Morphological Diversity, Protein Adsorption, and Cellular Uptake of

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Biological and Environmental Phenomena at the Interface

Morphological Diversity, Protein Adsorption and Cellular Uptake of Polydopamine-Coated Gold Nanoparticles Kwun Hei Samuel Sy, Lok Wai Cola Ho, Wilson Chun Yu Lau, Ho Ko, and Chung Hang Jonathan Choi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02572 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Morphological Diversity, Protein Adsorption and Cellular Uptake of Polydopamine-Coated Gold Nanoparticles Kwun Hei Samuel Sy,† Lok Wai Cola Ho,† Wilson Chun Yu Lau, ┴,ʭ,¶ Ho Ko, ‡, § Chung Hang Jonathan Choi*,†,ʭ,¶ Department of Biomedical Engineering, State Key Laboratory of Agrobiotechnology, ʭCentre

†

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for Cell and Developmental Biology, ¶School of Life Sciences, ‡Department of Medicine and Therapeutics, §Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

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ABSTRACT: Polydopamine (PDA)-coated nanoparticles are adhesive bionanomaterials widely utilized in intracellular applications, yet how their adhesiveness affects their colloidal stability and their interactions with serum proteins and mammalian cells remains unclear. In this work, we systematically investigate the combined effects of dopamine concentration and polymerization time (both reaction parameters spanning two orders of magnitude) on the morphological diversity of PDA-coated nanoparticles by coating PDA onto gold nanoparticle (AuNP) cores. Independent of DA concentration, Au@PDA NPs remain largely aggregated upon several hours of limited polymerization; interestingly, extended polymerization for 2 days or longer yield randomly aggregated NPs, nearly monodisperse NPs, or worm-like NP chains in ascending order of DA concentration. Upon exposure to serum proteins, the specific type of proteins adsorbed to the Au@PDA NPs strongly depends upon DA concentration. As DA concentration increases, less albumin and more hemoglobin subunits adhere. Moreover, cellular uptake is a strong function of polymerization time. Serum-stabilized Au@PDA NPs prepared by limited polymerization enter Neuro-2a and HeLa cancer cells more abundantly than those prepared by extended polymerization. Our data underscore the importance of dopamine concentration and polymerization time for tuning the morphology and degree of intracellular delivery of PDA-coated nanostructures.


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INTRODUCTION Polydopamine (PDA) is an emerging adhesive coating biomaterial derived from the in situ selfpolymerization of dopamine (DA) under alkaline conditions.1 DA is a structural analog of L-3,4dihydroxyphenylalanine (L-DOPA), the adhesive component of marine mussels.2-3 The strong adhesiveness of PDA facilitates its deposition onto the surface of nanoparticles (NPs), endowing the NPs with biocompatibility4 and the ability to enter mammalian cells in significantly more copious amounts than NPs coated by other types of biocompatible polymers, such as poly(ethylene glycol).5 In turn, the adhesive surface of PDA-coated NPs permits facile adsorption of biomolecules6 and their effective intracellular delivery to realize biomedical applications, such as molecular diagnostics,7 photothermal therapy,8 and receptor targeting.9 Nevertheless, the adhesiveness of PDA may increase the propensity of PDA-coated NPs to uncontrollably aggregate or agglomerate in water and cell culture medium,4, 10-11 hampering the experimental reproducibility of using PDA-coated NPs and their intracellular delivery of the NPs.12-13 While one may mitigate the aggregation of PDA-coated NPs in culture medium by stabilizing them with serum proteins,14 little is known about the nature of the protein corona surrounding PDA-coated NPs, an important knowledge gap to be filled because the adsorbed proteins influence colloidal stability15 and intracellular delivery of NPs in general.4, 16-17 Note that, depending on the surface coating, the adsorbed serum proteins may assist18 or obstruct5 the cellular uptake of NPs. Moreover, the composition of the serum proteins adsorbed to adhesive NPs like PDA-coated NPs may differ from those adsorbed to non-adhesive NPs.19-20 Our understanding in the interplay between aggregation, serum adsorption, and cellular uptake of PDA-coated NPs remains incomprehensive. A major challenge in systematically probing the “bio-nano” interactions of PDA-coated NPs lies in their inconsistent preparation methods. According to published protocols for coating PDA onto 3 ACS Paragon Plus Environment

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different substrates (e.g., bulk glass, bulk polymer, and inorganic NPs), the DA concentration used spans almost six orders of magnitude on a per unit substrate area basis, yet polymerization time typically does not exceed 24 h (Figure 1A and Table S1-2).1, 4, 7, 21-29 Currently, it remains unclear how polymerization time and initial DA concentration affect the depletion of DA monomers in the PDA coating during polymerization. Based on published structural models of PDA, polymerization of DA predominantly leads to the oxidation of amines to indoles and oxidation of diphenols to diquinones (Figure S1).30,31 Interestingly, Maier et al. previously suggested that the close proximity between the amine and catechol groups of L-DOPA synergistically contributes to the strong adhesiveness of mussel foot proteins.32 Rapp et al. also proved that the interaction between catechol and cationic groups in general (not restricted to amines) underpin the strong molecular adhesion.33 By considering these empirical evidence and structural models altogether, one may anticipate that the depletion of amines and catechols of DA during extended polymerization may influence the adhesiveness of the resultant PDA coating. Therefore, the aggregation or agglomeration of PDA-coated NPs depends on a balance between two opposing forces, (1) the electrostatic repulsion between the negatively charged PDA-coated NPs (to be verified in Table 2 below) and (2) the molecular adhesion contributed by the amine and catechol groups in the PDA coating. We hypothesize that polymerization time and DA concentration are two critical reaction parameters that govern the colloidal stability of PDA-coated NPs in water and culture medium. Such colloidal stability will, in turn, influence their adsorption by serum proteins and subsequent uptake by mammalian cells. In this work, we will investigate the “bio-nano” interactions of PDA-coated NPs at the culture medium, serum protein, and cellular levels. We choose gold nanoparticles (AuNPs)34 as the core substrate for the PDA coating, because the plasmon coupling effects due to close proximity of NPs 4 ACS Paragon Plus Environment

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permit colorimetric reporting of their state of aggregation or agglomeration.35 In this connection, we will prepare a collection of Au@PDA NPs by introducing varying amounts of DA into 50 pM of citrate-capped AuNPs of 30 nm in diameter (Figure S2 and Table S3) and allowing the polymerization reaction to proceed for different durations of time (Figure 1B). Both reaction parameters can be precisely controlled during polymerization, allowing us to derive insights into the design and biological applications of PDA-coated nanoparticles. All polymerization reactions take place in a moderately alkaline Tris buffer (pH = 8.5) to facilitate effective formation of the PDA coating, in agreement with the PDA literature.1,4,7-9 Recent studies further reveal that a moderately alkaline environment (with pH between 8 and 10) minimizes the detachment of PDA coating from Au substrates.36 To explore the design parameter space, both polymerization time and DA concentration will span almost two orders of magnitude in our ensuing investigations. We choose three relatively low DA concentrations (0.5 µg/mL, 20 µg/mL, and 65 µg/mL) when benchmarked against previously reported recipes for coating PDA, on the basis of DA per unit surface area (Table S1 and S2). Specifically, 20 µg/mL represents the optimal concentration to attain a thin PDA coating around AuNPs barely detectable by transmission electron microscopy (TEM) (to be demonstrated in Figure 2D). We will also address the two cases of incomplete PDA coating (0.5 µg/mL) and a thick PDA coating (65 µg/mL). To provide sufficient time for depletion of DA, our four chosen polymerization times will capture the extreme cases of limited polymerization (1 h and 6 h) and extended polymerization (48 h and 96 h). With this set of Au@PDA NPs, we will examine the roles of DA concentration and polymerization time on the (1) colloidal stability of Au@PDA NPs in water and culture medium (both serum-free and serumcontaining), (2) amounts and identities of serum proteins adsorbed onto the Au@PDA NPs, and


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(3) uptake of Au@PDA NPs by mammalian cells (Neuro-2a mouse neuroblastoma and HeLa human cervix adenocarcinoma) in serum-free and serum-containing medium.

Figure 1. Design space of the reaction parameters for coating PDA on AuNPs. (A) Summary of DA concentration per unit substrate surface area against polymerization time based on the preceding PDA literature (triangles) and this work (circles). The coating solutions of DA for different substrates are of comparable volumes (~mL). In case the same report contains multiple 6 ACS Paragon Plus Environment

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recipes, the average DA concentrations, coating surface areas, and polymerization times are chosen. Numbers in square brackets denote the references cited. (B) Schematic diagram for preparing the collection of PDA-coated gold nanoparticles (Au@PDA NPs) by using three different DA concentrations and four different polymerization times. EXPERIMENTAL SECTION Synthesis of Au@PDA NPs. 6 mL of 0.1 nM citrate-capped AuNPs were mixed with 0.6 mL of 0.01 mg/mL, 0.4 mg/mL, or 1.3 mg/mL of dopamine-hydrochloride (Aladdin) in 5.4 mL of 10 mM Tris buffer (pH = 8.5) for 1 h, 6 h, 48 h, or 96 h. Resultant concentration of citrate-capped AuNPs, three doses of dopamine-hydrochloride and Tris buffer are 0.05 nM, 0.5 µg/mL, 20 µg/mL, 65 µg/mL and 4.5 mM respectively. The reaction mixture was sonicated at 20 kHz (Branson) to maintain dispersity of the Au@PDA NPs. The as-coated Au@PDA NPs were centrifuged (Eppendorf) at 13 500 rpm for 10 min and resuspended in Nanopure water twice. UV-Vis Spectrometry. The absorption spectrum was measured by a Cary 6000 UV-Vis-NIR spectrophotometer. 1.5 mL of 0.1 nM Au@PDA NP solution [in water, DMEM without phenol red (Thermo Fisher), or DMEM without phenol red containing 10% fetal bovine serum (Gibco)] was loaded into a cuvette (Guanglianggaoke, China) for analysis. DLS. The hydrodynamic diameters of Au@PDA NPs (in water, PBS, or FBS-containing DMEM) were measured by dynamic light scattering (DelsaMax PRO, Beckman Coulter). All reported hydrodynamic sizes represent the “Z-average” values from three independent measurements. The ζ-potentials of NPs were measured by dynamic light scattering (DelsaMax PRO, Beckman Coulter) under a constant background of 1 mM KCl. Briefly, 100 mL of NPs in a 0.1 M stock solution was centrifuged and resuspended in 0.1 mL of 1 mM KCl, and the resultant solution was measured. 7 ACS Paragon Plus Environment

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Cryo-EM. 2.5 μL of 0.1 nM Au@PDA NPs were applied to Quantifoil grids (Electron Microscopy Sciences; EMS) that were glow-discharged in air for 15 s. The grids were blotted in a FEI Vitrobot for 0.5 s with ~100% relative humidity at 4 °C before plunge freezing in liquid ethane. Grids were imaged with a FEI Tecnai F20 electron microscope operating at 200 kV at 50 kX nominal magnification. Images were acquired with a 4k × 4k FEI Eagle CCD camera with an electron exposure of ~20 electrons/Å 2. Defocus values were varied between 2 and 4 μm. Serum Protein Adsorption and Desorption. 1 mL of Au@PDA NPs (0.1 nM) were centrifuged and resuspended in 250 µL of complete DMEM (Gibco) [i.e., DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco)] to result in 0.25 nM Au@PDA NPs and incubated for 1 h at 37 °C to allow for protein adsorption. Next, the serum-coated Au@PDA NPs were washed to remove unbounded serum proteins by three rounds of centrifugation (13,500 rpm at 4°C) and resuspension in PBS containing 0.05% v/v Tween20 (Sigma). Equal volumes of serum without NPs were treated in parallel as a control for non-specific protein adsorption. The washed NPs were pelleted by centrifugation. The supernatant was discarded, leaving ~30 µL of the NP pellet. 10 µL of 4X Laemmli sample buffer (Bio-Rad) was then added to the NP pellet, followed by incubation at 70 °C for 1 h with shaking to release the adsorbed proteins. The NPs were removed by centrifugation at 13,500 rpm at 4°C. The supernatant containing the desorbed proteins were transferred to a new tube. PAGE. 0.5 µL of mercaptoethanol (≥ 99.0%, Sigma) was added to 40 µL of the desorbed proteins in Laemmli sample buffer. The mixture was heated at 95 °C for 5 min with shaking. 6 µL of the denatured proteins, along with 6 µL of Precision Plus Protein Dual Color Standards (BioRad), was loaded onto a 4-20% mini-PROTEAN TGX Precast Protein Gradient Gel (Bio-Rad) in Tris-glycine SDS running buffer (Bio-Rad). After resolving the bands by electrophoresis at 200 V


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for 55 min, the gel was fixed with 40% (v/v) ethanol and 10% (v/v) acetic acid in water for 2 h, and stained with the Flamingo Fluorescent Gel Stain (Bio-Rad) for 3 h. The stained bands were visualized by the Chemi-Doc Touch gel imaging system (Bio-Rad). Purification of the Desorbed Proteins. 40 µL of the desorbed proteins was precipitated by adding 950 µL of 10% w/v trichloroacetic acid (TCA, Sigma) in acetone followed by overnight incubation at -80 °C. Precipitates were pelleted by centrifugation (18,000 rcf for 15 min at 4 °C) and the supernatant was discarded. The pellet was dissolved in 500 µL of 0.03% w/v sodium deoxycholate in water and precipitated by adding 100 µL of 72% w/v TCA in acetone followed by incubation for 30 min on ice. The precipitate was pelleted by centrifugation (18,000 × g for 15 min at 4 °C) and the supernatant was discarded. The pellet was washed once in 1 mL of acetone at -20 °C for 30 min, left to dry in the fume hood, and finally dissolved in 50 µL of 50 mM ammonium bicarbonate (Thermo Scientific) in water. Methods of protein digestion, LC-MS/MS and protein identification are provided in the Supporting Information. Protein Concentration. 25 µL of the purified desorbed proteins or different concentrations of serially diluted bovine serum albumin (BSA, Pierce) were transferred into a 96-well plate. Next, 200 µL of freshly prepared BCA Protein Assay working reagent (Pierce) was added to each well. The samples were incubated at 60 °C for 30 min. Absorbance at 562 nm was measured by a Multiskan GO UV-absorbance microplate reader (Thermo Scientific). Concentrations of the purified proteins were calculated according to the BSA standards. Cellular Uptake. Neuro-2a (mouse neuroblastoma; ATCC) cells and HeLa (human cervix adenocarcinoma; ATCC) cells were cultured in complete DMEM (Gibco) [i.e., DMEM supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco)] and maintained at 37 °C and 5% CO2. Before the uptake experiment, cells were seeded in 24-well plates until the cell


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population reached ~80% confluence. The cells were incubated with 0.25 nM NPs formulated in 0.2 mL of complete DMEM per well for 1 h. The short time interval allows us to disregard the effect of gravitational sedimentation.37 After the treatment, the NPs were removed, and the cells were rinsed twice with PBS, trypsinzed for cell counting by a hemacytometer. Cell pellets were collected by centrifugation at 4000 rpm for 5 min. ICP-OES. Cell pellets were digested by 0.25 mL of aqua regia overnight at RT, and further diluted to 10 mL by the matrix solution (2% HCl, 2% HNO3, with 10 ppb indium as internal standard) for ICP-OES measurements (Optima 43000DV, PerkinElmer). The number of AuNPs in each sample was obtained by computing the difference in gold content between the NP-treated and untreated cells based on the calibration curves derived from AuNP solutions of known concentrations. Reported data represent mean ± SD from three independent experiments. TEM. To visualize the morphologies of the Au@PDA NPs, 10 μL of 0.05 nM of Au@PDA NPs was blotted on TEM copper grids (200 mesh; Beijing Zhongjingkeyi Technology. The dried grids were imaged under a Techni TS12 electron microscope (FEI) at a beam voltage of 120 kV. To visualize the subcellular distribution of the Au@PDA NPs, freshly harvested cell pellets were fixed with glutaraldehyde (2.5% in phosphate buffer; pH = 7.2–7.4) for 2 h and stained by osmium tetroxide (1%) for another 2 h. Pellets were gradually dehydrated in increasing ethanol gradients and propylene oxide. They were embedded in Epon 812 resins (EMS) and polymerized at 55 °C for 48 h. Ultrathin sections of ~70 nm in thickness were deposited onto 200-mesh copper grids (EMS) and stained with 4% uranyl acetate (EMS, in 50% methanol/water) and Reynolds lead citrate (Sigma) for observation under a Hitachi H7700 TEM at a beam voltage of 100 kV.


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RESULTS AND DISCUSSION Colorimetric Analysis of Au@PDA NPs. Figure 2A portrays the palette of colors of our collection of Au@PDA NPs. Contrary to the reddish color for unmodified citrate-capped AuNPs in water, the color of the Au@PDA NP solution changes with polymerization time from pale to more solid colors, for all three DA concentrations considered. In the case of insufficient DA, the color changes only slightly across polymerization time from almost colorless after several hours of limited polymerization to faintly colored following longer polymerization of 48 h or longer. With optimal amounts of DA added, the color of Au@PDA NPs appears blue after 1 h of polymerization, purple after 6 h, and almost red after 48 h and 96 h. As the red color is similar to that of monodisperse, spherical citrate-capped AuNPs, this change of color across polymerization time suggests the importance of polymerization time on the colloidal stability of Au@PDA NPs. Finally, with excess amounts of DA added, the color of Au@PDA NPs is almost colorless upon limited polymerization but becomes dark blue upon extended polymerization. Such blue color resembles that of non-spherical, gold nanorods of small aspect ratios,38 as opposed to the red color of Au@PDA NPs prepared by using optimal amounts of PDA. By using slightly different polymerization times and DA concentrations than those used to prepare the Au@PDA NPs featured in Figure 2A, we reproduced the same palette of colors for our collection of Au@PDA NPs (Figure S3A). The data suggest the role of polymerization time (besides DA concentration) in governing the colloidal stability (or morphology) of Au@PDA NPs.


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Morphological Diversity of Au@PDA NPs. Motivated by the palette of colors observed, we next characterized the morphologies and physicochemical properties of our family of Au@PDA NPs as a function of initial DA concentration and polymerization time. Case I: Insufficient DA concentration: When insufficient concentrations of DA (0.5 µg/mL) are added to 50 pM of 30 nm citrate-capped AuNPs, we observed, by ultraviolet-visible (UV-vis) spectrometry, the disappearance of the plasmon peak of AuNPs at 520 nm and enhancement in the absorption at near-infrared wavelengths after 1 h and 6 h limited polymerization (Figure 2B). TEM images at lower magnifications portray clusters of Au@PDA NPs stacked together to form spatially unresolvable aggregates over expansive fields of view of 2 µm x 2 µm (Figure 2C) and 4 µm x 4 µm (Figure S4). This observation likely stems from the limited DA available in the polymerization reaction for yielding a PDA coating that can adequately protect the AuNPs from aggregation induced by DA (a salt), and the removal of citrate ions from the AuNP cores after multiple rinses by centrifugation. Higher-magnification TEM images do not reveal detectable PDA shell on the surface of AuNPs (Figure 2D). By DLS analysis, the mean hydrodynamic diameter of the Au@PDA NPs is 86 nm in water upon limited polymerization (Table 1), and their surface charge is weakly negative (Table 2). The apparent discrepancy in the size of the NP aggregates between our TEM and DLS data may be due to “drying effects” typically associated with NPs that are drop-cast onto the copper sample grid for TEM imaging, noting that DLS measurements were conducted in solution. In spite of the minute amounts of DA added for limited polymerization, the Fourier-transform infrared (FT-IR) spectrum of the Au@PDA NPs shows the disappearance of the characteristic peaks of monomeric DA in the range of 600-1200 cm-1 in conjunction with the emergence of bands at 3410 cm-1 (stretching vibration of phenolic O-H and N-H), 1605 cm-1 (stretching vibration of aromatic ring and bending vibration of N-H), and 1510 cm-1 (stretching 12 ACS Paragon Plus Environment

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vibration of phenolic C-O) (Figure S5A and S5B), indicating successful deposition of the PDA coating in accordance to the literature precedent.7, 39-41. Upon extended polymerization for 48 h or longer, these Au@PDA NPs still remain aggregated without any discernible PDA coating on the AuNP surface. Their UV-vis spectra depict the recovery of a small plasmon peak indicative of citrate-capped AuNPs at 525 nm, yet their strong near-infrared absorption still persists. By dynamic light scattering (DLS) analysis, the hydrodynamic size and zeta potential of the Au@PDA NPs prepared by extended polymerization are not much different than those of the Au@PDA NPs prepared by limited polymerization (Table 1 and 2). Additionally, the FT-IR spectra of the Au@PDA NPs prepared by limited and extended polymerization are largely consistent, reaffirming the consumption of DA (Figure S5B). In short, without sufficient amounts of DA added, the inadequately coated Au@PDA NPs irreversibly form large aggregates in as fast as 1 h of limited polymerization, and remain aggregated upon extended polymerization. Case II: Optimal DA concentration: In the second case when optimal amounts of DA are added (20 µg/mL) to 50 pM of 30 nm citrate-capped AuNPs, upon limited polymerization for 1 h or 6 h, the optical properties of the resultant Au@PDA NPs are characterized by the emergence of a secondary shoulder in the near-infrared region (at 650-700 nm) besides the primary surface plasmon resonance peak at 525 nm characteristic of unmodified AuNPs (Figure 2B). TEM imaging reveals pronounced aggregation of the NPs without evident formation of PDA shells due to aggregation induced by both DA (with its cationic amine group) and Tris salt in the reaction buffer (Figure 2C and S4). By DLS measurements, the average hydrodynamic size of the Au@PDA NPs is 261 nm after 1 h of reaction, a size larger than that obtained for Case I that reflects the initial addition of a higher concentration of DA (Table 1). The zeta potential of the Au@PDA NPs is ~17 mV, indicating an overall negative surface charge (Table 2). Provided limited polymerization 13 ACS Paragon Plus Environment

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and optimal amounts of DA, the resultant Au@PDA NPs are still largely aggregated without obvious spatial separation between the adjacent NPs. With extended polymerization for 48 h or 96 h, there is only one single absorption peak without any near-infrared shoulder (Figure 2B), a result corroborating the almost-red color observed and suggesting low polydispersity of the Au@PDA NPs (Figure 2A). Low-magnification TEM images show that the degree of aggregation declines with polymerization time (Figure 2C and S4). Upon extended polymerization, we can no longer detect micron-sized clusters of Au@PDAs NPs; instead, the TEM images illustrate predominantly isolated NPs and small clusters each consisting of no more than 10 NPs. High-magnification TEM images further reveal Au@PDA NPs with an intact PDA shell of 1-2 nm in thickness (Figure 2D). By DLS analysis, extended polymerization for 96 h led to a sharp decrease in the mean hydrodynamic diameter of Au@PDA NPs from 127 nm to 35 nm (Table 1), indicating the majority of the Au@PDA NPs prepared by extended polymerization is almost monodisperse. In terms of surface charge, the Au@PDA NPs become less negative upon extended polymerization (~-7 mV; Table 2). Finally, in agreement with the literature precedent, our FT-IR analysis of the PDA@AuNPs confirms the coating of PDA onto the AuNPs (Figure S5C).7, 39-41 In brief, provided optimal amounts of DA added, the Au@PDA NPs become less polydispersed upon extended polymerization. DA-induced aggregation becomes less prevalent, and the PDA coating on the AuNPs less adhesive. These data are in stark contrast to those for Case I, in which the Au@PDA NPs remain irreversibly clustered even after 96 h of extended reaction. Case III: Excess DA concentration: Finally, we addressed the scenario in which excess amounts of DA (65 µg/mL) are initially added to 50 pM of 30 nm citrate-capped AuNPs. Upon limited polymerization for 1 h or 6 h, UV-vis spectrometry reveals significant near-infrared absorption without a sharp plasmon peak at 525 nm (Figure 2B), echoing the colorless solution of the 14 ACS Paragon Plus Environment

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Au@PDA NPs and indicating NP aggregation (Figure 2A). While our TEM imaging data reveals aggregated Au@PDA NPs, we observed evident deposition of a PDA shell of 2-3 nm on the surface of the AuNPs, consistent with our addition of excess amounts of DA (Figure 2D). With higher amounts of DA initially added, DLS analysis shows that the mean hydrodynamic size of these NPs is 179 nm after 1 h of polymerization, larger than those achievable in Cases I and II (Table 1). This result highlights the most severe colloidal aggregation mediated by limited polymerization and the highest concentration of DA added. By DLS analysis, the Au@PDA NPs are negative in surface charge (~30 mV) upon 6 h of limited polymerization (Table 2), the most negatively charged sample in our collection of Au@PDA NPs. Interestingly, upon extended polymerization for 48 h, the solution of Au@PDA NPs switches from colorless to intensely blue (Figure 2A), accompanied by the emergence of a prominent and well-defined secondary absorption peak at 660 nm (Figure 2B). Strikingly, by low-magnification TEM imaging, we observed the assembly of “worm-like” NP chains comprised by interconnected Au@PDA NPs rather than aggregated clusters at the micrometer length scale (Figure 2C and S4). To exclude the possibility of drying effects associated with conventional TEM imaging, we performed cryo-EM imaging to confirm that these Au@PDA NPs indeed natively exist as “worm-like” chains in an aqueous solution (Figure 2E and S6), contrary to randomly oriented NP aggregates. DLS analysis reflects a drastic plunge in hydrodynamic diameter from 367 nm to 139 nm after 96 h of polymerization (Table 1). (While the DLS sizing data for such long chains may not be technically valid as DLS analysis typically assumes a near-spherical geometry of the NP to be measured, we believe that the 3-fold decline in size qualitatively suggests less severe aggregation of the NPs.) The Au@PDA NPs remain negatively charged upon 96 h of extended polymerization (Table 2), although their surface charge is slightly less negative (-12 mV) than that of the Au@PDA NPs prepared by


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limited polymerization (-30 mV). Moreover, because PDA does not absorb light in the nearinfrared region,42 the secondary plasmon peak at 645 nm appearing after 48 h of polymerization originates from the assembly of the Au@PDA NPs to form chains, in close resemblance to the plasmon peak arisen from the longitudinal mode of gold nanorods. After 96 h of polymerization, the Au@PDA NPs still self-assemble into “worm-like” chains, but the blue-shift of the secondary plasmon peak from 645 nm to 624 nm suggests shortening of the NP chains.43-44 High magnification TEM images reveal a PDA shell of 5-6 nm in thickness coated on the AuNPs after 96 h of reaction (Figure 2D), underscoring the effect of extended polymerization on the growth of the PDA shell. With the addition of higher concentrations of DA (in the form of DA hydrochloride salt) to unmodified citrate-capped AuNPs, we rationalize that the DA and/or HCl salt may trigger the aggregation of AuNPs, thus reducing the effective AuNP surface area available for the PDA coating and increasing the thickness of the resultant PDA shell. Finally, FT-IR analysis confirms the formation of the PDA shell on the AuNPs after 1 h and 96 h of polymerization (Figure S5D).7, 39-41

In summary, we have demonstrated the spontaneous self-assembly of PDA-coated AuNPs to

form “worm-like” NP chains. Our facile PDA-based approach is notable for (i) producing NP chains45-46 at a similar length scale with other previously reported methods47-49 and (ii) not requiring the aid of external forces or templates.50 Investigations into the mechanism of forming these chains are underway, but past related work on the assembly of chains comprised by AuNPs highlighted the involvement of short-range dipole-dipole attractions and long-range electrostatic charge repulsion between the NPs. 45, 51-52


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Table 1. Hydrodynamic Sizes of Au@PDA NPs as a Function of Initial DA Concentration and Polymerization Time in Water Hydrodynamic size in water (nm)a 1h 6h 48 h 96 h

0.5 µg/mL DA

20 µg/mL DA

65 µg/mL DA

86.03 ± 1.13 (0.232) 51.60 ± 0.57 (0.484) 73.10 ± 0.78 (0.238) 56.83 ± 0.38 (0.242)

127.40 ± 1.10 (0.232) 81.90 ± 1.59 (0.239) 36.70 ± 0.36 (0.236) 34.57 ± 0.12 (0.235)

179.23 ± 1.31 (0.234) 152.13 ± 2.17 (0.236) 250.47 ± 0.77 (0.232) 139.20 ± 0.41 (0.175)

a

Reported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refers to polydispersity index (PDI).

Table 2. Zeta Potentials of Au@PDA NPs as a Function of Initial DA Concentration and Polymerization Time in 1 mM KCl ζ-potential in 1 mM KCl (mV)a 1h 6h 48 h 96 h a

0.5 µg/mL DA

20 µg/mL DA

65 µg/mL DA

-12.02 ± 0.45 -8.69 ± 0.54 -17.99 ± 1.47 -7.78 ± 0.69

-17.59 ± 1.40 -15.88 ± 0.96 -5.26 ± 0.76 -8.32 ± 2.09

-27.76 ± 6.30 -30.94 ± 0.55 -22.78 ± 0.77 -12.81 ± 0.68

Reported data represent mean ± SD from three independent measurements.


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Figure 2. Collodial properties of Au@PDA NPs in water as a function of DA concentration and polymerization time. (A) Color of the Au@PDA NPs prepared by using different DA concentrations and polymerization times. (B) UV-vis spectra and (C) representative low18 ACS Paragon Plus Environment

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magnification TEM images of the collection of Au@PDA NPs. (D) Representative highmagnification TEM images of the Au@PDA NPs prepared by using three different initial DA concentrations after 1 h or 96 h of polymerization. (E) A representative cryo-EM image of the Au@PDA NPs prepared by using excess amounts of DA (65 µg/mL) and extended polymerization (96 h) shows their native “worm-like” chain morphology in water.

Physicochemical Characterization of Au@PDA NPs in Serum-Free Culture Medium. For intracellular applications, it is critical to address the colloidal stability of Au@PDA NPs in culture medium. To start with, we measured the physicochemical properties of our collection of Au@PDA NPs in serum-free culture medium. (We shall defer our analysis on the colloidal stability of the same series of Au@PDA NPs in serum-containing culture medium in the next section.) Upon incubation in serum-free Dulbecco’s Modified Eagle Medium (DMEM) devoid of phenol red at 37 °C for 1 h, we observed that the Au@PDA NPs prepared by using inadequate amounts of DA (0.5 µg/mL) becomes pale for all polymerization times analyzed (Figure 3A). Such pale color suggests aggregation possibly induced by both DA and salt in the culture medium, in line with their color in water due to incomplete coverage of the AuNP surface with the PDA coating. For the Au@PDA NPs prepared by using optimal (20 µg/mL) and excess (65 µg/mL) amounts of DA, the color of the NPs generally appears more pale for all polymerization times considered, contrary to their respective colors in water [i.e., sharp red for optimal amounts (Case II) and blue for excess amounts (Case III) after extended polymerization]. By DLS analysis, the hydrodynamic diameters of most members of the collection of Au@PDA NPs exceed 1 μm, indicating the formation of micron-sized aggregates in serum-free culture medium (Table 3). The two exceptions are those Au@PDA NPs prepared by using excess DA (65 µg/mL) and extended polymerization (48 h and 19 ACS Paragon Plus Environment

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96 h), with their hydrodynamic sizes falling in the sub-μm range (500-880 nm). Consistent with the DLS data, the UV-vis spectra of most members of the collection reveal depression or disappearance of the plasmon peak characteristic of citrate-capped AuNPs (~520 nm) (Figure 3B), another indicator of severe aggregation of Au@PDA NPs in serum-free culture medium. Interestingly, the UV-vis spectra of those Au@PDA NPs prepared by using excess amounts of DA and extended polymerization still exhibit a secondary plasmon peak around 650 nm, in agreement with the secondary plasmon peak observed when the same NPs are dispersed in water. By and large, our data suggest that Au@PDA NPs suffer from severe salt-induced aggregation in serumfree culture medium, for most polymerization times and DA concentrations tested. The Au@PDA NPs made by using extended polymerization and excess amounts of DA are still largely unstable in serum-free culture medium, yet less aggregated than other members in the collection.

Table 3. Hydrodynamic Sizes of Au@PDA NPs as a Function of Initial DA Concentration and Polymerization Time in Serum-Free Culture Medium Hydrodynamic size in serum-free culture medium (nm)a 1h 6h 48 h 96 h

0.5 µg/mL DA

20 µg/mL DA

65 µg/mL DA

1379.83 ± 10.44 (0.157) 1551.33 ± 37.13 (0.166) 1600.47 ± 48.22 (0.157) 1488.97 ± 40.14 (0.160)

1529.10 ± 65.09 (0.116) 1045.60 ± 232.87 (0.176) 1076.13 ± 365.94 (0.190) 808.97 ± 264.41 (0.235)

1097.63 ± 257.89 (0.330) 1025.67 ± 130.93 (0.035) 524.70 ± 16.41 (0) 727.10 ± 161.20 (0.286)

a



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Physicochemical Characterization of Au@PDA NPs in Serum-Containing Culture Medium. Given the adhesiveness of PDA-coated NPs and their instability in serum-free culture medium, we hypothesized that adsorption of serum proteins may stabilize the Au@PDA NPs in culture medium. We incubated our collection of Au@PDA NPs in DMEM containing 10% fetal bovine serum (FBS), devoid of phenol red, for 1 h at 37 °C. The colors of the Au@PDA NPs in serum-containing culture medium do not differ significantly from those in water. Specifically, the colors appear to be pale, red, and blue following the addition of inadequate, optimal, and excess amounts of DA for extended polymerization, respectively (Figure 3C). The UV-vis spectra of the serum-coated Au@PDA NPs are very similar to those for the same collection of NPs in water. In particular, we also detected the primary plasmon peak characteristic of citrate-capped AuNPs around 520 nm for those Au@PDA NPs prepared by using optimal amounts of DA and extended polymerization. Likewise, UV-vis spectrometry clearly shows the emergence of the secondary plasmon peak near 650 nm for those Au@PDA NPs prepared by using excess DA and extended polymerization (Figure 3D). By DLS measurements (Table 4), we observed two trends for the sizes of the Au@PDA NPs in serum-containing culture medium that corroborate those observed for the same collection of Au@PDA NPs in water: (1) smaller hydrodynamic sizes following extended polymerization for 2 d or longer and (2) larger hydrodynamic sizes due to the addition of the highest amounts of DA (65 µg/mL). For all DA concentrations and polymerization times tested, we observed an increase in hydrodynamic size of the Au@PDA NPs by up to 200 nm as a consequence of switching from water to serum-containing culture medium, suggesting the formation of protein corona on the NPs. Yet, our DLS data do not reveal a clear trend in the magnitude of increase in hydrodynamic sizes as a function of DA concentration and polymerization time. Importantly, we did not detect the formation of micron-sized aggregates of


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Au@PDA NPs in serum-containing culture medium, contrary to our observation for the same collection of NPs in serum-free culture medium. Taking the colorimetric, DLS, and UV-vis data altogether, introducing serum into the culture medium boosts the colloidal stability of our collection of Au@PDA NPs, protecting them from salt-induced aggregation. Apart from hydrodynamic size, we further measured the surface charge of the serum-coated Au@PDA NPs by DLS (Table 5). To do so, we incubated the set of Au@PDA NPs in serum-containing DMEM for 1 h, removed the excess unbound proteins, rinsed the NPs with phosphate buffered saline (PBS), and resuspended the serum-coated NPs in 1 mM KCl. Independent of DA concentration and polymerization time, the zeta potentials of our serum-coated Au@PDA NPs are negative, hovering between -15 mV and -9 mV. In short, the Au@PDA NPs remain largely negative in charge before and after incubation in serum-containing culture medium, although the range of zeta potentials for those Au@PDA NPs not incubated with serum is considerably wider (from -30 mV to -8 mV; Table 2) than that of their serum-coated counterparts.


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Figure 3. Effect of serum on the colloidal stability of Au@PDA NPs in cell culture medium. (A) Colors and (B) UV-vis absorption spectra of the collection of Au@PDA NPs after incubation in serum-free DMEM (without phenol red) at 37 °C for 1 h. (C) Colors and (D) UV-vis absorption spectra of the collection of Au@PDA NPs after incubation in serum-containing DMEM (without phenol red) at 37 °C for 1 h. 23 ACS Paragon Plus Environment

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Table 4. Hydrodynamic Sizes of Au@PDA NPs as a Function of Initial DA Concentration and Polymerization Time in Serum-Containing Culture Medium Hydrodynamic size in serum-containing culture medium (nm)a 1h 6h 48 h 96 h

0.5 µg/mL DA

20 µg/mL DA

65 µg/mL DA

124.20 ± 0.33 (0.236) 124.63 ± 1.97 (0.237) 102.73 ± 1.25 (0.157) 91.73 ± 0.78 (0.216)

130.27 ± 1.51 (0.203) 112.57 ± 0.68 (0.176) 84.17 ± 0.78 (0.173) 82.37 ± 0.82 (0.162)

276.57 ± 6.33 (0.571) 347.47 ± 6.35 (0.213) 276.00 ± 1.99 (0.204) 158.37 ± 1.37 (0.142)

a


Table 5. Zeta Potentials of Au@PDA NPs as a Function of Initial DA Concentration and Polymerization Time in 1 mM KCl after Incubation in Serum-Containing Medium ζ-potential in 1 mM KCl (mV)a 1h 6h 48 h 96 h a

0.5 µg/mL DA

20 µg/mL DA

65 µg/mL DA

-12.70 ± 2.16 -10.76 ± 2.69 -9.16 ± 1.86 -14.18 ± 2.34

-11.95 ± 2.89 -10.75 ± 1.66 -11.74 ± 2.38 -12.47 ± 1.89

-13.28 ± 1.91 -15.12 ± 1.72 -12.49 ±1.59 -14.97 ± 2.40

Reported data represent mean ± SD from three independent measurements.

Nature of the Protein Corona Surrounding the Au@PDA NPs. To explore the effects of DA concentration and polymerization time on the quantity and identity of the adsorbed proteins, we shortlisted the Au@PDA NPs prepared by using all three DA concentrations and two extreme polymerization times of 1 h (limited polymerization) and 96 h (extended polymerization). To obtain a qualitative overview of the adsorbed proteins onto the Au@PDA NPs, we isolated and denatured the adsorbed proteins, following by separating them based on their molecular weights 24 ACS Paragon Plus Environment

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by polyacrylamide gel electrophoresis (PAGE) coupled with a fluorescent protein stain (Figure 4A). For the NP-free control sample, the gel lane shows no detectable bands, confirming the lack of protein adsorption in the absence of NPs. By contrast, the lanes for all six types of Au@PDA NPs indicate the presence of adsorbed proteins, and their band patterns reveal multiple kinds of proteins. There are two notable sets of bands with molecular weights of 10-15 kDa and ~70 kDa. For both polymerization times considered, the ~70 kDa bands for the insufficient DA case (0.5 µg/mL) are significantly more intense than those for the optimal DA case (20 µg/mL) and excess DA case (65 µg/mL). Moreover, the 10-15 kDa bands for the excess DA case are noticeably stronger than those for the insufficient and optimal DA cases, a result more apparent upon extended polymerization (96 h) than limited polymerization (1 h). Next, we quantified the total amount of adsorbed proteins for the six shortlisted types of Au@PDA NPs by using the bicinchoninic acid (BCA) assay (Figure 4B). Upon limited polymerization, total protein adsorption for the Au@PDA NPs prepared by using insufficient amounts of DA (0.18 μg/cm2) is higher than that for Au@PDA NPs prepared by using optimal and excess amounts of DA (0.12 μg/cm2), consistent with our PAGE data. Upon extended polymerization, total protein adsorption for the Au@PDA NPs prepared by using optimal DA (0.12 μg/cm2) is the lowest compared to those prepared by using insufficient (0.25 μg/cm2) and excess DA (0.19 μg/cm2). These data show that protein adsorption onto the PDA coating is highly sensitive to both DA concentration and polymerization time. Next, we digested the total adsorbed proteins with trypsin and analyzed their identities by liquid chromatography tandem-mass spectrometry (LC-MS/MS; Table S4-S9). The identified proteins have an isoelectric point (pI) in the range of 5.0 and 9.4, with median and average values at 6.1 and 6.6 respectively. Considering the pH value of the culture medium (~7.4), the adsorbed proteins are slightly negative in net charge in DMEM. This result agrees with the negative zeta potential 25 ACS Paragon Plus Environment

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values for our collection of serum-coated AuNPs afforded by DLS measurements (Table 5). For five out of the six types of Au@PDA NPs studied, we found albumin (with a molecular weight of 66 kDa) as the most or second-most probable protein found in the corona, bolstering our PAGE data showing the ~70 kDa band detected independent of DA concentration and polymerization time. We next excised the strongest 10-15 kDa band (containing proteins adsorbed onto Au@PDA NPs prepared by using insufficient amounts of DA and extended polymerization) and the strongest 70 kDa band (containing proteins adsorbed onto Au@PDA NPs prepared by using excess amounts of DA and extended polymerization) from the original PAGE gel and subjected these two bands for additional proteomics analysis (Figure 4C). The most probable protein for the ~70 kDa band corresponds to albumin (Table S10). Our data corroborate with the preceding literature that shows (1) non-specific binding of albumin to citrate-capped AuNPs and (2) reduced adsorption of albumin due to denser polymeric coating of AuNPs.15, 18 For the 10-15 kDa band, we detected two types of hemoglobin subunit as the most probable proteins found in the corona of Au@PDA NPs (Table S11). Echoing the iron-containing function of hemoglobin, PDA, a structural analog of neuromelanin53 (an important iron-chelating polymer in the brain),54 can also chelate ferrous ions like neuromelanin.55 In light of the PAGE and proteomics data, we conclude that deposition of a thicker PDA shell onto AuNP cores (via addition of excess amounts of DA) promotes absorption of hemoglobin subunits onto the surface of Au@PDA NPs. Note that we did not explicitly address the effects of surface properties (e.g., surface charge and surface functionality) on the protein corona of Au@PDA NPs.56-57 Given the negative charge of our collection of Au@PDA NPs (Table 5), we do not expect surface charge to be a critical determinant of identity of the protein corona. On surface functionality, it remains challenging to determine the amounts of amine and catechol groups on the PDA coating as polymerization proceeds, especially when the chemical structure of


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PDA is still not completely clear.58-59 This knowledge gap precludes us from establishing a concrete connection between the surface functionality of Au@PDA NPs and their protein corona.

Figure 4. Analysis of the adsorbed serum proteins on Au@PDA NPs. (A) Polyacrylamide gel electrophoresis of the adsorbed serum proteins onto the collection of Au@PDA NPs as a function of initial DA concentration and polymerization time. CTRL refers to protein samples obtained by using the same experimental procedures without adding any NPs to the serum. (B) Quantification of the total adsorbed proteins onto Au@PDA NPs prepared by using DA concentrations of 0.5 µg/mL, 20 µg/mL, and 65 µg/mL with 1 h or 96 h polymerization. Data obtained from three independent measurements are presented as the average ± SD. (C) Identified proteins of the two distinct bands of interest by LC-MS/MS. The two bands are albumin and hemoglobin subunits.


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Interactions between Au@PDA NPs and the Cell in Serum-Containing Medium. We incubated two common mammalian model cell types, Neuro-2a and HeLa, with our collection of Au@PDA NPs prepared by using various DA concentrations and polymerization times in serumcontaining culture medium (Figure S7). To eliminate the effect of gravitational sedimentation37 and focus on the initial stage of cellular uptake, we limited the incubation time to 1 h. We then employed inductively coupled plasma mass spectrometry (ICP-OES) to quantify the cellular association of the Au@PDA NPs. Remarkably, polymerization time is a critical parameter of the association of Au@PDA NPs to both cell types. Irrespective of DA concentration, the Au@PDA NPs prepared by using limited polymerization of 1 h or 6 h associate with both cell types in significantly higher quantities than those prepared by extended polymerization of 48 h or 96 h (Figure 5A). For instance, for Au@PDA NPs prepared by using an initial DA concentration of 20 µg/mL, their association to HeLa and Neuro-2a cells plummets by 87% and 88%, respectively, when their polymerization time increases from 1 h to 96 h. Next, we captured light micrographs of Neuro-2a (Figure 5B) and HeLa (Figure S8) cells after their incubation with the collection of Au@PDA NPs. The micrographs generally do not reveal observable aggregation of the serumstabilized Au@PDA NPs on the cells, except for those Au@PDA NPs prepared by using excess amounts of DA (65 µg/mL) and limited polymerization time (1 h). To confirm their intracellular delivery, we further captured biological TEM images of the Neuro-2a cells after 1 h of incubation with our collection of Au@PDA NPs (Figures 5C and S9). Consistent with our ICP-OES data, we detected more abundant intracellular accumulation of the Au@PDA NPs prepared by using 1 h of limited polymerization than extended 96 h of polymerization in general (Figure 5C and S8). The Au@PDA NPs are mostly localized inside intracellular compartments, although a small portion is found in the cytosol. Of note, the morphologies of the Au@PDA NPs are well conserved in the


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extracellular matrix and inside the cell. Also, we consistently captured NP clusters, almost monodisperse NPs, and NP chains inside Neuro-2a cells when they are incubated with Au@PDA NPs prepared by using extended polymerization supplied with insufficient (0.5 µg/mL), optimal (20 µg/mL) and excess (65 µg/mL) amounts of DA, respectively. In addition, the intracellular compartments adopt the shapes of NP clusters and chains upon the entry of Au@PDA NPs into Neuro-2a cells. Note that we did not explicitly address the effects of NP physical parameters (such as size and shape) on the cellular uptake of Au@PDA NPs,60 despite the morphological diversity of our collection of Au@PDA NPs from randomly aggregated NPs to monodisperse particles and “worm-like” NP chains. Precise control over (1) the size of the random aggregate of PDA-coated nanoparticles and (2) the number of NPs per worm (let alone the aspect ratio and curvature of the worm) via bottom-up self-assembly remains a technical challenge to date, precluding us from establishing a concrete connection between the physical parameters of the assembled Au@PDA NPs and their cellular uptake. By contrast, our work focuses on tuning polymerization time and DA concentration, both defined and tunable reaction parameters, to produce a collection of Au@PDA NPs for subsequent analysis of their cellular uptake. In conclusion, our data reveal the crucial role of polymerization time on the cellular uptake of Au@PDA NPs. In serum-containing medium, the Au@PDA NPs prepared by limited polymerization enter the cell more effectively than those prepared by extended polymerization. Recall from Table 4 the similar hydrodynamic sizes of Au@PDA NPs prepared by limited polymerization (1 h) and extended polymerization (48 h) in serum-containing medium. We believe that our observed differences in cellular uptake of Au@PDA NPs between limited and extended polymerization is a result of polymerization time rather than physical aggregation. Unlike polymerization time, we did not observe any significant dependence of cellular uptake of Au@PDA NPs on DA concentration. Since we showed in Figure


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4 that DA concentration is a critical factor of the identity of protein corona, we believe that cellular uptake of Au@PDA NPs does not pronouncedly depend on the identity of the protein corona. Adsorption of albumin or haemoglobin subunits onto Au@PDA NPs does not offer a distinct advantage over one another for promoting cellular uptake.


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Figure 5. Cellular uptake of Au@PDA NPs as a function of DA concentration and polymerization time in serum-containing culture medium. In all experiments, the incubation time with the cells is 1 h. (A) Association of the Au@PDA NPs to HeLa and Neuro-2a cells. The Au content in the harvested cell pellets is determined by ICP-OES by using a standard curve. Data obtained from three independent measurements are presented as the average ± SD. Data were analyzed by the ttest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = no significant difference. (B) Representative light micrographs of Neuro-2a cells incubated with Au@PDA NPs prepared by 1 h and 96 h of polymerization. (C) Representative TEM images of Neuro-2a cells incubated with Au@PDA NPs prepared by 1 h and 96 h of polymerization. The lower, red-squared panel features the enlargement of the boxed area show in the upper panel. Black arrows indicate the PDA coating on the AuNPs. Legend: Nu = nucleus; Cy = cytosol; Ex = extracellular space.

Interactions between Au@PDA NPs and the Cell in Serum-Free Medium. To examine the role of serum proteins in cellular uptake of Au@PDA NPs, we finally analyzed the uptake of Au@PDA NPs by Neuro-2a and HeLa cells in culture medium devoid of serum proteins. By ICPOES measurements, we observed substantially higher association of Au@PDA NPs to both cell types in serum-free culture medium than serum-containing culture medium, for all DA concentrations and polymerization times tested (Figure 6A). However, we did not observe any correlation between cellular association to either polymerization time or DA concentration. Given our DLS data that indicate the generation of micron-sized aggregates of Au@PDA NPs upon their exposure to serum-free culture medium regardless of DA concentration and polymerization time (Table 4), we next addressed whether this elevated cellular association translates to intracellular delivery. Light micrographs show the localization of microscale aggregates of Au@PDA NPs on 31 ACS Paragon Plus Environment

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the membrane of both cell types (Figures 6B and S9). Bio-TEM images also capture the accumulation of tens of Au@PDA NPs either on the cell membrane or in the extracellular matrix of Neuro-2a cells, but rarely inside the cell. They also depict the thick PDA coating on the “wormlike” Au@PDA NP chains prepared by using excess amounts of DA and a polymerization time of 96 h (Figure 6C). These imaging data show that Au@PDA NPs, when immersed in serum-free medium, form microscale aggregates that merely stick to the cell membrane without actual cellular entry. This observation agrees with existing reports on the size-dependent uptake of NPs by nonphagocytic cells. For example, iron oxide nanoparticles up to 300 nm can effectively enter nonphagocytic T cells, but not their larger micron-sized counterparts.61 As Neuro2A and HeLa cells, our model cell lines, are both non-phagocytic, we believe that the major role of protein corona is to prevent aggregation of Au@PDA NPs into micron-sized entities in ion-containing culture medium. Our ICP-OES data also stress the importance of validating cellular association data by ICP-OES with imaging data to confirm actual intracellular delivery of Au@PDA NPs.


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Figure 6. Cellular uptake of Au@PDA NPs as a function of DA concentration and polymerization time in serum-free culture medium. In all experiments, the incubation time with the cells is 1 h. (A) Association of the Au@PDA NPs to HeLa and Neuro-2a cells. The Au content in the harvested cell pellets is determined by ICP-OES by using a standard curve. Data obtained from three independent measurements are presented as the average ± SD. Data were analyzed by the t-test. * 33 ACS Paragon Plus Environment

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p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = no significant difference. (B) Representative light microscopic images of Neuro-2a cells incubated with Au@PDA NPs prepared by 1 h and 96 h of polymerization. Purple dots (arrows) indicate clusters of Au@PDA NPs on the cell membrane. (C) Representative TEM images of Neuro-2a cells incubated with Au@PDA NPs prepared by 1 h and 96 h of polymerization. The lower, red-squared panel features the enlargement of the boxed area show in the upper panel. Black arrows indicate the PDA coating on the AuNPs. Legend: Nu = nucleus; Cy = cytosol; Ex = extracellular space.

CONCLUSIONS We have prepared a collection of Au@PDA NPs by systematically varying two parameters, DA concentration and polymerization time, over two orders of magnitude. At the nanomaterials level, our results show extended polymerization as a viable approach to realizing PDA-coated NPs of diverse morphologies, including aggregated NPs, almost monodisperse NPs, and NP chains depending on DA concentration. In serum-free culture medium, Au@PDA NPs of all DA concentrations and polymerization times face salt-induced aggregation and become microscale aggregates. In serum-containing medium, however, the Au@PDA NPs are more stable than in serum-free medium, only becoming slightly larger than their original, non-coated forms. In terms of the protein corona around the Au@PDA NPs, the type of proteins depends strongly on DA concentration but not polymerization time. Au@PDA NPs made with increasing amounts of DA typically favor adsorption by hemoglobin subunits. Finally, serum coating and polymerization time dictate the intracellular delivery of Au@PDA NPs. In serum-free culture medium, Au@PDA NPs prepared by using all DA concentrations and polymerization times mostly adhere onto the cell


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membrane as micron-sized aggregates without obvious cellular entry shortly after 1 h of incubation. Instead, serum-stabilized Au@PDA NPs can more effectively enter the cell and preserve their original NP morphologies (i.e., aggregated, almost-monodisperse and “worm-like” NPs) inside the cell. The degree of cellular uptake of serum-stabilized Au@PDA NPs is a strong function of polymerization time but not DA concentration. Au@PDA NPs prepared by using limited polymerization enter the cell at substantially higher amounts than those prepared by using extended polymerization. Our data provide insights into the interactions between PDA-coated NPs and cells, emphasizing DA concentration and polymerization time as two critical parameters for designing PDA-coated NPs with colloidal stability for effective intracellular delivery.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Additional experimental details (including methods of protein digestion, LC-MS/MS and protein identification), a summary of past PDA-related literature, FT-IR data of Au@PDA NPs, additional UV-vis spectra and TEM images of Au@PDA NPs, more cryo-EM images of “worm-like” Au@PDA NP chains, original pellets of HeLa and Neuro-2a cells collected after incubation with Au@PDA NPs in the presence of serum, extra biological TEM images showing the cellular uptake of Au@PDA NPs, and microscopic images of HeLa cells incubated with Au@PDA NPs in the presence and absence of serum. (PDF)


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AUTHOR INFORMATION Corresponding Author *E-mail (C. H. J. Choi): [email protected] ORCID Chung Hang Jonathan Choi: 0000-0003-2935-7217 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by an Early Career Scheme grant (Project No. 24300014) and a General Research Fund (Project No. 14302916) from the Research Grants Council. It was also supported by the Chow Yuk Ho Technology Centre for Innovative Medicine at The Chinese University of Hong Kong (CUHK). C.H.J.C. acknowledges a Croucher Startup Allowance and a Croucher Innovation Award from the Croucher Foundation. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS We thank Liwen Jiang (School of Life Sciences, CUHK) for supporting cryo-EM measurements, Melanie Wong (Department of Biomedical Engineering, CUHK) and Cecilia Chan (Department of Surgery, CUHK) for helpful discussions, Josie Lai (School of Biomedical Sciences, CUHK) for guidance in ultramicrotomy and biological TEM, Yick Keung Suen for assistance in LC-MS/MS


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TOC:


Morphological Diversity, Protein Adsorption, and Cellular Uptake of

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