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. Author manuscript; available in PMC: 2019 Apr 15.
Published in final edited form as: Carbohydr Polym. 2018 Feb 2;186:243–251. doi: 10.1016/j.carbpol.2018.01.046

Hyaluronate Coating Enhances the Delivery and Biocompatibility of Gold Nanoparticles

Bedia Begüm Karakocak 1, Jue Liang 2,3, Pratim Biswas 1, Nathan Ravi 1,2,3,*
PMCID: PMC5821145  NIHMSID: NIHMS937047  PMID: 29455984

Abstract

For targeted delivery with nanoparticles (NPs) as drug carriers, it is imperative that the NPs are internalized into the targeted cell. Surface properties of NPs influence their interactions with cells. We examined the responses of retinal pigment epithelial cells, NIH 3T3 fibroblast cells, and Chinese hamster ovary cells to gold nanoparticles (Au NPs) in their nascent form as well as coated with end-thiolated hyaluronate (HS-HA). The grafting density of HS-HA on Au NPs was calculated based on total organic carbon measurements and thermal gravimetric analysis. We imaged the intracellular NPs by 3D confocal microscopy. We quantified viability and generation of reactive oxygen species (ROS) of the cells to Au NPs and monitored cell-surface attachment via electrical cell-substrate impedance sensing. The results confirmed that receptors on cell surfaces, for HA, are critical in internalizing HS-HA-Au NPs, and HA may mitigate ROS pathways known to lead to cell death. The 50- and 100-nm HS-HA-Au NPs were able to enter the cells; however, their nascent forms could not. This study shows that the delivery of larger Au NPs is enhanced with HS-HA coating and illustrates the potential of HA-coated NPs as a drug delivery agent for inflamed, proliferating, and cancer cells that express CD44 receptors.

Keywords: Thiolated-hyaluronate, gold nanoparticles, internalization, CD44, 3D confocal

1. Introduction

Gold nanoparticles (Au NPs) have been widely exploredin medicine as potential delivery agents for various biopharmaceuticals(Kim et al. 2011; Kumar et al. 2015). Although Au NPs are usually more biocompatible than other metal or metal oxide nanoparticles, their inherent positively charged surface disrupts the negatively charged cell membrane, while their production of reactive oxygen species (ROS) causes cytotoxicity (Moghadam et al. 2012). Moreover, plasma proteins can spontaneously adsorb to nascent Au NPs, affecting the surface properties of the particles and their interaction with cells(Nel et al. 2009). As a result, surface modification is essential ideally to prevent protein adsorption(Larson et al. 2012), and nonspecific delivery of Au NPs(Rana et al. 2012), to decrease opsonization by the immune system(Papasani et al. 2012), and finally mitigate their toxicities.

Many coating conjugates, ranging from synthetic ligands to natural biomolecules, have been used to improve the stability of particles and their delivery to specific cells or tissues (Lin et al. 2017; Yilmaz et al. 2016). Our focus is on hyaluronic acid (HA) as a coating material. HA is a bioactive linear polysaccharide that prevents adsorption of proteins on surfaces of biomaterials(Hans and Lowman 2002) and has an antifouling effect that arises from its hydrophilic and polyanionic characteristics(Lee et al. 2008; Santhanam et al. 2015). HA also scavenges free radicals and chelates pro-oxidant metals(Glucksam-Galnoy et al. 2012). HA serves as a ligand for several cell-surface receptors and thereby has an important physiological role. Cluster of differentiation 44 (CD44) is the most well-studied cell membrane receptor(Jaggupilli and Elkord 2012). It is present on, for example, the lymphatic vessel endothelial HA receptor (LYVE-1) (Chen et al. 2005), the receptor for hyaluronate-mediated motility (RHAMM)(Nedvetzki et al. 2005), and the HA receptor for endocytosis (HARE) (Pandey and Weigel 2014). The type of HA receptor on the cell membrane depends on the function of the tissue.

The role of HA receptors in healthy and diseased cells is not well understood. CD44 had been identified during the development, differentiation, and proliferation of cells, for example, the neural retina and in Muller cells (Chaitin and Davis 1995). CD44 has also been found under pathologic conditions, such as inflammation and proliferative vitreoretinopathy, where retinal pigment epithelial( RPE) cells actively proliferate in the subretinal space, on the surface and undersurface of the retina, and in the vitreous cavity. Hence, RPE cells exhibit significantly higher CD44 receptors compared to their quiescent and stationary state (Moysidis et al. 2012). Furthermore, it has been reported that cancer cells also express CD44 receptors and have a significant regulatory role in almost all cancer types (Kesharwani et al. 2015; Wang et al. 2016). It is now well recognized that a threshold of CD44 expression is required before HA binding is observed (Perschl et al. 1995; Tzircotis et al. 2005). Therefore, cells like NIH 3T3, which express relatively less number of CD44 receptors, may lack HA binding while cells like Chinese hamster ovary (CHO) cells with high CD44 receptor density show strong HA binding (Katoh et al. 1995; Tzircotis et al. 2005).

In our previous study, we have found that for a fixed total surface area, Au NPs (particle diameter, dp < 50 nm) were toxic to retinal pigment epithelial cells, independent from the particle size(Karakocak et al. 2016). The toxicity of Au NPs arises from reactive oxygen species (ROS) activity, which was shown to be correlated to the available total surface area (Jiang et al. 2008; Pan et al. 2007). HA is a known free radical quencher; it is natural and consequently biocompatible(Balogh et al. 2003). Our objective in the current work is to expand the usefulness of Au NPs by mitigating their toxicity via coating them with HA. As expected, we observed that HA coating significantly enhanced the biocompatibility of Au NPs; unexpectedly, the coating also greatly improved the internalization of larger Au NPs, which in their nascent form could not enter the cell. The outcomes of this study could be valuable in treating inflamed, proliferating, or cancer cells that express CD44 receptors.

2. Materials and Methods

2.1. Reagents

1-Ethyl-3-[3-(dimethylamino) propylcarbodiimide (EDC), sodium citrate, hydrogen tetrachloroaurate (HAuCl4), cystamine dihydrochloride, sodium cyanoborohydride (NaBH3CN), Dulbecco’s modified Eagles’s medium/nutrient mixture F-12 Ham (DMEM/F12), trypsin-ethylenediaminetetraacetic acid (EDTA) solution 10x, and fetal calf serum (FCS), and DAPI for nucleic acid staining were obtained from Sigma-Aldrich (St. Louis, MO). ARPE-19 (Retinal pigment epithelial)(ATCCR® CRL-2302™), NIH 3T3 (ATCCR® CRL-1658 ™) and CHO(CCL-61™) cells were purchased from American Type Culture Collection (Manassas, VA). Dithiothreitol (DTT) was obtained from Duchefa Biochemie (Haarlem, Netherlands). HiLyte-Fluor™ 647 amine was purchased from AnaSpec (San Jose, CA). Sodium hyaluronate having a molecular weight of 10,000 Da was obtained from Lifecore (Chaska, MN). The Apo Tox-Glo™ and ROS -Glo™ H2O2 assay kits were purchased from Promega Biosciences (San Luis Obispo, CA). All chemicals and reagents were of analytical grade.

2.2. Synthesis of Au NPs

We synthesized Au NPs by the citrate reduction method(Frens 1973; Kimling et al. 2006; Tyagi et al. 2016). HAuCl4(10 mg) was dissolved in 90 ml of deionized (DI) water, and this solution was heated to boiling. We added sodium citrate solution (250 mM) to the boiling solution in amounts ranging from 400 μl to 900 μl and stirred for 20–30 min until the solution became wine-red. The solution was then left undisturbed in the dark for 24 h at room temperature. Subsequently, Au NP solutions were centrifuged at 10,000 rpm for 20 min and suspended in DI water. We confirmed the final size using transmission electron microscopy (TEM) analysis and dynamic light scattering (DLS)measurements. The detailed procedure for particle size measurements is given as supporting information.

2.3. Preparation of end-thiol modified HA (HS-HA)

We prepared HS-HA by reductive amination as described elsewhere (Kumar et al. 2015; Lee et al. 2012). HA (MW: 10,000 Da, 100 mg) and cystamine dichloride (60 mg) were dissolved in 0.1 M borate buffer (10 ml, pH 8.5) with 0.4 M NaCl and stirred for 2 h. NaBH3CN was added to the solution at a final concentration of 0.2 M and reacted at 50°C for three days. The reaction mixture was then incubated with 0.1 M DTT for 12 h to introduce a free thiol group (Fig. 1). The resulting solution was dialyzed against a large excess of 0.1 M NaCl solution for one day, 25% ethanol for one day, and pure water for one day to remove unreacted chemicals. The efficiency of thiol-end functionalization of HA was higher than 90%, and the ratio of [SH] to [HA repeating units] was 0.025 as determined by Ellman’s assay. Physicochemical properties of the HS-HA were obtained using Proton Nuclear Magnetic Resonance (1H-NMR), Fourier Transform Infrared (FTIR) spectroscopy, and Gel Permeation Chromatography (GPC) analysis. Please refer to supporting information for details of Ellman’s assay, 1H-NMR, FTIR, and GPC analysis.

Fig. 1.

Fig. 1

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HS-HA synthesis chemistry. In the presence of borate buffer at pH9, the terminal cyclic hemiacetal ring opens and changes to a linear aldehyde form. A Schiff base forms with cystamine. Thus, the terminal saccharide reverses between two states to reach the final form. The Amadori compound, sodium cyanoborohydride (NaBH3CN), is added, and then Dithiothreitol (DTT) is added to reduce the disulfide and produce HS -HAs.

2.4. Conjugation of HS-HA with Au NPs and quantification of the coating

Before initiating the conjugation process, we calculated the average number of HA molecules (MW: 19.69 k Da, 5.42 nm hydrodynamic radius (Rh))that can bind to one Au NP (particle size based on the TEM and DLS measurements). From that, we calculated the approximate HA to Au NP conjugation mass ratio and ensured that we used at least 1.5 x in excess of this amount. HS-HA was added dropwise to the dispersed Au NPs in DI water at room temperature with moderate stirring. The solution was stirred overnight. The HA -Au NP conjugate was further purified by centrifuging it twice at 10,000 rpm for 20 min to remove unbounded HA molecules and was finally redispersed in DI water. To take further measurements in the solid phase, the coated nanoparticle solution was lyophilized for two days at −105 °C. We used total organic carbon (TOC) and thermal gravimetric analysis (TGA) to determine the amount of coating around the nanoparticles. Please refer to supporting information for details of TOC and TGA analysis.

The number of bound HA molecules per unit surface area, grafting density (σ) was calculated based on both TOC (σTOC) and TGA (σTGA) measurements as described previously (Benoit et al. 2012). In summary, first, the number of Au NPs in a sample was calculated based on the hydrodynamic diameter of the Au NP and the density of Au. Next, the total available surface area of Au NPs in a sample for HA molecules to bind was determined. The number of HA molecules present in a sample was calculated taking the molecular weight of HA which is determined by GPC analysis. Finally, grafting density was calculated by dividing the number of HA molecules in a sample to the available surface area of Au NPs.

2.5. Investigation of the internalization of NPs

The cellular internalization of coated and nascent Au NPs was determined by exposing NPs to ARPE-19, NIH 3T3, and CHO cell lines. This was followed by careful washing to remove NPs that may have reached and been adsorbed on the cell surface (Cho et al. 2011). The intracellular concentrations of Au were quantified with an ELAN DRC II Inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer, Inc., USA). To identify the difference between the administered and delivered doses, the number of nanoparticles that failed to enter the cell, as well as the specific Au NP intracellular concentrations, were quantified in aliquots from the phosphate buffer saline (PBS) washes done before confocal imaging. Using ICP-MS, the aliquots were tested for the presence of Au NPs.

2.6. In-vitro cytotoxicity measurements on ARPE-19 cells with MTT, ApoTox-Glo™, and ROS-Glo™ H2O2 assays

Cell metabolic activity, and hence the viability of retinal cells in the presence of Au NPs, was assessed with MTT(3 -[4, 5 dimethyl-thiazoly-2-yl] 2–5 diphenyl tetrazolium bromide). The relative amount of apoptotic cells and ROS generation were measured, respectively, by ApoTox-Glo™ and ROS Glo™ H2O2 assays. See the supporting information for the details.

2.7. Confocal microscopy

To confirm the presence of NPs inside cells, we investigated the distribution of Au NPs at their critical mass concentrations (LD50, M), determined previously (Karakocak et al. 2016), using a nanoplasmonic confocal laser scanning microscope (Leica TCS-SP8). Au NPs have a surface plasmon resonance property (SPR). For Au NPs in size range of 5–100 nm, the SPR wavelength is around 520 nm (Huang et al. 2008), depending on the sizes of the nanoparticles. See the supporting information for the details.

2.8. Cell attachment measurements by electrical impedance spectrometry (ECIS)

We analyzed the biocompatibility of Au NPs as they were exposed to ARPE -19 cells using ECIS, a noninvasive technique that measures the impedance across gold electrodes at the bottom of tissue culture wells, using a range of frequencies of alternating current (Arndt et al. 2004; Wegener et al. 2000). As adhesion proteins changed the cell morphology and attachment to the electrodes in the bottom of wells, the resistance across the electrodes also changed(Borradori and Sonnenberg 1999; Santhanam et al. 2016). The change in resistance at frequencies ranging from 400 to 64,000 Hz was measured over time. Low-frequency impedance can be used to monitor the solution paths around the cells, and hence the layer’s cell-to-cell barrier functions(Wegener et al. 2000). The addition of Au NPs complicates the impedance of the system. However, at a frequency of 4,000 Hz, the contribution to resistance from cells was dominant over the contribution to resistance from Au NPs with the medium (Kandasamy et al. 2010). Therefore, we chose a frequency of 4,000 Hz to monitor cell growth and biocompatibility.

2.9. Statistical Analysis

Results are presented as means ± standard deviation on the graphs for experiments done at least with three biological replicates per condition or group. First, each test result (viability measurements, the relative amount of apoptotic cells, and ROS generation) was compared with the corresponding negative (untreated cells) and positive(NPs without the cells) control groups with Student’s t-test. P* < 0.05, the significance level, was statistically acceptable.

Second, Two-Factor ANOVA (analysis of variance) was used to evaluate the results of viability measurements, the relative number of apoptotic cells, and ROS generation.

For cell attachment measurements, eight replicates were performed for each independent experiment. Statistical significance was evaluated using ANOVA to compare the results with the respective negative (untreated cells) and positive control (only cell media) groups. P* < 0.05, the significance level, was statistically acceptable.

3. Results and discussion

3.1. Synthesis of end-thiolated hyaluronic acid (HS-HA)

HS-HA was synthesized by a previously reported method(Lee et al. 2012), outlined in Fig. 1. HA has multiple hydroxyl and carboxyl groups, but only the end six-member ring can open up and form an aldehyde group.

We took advantage of this lone functional group, modifying it with an amine to introduce an end thiol group and covalently binding it to Au (Fig. 2). Ellman’s assay results confirmed ~90% of thiolation of HA, and the molar ratio of SH to HA was found to be ~0.025. However, 1H-NMR and FTIR analysis could not clearly detect the end-thiol content of hyaluronate as [SH]/[HA repeating units ] is low (+Fig. S1 and Fig. S2). On the other hand, based on gel permeation chromatography (GPC) analysis, we determined that the hydrodynamic radius of HS-HA was 5.42 nm. Its number average molecular weight was 15.51 kDa, its polydispersity 1.27, and its intrinsic viscosity was 0.58 dl/g(Table 1). The Mark-Houwink constant changes linearly with the logarithm of MW of HA which can correspond to a change in conformation of HA from a random coil to a rigid structure (La Gatta et al. 2010). The Mark-Houwink constant, based on GPC analysis of the HS-HA, was 0.70, suggesting the presence of a random coil.

Fig. 2.

Fig. 2

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Illustration of coated Au NPs with end-thiolated HA (HS-HA), where the thiol group at the end of each HA chain is attracted to the Au NP surface.

Table 1.

Gel permeation chromatography results for HS-HA

Parameter Value
Number average molecular weight (Mn) 15.51 kDa
Weight average molecular weight (Mw) 19.69 kDa
Polydispersity 1.27
Hydrodynamic radius 5.42 nm
Intrinsic viscosity 0.58 dl/g
Mark-Houwink constant 0.70
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3.2. Synthesis of Au NPs conjugated with HS-HA and confirmation of the presence of the coating

We synthesized Au NPs with sizes of 5-, 10-, 20-, 50-, and 100-nm, the sizes most preferred for targeted drug delivery studies(Karthikeyan et al. 2010; Ngwa et al. 2012). The high-resolution TEM (HR-TEM) images of the uncoated Au NPs can ben found in supporting information (Fig. S3). The Au NPs were conjugated with HS-HA (see Methods). The amount of coating on Au NPs was determined with TOC analysis and TGA. These two approaches are well suited to probe the content of inorganic and organic compounds on nanoparticle surfaces(Benoit et al. 2012). The presence of the coating was first confirmed and quantified by TOC measurements by determining the extent of organic and inorganic carbon. Because inorganic carbon was negligible, the total carbon content was equivalent to the organic carbon content which was attributed to HA coating. To further confirm and quantify the presence of the coating, we used TGA which records the change in mass as a function of temperature. The peak at ~280 °C corresponded to the decomposition of HA (Fig. 3). The exact quantity of the coating was determined by calculating the change in mass around the temperature at which HA is known to decompose (Ahire et al. 2016).

Fig. 3.

Fig. 3

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Thermal gravimetric analysis plot for the HS-HA coated Au NPs. HA decomposition occurs at around 280 °C, and Au NPs melt approximately at 500 °C. Au NP grafting density (the number of HA molecules/nm2) determined by TOC and TGA measurements. Errors provided reflect standard deviation of the replicate error in repeated measurements of the sample. At least two measurements were taken for each analysis technique.

Inline graphic 5 nm, Inline graphic 10 nm, Inline graphic 20 nm, Inline graphic 50 nm, Inline graphic 100 nm

As detailed in a previous study(Benoit et al. 2012), we calculated the grafting density of HA molecules, the number of bound HA per unit surface area (# HA molecules/nm2) based on results of both TOC (σTOC) and TGA (σTGA) measurements(Fig. 3 and Table S1). The results showed that, for both measurement techniques, the number of HA molecules adsorbed per unit surface area of Au NP decreases as the particle size increases. This trend may be attributed to the HA-HA interactions which could be geometrically minimized due to the increased curvature of the smaller particles (Nasir et al. 2015). One should note that these calculations were based on two main assumptions: Au surface is smooth, even though it is known that the surface of Au has irregularities at the atomic scale (Loskutov et al. 2009) and secondly, end-thiolation allows only a single covalent attachment per HA molecule. We do not have any evidence on the orientation of HA on the surface of Au. However, polymers with a single point attachment to the core nanoparticle generally take on brush-like configuration (Benoit et al. 2012 and Dukes et al. 2010).

3.3. Characterization of HS-HA conjugated Au NPs

The hydrodynamic sizes and zeta potential of both nascent and coated Au NPs in DI water and DMEM were measured by dynamic light scattering (DLS) analysis (Figs. S4 and S5). When the nascent particles were dispersed in cell medium, the results indicated a substantial increase in particle size and a decrease in zeta potential. The HS-HA coating also had a major effect on particle size. The increase in size for nascent NPs in cell culture media was attributed to nonspecific protein adsorption on the Au NP. When the Au NPs are coated with HS-HA, and then dispersed in DMEM, the extent of protein adsorption was less, which is consistent with the known property of HA to decrease protein adsorption (Pitarresi et al. 2007).

3.4. Cellular uptake of nascent and coated Au NPs

Our uptake studies were based on the measurement of intracellular gold concentration. We had previously determined the critical mass (LD50, M)concentration of Au nanoparticles of different sizes and used these critical mass concentrations in the rest of the cellular experiments, including the uptake study(Karakocak et al. 2016). Our earlier study with RPE cells indicated that the LD50, M concentrations of Au NPs with sizes of 5 -, 10-, 20-nm were, 0.03 mg/ml, 0.08 mg/ml, and 0.11 mg/ml respectively(Karakocak et al. 2016). Furthermore, Au NPs 50 nm and 100 nm in diameter did not exert any toxic effect up to 5 mg/ml, because they did not enter the cells. Here, we extended our study using two additional different cell lines: ARPE-19 cells and CHO cells with high-density CD44 receptors (Jaggupilli and Elkord 2012)and NIH 3T3 cells with relatively less CD44 receptors (Culty et al. 1992) to investigate the CD44 receptor-mediated internalization of 50-nm Au NPs.

According to the ICP-MS results, the number of NPs internalized by ARPE -19 cells changed with the presence of the coating. Indeed, the coating played a significant role, especially for particles with diameters larger than 50 nm. Nascent particles with a diameter larger than 50 nm were not able to enter cells (Fig. 4A). However, when the same particles were coated with HS -HA, their size increased to 58.41 nm in DMEM, and the number of particles internalized by the retinal cells increased considerably (Figs. S4 and S6). Conversely, HS-HA coating increased the size of smaller Au NPs significantly and slightly decreased the number of intracellular Au NPs(Figs. S4 and S6). This difference may be attributed to increase of the coated NP size and the presence of HA coating, therefore the internalization pathway of nanoparticles is predominantly CD44 receptor -mediated endocytosis(Almalik et al. 2013).

Fig. 4.

Fig. 4

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(A) Cellular uptake of Au NPs of different sizes, with and without HS-HA coating by ARPE-19 cells. (B) 50 nm Au NPs (0.11 mg/ml) exposed to ARPE-19, NIH 3T3, and CHO cells with and without pretreatment with 10x HS-HA. CD44 receptor density of NIH 3T3 cells is significantly less than that of ARPE-19 cells; therefore, blocking the CD44 receptors of NIH 3T3 cells did not significantly affect internalization of HS-HA-Au NPs, unlike the results for ARPE-19 and CHO cells. Overall, cells like ARPE-19 and CHO cells, which express high enough number of CD44 receptors and meet the threshold for HA binding, internalize more coated NPs than NIH 3T3 cells. ARPE-19 cells exposed to ● HS-HA-Au NPs, ○ Nascent Au NPs, Inline graphic ARPE-19 cells treated with 10x HS-HA, and then exposed to HS-HA-Au NPs.

To test whether the internalization was CD44 receptor-mediated, nascent( 53.81-nm) and coated (58.41-nm) Au NPs in DMEM were exposed to two more cell lines: NIH 3T3 cells, which have significantly fewer CD44 receptors than ARPE-19 cells, and CHO cells which are also known to express CD44 but a cancer cell line, unlike ARPE-19 cells(Culty et al. 1992). The amount of HS-HA-Au NPs internalized by NIH 3T3 cells was significantly less than the amount internalized by ARPE -19 and CHO cells. Therefore, it can be concluded that the internalization of Au NPs coated with HS -HA depends on the density of cell -surface receptors for HA, in this case, CD44 (Fig. 4B).

As a step toward confirming this finding, all cells were treated with 10x HS-HA, to saturate the CD44 receptors, and then exposed to 50 -nm HS-HA-Au NPs. As expected, we found that the number of internalized particles was significantly decreased(Fig. 4B). Cell membrane receptors and lipid bilayer barriers are specific to cell type. Because of the presence of CD44 receptors, the amount of internalized HS -HA-Au NPs was significantly different in various types of cells. As the size of the nanoparticles decreases, the internalization pathway may involve passive diffusion; however, as particle size increases, receptor-mediated endocytosis may become more prominent(Gupta and Rai 2017; Panariti et al. 2012; Zhang et al. 2009). For smaller particles, there are alternate size -dependent internalization paths that do not involve CD44 receptors. Therefore, the internalized number of coated nanoparticles may slightly decrease because of the increased size of coated Au NPs (Figs. S5 and S7). For RPE cells, we found that internalization of 5-, 10-, and 20-nm particles was only minimally decreased by the presence of the coating. In contrast, internalization of 50-and 100 -nm HS-HA coated Au NPs was significantly increased (Fig. 4A). Furthermore, when we blocked the receptors for HA by adding excess HA to saturate the receptors before administering HS-HA-Au, there was less internalization(Fig. 4B). Thus, although several pathways exist for internalization of nanoparticles, CD44 -mediated endocytosis is particularly important for larger particles. This finding is noteworthy in that coating with HA provides a “Trojan horse” approach to the delivery of large Au NPs and possibly to other hydrophobic particles into cells with receptors for HA.

3.5. The fate of nascent and coated Au NPs in the cells

To confirm the presence of NPs inside cells, we investigated the distribution of Au NPs using a nano-plasmonic confocal laser scanning microscope (Leica TCS-SP8) (see Methods). We first studied the effect of exposure of different sizes of nascent and coated Au NPs to ARPE-19 cells.

Per the 2D and 3D confocal images (Figs S6 and S7, respectively), it can be inferred that for smaller NPs (dp < 50 nm), the presence of an HA coating slightly decreased the number of Au NPs inside retinal cells. Nascent particles with diameters of 50 nm and above could not enter retinal cells (Figs. S6H/S7H and S6J/S7J). However, when these particles were coated with HA, their presence within cells significantly increased (Figs. S6I/S7I and S6K/S7K), as also confirmed by the ICP-MS results shown in Fig. 4. In other words, the HA coating enabled NPs larger than 50 -nm (58.41 nm in DMEM) to enter retinal cells(Fig. 5), even though DLS measurements showed that the size of these particles had significantly increased (Figs S4 and S5). This lends credence to the hypothesis that internalization of larger HS -HA coated Au NPs occurs via the HA receptor-mediated CD44 pathway. Because large HS-HA-coated particles can enter cells, they can efficiently deliver a greater payload than previously reported targeted drug delivery vehicles(Diebold and Calonge 2010).

Fig. 5.

Fig. 5

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3D Confocal microscopy images of Au NPs with ARPE-19 cells. The red staining corresponds to actin, the nucleus is blue, and Au NPs are green. ARPE-19 cells exposed to (A) no particles, the negative control. 50-nm (B) nascent Au NPs (C) HS-HA-Au NPs (D) ARPE-19 cells pretreated with 10x HS-HA and then exposed to 50-nm HS-HA-Au NPs. The HS-HA coating significantly enhanced the intracellular concentration of Au NPs, whereas when the same particles were exposed to the retinal cells after blocking the CD44 receptors, the amount internalized is significantly decreased. The exposure concentration was selected as 0.11 mg/ml. Scale bars are 20 μm.

Additionally, our observations also extend to non-ocular tissues. Nascent and HS-HA-coated50 -nm Au NPs were administered to NIH 3T3 cells and CHO cells. Both IC P-MS findings (Fig. 4B) and 3D confocal microscopy (Figs. S8 and S9) showed that significantly increased number of particles entered NIH 3T3 and CHO cells. This observation confirmed our hypothesis that the number of HS -HA-Au NPs internalized is dictated by the presence or absence of the relevant receptors on the cell membrane (in this case, CD44).

3.6. Biocompatibility assessment of HS-HA-coated Au NPs with ARPE-19 cells

We next studied the biocompatibility of HS-HA Au NPs with ARPE-19 cells. Three different independent end-point assays were used: viability based on enzymatic activity in mitochondria; the percent of apoptotic cells based on caspase 3/7, and activity ROS generation, which is proportional to luciferase activity (see Methods). The results indicated that nascent Au NPs with diameters smaller than 50 nm were not biocompatible above 0.03 mg/ml (Fig. 6A), initiated apoptotic cell death (Fig. 7A), and induced ROS generation (Fig. 7C). On the other hand, no toxicity was observed for HS -HA-Au NPs with a concomitant negligible generation of ROS(Figs. 6B, 7B, and 7D). Most of the toxicity of Au originate from ROS production because of its surface chemistry in the cell’s cytoplasm (Balogh et al. 2003). Coating with HA significantly decreases the available surface on Au by steric hindrance. In addition, HA and its degradation products (tetrasaccharides) have an exceptional ability to quench free radicals(Balogh et al. 2003). Thus, any ROS formed at the surface of Au NPs has very little opportunity to escape the HA coating, as was confirmed by our ROS Glo™ H2O2 assay results (Figs. 7C and 7D ).

Fig. 6.

Fig. 6

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Biocompatibility results of (A) nascent Au NPs and (B) HS-HA-Au NPs. HS-HA coating significantly increased the compatibility of NPs with ARPE-19 cells. Values are expressed in mean ± SEM for each condition; three independent experiments were done with eight replicates (n = 24; Student’s t-test, *P < 0.05 versus controls). The results were compared to each other using two-factor ANOVA analysis. For nascent particles, in terms of both particle size and exposure concentration, the results were significantly different from each other (*P < 0.05). For coated particles, the results were found to be independent of the exposure concentration (**P > 0.05) but statistically different among each nanoparticle size tested (P* < 0.05).

Inline graphic 5nm, Inline graphic 10 nm, Inline graphic 20 nm, Inline graphic 50 nm, Inline graphic 100 nm

Inline graphic5nm, Inline graphic 10 nm, Inline graphic 20 nm, Inline graphic 50 nm, Inline graphic 100 nm

Inline graphic untreated cells, Inline graphic media (DMEM) only

Fig. 7.

Fig. 7

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Measurement of Apoptotic cells amounts using the Apo Tox-Glo™ assay when the cells are exposed to (A) nascent Au NPs and (B) HS-HA-Au NPs. Measurement of ROS levels using the ROS Glo™ H2O2 assay, when the cells are exposed to (C) nascent Au NPs and (D) HS-HA-Au NPs. The HS-HA coating prevented both apoptosis and ROS generation in ARPE-19 cells for all particle sizes tested. (n = 24; Student’s t-test, *P < 0.05 versus controls). The results were compared to each other using two-factor ANOVA analysis. In both measurements, for nascent particles, in terms of both particle size and exposure concentration, the results were significantly different from each other (*P < 0.05). For coated particles, the results were found to be independent of the exposure concentration (**P > 0.05) but statistically different among each nanoparticle size tested (*P < 0.05).

Inline graphic 5nm, Inline graphic 10 nm, Inline graphic 20 nm, Inline graphic 50 nm, Inline graphic 100 nm

Cell morphology was observed by 3D confocal microscopy. There was a significant structural change in cytoskeleton fibers (actin) in retinal cells exposed to nascent NPs (dp < 50 nm) (Fig. S7). The nuclear morphology was disrupted, and the density of actin significantly decreased (see supporting information, Figs. S7B, S7D, S7F). In a previous study degradation of actin was linked to cell death (Tezel et al. 1999), consistent with our confocal imaging observations. Cells exposed to HS-HA-Au NPs maintained good actin density and intact nuclei (Figs. S7C, S7E, S7G, S7I, S7K). These observations are consistent with our biocompatibility results, which indicated that coated Au NPs are not toxic and do not initiate apoptosis.

3.7. Cell attachment behavior with ECIS measurements in response to Au NPs

We monitored retinal cells’ attachment in the presence of both nascent and coated Au NPs via electrical impedance measurements. Retinal cells exposed to nascent NPs less than 50 nm in diameter, at their critical LD50, M concentrations, almost immediately started to detach from the plate (Fig. 6C). On the other hand, cell attachment was not hindered when the ARPE-19 cells were exposed to HS-HA-Au NPs (Fig. 6D). Following the addition of HS-HA-Au NPs, the impedance curves, which show the real-time cellular response, fall in line with the negative control group, in which neither coated nor nascent NPs were exposed to the cells (Fig. 6D). The cell attachment behavior is directly correlated to cell viability. Therefore, it can be inferred that cell viability is not compromised in the presence of HS-HA-Au NPs. As a result, it is clear that not only HA coating plays a significant role in the internalization of Au NPs, but also that its presence, independent of particle size and the amount of Au NPs inside a cell, protects the cell from ROS damage, does not initiate cell death.

4. Conclusion

Our TOC and TGA results indicated that we successfully coated Au NPs with HS-HA. The grafting density varied from 0.18 to 0.03 # of HA molecules/nm2 as the size of Au NPs increased from 5 to 100 nm. HS-HA coating decreased Au NPs’ toxicity to the cells. The improved biocompatibility is associated with HA’s ability to act as a free-radical quencher as revealed by our ROS measurements. The native 50- and 100-nm Au NPs could not enter the cell; however, the HS-HA coating enabled their entry in the cells via CD44 receptors, acting as a Trojan horse for the larger particles. The endocytosis of large particles coated with HS-HA is a significant observation for targeted drug and gene delivery applications.

Internalization was a function of the density of CD44 receptors as evidenced by percent internalization results with three different cell lines: CHO>ARPE-19>NIH 3T3. In conclusion, we showed that HS-HA coating facilitates entry of Au NPs in size range of 5–100 nm into cells that express CD44 receptors and renders the NPs biocompatible. This observation portends that HS-HA can be used as a biocompatible carrier to deliver larger payloads for treating inflamed, proliferating, and cancer cells that are associated with increased expression of CD44 receptors.

Supplementary Material

supplement

Highlights.

  • Gold nanoparticles (Au NPs) were coated with end-thiolated hyaluronic acid (HS-HA) to achieve an efficient homogeneous coating layer.

  • HA specific CD44-rich cells (retinal epithelial and Chinese hamster ovary), as well as cells lacking CD44 receptors (NIH 3T3), were used in the study.

  • Internalization of five different sizes of Au NPs (with and without HS-HA coating) was assessed.

  • 2D and 3D confocal microscopy images showed that the 50- and 100-nm HS-HA-Au NPs were able to enter the cells; however, their nascent forms could not.

  • When coated with HS-HA, 5-, 10-, and 20-nm gold nanoparticles did not initiate apoptosis, and the amount of reactive oxygen species generation was significantly reduced.

Acknowledgments

This work was supported by NIH RO1 ECS-0335765, a Lacey and Nelson grant, a Core grant from the Association for the Prevention of Blindness, and a Veteran Affairs Merit review grant. Veteran Affairs Office of Research and Development IO1BX007080 grant to Kelle H. Moley supported confocal imaging. (NR). Partial support from McDonnell Academy Global Energy and Environment Partnership (MAGEEP) and the Lopata Endowment are gratefully acknowledged (PB).

Appendix A. Supporting data

Comprehensive experimental details on HR-TEM imaging, 1H NMR, FTIR, GPC analysis, particle size measurements, in-vitro toxicity analysis, and confocal microscopy imaging. Dynamic Light Scattering (DLS) analysis results: Particle size distribution graphs and zeta potential measurement results of gold nanoparticles used in the study. Additional 2D/3D confocal images of ARPE-19 NIH 3T3, and CHO cells exposed to Au NPs with and without HS-HA coating.

Footnotes

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