Zwitterionic Gel Coating Endows Gold Nanoparticles with Ultrastability

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Zwitterionic gel coating endows gold nanoparticles with ultrastability Wenchen Li, Kuanwu Chu, and Lingyun Liu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01600 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Zwitterionic gel coating endows gold nanoparticles with ultrastability

Wenchen Li, Kuanwu Chu, and Lingyun Liu*

Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, 44325

*Correspondence to: Lingyun Liu, Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325; e-mail: [email protected]; Phone: (330) 972-6187

ACS Paragon Plus Environment

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ABSTRACT

Colloidal stability of gold nanoparticles (GNPs) is of great importance for their applications. Although a number of ligands have been developed to protect GNPs, irreversible aggregation still happens under some particular conditions. Herein, we report a zwitterionic encapsulation strategy to endow GNPs with ultrastability. Each single GNP is coated with a crosslinked zwitterionic poly(ornithine methacrylamide) hydrogel thin-layer, which prevents their aggregation under extremely harsh conditions, including lyophilization, strong acid solution, saturated salt solution, and concentrated alkali solution. In addition, the zwitterionic gel layer resists protein adsorption in biological milieu, keeping the nanoparticles stable in full human blood serum. Furthermore, the amino acid derived polymer gel provides numerous conjugation sites for on-demand biomolecular functionalization. With all these merits, the zwitterionic gel coated GNPs hold great potential in many applications such as sensing and theragnostics.

Keywords: gold nanoparticles, ultrastability, poly(ornithine methacrylamide), zwitterionic, hydrogel coating

1


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Introduction Gold nanoparticles (GNPs) hold great potential in many biomedical applications, including drug and gene delivery1-2 and diagnostics3-4, due to their biocompatibility, ease of synthesis, and unique physical and chemical properties5-6. To fulfill these applications, surface chemistry of GNPs needs to be engineered to support their colloidal stability7, antifouling property8, and functionalization9. The first priority for GNPs to be used in any in vitro or in vivo application is to maintain their colloidal stability.10 Changes of environment or processing conditions, such as solution ionic strength, solution pH, or freeze-drying, could easily trigger irreversible aggregation of GNPs and limit their further processing and applications. When GNPs are applied to complex biological media (e.g. blood or tissue culture media), proteins may adsorb onto nanoparticle surfaces, destabilize particles, and impair their functions11. Furthermore, GNP surface functionalization, for example through conjugation of targeting ligands12, antibody13, aptamer14, and drug15, is typically necessary for further applications. Therefore, a protecting layer on GNP surfaces that can maintain colloidal stability, resist non-specific protein adsorption, and enable functionalization is greatly desired.

Classic GNP protecting layers usually fall into two categories: small molecule ligands and polymer brushes. For example, zwitterionic ligands bearing phosphorylcholine16 or carboxybetaine17 groups have been proven to stabilize GNPs through their strong hydration effect. Polyethylene glycol (PEG)18 or zwitterionic polymer brushes19 were “grafted to” or “grafted from” GNPs to enhance their stability. Although significant advances have been made to stabilize GNPs, some challenges still remain with the existing methods, such as insufficient resistance to nonspecific protein adsorption leading to instability in complex biological environments20, absence of conjugation sites on capping agents for further functionalization21, and complicated modification procedures such as atom transfer radical polymerization (ATRP) which could be further perplexed by large polarity difference between hydrophobic ATRP initiator and hydrophilic monomer

19-20, 22

. Also when drug is

linked or incorporated into the protecting monolayer of GNPs, total drug loading is limited due to space restriction2, 15. Thus, a robust and simple coating strategy to overcome all these 2


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limitations is highly desirable.

Herein, we report an ultra-stable GNP construction by coating a thin zwitterionic hydrogel layer on single nanoparticle surface. Through in situ free radial polymerization, each gold nanoparticle was trapped within a zwitterionic “nanocage” to avoid the possibility of aggregation. An amino acid based zwitterionic polymer poly(ornithine methacrylamide) (pOrnAA) was used in this study to form the protecting nanogel shell. The natural amino acid based zwitterionic polymers (pAAZ) have recently emerged as a new class of antifouling materials, not only exhibiting excellent biofouling resistance attributed to their strong electrostatic interaction with water, but also providing a platform for further functionalization using abundant carboxyl and amine groups.23-27 It is hypothesized here that by encapsulating GNPs with a thin layer of pOrnAA nanogel, the colloidal stability of particles can be significantly improved. In this work, we investigated the dispersion stability of pOrnAA-protected

GNPs

under

a

wide

range

of

harsh

conditions,

including

phosphate-buffered saline, lyophilization, broad range of pH, highly concentrated salt solutions, strong base solutions, and human blood serum. Furthermore, folic acid (FA) was used as a model targeting ligand28 to functionalize GNPs by conjugating onto the pOrnAA layer. Cellular uptake of GNPs with or without FA conjugation was examined and compared in both ovarian cancer cells and normal cells.

Experimental Section Materials L-ornithine hydrochloride (99%), methacryloyl chloride (97%), 8-hydroxyquinoline, glutathione (GSH), N-hydroxysulfosuccinimide (NHS), and folic acid were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) carbonate basic, gold(III) chloride trihydrate (HAuCl4), N-acryloxysuccinimide, N,N’-methylene bisacrylamide (MBAA), ammonium persulphate (APS), N, N, N’, N’-tetramethylethylenediamine (TEMED), sodium hydroxide (NaOH), sodium chloride (NaCl), and phosphate-buffered saline (PBS; pH 7.4, 10 mM, 138mM NaCl, 2.7mM KCl) were purchased from Sigma-Aldrich (Milwaukee, WI). Sodium citrate

tribasic

dehydrate

was

bought

from

Fisher

3


Scientific

(Waltham,

MA).

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1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDC) was purchased from Pierce Biotechnology (Rockford, IL). Pooled human blood serum was purchased from BioChemed Services. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Life Technologies (Carlsbad, CA). Dulbecco’s modified Eagle’s medium (DMEM; with 4.5 g/L glucose, 4mM L-glutamine and 110 mg/L sodium pyruvate) was obtained from Thermo Scientific (Waltham, MA). Nonessential amino acids and sodium pyruvate were purchased from Lonza (Walkersville, MD). Penicillin streptomycin was purchased from Invitrogen (Grand Island, NY). N-δ-methacryloyl ornithine (i.e. ornithine methacrylamide, OrnAA) was synthesized by the reaction of L-ornithine hydrochloride with methacryloyl chloride using the method we published before. 25

Synthesis of citrate-capped gold nanoparticles (Citr-GNPs) Citrate-capped gold nanoparticles were prepared by the reduction of HAuCl4 with sodium citrate29. 250 ml HAuCl4 (1mM) aqueous solution was stirred vigorously under heating. When the solution was heated to boiling, 25 mL sodium citrate solution (38.8 mM) was added quickly. The reaction system was kept boiling for another 20 min and then cooled down to room temperature.

Preparation of pOrnAA hydrogel coated gold nanoparticles (pOrn-GNPs) Glutathione solution (300 µl, 2 mg/ml in PBS) was adjusted to pH of 8.5 and reacted with 3 mg N-acryloxysuccinimide in 30 µl dimethyl sulfoxide (DMSO) for 2 h. The acryloylated glutathione solution was then transferred into 1 ml Citr-GNP solution (0.35 mg/ml, 26.6 nM) to exchange the capping citrate molecules. The reaction was carried at room temperature for 4 h to introduce vinyl groups on GNP surfaces.

Zwitterionic pOrnAA nanogel coated gold nanoparticles were formed by in situ free radical polymerization from surfaces of the acryloylated GNPs, as described below. OrnAA (120 mg, monomer) and MBAA (7.2 mg, crosslinker) were first dissolved in 0.7 ml PBS with 5 µl TEMED. The mixture was then added into the acryloylated GNP solution made above. The polymerization was initiated by adding 0.5 mg APS in 50 µl PBS. The reaction was allowed 4


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to proceed for 2 h under nitrogen atmosphere. To purify the pOrnAA-coated GNPs (denoted as pOrn-GNPs), Amicon Ultra centrifugal filter device with 100 kD molecular weight cutoff (Millipore, Burlington, MA) was used to remove the unreacted monomers and other molecules. The product was further purified by hydrophobic interaction chromatography (Phenyl Sepharose™ CL-4B Gel, GE) using PBS as eluent. The final solution was concentrated and dispersed in 1 ml DI-water or PBS for further use.

Characterization of gold nanoparticles Fourier transform infrared (FTIR) spectrum of pOrn-GNPs was recorded with a Thermo Scientific Nicolet 380 series spectrometer. Hydrodynamic diameter and zeta potential distribution of Citr-GNPs and pOrn-GNPs were characterized by a Zetasizer NanoZS dynamic light scattering (DLS) instrument (Malvern, U.K.) at a wavelength of 633 nm and a scattering angle of 173°. The dispersant refractive index and viscosity of PBS or water were taken as 1.330 and 0.8872 cP, respectively. Transmission electron microscope (JEOL, JSM-1230) was used to image Citr-GNPs and pOrn-GNPs. UV-Vis absorption was measured on a UV-1601 Shimadzu spectrophotometer.

Particle stability evaluation Stability of citrate or pOrnAA protected GNPs under various conditions was characterized by UV-vis spectra. To test the stability of pOrn-GNPs and Citr-GNPs in PBS, particles were centrifuged from water and redispersed in PBS. Since well dispersion of nanoparticles in buffer (e.g. PBS) is a prerequisite for biomedical applications, PBS was used to disperse pOrn-GNPs in subsequent experiments unless otherwise noted. Citr-GNPs were still dispersed in water to ensure their stability before conditions changed, since Citr-GNPs aggregated in PBS (results shown later). To determine particle stability upon lyophilization, pOrn-GNP and Citr-GNP solutions (both in water) were lyophilized and redispersed in PBS and water, respectively. The effect of pH on the stability of pOrn-GNPs and Citr-GNPs were measured after adjusting the solution pH to different desired values, ranging from 1 to 12, by 1M HCl or NaOH. For the salt effect on particle stability, pOrn-GNPs and Citr-GNPs were dispersed in NaCl solutions with different concentrations (0.2, 0.5, 1, 2, or 5 M). POrn-GNPs 5


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was also dispersed in a saturated NaCl solution (~ 6.1 M), with salt crystals clearly visible at the bottom of the vial; the top solution was used for the UV-vis characterization. For the stability of pOrn-GNPs and Citr-GNPs under extreme basic conditions, 4%, 10% or 20% (w/v) NaOH solutions were mixed with pOrn-GNPs or Citr-GNPs solutions in 1:1 volume ratio, leading to particle dispersions in 2%, 5%, or 10% NaOH solutions for UV-vis characterization. To test the particle stability in serum, pOrn-GNPs and Citr-GNPs were centrifuged and resuspended in undiluted human blood serum (4 °C) for incubation. At various time points, samples were centrifuged, washed twice, and then re-dispersed in PBS and water respectively for DLS measurements and UV-vis characterization.

Folic acid conjugation Folic acid (FA) was activated by the EDC/NHS chemistry first and then conjugated onto the pOrn-GNP surfaces. Specifically, 4 mg FA was dissolved in 4 ml of water/DMSO mixture (3:2, v/v), followed by addition of 2.4 mg EDC and 1.4 mg NHS. The reaction was kept at room temperature for 2 h. The activated FA solution was then added dropwise into 2 ml of pOrn-GNP solution in PBS (adjusted to pH of 8-9). After an overnight reaction, the solution pH was adjusted to 9-10. An ultra-centrifuge purification step was then used to remove the unreacted FA and other reactants and repeated three times. Another washing step with DI water followed and was repeated five times. The solution was finally freeze-dried to get pOrn-GNPs conjugated with FA (denoted as FA-pOrn-GNPs).

Cytotoxicity assay Cytotoxicity of Citr-GNPs, pOrn-GNPs and FA-pOrn-GNPs was assessed against NIH/3T3 fibroblasts through the MTT assay. NIH/3T3 cells were seeded in a 96-well culture plate at 104 cells/well, in culture medium composed of DMEM, 10% fetal bovine serum, 1% nonessential amino acids, 1% sodium pyruvate, and 2% penicillin streptomycin. After 24-h incubation at 37 °C, the medium was refreshed and different particles at various concentrations (20, 50, 200, or 500 µg/ml) were added. After culturing for another 24 h, the metabolic activity of cells was determined by a MTT test. Briefly, the medium was removed and 100 µl of phenol red-free medium containing 10 µl MTT solution (12 mM in PBS) was 6


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added and incubated with cells for 4 h. Medium was then removed and 150 µl DMSO was added into each well, followed by 10-min incubation and 15-min shaking. The absorbance of each well was read at 570 nm using a 96-well plated reader (SpectraMax M5, Molecular Devices). Each measurement had 6 replicates. The cell viability percentage was determined by normalizing to the absorbance when no nanoparticles were added in the culture medium. Statistical analysis was performed using Student’s t-test, defining p < 0.05 as significant difference.

In-vitro cellular uptake SKOV3 ovarian carcinoma cells (ATCC, St. Cloud, MN) and bovine aortic endothelial cells (BAECs) were grown separately in 75-cm2 culture flasks to 90% confluency. The medium in each flask was then refreshed with culture medium containing 200 µg/ml pOrn-GNPs or FA-pOrn-GNPs. After 12-h incubation, culture medium was removed and flasks were washed with PBS twice. Cells were harvested by trypsinization and collected by centrifugation. Cell pellets were then treated by aqua regia (HNO3: HCl = 1:3) for 2 h and diluted to 5% acid concentration with Millipore water. The Au3+ content in the solution was finally determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). Cells without treatment of nanoparticles were used as control. The total cell number was determined with a Neubauer Chamber.

Results and Discussion GNP fabrication, modification, and characterization Citr-GNPs were synthesized by reducing HAuCl4 with sodium citrate in a boiled aqueous solution following a widely used protocol. POrn-GNPs were prepared by coating a zwitterionic pOrnAA polymer shell on GNPs. As shown in scheme 1, the nanoparticles were first modified with acryloylated GSH via ligand exchange reaction to introduce polymerizable vinyl groups onto particle surfaces. In situ polymerization of OrnAA monomer and MBAA crosslinker was subsequently carried out in the presence of the acryloylated GNPs, resulting in the pOrnAA-gel-encapsulated GNPs. FTIR analysis was used to verify the formation of pOrnAA layers on GNP surfaces, as shown in Figure S1. It reveals the 7


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absorption peaks corresponding to the functional groups of the pOrnAA polymer (3300-2900, ν-OH; 1562, ν-amide; 1462, δ-CH2 & CH3; 1427, δ-OH; 1367, ν-CN & δ-CH3; 1234, ν-CO; 830, δ-NH). After the polymer gel coating, the nanoparticle hydrodynamic size increased from ~18 to ~28 nm, as measured by DLS (Figure 1a). The size growth is attributed to the presence of the polymer network as well as its hydration layer. Zeta-potential of Citr-GNPs was -34.5 mV (Figure 1b), corresponding to the acidic nature of the citrate capping agent. After pOrnAA encapsulation, zeta-potential of pOrn-GNPs increased to -7.9 mV, which is consistent with the previously reported potential of pOrnAA nanogels27. Change of nanoparticle size and zeta-potential both demonstrates successful formation of the zwitterionic pOrnAA gel layers encapsulating GNPs. Transmission electron microscopy (TEM) images of Citr-GNPs and pOrn-GNPs are shown in Figure 1c and Figure S2. In contrast to Citr-GNPs, a thin polymer layer is clearly revealed around each single core of pOrn-GNPs. The characteristic absorption of GNPs was recorded using UV-Vis spectroscopy (Figure 1d). Gel encapsulation did not change the surface plasmon resonance (SPR) peak of nanoparticles5, with the absorption peak of both Citr-GNPs and pOrn-GNPs appearing at ~ 521 nm. Photographs in Figure 1d show that the gel encapsulation did not alter the appearance of aqueous solutions of gold nanoparticles. Both Citr-GNP and pOrn-GNP solutions remained bright red and transparent.

Stability evaluation Remaining stable in buffer solutions such as PBS is a prerequisite for biomedical applications of GNPs. Citr-GNPs aggregated immediately after being dispersed in PBS, as demonstrated by disappearance of the SPR peak (Figure 2). Aggregation of Citr-GNPs in PBS is because of the screening of electrostatic repulsion between particles by salt. In contrast, pOrnAA gel encapsulated GNPs dispersed well both in water and in PBS without aggregation (Figure 2). Considering that PBS is a widely used buffer for biological applications and a more favorable solvent for pOrn-GNPs because it facilitates the ionization of both carboxyl and amino groups of the pOrnAA polymer, in the subsequent experiments, pOrn-GNPs were dissolved in PBS while Citr-GNPs were dissolved in water unless specifically noted.

Stability in dry state is another desirable merit to promote applications of nanoparticles. It is 8


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much more convenient to store, transport and use them in a solid form. Moreover when nanoparticles are conjugated to biomolecules (e.g., antibodies, enzymes, and other proteins) which have superior stability as dry powder compared to solution, the solid form of the conjugate has much extended storage shelf life. Commercial GNPs are typically stored in solution due to the instability of particles in dry state. Absence of water molecules would cause irreversible aggregation of GNPs with insufficient ligand protection. As shown in Figure 3, disappearance of the SPR peak in the UV-vis spectrum of the re-dispersed Citr-GNPs after freeze-drying indicates their aggregation is substantial and irreversible. In contrast, pOrn-GNPs were easily dissolved in PBS after lyophilization; the solution appearance and absorption spectrum do not show significant changes compared to that of the original colloidal solution (Figures 3 and S3). Although a marginal red-shift was observed, tapping-mode AFM images show that pOrn-GNPs remained mostly isolated after freeze-drying (Figure S4). It is possible that the lyophilization process caused structural changes (e.g., arising pores) of the thin hydrogel coating, thus affecting the optical properties of the particles. We verified that a pOrnAA hydrogel disk (diameter ~ 1 cm) turned very porous and cloudy after freeze-drying (data not shown). During freeze-drying, the pOrnAA gel layer functioned as a nanocage to isolate each gold core thus protected them from aggregation. When re-dissolving, the superhydrophilic property and zwitterionic nature of the polymer ensured fast water penetration and quick dispersion of GNPs in aqueous solutions.

Other strategies were also developed to protect GNPs against stresses from lyophilization. For example, the freeze-dried GNPs coated with PEG (MW 5000) could easily be dissolved in water and maintained the plasmonic properties of parent colloids without forming aggregations.30 GNPs protected with cryoprotectants (e.g., saccharide) exhibited remarkable stability upon lyophilization, attributed to the formation of an amorphous glassy matrix which inhibited nanoparticle mobility thus aggregation.31 However further functionalization of GNPs is not straightforward, if possible, for both cases. Another study shows that GNPs capped with PEG (average Mn=800) displayed significant shift and broadening in the SPR band indicating aggregation after freeze-drying. 17 9


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In many applications of GNPs (e.g., pH-responsive drug delivery systems32 and protein-colloidal gold conjugates33), the solution pH often needs to be adjusted for different purposes, such as controlling the stimuli pH, or optimizing the reaction conditions. Therefore, it is necessary to maintain stability of GNPs under different pH values. Figure 4a shows the UV-Vis spectra of pOrn-GNP solutions with pH from 1 to 12. Interestingly, it is noted that the absorbance peak was at ~ 521 nm when the solution pH was 6 or above, but red-shifted to ~ 537 nm when pH was 4 or below. Such change was unlikely due to the aggregation or assembly phenomenon that occurred in other GNP systems, since aggregation would usually result in a significant decrease of the absorbance peak intensity and a much broader peak shape. Instead, the absorbance spectra of pOrn-GNPs here well maintained their shape with a slightly higher intensity under acidic conditions. The hydrodynamic size of pOrn-GNPs measured by DLS further confirms that no aggregation occurred when pH was changed, as shown in Figure 4b.

For GNPs, the plasmon resonance peak position ( ) is governed by34: = + 2

(1)

where ϵ is the high-frequency dielectric constant of gold due to interband and core transitions, ϵ is the dielectric constant of the surrounding medium, and λ is the bulk plasmon wavelength of gold given by = 2/( ⁄ )/

(2)

in which is the speed of light in vacuum, is the density of free electrons in the nanoparticle, is the electronic charge, is the effective mass of the electron, and ϵ is the permittivity in vacuum. Accordingly, the red shift of the plasmon resonance peak wavelength can result from the increase of the surrounding medium refractive index and/or the decreased free electron density of GNPs. Considering the insignificant change of the medium refractive index as pH changes, the extent of the spectrum wavelength shift (~16 nm) is most possibly attributed to the decreased free electron density of particles, which could result from the coordination bonds between gold and protonated sulfur, inductive effect, and the non-covalent interactions with gold through chemical groups in the coating (e.g., NH3+) at 10


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lower acidic pH values.35-36 Also notice that there is a slight red shift in the plasmon band (~ 2 nm) when pH was changed from 5 to 4 (Figure S5), explained likewise by the above reasons. As the pH became lower, more H+ resulted in the Au-SH coordination bonds and stronger inductive effect, causing decreased free electron density of particles. The shift may also be related to the close by isoelectric point of the coating material. When pH was tuned above 5, the absorbance spectrum of pOrn-GNPs shifted back to its original position at 521 nm (Figure 4c). The spectrum peak position alternated between 537 and 521 nm during four pH change cycles (Figure 4d), demonstrating robust protection of GNPs using pOrnAA polymer layer. In contrast, Citr-GNPs aggregated when exposed to acidic environment (pH = 2), and such change was irreversible since the absorption peak did not shift back when pH was adjusted from 2 to 9 (Figure 4e).

To challenge the limit of ionic strength that pOrn-GNPs can tolerate, stability of pOrn-GNPs was tested in NaCl solutions with various concentrations (0.2 M to saturation). The UV-Vis spectra of pOrn-GNPs (Figure 5) show that the spectrum shape and width were generally unchanged with the increasing salinity. The SPR peak position remained mostly same, except a slight red shift under extreme salt concentrations (above 5 M). In contrast, Citr-GNPs aggregated severely even at the lowest tested NaCl concentration (200 mM), as evidenced by the solution color change (to purple or black) and abnormal UV-Vis spectra (Figure S6). This is in consistency with literature reports that citrate-protected GNPs tend to aggregate at salt concentrations higher than 10 mM 6, 17. The ultra-stability of pOrn-GNPs is attributed to both polymer property and gel physical structure. The superhydrophilic and zwitterionic pOrnAA layer promotes nanoparticle solubility due to its electrostatically induced strong hydration effect. On the other hand, entrapment of GNP in a crosslinked polymer network isolates each single nanoparticle thus avoiding the aggregation tendency. In short, pOrnAA gel coating is capable of conferring stability to GNPs even at an extreme saturated salt concentration.

Compared to the stability of GNPs with other capped agents like mixed charged zwitterionic self-assembled monolayers

21

, chitosan with phosphorylcholine group37, or carboxybetaine

ligand17, pOrn-GNPs exhibited similar or even better stability in highly concentrated saline 11


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solutions. Notice that the concentration of NaCl used to challenge stability of GNPs in other reports mostly ranges from 0.5 to 4 M 16, 18-19, 21, 37-39. Herein, thin pOrnAA gel-coated GNPs remained stable even in the saturated salt solution. Also notice that even if PEG is commonly used to cap GNPs, PEG-coated nanoparticles tend to aggregate under high salt conditions8. PEG800-capped GNPs were stable at 1 M NaCl but showed signs of aggregation at 5M NaCl 17

. In another work, GNPs protected by PEG5000 aggregated at 20% NaCl19.

The observed slight red shift of the adsorption peak at high salt concentrations can be attributed to the refractive index (n) increase of the concentrated NaCl solutions. From Equation 1, the adsorption peak wavelength depends on not only , but also the dielectric constant of the surrounding medium ( = ). NaCl solutions at different concentrations have different refractive index. The refractive index of 1 M and 5M NaCl solutions is about 1.343 and 1.377, respectively, much larger than that of pure water (n = 1.330). In fact, using a commercial SPR sensor24, we observed that SPR wavelength increased dramatically, to beyond the sensor detection range, when the solution past the sensor chip (a flat gold-coated glass substrate) was switched from 0.2 to 2 M NaCl (data not shown), which resulted from the bulk fluid refractive index increase.

Stability of pOrn-GNPs was further tested under harsh alkali conditions (0 ~10% NaOH solutions). No apparent change in solution color was observed. As shown in Figure 6, UV-vis spectrum shape remained mostly unchanged, except a slight red shift under higher NaOH concentrations, similar to the case with extreme salinity, resulting from the refractive index increase when NaOH concentration increases. On the other hand, Citr-GNPs treated with NaOH solutions showed severe aggregation along with abnormal spectra (Figure S6), demonstrating the instability of Citr-GNPs under such harsh conditions.

GNPs are inevitably exposed to complex media if used for biomedical applications. The nonspecific protein adsorption from biological media could destabilize nanoparticles, cause particle aggregation, and impair their functions. If used in vivo, nanoparticles’ interaction with blood proteins (i.e., opsonization) would result in recognition and clearance by immune 12


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cells, hence decreased delivery to the intended target sites.40 Thus the ability to remain stable and resist protein adsorption from complex media is a key merit for a successful GNP surface protection. Our previous work shows that nanogels prepared from pOrnAA matrix (diameter ~ 115 nm) have great stability in fibrinogen solution, bovine serum albumin solution, and fetal bovine serum25, 27. Here, to test the stability of pOrnAA gel coated GNPs in complex media, pOrn-GNPs were incubated with human blood serum at 4 °C up to 24 hours. At each time point, samples were separated from serum, washed and re-dispersed in PBS, followed by the UV-Vis and DLS measurements. The UV-Vis absorbance spectra of pOrn-GNPs remained identical throughout the 24-h incubation period (Figure 7a). The size change of pOrn-GNPs was also monitored by DLS and compared to that of Citr-GNPs. As depicted in Figure 7b, pOrn-GNPs kept the original size without aggregation while Citr-GNPs aggregated quickly. Upon addition of serum, Citr-GNP solution turned much darker, while pOrn-GNP solution still maintained the clear red color (Figure 7c). Actually even being in serum for 2 months (in fridge at 4°C), pOrn-GNPs still remained stable and no obvious color change was observed in the solution (data not shown). Overall, results show that the pOrnAA gel coatings on GNPs prevent particle agglomeration and render them great dispersion stability in serum. Consistently, several other works also report that the zwitterionic small molecule or polymer coated GNPs17, 19 and quantum dots41 as stealth nanoparticles demonstrated improved stability in biological environment. Though PEG is commonly used to stabilize GNPs, PEG5000-thiol coated GNPs remained stable only in 10% blood serum, but aggregated significantly in 100% blood serum19. GNPs protected with PEG 800 or citrate formed large aggregates in 50% serum17. Autoxidation of PEG into shorter chains is another limit resulting in the aggregation of GNPs.21

Functionalization and cellular uptake Surface conjugation with functional molecules further increases versatility of nanoparticles. The pOrnAA layer coated on the GNP surface possesses abundant carboxyl and amino groups suitable for chemical conjugation. In this study, a model ligand, folic acid (FA), was conjugated to pOrn-GNPs through standard EDC/NHS coupling chemistry. Successful conjugation of FA was proved by an absorption shoulder appearing at around 365 nm in the 13


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UV-Vis spectrum, which is characteristic of FA (Figure 8). The amount of FA conjugated on pOrn-GNPs was calculated as ~ 0.11 wt%, by comparison with the UV calibration curve of FA.

Cellular uptake experiments were then carried out to test the specific targeting ability of FA-pOrn-GNPs to cancer cells. SKOV3 ovarian carcinoma cells were used as the model cancer cells because of their overexpressed folate receptors. Bovine aortic endothelial cells (BAECs) that do not overexpress folate receptors were used for comparison. Cells cultured in normal medium without exposure to any gold nanoparticles served as negative control. The amount of cell-internalized GNPs was measured via ICP-OES. As demonstrated in Figure 9, pOrn-GNPs, without FA conjugation, were not significantly internalized by both types of cells after incubation for 12 hours (comparable to the negative control), because the zwitterionic pOrnAA surface suppressed the interaction between nanoparticle and cell membrane through its “stealth” property. In contrast, the uptake of FA-pOrn-GNPs was cell type dependent. The amount of FA-conjugated pOrn-GNPs internalized by SKOV3 cells (5.9 ± 0.7 ng/103 cells) was significantly higher than that in BAEC cells (0.7 ± 0.2 ng/103 cells), and also significantly greater than uptake of the unconjugated pOrn-GNPs in SKOV3 (1.1 ± 0.1 ng/103 cells). No significant difference (p>0.05) was found for gold internalization between FA-pOrn-GNPs and pOrn-GNPs in BAEC cells. All these results prove that surface modification with FA endows the pOrn-GNPs with the cancer cell targeting specificity without sacrificing the “stealth” property of the particles. Our early work also shows that the FA-functionalized pOrnAA nanogels remained dispersed without aggregation in protein solutions and can be selectively uptaken by cancer cells but not fibroblasts27. Dixit et al. reported selective uptake of folate-PEG grafted GNPs by KB cells (a folate receptor-positive nasopharyngeal cancer cell line), whereas the uptake was minimal in cells that do not overexpress folate receptors or when GNPs were coated with PEG only38. A peptide containing a stealth glutamic acid/lysine (EK) portion and a cell recognition sequence (cRGD) was designed by Nowinski to coat GNPs. High specific cell uptake of the particles was observed, in contrast to the low uptake of GNPs capped with peptides containing scrambled cRDG or no targeting sequence39. Together, these work demonstrate that antifouling materials 14


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can be designed to resist non-specific cell uptake as well as target specific cells for uptake at the same time.

Particle cytotoxicity Cytotoxicity of Citr-GNPs, pOrn-GNPs and FA-pOrn-GNPs was evaluated against NIH-3T3 cells for 24 h by MTT assay at various nanoparticle concentrations. As presented in Figure 10, viability of cells treated with Citr-GNPs remained above 80% at all tested concentrations, considering the gold core is essentially inert and non-toxic. Viability of cells treated with pOrn-GNPs or FA-pOrn-GNPs was all above 90%, and statistical analysis revealed no significant difference between the two groups (p > 0.05). These results indicate that pOrnAA, FA, as well as GNPs were all non-toxic to cells. In a word, pOrn-GNPs, with or without FA treatment, both exhibited great biocompatibility.

Conclusions Ultra-stable GNPs were fabricated by encapsulating each single nanoparticle into an amino acid derived zwitterionic pOrnAA nanocage. The pOrnAA gel layer provided both physical and chemical protection for GNPs, without affecting their optical properties. The superhydrophilic gel coating stabilized GNPs over a wide range of harsh conditions, including lyophilization, PBS buffer, an extended range of pHs, salty solutions (till saturated), strong base solutions, as well as human serum. Furthermore, the pOrnAA gel surface provided a convenient platform to multivalently conjugate functional molecules onto GNPs. As a proof of concept, FA was successfully attached onto the nanoparticle surface. The resulting FA-pOrn-GNPs were shown to be selectively internalized in folate receptor overexpressed cancer cells, but not normal cells, suggesting that the functionalization leads to cancer cell targeting specificity but does not affect particles’ stealth property. This facile protection and functionalization strategy offers great potential to ease and broaden biomedical applications of gold nanoparticles.

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Acknowledgements We would like to thank Dr. Gang Cheng for the use of DLS instrument, Dr. Jie Zheng for the use of atomic force microscope, Dr. Jiahua Zhu for the use of UV-vis spectrophotometer, and Mr. Tuo Ji for his help with the TEM characterization. This work is financially supported by the University of Akron faculty start-up fund.

Supporting Information. FTIR spectrum of pOrn-GNPs; TEM images of Citr-GNPs and pOrn-GNPs with wider view; Pictures of the pOrn-GNP suspension before and after freeze-drying; Tapping-mode atomic force microscopy (AFM) images of pOrn-GNPs before and after freeze-drying; Enlarged UV−vis spectra of pOrn-GNPs under pH = 1 – 5; UV-vis spectra of Citr-GNPs in NaCl and NaOH solutions with different concentrations.

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(20) Yang, W.; Liu, S.; Bai, T.; Keefe, A. J.; Zhang, L.; Ella-Menye, J.-R.; Li, Y.; Jiang, S. Poly(Carboxybetaine) Nanomaterials Enable Long Circulation and Prevent Polymer-Specific Antibody Production. Nano Today 2014, 9, 10-16. (21) Liu, X.; Huang, H.; Jin, Q.; Ji, J. Mixed Charged Zwitterionic Self-Assembled Monolayers as a Facile Way to Stabilize Large Gold Nanoparticles. Langmuir 2011, 27, 5242-5251. (22) Zhao, C.; Zheng, J. Synthesis and Characterization of Poly (N-Hydroxyethylacrylamide) for Long-Term Antifouling Ability. Biomacromolecules 2011, 12, 4071-4079. (23) Li, W.; Liu, Q.; Liu, L. Antifouling Gold Surfaces Grafted with Aspartic Acid and Glutamic Acid Based Zwitterionic Polymer Brushes. Langmuir 2014, 30, 12619-12626. (24) Liu, Q.; Singh, A.; Liu, L. Amino Acid-Based Zwitterionic Poly (Serine Methacrylate) as an Antifouling Material. Biomacromolecules 2012, 14, 226-231. (25) Liu, Q.; Li, W.; Singh, A.; Cheng, G.; Liu, L. Two Amino Acid-Based Superlow Fouling Polymers: Poly (Lysine Methacrylamide) and Poly (Ornithine Methacrylamide). Acta Biomater. 2014, 10, 2956-2964. (26) Li, W.; Liu, Q.; Liu, L. Amino Acid-Based Zwitterionic Polymers: Antifouling Properties and Low Cytotoxicity. J Biomater Sci Polym Ed 2014, 25, 1730-1742. (27) Li, W.; Liu, Q.; Zhang, P.; Liu, L. Zwitterionic Nanogels Crosslinked by Fluorescent Carbon Dots for Targeted Drug Delivery and Simultaneous Bioimaging. Acta Biomater 2016, 40, 254-262. (28) Low, P. S.; Kularatne, S. A. Folate-Targeted Therapeutic and Imaging Agents for Cancer. Curr Opin Chem Biol 2009, 13, 256-262. (29) Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nature 1973, 241, 20-22. (30) Khlebtsov, B. N.; Panfilova, E. V.; Terentyuk, G. S.; Maksimova, I. L.; Ivanov, A. V.; Khlebtsov, N. G. Plasmonic Nanopowders for Photothermal Therapy of Tumors. Langmuir 2012, 28, 8994-9002. (31) Alkilany, A. M.; Abulateefeh, S. R.; Mills, K. K.; Yaseen, A. I.; Hamaly, M. A.; Alkhatib, H. S.; Aiedeh, K. M.; Stone, J. W. Colloidal Stability of Citrate and Mercaptoacetic Acid Capped Gold Nanoparticles Upon Lyophilization: Effect of Capping Ligand Attachment and Type of Cryoprotectants. Langmuir 2014, 30, 13799-13808. (32) Wang, F.; Wang, Y.-C.; Dou, S.; Xiong, M.-H.; Sun, T.-M.; Wang, J. Doxorubicin-Tethered Responsive Gold Nanoparticles Facilitate Intracellular Drug Delivery for Overcoming Multidrug Resistance in Cancer Cells. ACS Nano 2011, 5, 3679-3692. (33) Thobhani, S.; Attree, S.; Boyd, R.; Kumarswami, N.; Noble, J.; Szymanski, M.; Porter, R. A. Bioconjugation and Characterisation of Gold Colloid-Labelled Proteins. J Immunol Methods 2010, 356, 60-69. (34) Jain, P. K.; Qian, W.; El-Sayed, M. A. Ultrafast Cooling of Photoexcited Electrons in Gold Nanoparticle-Thiolated DNA Conjugates Involves the Dissociation of the Gold-Thiol Bond. J. Am. Chem. Soc. 2006, 128, 2426-2433. (35) Xue, Y.; Li, X.; Li, H.; Zhang, W. Quantifying Thiol–Gold Interactions Towards the Efficient Strength Control. Nat. Commun. 2014, 5, 4348. (36) Häkkinen, H. The Gold–Sulfur Interface at the Nanoscale. Nature chemistry 2012, 4, 443-. (37) Liu, X.; Huang, H.; Liu, G.; Zhou, W.; Chen, Y.; Jin, Q.; Ji, J. Multidentate Zwitterionic Chitosan Oligosaccharide Modified Gold Nanoparticles: Stability, Biocompatibility and Cell Interactions. Nanoscale 2013, 5, 3982-3991. (38) Dixit, V.; Van den Bossche, J.; Sherman, D. M.; Thompson, D. H.; Andres, R. P. Synthesis and Grafting of Thioctic Acid− Peg− Folate Conjugates onto Au NanoparJcles for SelecJve TargeJng of Folate Receptor-Positive Tumor Cells. Bioconjugate Chem. 2006, 17, 603-609. 18


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(39) Nowinski, A. K.; White, A. D.; Keefe, A. J.; Jiang, S. Biologically Inspired Stealth Peptide-Capped Gold Nanoparticles. Langmuir 2014, 30, 1864-1870. (40) Dobrovolskaia, M. A.; Aggarwal, P.; Hall, J. B.; McNeil, S. E. Preclinical Studies to Understand Nanoparticle Interaction with the Immune System and Its Potential Effects on Nanoparticle Biodistribution. Mol Pharm 2008, 5, 487-495. (41) Liu, X.; Zhu, H.; Jin, Q.; Zhou, W.; Colvin, V. L.; Ji, J. Small and Stable Phosphorylcholine Zwitterionic Quantum Dots for Weak Nonspecific Phagocytosis and Effective Tat Peptide Functionalization. Adv Healthc Mater 2013, 2, 352-360.

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Figure Captions Scheme 1. Preparation of pOrn-GNPs and FA-pOrn-GNPs. Figure 1. (a) Hydrodynamic diameter and (b) Zeta-potential distribution of Citr-GNPs and pOrn-GNPs, measured by DLS. (c) TEM images of Citr-GNPs (left) and pOrn-GNPs (right). (d) UV−vis spectra of Citr-GNPs and pOrn-GNPs; the insets are optical images of Citr-GNP and pOrn-GNP solutions in water. Figure 2. UV-vis spectra of Citr-GNPs and pOrn-GNPs in water and PBS. Figure 3. Effect of lyophilization on stability of Citr-GNPs and pOrn-GNPs. After freeze-drying, Citr-GNPs and pOrn-GNPs were re-dispersed in water and PBS, respectively. Figure 4. (a) UV−vis spectra of pOrn-GNPs under various pH values. (b) Hydrodynamic diameter (nm) of pOrn-GNPs measured by DLS at pH=7.4 and pH=3. (c) Responsive behavior of pOrn-GNPs when switching pH between 2 and 9. (d) Switch of the plasmon resonance peak wavelength (λmax) of pOrn-GNPs by changing pH between 2 and 9. (e) UV−vis spectra of Citr-GNPs in water, under pH of 2, and under pH of 9. Figure 5. UV-vis spectra of pOrn-GNPs in NaCl solutions with different concentrations. Figure 6. UV-vis spectra of pOrn-GNPs in NaOH solutions with different concentrations. Figure 7. (a) UV-vis spectra of pOrn-GNPs after incubation in human blood serum for different periods of time. (b) Stability, as characterized by hydrodynamic size, of pOrn-GNPs and Citr-GNPs in human blood serum for 24 h. (c) Optical images of pOrn-GNPs (left) and Citr-GNPs (right) in human blood serum. For (a) and (b), all samples were centrifuged and reconstituted in PBS before measurement. Figure 8. UV-vis spectra of FA-pOrn-GNPs, pOrn-GNPs and folic acid (FA). Figure 9. ICP-OES measurements for cellular uptake of FA-pOrn-GNPs and pOrn-GNPs in SKOV3 and BAEC cells. Control samples did not have any nanoparticles in the culture medium. Figure 10. Viability of NIH/3T3 fibroblasts after treatment with pOrn-GNPs, FA-pOrn-GNPs, and Citr-GNPs for 24 hours.

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Scheme 1

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(a)

(b)

20

5

3.0x10

Citr-GNPs pOrn-GNPs

Citr-GNPs pOrn-GNPs

5

2.5x10 15

5

2.0x10

Intensity

Volumes (%)

10

5

1.5x10

5

1.0x10 5

4

5.0x10 0

0.0 1

10

100

1000

-80

-60

Size (nm)

(c)

-40

-20

0

20

Zeta potential (mV)

(d) 1.2

Absorbance (Normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Citr-GNPs pOrn-GNPs

Citr-GNPs

pOrn-GNPs

1.0 0.8 0.6 0.4 0.2 0.0 400

500

600

Wavelength (nm)

Figure 1

22


700

800

40

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Citr-GNPs-Water Citr-GNPs-PBS pOrn-GNPs-Water pOrn-GNPs-PBS

1.0 Absorbance (Normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8 0.6 0.4 0.2 0.0 400

500

600

700

Wavelength (nm)

Figure 2

23


800

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Citr-GNPs Citr-GNPs-Freeze-Dry pOrn-GNPs pOrn-GNPs-Freeze-Dry

1.0 Absorbance (Normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8 0.6 0.4 0.2 0.0 400

500

600

700

Wavelength (nm)

Figure 3

24


800

Langmuir

(b) 0.8

16

pH=1 pH=2 pH=3 pH=4 pH=5

0.7 0.6 0.5

pH=6 pH=7.4 pH=8 pH=9 pH=10 pH=11 pH=12

0.4 0.3 0.2 0.1

Orn-GNPs at pH=7.4 Orn-GNPs at pH=3

14

Volumes (%)

(a)

12 10 8 6 4 2 0

0.0

-2 400

500

600

700

10

800

100

Size (nm)

Wavelength (nm)

(c)

pH=9

(d)

pH=2

1.0 Cycle 1 Cycle 2 Cycle 3 Cycle 4

0.8

pH=2 pH=2 pH=2 pH=2

540

pH=9 pH=9 pH=9 pH=9

536

λ max (nm)

0.6 0.4

528

520

0.0 400

532

524

0.2

500

600

700

800

2

9

2

Wavelength (nm)

(e)

9

2

pH

Citr-GNPs (H2O)

0.55

Citr-GNPs (pH=2)

0.50

Citr-GNPs (pH change from 2 to 9)

0.45

Adsorbance

Absorbance(Normalized)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.40 0.35 0.30 0.25 0.20 0.15 0.10 400

500

600

700

800

Wavelength(nm)

Figure 4

25


9

2

9

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1.2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0M 0.2 M 0.5 M 1M 2M 5M Saturated

1.0 0.8 0.6 0.4 0.2 0.0 400

500

600

700

Wavelength (nm)

Figure 5

26


800

Langmuir

PBS 2% NaOH 5% NaOH 10% NaOH

1.0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8 0.6 0.4 0.2 0.0 400

500

600

700

Wavelength (nm)

Figure 6

27


800

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(a)

(b)

0h 1h 2h 8h 18 h 20 h 24 h

0.8 0.6

180

Citr-GNPs pOrn-GNPs

160

Hydrodymanic size (nm)

1.0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.4 0.2

140 120 100 80 60 40 20

0.0 400

0

500

600

700

800

0

5

Wavelength (nm)

10

15

Time (h)

(c)

Figure 7

28


20

25

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1.4 FA-pOrn-GNPs pOrn-GNPs FA

1.2 365 nm

1.0

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8 0.6 0.4 0.2 0.0 300

400

500

600

Wavelength (nm)

Figure 8

29


700

800

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7

SKOV3 BAEC

6

3

ng Au / 10 cells

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5 4 3 2 1 0 FA-pOrn-GNPs

pOrn-GNPs

Figure 9

30


Control

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20 µg/ml 50 µg/ml 200 µg/ml 500 µg/ml

120 100

Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0

pOrn-GNPs

FA-pOrn-GNPs

Figure 10

31


Citr-GNPs

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Table of Contents (TOC) Graphic

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Zwitterionic Gel Coating Endows Gold Nanoparticles with Ultrastability

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