) on the other hand promotes swelling, due to the effective electrostatic repulsion between the cationic NPs. In this regime, each HA chain likely interconnects many NPs, and the electrostatic repulsion between NPs, likely aided by the relatively high intrinsic chain stiffness of HA (the intrinsic persistence length is 4 to 8 nm,31,35 i.e. similar to or larger than the NP size), promotes the formation of elongated assemblies. Under these conditions, multiple HA chains can apparently also interconnect and form fiberlike assemblies of many micrometers in length (Figure 7). Although the conditions of preparation are distinct, we notice a remote resemblance with so-called “HA cables”, that is, monocyte-adhesive HA structures that are formed by cells under certain pathological conditions.58−60 Perhaps the most notable observation is the formation of soft, spherical complexes when ionizable groups on HA and TMA-NPs are balanced. The TEM data (Figures 4 and 5) indicate that the cationic NPs are well dispersed within the complex. Moreover, they strongly suggest that stable complexes containing a single HA chain can be formed, and thus that the volume of these complexes scales with the size of HA. Adjusting the size of HA thus provides a simple and direct measure to tune the size of the complexes. When adsorbed to a surface, the interparticle distance within the complex is 10 nm, that is, much larger than the particle size. This suggests that solvent makes up a large volume fraction of the complexes, and it is likely that this solvation aids in the stabilization of the complexes. This raises the interesting perspective that complexes of defined and tunable size can be formed by mixing of cationic NPs and HA at a defined ratio.
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ASSOCIATED CONTENT
S Supporting Information *
Details of TMA-NP preparation (Figure S1) and AFM height profiles of TMA-NPs and TMA-NP/HA complexes (Figure S2). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01505.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge fruitful discussions with Soledad Penadés (CIC biomaGUNE and CIBER-BNN) on the design and analysis of experiments, and the manuscript. This work was supported by the Spanish Ministry of Economy and Competitiveness Plan Nacional (MAT2011-24306) to R.P.R. M.C. acknowledges a Research Fellow Grant from Ikerbasque, Basque Foundation for Science.
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ABBREVIATIONS HA, hyaluronan; b-HA, HA with reducing-end biotin; SAv, streptavidin; SLB, supported lipid bilayer; SUV, small unilamellar vesicle; b-SUV, SUV containing biotinylated lipids; NP, nanoparticle; TMA, trimethylammonium; QCM-D, quartz crystal microbalance with dissipation monitoring; TEM, transmission electron microscopy; AFM, atomic force microscopy
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REFERENCES
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CONCLUSIONS We demonstrate that HA can establish interactions with cationic nanoparticles that lead to complexes of distinct morphologies, and illustrate the versatile use of TEM, AFM and QCM-D for the characterization of such complexes. Based on the characterization of HA films by QCM-D, and of HA/ TMA-NP complexes formed in solution by TEM and AFM, we conclude that cationic NPs profoundly affect the morphology 8418
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