Effect of Alkylation on the Cellular Uptake of Polyethylene Glycol

Additionally, alkyl-PEG-AuNPs can rapidly enter Kera-308 cells via the ... how alkyl as a functional group can affect the interactions between NPs and...
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Effect of Alkylation on the Cellular Uptake of Polyethylene Glycol-Coated Gold Nanoparticles Lok Wai Cola Ho,† Wing-Yin Yung,‡ Kwun Hei Samuel Sy,† Ho Yin Li,§ Chun Kit K. Choi,† Ken Cham-Fai Leung,‡ Thomas W. Y. Lee,§ and Chung Hang Jonathan Choi*,†,⊥ †

Department of Electronic Engineering (Biomedical Engineering), §School of Pharmacy,⊥Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong, Shatin, New Territories, and ‡Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong, China S Supporting Information *

ABSTRACT: Alkyl groups (CnH2n+1) are prevalent in engineered bionanomaterials used for many intracellular applications, yet how alkyl groups dictate the interactions between nanoparticles and mammalian cells remains incomprehensively investigated. In this work, we report the effect of alkylation on the cellular uptake of densely polyethylene glycol-coated nanoparticles, which are characterized by their limited entry into mammalian cells. Specifically, we prepare densely PEGylated gold nanoparticles that bear alkyl chains of varying carbon chain lengths (n) and loading densities (termed “alkyl-PEG-AuNPs”), followed by investigating their uptake by Kera308 keratinocytes. Strikingly, provided a modest alkyl mass percentage of 0.2% (2 orders of magnitude lower than that of conventional lipid-based NPs) in their PEG shells, dodecyl-PEG-AuNPs (n = 12) and octadecyl-PEG-AuNPs (n = 18) can enter Kera-308 cells 30-fold more than methoxy-PEG-AuNPs (no alkyl groups) and hexylPEG-AuNPs (n = 6) after 24 h of incubation. Such strong dependence on n is valid for all serum concentrations considered (even under serum-free conditions), although enhanced serum levels can trigger the agglomeration of alkyl-PEG-AuNPs (without permanent aggregation of the AuNP cores) and can attenuate their cellular uptake. Additionally, alkyl-PEG-AuNPs can rapidly enter Kera-308 cells via the filipodia-mediated pathway, engaging the tips of membrane protrusions and accumulating within interdigital folds. Most alkyl-PEG-AuNPs adopt the “endo-lysosomal” route of trafficking, but ∼15% of them accumulate in the cytosol. Regardless of intracellular location, alkyl-PEG-AuNPs predominantly appear as individual entities after 24 h of incubation. Our work offers insights into the incorporation of alkyl groups for designing bionanomaterials for cellular uptake and cytosolic accumulation with intracellular stability. KEYWORDS: alkylation, cellular uptake, gold nanoparticles, polyethylene glycol, filopodia

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interactions between alkyl-containing NPs and the cell, the primary effects of alkyl groups on these interactions were inconclusive because the emphasis of these comparative studies did not lie on alkyl groups alone.10−13 For example, Rotello and co-workers compared the exocytosis properties and immune responses of NPs bearing various functional groups. Notably, they observed that NPs functionalized with cyclohexyl groups can trigger more secretion of pro-inflammatory cytokines by splenocytes than those with alkyl groups.11 They later showed that benzyl-containing NPs are more effectively exocytosed by breast cancer cells than their alkyl-containing counterparts.12 In another comparative study, Irvine and Stellacci explored how

lkyl groups (CnH2n+1) are prevalent in engineered bionanomaterials that are utilized in a multitude of intracellular applications, such as phospholipid-coated quantum dots for molecular imaging,1,2 lipid nanoparticles (NPs) for gene delivery,3,4 and cholesterol-coated5,6 or vitamin E-coated7,8 NPs for drug delivery. Motivated by the ability of alkyl-containing NPs to enter mammalian cells, previous researchers extensively elucidated the mechanisms for their endocytosis. Poste et al. showed that liposomes can enter fibroblasts via endocytosis or fusion with the cell membrane.9 Recently, Gilleron et al. proved that lipid NPs can enter cancer cells by clathrin-mediated endocytosis and micropinocytosis.4 These investigations treated the alkyl-containing NP as a single entity, yet fell short of addressing, at the functional group level, how its constituent alkyl groups influence the cellular uptake of the NP. While other related studies tangentially covered the © 2017 American Chemical Society

Received: March 24, 2017 Accepted: May 31, 2017 Published: May 31, 2017 6085

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Figure 1. Preparation of alkyl-terminated, densely polyethylene glycol-coated gold NPs (alkylx%-PEG-AuNPs). (A) Synthesis of thiol- and alkyl-terminated PEG strands (HS-PEG-CnH2n+1). Alkylamine (H2N-CnH2n+1) of various carbon chain lengths (n = 6, hexyl; n = 12, dodecyl; n = 18, octadecyl) is covalently linked to a thiol- and carboxy-terminated bifunctional PEG linker (HS-PEG-COOH) via EDC/NHS chemistry. (B) Preparation of alkylx%-PEG-AuNPs. Different mole ratios of thiol- and alkyl-terminated PEG [HS-PEG-alkyl; x mol %] to thiol- and methoxy-terminated PEG [HS-PEG-OCH3; (100 − x) mol %] are coupled to the surface of citrate-capped AuNPs of 25 nm in diameter via gold-sulfur linkages. UV−vis spectra and representative TEM images of (C) citrate-capped AuNPs and methoxy-PEG-AuNPs, (D) hexylx%PEG-AuNPs, (E) dodecylx%-PEG-AuNPs, and (F) octadecylx%-PEG-AuNPs as a function of loading density of alkyl groups (in terms of mole percentage: x mol %). Negative staining of methoxy-PEG-AuNPs and alkyl4%-PEG-AuNPs by phosphotungstic acid reveals the dense PEG shell around the AuNP core. All PEG strands used have a molecular weight of around 5 kDa.

will prepare a series of alkyl-terminated, densely polyethylene glycol-coated gold NPs (termed “alkyl-PEG-AuNPs”). These NPs consist of a 25 nm AuNP core, a dense shell of PEG strands covalently attached to the surface of the AuNP, and defined amounts of alkyl groups connected to the distal end of the tethered PEG strands (Figure 1). In our NP design, the spatial distribution of alkyl chains differs from that of other conventional alkyl-containing NPs. For liposomes, the alkyl chains are buried in the interior of the phospholipid bilayer.14 For lipid NPs, the alkyl groups are mostly located inside the

the spatial distribution of alkyl groups on the NP surface may affect cellular uptake. Interestingly, they observed that those with uncharged alkyl groups orderly patterned on their surface can enter dendritic cells by directly penetrating the cell membrane, but those containing randomly distributed uncharged alkyl groups on their surface cannot.13 Nevertheless, these previous studies did not address how alkyl as a functional group can affect the interactions between NPs and the cell. In this work, we will derive mechanistic understanding in the role of alkylation in the cellular uptake of NPs. To do so, we 6086

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ACS Nano Table 1. Hydrodynamic Sizes of Alkylx%-PEG-AuNPs as a Function of Loading Densitya hydrodynamic size in water (nm)b sample

1 mol %

1.3 mol %

2 mol %

4 mol %

10 mol %

100 mol %

hexylx%-PEG-AuNPs dodecylx%-PEG-AuNPs octadecylx%-PEG-AuNPs

44.0 ± 0.4 (0.1) 44.2 ± 0.8 (0.1) 46.7 ± 1.5 (0.2)

44.2 ± 1.3 (0.1) 45.2 ± 1.8 (0.1) 47.9 ± 0.8 (0.2)

45.4 ± 0.5 (0.1) 47.2 ± 1.8 (0.1) 47.8 ± 3.6 (0.3)

45.5 ± 0.8 (0.2) 45.1 ± 0.9 (0.2) 44.3 ± 0.8 (0.2)

43.0 ± 1.8 (0.1) 67.6 ± 2.1 (0.2) 55.5 ± 1.5 (0.3)

45.2 ± 1.3 (0.2) 59.3 ± 2.4 (0.3) 111.5 ± 2.4 (0.3)

In terms of mole percentage of alkyl groups: x mol %. bReported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refer to the polydispersity index (PDI). a

solid lipid core matrix.15 Unlike liposomes and lipid NPs, the alkyl groups of our alkyl-PEG-AuNPs are exposed to the aqueous environment, tagged to the distal ends of the PEG strands that are densely tethered onto the surface of AuNPs. The AuNP core allows for measurement of their intracellular quantities by inductively coupled plasma mass spectrometry (ICP-MS) as well as visualization of their intracellular distribution by dark-field microscopy16 and transmission electron microscopy (TEM).17 As serum proteins in the cell culture medium can bind to alkyl groups,18,19 the plasmonic properties of AuNPs can empower us to address the possibility of serum-induced aggregation or agglomeration of alkyl-PEGAuNPs by UV−vis spectrophotometry and dynamic light scattering (DLS), and how such aggregation or agglomeration may influence intracellular delivery. Next, the dense PEG shell surrounding the AuNP cores, at a density of 2−3 PEG molecules per nm2 of AuNP surface, serves two purposes, stability and benchmarking. Whereas many types of emergent bionanomaterials (such as spherical nucleic acids or lipid NPs) can effectively enter mammalian cells, they often face premature disassembly or degradation inside the cell.20,21 Here, we will show that the dense PEG shell endows the AuNP core with intracellular stability (in the cytosol and intracellular compartments) for up to 24 h of incubation, a desirable feature yet unavailable in many conventional NP-based delivery systems. It is also imperative to note that densely PEGylated NPs do not typically enter mammalian cells at high amounts.22−24 Chan and co-workers previously proved that AuNPs decorated by a dense shell of PEG strands of 5 kDa in molecular weight at a density higher than ∼1 PEG strand per nm2 of the AuNP surface can significantly curb adsorption by serum proteins and reduce serum-induced uptake by macrophages.23 Likewise, Parak and co-workers reported that inorganic NPs coated by 10 kDa-PEG molecules exhibit a drastically attenuated ability to bind to serum proteins and enter fibroblasts.24 Therefore, conjugation of alkyl groups to the dense PEG shell can permit us to objectively assess any enhancement in cellular uptake due to alkylation. In this connection, we also will select AuNPs coated by 5 kDa methoxy-terminated PEG strands (denoted “methoxy-PEG-AuNPs”) as our “benchmark” NPs against the alkyl-PEG-AuNPs. To ascertain the effect of alkylation, we will evaluate how two important parameters, (1) loading density of alkyl groups on the dense PEG shell and (2) carbon chain length of the alkyl groups, collectively dictate the internalization of NPs by Kera-308 cells (mouse keratinocytes). In this study, we covalently attached hexyl (n = 6), dodecyl (n = 12), or octadecyl (n = 18) groups to the dense PEG shells and at different loading densities. Note that the key ingredients of conventional alkyl-containing NPs [e.g., distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and cholesterol; see Table S1] bear alkyl chains of 6−20 carbon atoms long. We demonstrate that incorporation of a modest alkyl mass percentage of 0.2% in the

dense PEG shell can lead to a 30-fold increase in the uptake of densely PEGylated AuNPs by Kera-308 cells after 24 h of incubation. We further interrogate the endocytosis pathway of alkyl-PEG-AuNPs by Kera-308 cells and mapped their route of intracellular trafficking.

RESULTS AND DISCUSSION Preparation and Characterization of Alkyl-Terminated, Densely PEGylated AuNPs. Initially, we synthesized thiol- and alkyl-terminated PEG strands (HS-PEG-CnH2n+1) by covalently conjugating alkylamine (H2N-CnH2n+1) of various carbon chain lengths (n) to a commercially available thiol- and carboxy-terminated bifunctional PEG linker strand (HS-PEGCOOH) via 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide/ N-hydroxysuccinimide (EDS/NHS) chemistry (Figure 1A). By literature precedent, NPs containing positively charged functional groups (e.g., lysine-coated micelles or amine-coated quantum dots)25,26 may nonspecifically enter cells more pronouncedly than their negatively charged or neutrally charged counterparts.27 To prevent the presence of free unreacted alkylamines associated with our alkyl-PEG-AuNPs from interfering with the cellular uptake data, we extensively purified the HS-PEG-CnH2n+1 product by solvent extraction and dialysis, followed by performing the ninhydrin test to confirm the removal of almost 98% of the free alkylamines which was initially introduced to the reaction mixture (Figure S1B). To deposit the PEG coating onto the surface of AuNPs to form alkylx%-PEG-AuNPs via gold-sulfur linkages, we next added a mixture of x mol % of HS-PEG-alkyl and (100 − x) mol % of HS-PEG-OCH3 to an aqueous suspension of citrate-capped AuNPs of 25 nm in diameter (Figure 1B). Based on previous studies, the ratio of two types of thiolated ligands in the reaction mixture corresponds to the display ratio of the ligands on the AuNP surface.28,29 We therefore assume that the display ratio of the methoxy-PEG and alkyl-PEG strands will correspond to the mole ratio of the two PEG types initially added to the AuNP solution. In this connection, we carefully controlled the mole ratio of alkyl-PEG-SH to methoxy-PEG-SH added from 0 mol % (only methoxy-PEG-SH added without alkyl-PEG-SH) to 100 mol % (only alkyl-PEG-SH added without methoxy-PEG-SH). After 1 h of PEGylation, we rinsed off the excess PEG strands by several rounds of centrifugation and resuspended the resultant methoxy-PEG-AuNPs and alkylx%-PEG-AuNPs in deionized water. To start with, we measured the UV−vis absorbance of methoxy-PEG-AuNPs, hexylx%-PEG-AuNPs, dodecylx%-PEG-AuNPs, and octadecylx%PEG-AuNPs in water (Figure 1 C−F and Table S2). In general, attachment of methoxy-terminated PEG strands to AuNPs led to a redshift in the surface plasmon reasonance (SPR) peak from 522 to 525 nm in water. For hexylx%-PEG-AuNPs, their SPR peaks are at 524−525 nm in water for most hexyl contents considered. The only exception is hexyl100%-PEG-AuNPs (i.e., with all PEG strands containing a hexyl group), whose SPR 6087

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ACS Nano Table 2. Other Physicochemical Parameters of Alkyl4%-PEG-AuNPs sample citrate-capped AuNPs methoxy-PEGAuNPs hexyl4%-PEGAuNPs dodecyl4%-PEGAuNPs octadecyl4%-PEGAuNPs

hydrodynamic size in water (nm)a

ζ-potential (mV)b

24.9 ± 0.3 (0.1)

−33.4 ± 0.7

N.A.

N.A.

N.A.

45.9 ± 0.7 (0.2)

−16.3 ± 1.1

4733 ± 125

2.41 ± 0.06

0

45.5 ± 0.8 (0.2)

−23.0 ± 0.1

4407 ± 500

2.24 ± 0.25

0.07

45.1 ± 0.9 (0.2)

−21.3 ± 0.0

4177 ± 203

2.42 ± 0.10

0.14

44.3 ± 0.8 (0.2)

−17.1 ± 1.4

4953 ± 334

2.52 ± 0.17

0.20

no. of PEG strands/AuNP

no. of PEG strands/nm2 of AuNP surface

mass percentage of alkyl groups in the total PEG coating (%)c

Reported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refer to the polydispersity index (PDI). bReported data represent mean ± SD from three independent measurements. cMass percentage of alkyl groups in total PEG coating = [(mass of alkyl group × total number of alkyl-terminated PEG strands)/(mass of PEG × total number of alkyl- and methoxy-terminated PEG stands)] × 100%. a

alkyl4%-PEG-AuNPs by DLS (Tables 2 and S3 and Figures S3 and S4). Attachment of methoxy-PEG strands to the surface of citrate-capped AuNPs led to an increase in ζ-potential from −33 mV to −16 mV, an observation consistent with the notion of charge screening.30 For alkyl4%-PEG-AuNPs, their ζpotentials are not significantly different from that of methoxyPEG-AuNPs, ranging between −23 mV and −17 mV for all carbon chain lengths considered. Furthermore, we quantified the total number of PEG strands attached to each AuNP by the Ellman’s assay.34,35 Each methoxy-PEG-AuNP contains approximately 4700 PEG strands, amounting to 2.4 strands per nm2 of AuNP surface. There are roughly 4500 PEG strands per alkyl4%PEG-AuNP (amounting to 2.4 strands per nm2 of AuNP surface), regardless of carbon chain length. Note that this PEG loading density is on par with the saturation loading density of 5 kDa PEG molecules on the surface of a 30 nm AuNP (i.e., 2− 3 PEG molecules per nm2 of AuNP surface).23,35 Our TEM, DLS, and Ellman’s assay data demonstrate the successful deposition of a dense PEG shell that constitutes methoxy-PEGAuNPs and alkyl4%-PEG-AuNPs of all carbon chain lengths. Chain Length-Dependent Cellular Uptake of AlkylPEG-AuNPs. Armed with the collection of alkylx%-PEGAuNPs, we systemically investigated the combined effects of carbon chain length (n) and alkyl chain loading (x mol %) on the uptake by Kera-308 keratinocytes (Figure 2). Unless specified, we performed our cellular uptake experiments by incubating cells in Opti-MEM I reduced serum medium (OptiMEM), in order to preliminarily focus our attention on elucidating the effects of n and x mol % on cellular uptake without a significant effect by serum proteins. With full recognition of the critical role of serum adsorption on the cellular uptake of NPs,23,24,36−38 we shall defer our examination of the effect of serum on cellular uptake to later sections of this work (Figure 4). In view of previous studies that documented the potential cytotoxicity of aliphatic hydrocarbons39 and carbon-based nanomaterials40 (e.g., single-walled carbon nanotubes, multiwalled carbon nanotubes, and fullerenes),41−43 we first evaluated the cytotoxicity of our alkyl-PEG-AuNPs in Kera308 cells (Figure S5). By the alamarBlue assay, the alkylx%PEG-AuNPs display high biocompatibility across all carbon chain lengths and loading contents of alkyl groups considered. Live/dead staining of the cells treated with alkyl4%-PEG-AuNPs further supports that the cells remain largely viable after 24 h of incubation, irrespective of carbon chain length. We subsequently conducted the cellular uptake studies by incubating Kera-308 cells with 1 nM methoxy-PEG-AuNPs or alkylx%-

peak is at 527 nm. For dodecylx%-PEG-AuNPs, their peaks are at 524−525 nm for the majority of dodecyl contents considered except for dodecyl100%-PEG-AuNPs (i.e., with all PEG strands containing a dodecyl chain), whose SPR peak lies at 529 nm. For most octadecyl contents considered, the SPR peaks of octadecyl x%-PEG-AuNPs are at 522−523 nm. Notable exceptions include octadecyl10%-PEG-AuNPs and octadecyl100%-PEG-AuNPs, whose SPR peaks reveal obvious broadening and redshift to 530 and 532 nm, respectively. Besides UV−vis spectrometry, we next utilized DLS to characterize the same collection of alkylx%-PEG-AuNPs in water (Table 1). Engraftment of methoxy-PEG-SH strands to the surface of citrate-capped AuNPs increases the overall hydrodynamic diameter from 24.9 to 45.9 nm, which implies a thickness of roughly 10 nm for the PEG coating in agreement with previous studies.30 For all values of x considered, the hydrodynamic diameter of hexylx%-PEG-AuNPs is 43−45 nm, a size similar to that of methoxy-PEG-AuNPs. For dodecylx%-PEG-AuNPs and octadecylx%-PEG-AuNPs, their hydrodynamic sizes are similar to that of methoxy-PEG-AuNPs for x < 10%. On the contrary, the hydrodynamic sizes of dodecyl100%-PEG-AuNPs and octadecyl100%-PEG-AuNPs increase to 59 and 112 nm, respectively, when all PEG chains contain alkyl chains. Taken together, these UV−vis and DLS results underscore colloidal instability of dodecylx%-PEG-AuNPs and octadecylx%-PEGAuNPs in water for x = 10 or above, possibly due to strong interparticle hydrophobic interactions.31 The log P values (where P refers to the octanol/water partition coefficient) of hexane, dodecane, and octadecane are estimated to be 3.66, 6.99, and 9.00, respectively, indicating that these alkyl groups are hydrophobic. At this point, we performed additional physicochemical characterization studies of alkyl4%-PEG-AuNPs only, noting their higher stability in water than alkyl10%-PEG-AuNPs and alkyl100%-PEG-AuNPs. We confirmed the presence of the PEG shell by negatively staining the methoxy-PEG-AuNPs and alkyl4%-PEG-AuNPs with phosphotungstic acid. By TEM imaging, we observed a homogeneous layer of PEG coating (negatively stained by phosphotungstic acid) on the surface of methoxy-PEG-AuNPs and alkyl4%-PEG-AuNPs of around 4.5 nm in thickness irrespective of carbon chain length (Figure 1C−F, inset), a result consistent with previous reports on methoxy-PEG5000-coated AuNPs.32,33 Negative staining by uranyl acetate reveals a PEG layer of around 2.5 nm in thickness on the surface of AuNPs (Figure S2). Moreover, we determined the ζ-potential of methoxy-PEG-AuNPs and 6088

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Figure 2. Cellular uptake of alkylx%-PEG-AuNPs of varying alkyl chain lengths and alkyl amounts. The incubation time and concentration were 24 h and 1 nM, respectively. (A) Kera-308 cells (mouse keratinocytes) were incubated with either methoxy-PEG-AuNPs or alkylx%-PEGAuNPs containing 1%, 1.3%, 2%, 4%, 10%, and 100% (all in terms of mol %) hexyl, dodecyl, or octadecyl groups in the dense PEG coating. By ICP-MS measurements, cellular association of NPs increases drastically with carbon chain length and alkyl amounts, with dodecyl- and octadecyl-terminated PEG-AuNPs outperforming methoxy- and hexyl-terminated PEG-AuNPs. (B) Representative dark-field scattering images of Kera-308 cells. Cells treated with dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs scatter strong reddish light (an indicator of substantial cellular uptake), but methoxy-PEG-AuNPs and hexyl4%-PEG-AuNPs do not. (C) Representative TEM images of Kera-308 cells. Dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs can enter the cell more pronouncedly than methoxy-PEG-AuNPs and hexyl4%-PEGAuNPs, mostly accumulating inside intracellular compartments (red arrows). The bottom row shows the enlargement of the boxed area of the top row. (Legend: Nu = nucleus; Cy = cytosol; and Ex = extracellular space.) (D) Kera-308, HeLa (human cervical cancer), C166 (mouse endothelial), and RAW 264.7 (mouse macrophage) cells were incubated with 1 nM alkyl4%-PEG-AuNPs of different carbon chain lengths for 24 h. By ICP-MS measurements, we observed in four cell types that dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs exhibit more pronounced cellular uptake than that of methoxy-PEG-AuNPs and hexyl4%-PEG-AuNPs. Data were analyzed by the t test. **** p < 0.0001 and ns = no significant difference.

cells by TEM imaging.44 We also did not choose exceedingly high NP concentrations (in the μM range), because higher concentrations of AuNPs may induce cytotoxicity to

PEG-AuNPs for 24 h. We selected 1 nM rather than exceedingly low NP concentrations (in the pM range) in order to more readily identify the internalized AuNPs inside the 6089

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ACS Nano keratinocytes.45 After the experiment, we rinsed the treated cells with phosphate-buffered saline (PBS) and harvested the cell pellets. Direct inspection of the cell pellets reveals two clear trends in their red color, which stems from the SPR of AuNPs (Figure S6). On the effect of carbon chain length, the cell pellets resulting from incubation with dodecylx%-PEG-AuNPs and octadecylx%-PEG-AuNPs are substantially more intense in their red color than those resulting from incubation with methoxy-PEG-AuNPs and hexylx%-PEG-AuNPs. Moreover, the red color becomes more intense with an increasing amount of alkyl groups in the PEG coating, a trend applicable to the cell pellets treated with dodecylx%-PEG-AuNPs and octadecylx%PEG-AuNPs. These trends highlight that the cellular association of alkylx%-PEG-AuNPs increases with carbon chain length and alkyl loading density. By subsequently analyzing the Au content of the harvested cell pellets by ICPMS, we observed that methoxy-PEG-AuNPs and hexylx%-PEGAuNPs exhibit low levels of association to Kera-308 cells regardless of alkyl content, both achieving around 5 × 103 NPs per cell after 24 h of incubation. By stark contrast, dodecylx%PEG-AuNPs and octadecylx%-PEG-AuNPs associate with cells more effectively than methoxy-PEG-AuNPs and hexylx%-PEGAuNPs. For example, in the presence of low levels of alkyl groups in the PEG coating (∼1 mol %), the cellular association of dodecyl1%-PEG-AuNPs and octadecyl1%-PEG-AuNPs is already 2-fold and 7-fold higher than that of methoxy-PEGAuNPs, respectively. Next, given a medium alkyl loading (4 mol %), the cellular association of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs amounts to 1 × 105 and 1.5 × 105 NPs per cell, respectively, which are ∼20-fold and 30-fold higher than that of methoxy-PEG-AuNPs. Incidentally, the cellular association of octadecyl-PEG-AuNPs is consistently higher than that of dodecyl-PEG-AuNPs when we maintained the alkyl content to be below 10 mol %. Finally, when saturated levels of alkyl chains are added to the PEG shell (100 mol %), the cellular association of both dodecyl100%-PEG-AuNPs and octadecyl100%-PEG-AuNPs amount to 2.5 × 105 NPs per cell (∼50-fold higher than that of methoxy-PEG-AuNPs), the most pronounced degree of association ever observed for Kera-308 cells in our study. Indeed, the pellet of cells incubated with dodecyl100%-PEG-AuNPs and octadecyl100%-PEG-AuNPs appears intensely purple in color, pointing to substantial cellular uptake of aggregated NPs induced by interparticle hydrophobic interactions (Table 1 and Figure S6). Both the purple-blue color of dodecyl100%-PEG-AuNPs and octadecyl100%-PEGAuNPs in water (data not shown) and the purple color of the cell pellets derived from cells incubated with dodecyl100%PEG-AuNPs and octadecyl100%-PEG-AuNPs suggest aggregation of the AuNP cores, to the extent that interparticle distance becomes smaller than average AuNP diameter.46 [Beyond this point, we deliberately subjected only alkyl4%-PEG-AuNPs to further cellular uptake studies. Besides the said requirement for maintaining colloidal stability, the primary objective of this study is to elucidate the effect of alkylation instead of hydrophobicity per se. By incorporating minute amounts of alkyl chains attached to the PEG shell (i.e., no more than 4 mol %), we anticipated that the alkyl-PEG-AuNPs are hydrophilic, rendering the issue of hydrophobicity on cellular uptake less relevant to this work. To gain some preliminary insights into the hydrophobicity of the alkyl-PEG-AuNPs, we performed an equilibrium partition experiment based on the water/1-octanol solvent system, a simple tool for assessing the hydrophobicity of carbon-rich NPs such as fullerenes47 and multiwalled carbon

nanotubes.48 After 24 h of mixing the NPs in the solvent system, most methoxy-PEG-AuNPs and alkyl4%-PEG-AuNPs still remain in the aqueous phase with little transfer to the nonpolar phase (Figure S7). Whereas this partition assay points to the general hydrophilic nature of our alkyl-PEG-AuNPs, more sophisticated measurements (e.g., dynamic interfacial tension) are necessary to offer more precise characterization of the hydrophobicity of the NPs.49] We further confirmed the internalization of alkyl4%-PEGAuNPs by Kera-308 cells by imaging. To start with, we used dark-field microscopy to image the NP-treated Kera-308 cells after 24 h of incubation (Figure 2B). Consistent with the ICPMS data, our dark-field images capture intense reddish scattered light (an indicator of substantial cellular uptake)16 from the cells treated with dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs. On the contrary, we could not detect significant dark-field scattered light from the cells treated with methoxy-PEG-AuNPs or hexyl4%-PEG-AuNPs. Next, we performed TEM imaging to monitor the subcellular localization of alkyl-PEG-AuNPs inside Kera-308 cells (Figure 2C). After 24 h of incubation, most dodecyl4% -PEG-AuNPs and octadecyl4%-PEG-AuNPs accumulate inside intracellular compartments, whereas methoxy-PEG-AuNPs and hexyl4%-PEGAuNPs are rarely found inside the cell. These TEM images corroborate with our ICP-MS analysis and dark-field images, underscoring the effect of carbon chain number on the endocytosis of alkyl-PEG-AuNPs. Finally, we addressed the generalizability of our cellular uptake data in cell types besides Kera-308 cells. To this end, we incubated three additional types of mammalian cells, including RAW 264.7 (macrophage), HeLa (epithelial), and C166 (endothelial) cells with 1 nM alkyl4%PEG-AuNPs for 24 h (Figure 2D). For these four cell types, we proved by ICP-MS analysis that dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs exhibit more significant cellular uptake than methoxy-PEG-AuNPs and hexyl4%-PEG-AuNPs. For instance, the cellular association of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs with C166 cells is 1.0 × 105 NPs per cell and 3.5 × 105 NPs per cell, respectively, almost 30-fold and 100-fold higher than that of methoxy-PEG-AuNPs and hexyl4%-PEG-AuNPs (∼3.5 × 103 NPs per cell), respectively. On average, the cellular association of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs to each C166 and HeLa cell is up to 3-fold and 9-fold higher than that of each RAW 264.7 and Kera-308 cell, respectively. On a per cell surface area basis, however, the number of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs associated with all four cell types is actually comparable because RAW 264.7 and Kera-308 cells are noticeably smaller than C166 and HeLa cells (Figure S8). These data further reaffirm that carbon chain length of the alkyl chains is a critically important parameter for the cellular uptake of alkyl-PEG-AuNPs. In summary, alkylx%-PEG-AuNPs enter the cell effectively when (1) the carbon chain length of alkyl chains is at least 12 and (2) the fraction of alkyl chains in the PEG shell is more than 1 mol %. Alkylx%-PEG-AuNPs with higher alkyl contents can enter cells more significantly, yet suffer from colloidal instability possibly due to interparticle aggregation. Note that our imaging data reveal clear abundance of dodecyl4%-PEGAuNPs and octadecyl4%-PEG-AuNPs inside the cell. Four mol % of dodecyl-PEG and octadecyl-PEG strands tethered on the AuNP translates to a mass percentage of only 0.14% for dodecyl groups and 0.20% for octadecyl groups in the dense PEG shell, respectively (Table S4). Remarkably, such a modest 6090

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Figure 3. Cellular uptake kinetics of alkyl-PEG-AuNPs. (A) By ICP-MS measurements, dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs rapidly associate with Kera-308 cells after 0.25 h of incubation. After 24 h of incubation, their cellular association was 30-fold and 40-fold higher than that of methoxy-PEG-AuNPs, respectively. Error bars denote standard deviations resulting from triplicate experiments. (B) Kera308 cells were incubated with Cy5-tagged alkyl4%-PEG-AuNPs for different durations of time. By confocal imaging, we observed gradual increase in intracellular Cy5 fluorescence in cells treated with dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs. By contrast, we could not detect appreciable intracellular Cy5 fluorescence after incubating the cells with Cy5-labeled hexyl4%-PEG-AuNPs for 24 h.

contains a tiny portion of PEG strands tagged with a Cyanine 5 (Cy5) molecule at their distal ends for confocal imaging. These Cy5-labeled, alkyl4%-PEG-AuNPs contain merely sufficient amounts of Cy5 molecules (∼0.13 mol %) in the PEG shell to facilitate confocal imaging, but not excessive amounts that would severely modify the surface functionality of the alkylPEG-AuNPs. To this end, we introduced varying ratios of methoxy-PEG-SH, alkyl-PEG-SH, and Cy5-PEG-SH strands to an aqueous suspension of 25 nm citrate-capped AuNPs and allowed the reaction to proceed for 1 h (Figure S9A). We confirmed by DLS analysis that introduction of Cy5 molecules to the PEG coating does not drastically change the hydrodynamic size in OptiMEM and ζ-potential of alkyl4%-PEGAuNPs (Figure S9C). Next, we performed three control experiments to exclude the role of Cy5 in cellular uptake. In the first experiment, we incubated Kera-308 cells with Cy5-labeled, methoxy-PEG-AuNPs for 24 h, followed by detecting the intracellular Cy5 fluorescence by confocal imaging. Our confocal images do not reveal detectable amounts of Cy5labeled methoxy-PEG-AuNPs inside the cell (Figure S9B). These data verify that, contrary to alkyl groups, incorporation of Cy5 molecules to the dense PEG coating does not significantly promote cellular entry. In the second experiment, we incubated Kera-308 cells with Cy5-labeled, dodecyl4%-PEGAuNPs. In this case, we observed intense intracellular Cy5 fluorescence signals due to the efficient cellular entry of Cy5labeled, dodecyl4%-PEG-AuNPs. In agreement with the data presented in Figure 2, the intracellular Cy5 fluorescence signals intensify with increasing amounts of Cy5 in the dense PEG coating. In the third experiment, we added methoxy-PEGAuNPs and alkyl4%-PEG-AuNPs, with and without Cy5 modification, to Kera-308 cells for 3 and 24 h. By ICP-MS measurements, we confirmed that Cy5 modification does not alter the cellular uptake kinetics of the NPs (Figure S9D). Ultimately, we returned to our original confocal imaging studies on the cellular uptake kinetics of Cy5-labeled, alkyl4%-PEGAuNPs of Kera-308 cells. We observed a gradual increase in

alkyl mass percentage of 0.2% is 2 orders of magnitude lower that of conventional lipid-based NPs, like liposomes for drug delivery, lipid NPs for gene delivery, and phospholipid-coated quantum dots for imaging (Tables S1).1,4,50 In view of the widely acknowledged difficulty for densely PEGylated NPs to enter mammalian cells, our data accentuate the promise of incorporating minute amounts of alkyl groups (≤0.2 mass percent) as a viable strategy for boosting the intracellular delivery of NPs. Cellular Uptake Kinetics of Alkyl-PEG-AuNPs. To investigate the cellular uptake kinetics of alkyl4%-PEG-AuNPs, we incubated Kera-308 cells with 1 nM methoxy-PEG-AuNPs or alkyl4%-PEG-AuNPs for different durations of time, followed by harvesting the cell pellets for ICP-MS measurements (Figure 3A). Methoxy-PEG-AuNPs do not exhibit any appreciable increase in cellular association over the 24-h observation time window, with their intracellular Au content hitting an equilibrium value of 3 × 103 NPs per cell after only 15 min of incubation. The cellular uptake of hexyl4%-PEG-AuNPs narrowly increases over time, reaching 6 × 103 NPs per cell (or 2-fold higher than methoxy-PEG-AuNPs) after 24 h of incubation. On the contrary, the cellular uptake kinetics of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs are much more rapid than those of methoxy-PEG-AuNPs and hexyl4%PEG-AuNPs, netting Au contents of 4−5 times higher than that of methoxy-PEG-AuNPs (i.e., 1.2−1.5 × 104 NPs per cell) after 15 min of incubation. Strikingly, after 24 h of incubation, the intracellular Au contents of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs become 9 × 104 NPs per cell (i.e., 30-fold higher than methoxy-PEG-AuNPs) and 1.2 × 105 NPs per cell (i.e., 40-fold higher than methoxy-PEG-AuNPs), respectively. Note that the amount of octadecyl4%-PEGAuNPs internalized by Kera-308 cells consistently leads that of dodecyl4%-PEG-AuNPs over the 24 h observation time window. To visualize the cellular uptake kinetics of alkyl4%-PEGAuNPs, we prepared a collection of alkyl4%-PEG-AuNPs that 6091

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ACS Nano Table 3. Hydrodynamic Sizes of Alkyl4%-PEG-AuNPs in Different Media hydrodynamic size of alkyl4%-PEG-AuNPs (nm)a b

water methoxy-PEG-AuNPs hexyl4%-PEG-AuNPs dodecyl4%-PEG-AuNPs octadecyl4%-PEG-AuNPs

46.6 47.3 45.3 45.0

± ± ± ±

0.3 0.3 0.2 0.2

PBS (0.2) (0.2) (0.2) (0.2)

47.3 42.5 46.5 83.2

± ± ± ±

0.9 1.2 0.0 1.5

(0.2) (0.1) (0.1) (0.4)

OptiMEM

DMEM + 0% FBSc

DMEM + 10% FBS

45.2 ± 0.4 (0.2) 45.6 ± 1.2 (0.2) 83.9 ± 2.5 (0.3) 164.7 ± 1.8 (0.2)

48.6 ± 0.8 (0.2) 48.4 ± 0.1 (0.2) 46.2 ± 0.7 (0.2) 108.3 ± 0.3 (0.2)

44.0 ± 1.9 (0.4) 42.7 ± 0.9 (0.3) 126.7 ± 1.1 (0.5) 575.6 ± 34.2 (0.4)

a Reported data represent mean ± SD from three independent measurements of Z-average sizes. Numbers in parentheses refer to the polydispersity index (PDI). bPBS = phosphate-buffered saline. cFBS = fetal bovine serum.

Figure 4. Effect of serum on the cellular uptake of alkyl-PEG-AuNPs. (A) UV−vis spectra of methoxy-PEG-AuNPs and alkyl4%-PEG-AuNPs incubated in PBS, OptiMEM, DMEM without FBS, or DMEM with 10% FBS at 37 °C for 24 h show a single peak without significant red-shift of the surface plasmon resonance peak of the AuNPs, supporting the lack of obvious NP aggregation across the 24 h incubation time window. (B) Kera-308 cells were incubated with 1 nM methoxy-PEG-AuNPs or alkyl4%-PEG-AuNPs in OptiMEM, DMEM without FBS, or DMEM with 10% FBS for 24 h. By ICP-MS analysis, incubation in DMEM with 10% FBS led to 80% and 60% reduction in the cellular association of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs when compared to the case of incubation in OptiMEM. Error bars denote standard deviations resulting from triplicate experiments.

Cy5 fluorescence signals in cells treated with dodecyl4%-PEGAuNPs and octadecyl4%-PEG-AuNPs over the entire 24 h observation window (Figure 3B). By contrast, we could not detect appreciable intracellular Cy5 fluorescence signals after incubating the cells with Cy5-labeled, hexyl4%-PEG-AuNPs, in close agreement with our dark-field imaging data presented in Figure 2. In brief, our ICP-MS and confocal imaging data demonstrate that dodecyl4%-PEG-AuNPs and octadecyl4%-PEGAuNPs can enter Kera-308 cells at markedly higher rates and in larger amounts than methoxy-PEG-AuNPs and hexyl4%-PEGAuNPs, reinforcing the pivotal role of alkyl chain length on cellular uptake kinetics. Effect of Serum on the Cellular Uptake of Alkyl-PEGAuNPs. At the NP level, we have established that alkyl chain number and loading density of alkyl chains dictate the cellular uptake of alkyl-PEG-AuNPs. We next questioned, at the cell culture medium level, whether serum would also influence cellular uptake, because serum proteins may adsorb onto the surface of NPs and alter their cellular uptake properties.23,24,36−38 To this end, we incubated alkyl4%-PEG-AuNPs in a medium containing different concentrations of serum proteins, including water, PBS, DMEM without fetal bovine serum (FBS), OptiMEM, and DMEM with 10% FBS (the complete medium for culturing Kera-308 cells) at 37 °C. We ascertained a priori, by the bicinchoninic acid (BCA) protein assay, that the concentration of proteins in OptiMEM is

equivalent to that of 1% FBS. After 24 h of incubation, we characterized the physicochemical properties of the NPs and correlated these properties to the cellular uptake of the NPs by Kera-308 cells. For our characterization studies, we performed DLS measurements of the NPs after incubation in different types of medium (Tables 3 and Table S5 and Figures S10− S13). At all serum concentrations up to 10% FBS, the hydrodynamic diameters of methoxy-PEG-AuNPs and hexyl4%-PEG-AuNPs constantly hover in the range of 43−49 nm, in agreement to our earlier DLS data presented in Table 2. For dodecyl4%-PEG-AuNPs, their sizes in nonserum containing media (i.e., water, PBS, and DMEM without FBS) are typically around 45 nm; yet, the NPs become progressively larger upon incubation in a medium with increasing serum contents, from 84 nm in OptiMEM to 127 nm in DMEM with 10% FBS. For octadecyl4%-PEG-AuNPs, their sizes in OptiMEM, DMEM without FBS, and DMEM with 10% FBS are 165, 108, and 576 nm, respectively. Taken together, our DLS results appear to highlight severe serum-induced aggregation of dodecyl4%-PEGAuNPs and octadecyl4%-PEG-AuNPs. Paradoxically, the UV− vis spectra of these alkyl4%-PEG-AuNP samples reveal one single SPR peak without redshifting nor broadening when the NPs are incubated in all types of medium considered (Figures 4A and S14A,B). This observation fundamentally differs from the broadened absorption spectrum of the aggregated NPs after being exposed to protein-containing medium reported in other 6092

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Figure 5. Pathway for the uptake of dodecyl4%-PEG-AuNPs by Kera-308 cells. (A) Representative TEM images depict the initial contact of dodecyl4%-PEG-AuNPs with the cell membrane within the first 0.5 h of incubation. (i−ii) Note the two modes of involvement by the filopodium layer, tip engagement (red arrow) and formation of invaginations between adjacent filopodia (asterisks). (iii) The “crisscrossing” of protrusions in these hot spots triggers the formation of invaginations that facilitate the cellular entry of dodecyl4%-PEG-AuNPs and (iv) accumulation inside intracellular compartments. The bottom row shows the enlargement of the boxed area in their top row. (Legend: Nu = nucleus; Cy = cytosol; and Ex = extracellular space.) (B) Upon pretreatment of Kera-308 cells with dynasore and methyl-β-cyclodextrin (mβCD), the intracellular fluorescence signal of Cy5-labeled, dodecyl4%-PEG-AuNPs significantly plunges. The cell pellets reveal reduced cellular uptake after dynasore and m-βCD treatment, as seen by the diminished red color due to the surface plasmon resonance of AuNPs. (C) By ICP-MS measurements, pretreatment with dynasore or m-βCD and placement of cells at 4 °C suppress the association of dodecyl4%-PEGAuNPs with Kera-308 cells by over 60%. (D) By ICP-MS measurements, RNAi-mediated genetic knockdown of caveolin-1 (Cav-1), clathrin heavy chain 1 (Clhc-1), and dynamin-2 (Dnm-2) does not severely attenuate the association of dodecyl4%-PEG-AuNPs to Kera-308 cells. Error bars denote standard deviations resulting from triplicate experiments.

studies.51−54 Additionally, the solutions of alkyl4%-PEG-AuNPs in all types of medium remain red in color after 24 h of incubation, accompanied by the lack of large visible aggregates of NPs sedimenting to the bottom of the reaction vessels (Figure S14C). These UV−vis data and colorimetric observations suggest no obvious aggregation between the adjacent NPs in all types of medium considered. The red color indicates that the interparticle distance between the AuNP cores is far greater than the average AuNP diameter.46 Thus, we envision that the increase in hydrodynamic diameter of alkyl4%PEG-AuNPs upon incubation in serum reflects agglomeration of alkyl-PEG-AuNPs51 rather than irreversible aggregation of the AuNP cores,36,52 a point later proven by TEM imaging in our intracellular trafficking studies (Figure 6). As such, the dense PEG shells of the NPs only touch one another, whereas

the NP cores are not in physical contact. Previously, Spector showed that fatty acids that contain 18 carbons can associate with albumin, the most abundant protein in serum, with 8-fold and 5000-fold higher affinity than those fatty acids containing 12 and 6 carbons, respectively.18 Wolfrum et al. reported that siRNAs conjugated to alkyl chains with 12 carbon atoms (lauryl) and 18 carbon atoms (stearyl) can also bind to albumin.19 Given that the average size of a methoxy-PEGAuNP is around 45 nm, we believe that the hexyl4%-PEGAuNPs exist as monodisperse NPs in OptiMEM. By estimation, dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs manifest themselves as a loose agglomerated cluster of 2−3 NPs and 4− 6 NPs in OptiMEM, respectively. In summary, alkyl-PEGAuNPs can form loose NP clusters in serum-containing cell culture medium, and the degree of agglomeration increases 6093

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PEG-AuNPs (Figure 2 and Table 3). To begin with, we investigated the ultrastructural details of the endocytosis process by capturing the initial contact of dodecyl4%-PEGAuNPs with the cell membrane of Kera-308 cells within the first 2 h of incubation by TEM imaging. Representative TEM images of the extracellular region depict the abundance of individual NP cores that resemble colloidally stable PEGylated NPs60−62 rather than aggregated NP cores,52,60,63 indicating that the alkyl-PEG-NPs primarily enter Kera-308 cells as individual entities instead of aggregated clusters. This observation supports our hypothesis that the alkyl-PEGAuNPs remain loosely interconnected in serum-containing medium (Figures 5A and S6) and underscores the merit of incorporating a low mass percentage of alkyl groups to the dense PEG shell to achieve effective intracellular delivery and to maintain colloidal stability in the extracellular region. These TEM images repeatedly depict association of dodecyl4%-PEGAuNPs with finger-like cell membrane protrusions of 60−200 nm in diameter, matching past literature reports on the length characteristic of filopoida64 (Figures 5A and S15). Found in many different cell types, (e.g., keratinocytes, neurons, endothelial cells and invasive cancer cells), filopodia are highly dynamic cellular protrusions that constantly probe the extracellular environment for controlling cell adhesion and migration.64,65 Association of NPs with the filopodia manifests in two distinct modes: (1) engagement by the tip of membrane protrusions and (2) accumulation in the “interdigital folds” on the cell membrane (i.e., space between adjacent membrane protrusions). These observations support a filopodia-dependent pathway of endocytosis, whereby biological objects (e.g., exosome and virus) are grabbed by the protrusions and then pulled toward the “endocytic hot spots” located in the filopodia base.66,67 “Crisscrossing” of protrusions in these hot spots triggers the formation of invaginations that facilitate the cellular entry of alkyl-PEG-AuNPs and accumulation inside intracellular compartments (Figure 5A). We also confirmed the abundance of filopodia by TEM imaging in untreated Kera-308 cells, illustrating that the presence of filopodia is not a consequence of NP incubation (Figure S16). The above TEM imaging data appear to reveal the dearth of honeycomb-shaped lattices that resemble clathrin-like structures68 or flask-shaped invaginations that resemble caveolae69 in close proximity to the cell membrane. We further used a combination of pharmacological inhibition and genetic knockdown approaches to address whether the internalization of alkyl-PEG-AuNPs by Kera-308 cells is clathrin-dependent or lipid raft/caveolae-dependent. In one set of studies, we asked whether pretreatment of Kera-308 cells with a series of pharmacological inhibitors of key pathways may adversely affect the cellular uptake of dodecyl4%-PEG-AuNPs (Figure S17A). We confirmed in advance that incubation of Kera-308 cells with these inhibitors for 6 h does not substantially reduce their viability (Figure S17B). As our first inhibitor, we pretreated Kera-308 cells with methyl-β-cyclodextrin (mβCD), a widely used inhibitor that alters the structure of cholesterol-rich domains in the cell membrane,70 before adding dodecyl4%-PEG-AuNPs to the cells. By confocal imaging, pretreatment with m-βCD drastically attenuated the cellular uptake of Cy5-labeled, dodecyl4%-PEG-AuNPs, as seen by the significant reduction in the intracellular Cy5 fluorescence signals (Figure 5B). While we also detected by ICP-MS analysis a 95% decrease in the cellular association of dodecyl4%PEG-AuNPs following pretreatment with m-βCD (Figure 5C),

with carbon chain length and serum concentration. Notably, due to their dense PEG shell, these alkyl-PEG-AuNPs still maintain considerable colloidal stability as their cores do not aggregate after prolonged incubation for 24 h in serumcontaining medium. For most cellular uptake experiments in this study, we opted for the use of OptiMEM as the incubation medium, because it allows us to investigate the cellular uptake of alkyl-PEG-AuNPs without significantly impacted by serum while maintaining the health of the cells over an incubation time of 24 h. After clarifying the effect of serum on NP agglomeration, we turned to investigate how the cellular uptake of alkylx%-PEGAuNPs depends on serum concentration. We incubated Kera308 cells with 1 nM alkyl4%-PEG-AuNPs in OptiMEM, DMEM without FBS, or DMEM with 10% FBS for 24 h (Figure 4B). By ICP-MS analysis, we found that cellular uptake in OptiMEM is similar to that in DMEM without serum, with the latter incubation condition being around 15% lower (12 × 104 NPs per cell versus 10 × 104 NPs per cell for dodecyl4%-PEG-AuNPs and 15 × 104 NPs per cell versus 13 × 104 NPs per cell for octadecyl4%-PEG-AuNPs). Note that this observation applies to all carbon chain lengths considered. The slightly attenuated cellular uptake in DMEM without serum may arise from prolonged serum starvation of the treated cells.55 More importantly, even in the absence of serum (DMEM without FBS), dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs still enter Kera-308 cells more pronouncedly than methoxy-PEGAuNPs or hexyl4%-PEG-AuNPs, driving home the point that the cellular uptake of alkyl-PEG-AuNPs does not require the aid of serum proteins. While our DLS and UV−vis data suggest mild serum-induced agglomeration of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs in OptiMEM, the ICP-MS data demonstrate that their cellular entry does not significantly depend upon serum adsorption when serum concentration is 1%. However, higher amounts of serum (i.e., DMEM with 10% FBS) cause sharp attenuation in the cellular uptake of the same collection of alkyl-PEG-AuNPs. When compared to the OptiMEM case, the cellular uptake of dodecyl4%-PEG-AuNPs and octadecyl4%-PEG-AuNPs in DMEM with 10% FBS decreases by nearly 80% and 60%, respectively. By literature precedent, we speculate two possible explanations to the drastically reduced uptake of alkyl-PEG-AuNPs in the presence of higher amounts of serum (10% FBS): (1) NPs or NP clusters larger than 100 nm generally enter nonmacrophage cells less effectively than their smaller counterparts;56−58 and (2) serum adsorption reduces the ability of NPs to adhere to the cell membrane or interact with membrane receptors for their subsequent entry to the cell.36,59 Interestingly, despite the overall decline in the cellular uptake of alkyl4%-PEG-AuNPs in DMEM with 10% FBS, our ICP-MS data shows that the degree of cellular uptake increases with alkyl chain number, a trend similar to that observed for the cases of OptiMEM and DMEM without serum. In conclusion, the cellular uptake of alkyl-PEGAuNPs does not necessitate the involvement of serum protein, although it markedly decreases at higher serum concentrations. With or without serum, cellular uptake increases with the carbon chain number of the alkyl groups. Pathway for the Cellular Uptake of Alkyl-PEG-AuNPs. We further elucidated the mechanism for the cellular uptake of alkyl-PEG-AuNPs at the pathway level. We selected dodecyl4%PEG-AuNPs as our representative alkyl-PEG-AuNP because they exhibit an optimal balance between cellular uptake and colloidal stability in OptiMEM among our collection of alkyl6094

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Figure 6. Intracellular trafficking of dodecyl4%-PEG-AuNPs. Kera-308 cells were incubated with Cy5-labeled, dodecyl4%-PEG-AuNPs for confocal imaging or dodecyl4%-PEG-AuNPs for TEM imaging. (A) By confocal imaging, the fluorescence signals of NPs (red) do not colocalize with those of intracellular acidic compartments (LysoTracker Green DND-26; green) after 2 h of incubation. After 10 and 24 h of incubation, the Cy5 fluorescence signals substantially colocalize with the those of LysoTracker (yellow) with a Mander’s coefficient of 0.8 (bottom-left corner of pictures).18,95 (B) After 2 h of incubation, representative TEM images show the accumulation of dodecyl4%-PEGAuNPs inside vesicles of around 200 nm in diameter (red arrows) or the cytosol (blue arrows). After 10 h of incubation, most NPs accumulate in late endosome, as evidenced by the presence of multivesicular bodies of ∼100 nm in diameter (MVBs; red arrows). After 24 h of incubation, most NPs localize in the lysosome, which are characterized by the presence of multilamellar bodies (MLBs; red arrows) and its high electron density. (C) Representative TEM images capture the localization of dodecyl4%-PEG-AuNPs in the cytosol (blue arrows) of Kera308 cells consistently after 2, 10, and 24 h of incubation. The lower images show the enlargement of the boxed areas on the upper panels. (Legend: Nu = nucleus; Cy = cytosol; and Ex = extracellular space.)

nonspecifically inhibit lipid raft or caveolae-mediated uptake,71 it also blocks clathrin-mediated71,72 uptake and fluid-phase

we could not conclusively pinpoint the involvement of a specific pathway of endocytosis. Not only does m-βCD 6095

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ACS Nano endocytosis.73 Further complicating our analysis, the decrease in cellular uptake may arise from the interaction between mβCD and dodecyl4%-PEG-AuNPs besides the customary role of m-βCD in depleting the cholesterol in the cell membrane, because m-βCD can bind to alkyl-containing molecules.74 Moving on to other inhibitors, we noticed that pretreatment of Kera-308 cells with chlorpromazine (a relatively specific inhibitor of clathrin-mediated endocytosis)68,75 or filipin III (a relatively specific inhibitor of lipid raft/caveolae-meidated endocytosis)68,76 does not appreciably interfere with the cellular uptake of dodecyl4%-PEG-AuNPs (Figure 5C). In another set of studies, we transfected Kera-308 cells with siRNA that specifically suppresses the expression of key proteins affiliated with the canonical cellular uptake pathways such as heavy chain 1 of clathrin (Clhc-1) and caveolin-1 (Cav1), followed by measuring the cellular uptake of dodecyl4%PEG-AuNPs. We confirmed the reduction in expression of both proteins after transfection by Western blotting (Figure S17C). In line with our pharmacological inhibition studies, genetic knockdown of Clhc-1 or Cav-1 failed to attenuate the association of NPs to Kera-308 cells by a wide margin (Figure 5D). Summing up the TEM imaging, pharmacological inhibition, and genetic knockdown data, we conclude that the uptake of dodecyl4%-PEG-AuNPs by Kera-308 cells is not primarily clathrin- or caveolae-mediated. Moreover, we pretreated Kera-308 cells with amiloride (an inhibitor of macropinocytosis and phagocytosis),77,78 mannan (a ligand of the phagocytosis-related mannose receptor), fucoidan (a ligand of the phagocytosis-related scavenger receptor),79 or cytochalasin D (which disrupts the polymerization of actin filaments).68,80 As we did not detect a considerable decrease in the cellular uptake of dodecyl4%-PEG-AuNPs following pretreatment by these four inhibitors (Figure 5C), we conclude limited involvement of micropinocytosis and phagocytosis. Furthermore, we inquired the potential relevance of dynamin to the cellular uptake of alkyl-PEG-AuNPs. Since our TEM imaging data show that the dodecyl4%-PEG-AuNPs NPs enter Kera-308 cells by interacting extensively with their filopodia (Figure 5A), we speculate that dynamin may mediate the uptake of these NPs because dynamin can regulate actin-based cytoskeleton,81 such as lammellipodia and filopodia.82,83 Nevertheless, existing literature on dynamin mostly indicates that dynamin is essential for the formation of membrane invaginations that support clathrin-mediated endocytosis,84,85 a pathway with little involvement in the internalization of alkylPEG-AuNPs by Kera-308 cells. Therefore, we used both pharmacological inhibition and genetic knockdown approaches to interrogate the role of dynamin. We selected dynasore, a frequently used inhibitor to probe the relevance of the dynamin-mediated pathway to the cellular uptake of many biomolecules80 and NPs.81 Notably, by confocal imaging and ICP-MS analysis, pretreatment with dynasore led to a sharp 72% decline in the cellular uptake of dodecyl4%-PEG-AuNPs (Figure 5B,C), a result that purportedly supports the notion of dynamin-mediated uptake. Nonetheless, dynasore has become a debatable inhibitor of dynamin in recent years, plagued by its frequently reported off-target effects.86 For example, Camilli and co-workers found that in dynamin-knockout fibroblasts, dynasore can inhibit dynamin-mediated endocytosis and nonspecifically impair fluid-phase endocytosis and peripheral membrane ruffling fluid-phase.87 In view of these recently reported off-target effects of dynasore on cellular cholesterol, lipid raft, and actin,86 we further employed RNA interference to

suppress the expression of dynamin-2 (Dnm-2), a key constituent protein of dynamin. Dnm-2 is ubiquitously expressed in most cell types, whereas dynamin-1 and dynamin-3 are specifically expressed in neurons.85 Surprisingly, we observed only 12% reduction in the cellular uptake of dodecyl4%-PEG-AuNPs upon genetic inhibition of Dnm-2 knockdown cells (Figure 5D). By juxtaposing the severe decrease in cellular uptake due to dynasore pretreatment and the modest decrease arising from Dmn-2 genetic knockdown, we cannot establish a specific role of dynamin in the endocytosis of alkyl-PEG-AuNPs by Kera-308 cells. Instead, we can only speculate the presence of off-target effects associated with dynasore that ultimately interfere with the uptake process. Intracellular Trafficking of Alkyl-PEG-AuNPs. Lastly, we examined the intracellular trafficking of dodecyl4%-PEG-AuNPs following their initial cellular entry. On one hand, we incubated Kera-308 cells with Cy5-labeled, dodecyl4%-PEG-AuNPs for different durations of time, followed by staining the cells with LysoTracker, a green fluorescent dye that stains intracellular acidic compartments like late endosome or lysosome, and visualizing the intracellular distribution of the NPs by confocal microscopy. On the other hand, we performed TEM imaging of the Kera-308 cells treated with dodecyl4%-PEG-AuNPs as a function of incubation time. After 2 h of incubation, we detected by confocal imaging that most NPs are located inside the cell without entering acidic compartments (Figure 6A). Representative TEM images reveal that the NPs accumulate inside vesicles of around 200 nm in diameter (Figure 6B). Also, they depict localization of ∼14.4% of the NPs in the cytosol (Figures 6C and S18). After 10 h of incubation, confocal images portray strong overlapping fluorescence signals of the NPs and LysoTracker with a Manders coefficient of 0.8, implying significant accumulation of dodecyl4%-PEG-AuNPs inside intracellular acidic compartments. TEM images further depict conspicuous accumulation of NPs inside multivesicular bodies (MVBs) of ∼100 nm in diameter,20,88 hallmark features of the late endosome (Figure 6B).89 We still identified considerable accumulation of NPs in the cytosol after 10 h of incubation (Figure 6C). After 24 h of incubation, most dodecyl4%-PEG-AuNPs accumulate inside intracellular acidic compartments, as evidenced by prominent colocalization of the fluorescence signals of Cy5 and LysoTracker also with a Manders coefficient of 0.8. By TEM imaging, most NPs localize in the lysosome, which are characterized by the presence of multilamellar bodies of around 100 nm to 2.4 μm in diameter (MLBs; refer to Figure S19 for untreated Kera-308 cells) and its high electron density (Figure 6B).90,91 In line with our 2 h data, 18.9% of the alkyl-PEG-AuNPs still accumulate freely in the cytosol after 24 h of incubation. Of note, these NPs exist as discrete entities inside the cell and show negligible aggregation of the AuNP cores throughout the 24 h observation time window. In fact, we can still appreciate the PEG coating of the NPs (positively stained by the osmium tetroxide92 added to the cell samples in preparation for TEM imaging) in highmagnification TEM images taken from cells after 24 h of incubation (Figure S20). Thus, our alkyl-PEG-AuNPs are markedly different from other NP-based delivery systems that can enter cells pronouncedly but aggregate irreversibly inside intracellular compartments.20,60 The PEG shell of our alkylPEG-AuNPs probably suffers from inappreciable enzymatic degradation,93 preventing the AuNPs from aggregating inside the cell even after 24 h. In short, alkyl-PEG-AuNPs 6096

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ACS Nano predominantly adopt the conventional “endo-lysosomal” trafficking route upon cellular entry, leaving ∼15% of them freely accumulating in the cytosol. This degree of cytosolic accumulation is consistent with previous reports on the trafficking of PEG-coated AuNPs. Brandenberger et al. observed that ∼80% of the PEG-AuNPs accumulates inside vesicles and 10−15% of them in the cytosol of alveolar epithelial cells, a trend consistently observed after 1, 4, and 24 h of incubation.94 TEM micrographs by Oh and Park captured the migration of PEG-AuNPs in the cytosol of macrophages and their ultimate exocytosis.60 Therefore, we believe that weak alkylation (via incorporation of a modest alkyl mass percentage of no more than 0.2%) does not significantly bias the previously published intracellular distribution of PEG-AuNPs.

and briefly dried. Finally, the product was dialyzed against methanol for 1 wk, against 0.1% acetic acid in water for 1 d, and against deionized water for 1 more d. Successful conjugation of alkylamine to the bifunctional PEG linker was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) (Bruker). The octanol−water partition coefficient (log P) values of the alkanes were calculated using the Molinspiration Cheminfomatics open source module for molecular properties and bioactivity score calculation. Preparation of Alkyl-Terminated PEG-Coated NPs. Citratecapped AuNPs of 25 nm in diameter were prepared by an established seed-mediated growth method.96 In brief, 150 mL of 2.2 mM anhydrous sodium citrate (Alfa Aesar) was brought to boil in a threenecked round-bottom flask with rapid stirring, followed by injection of 1 mL of 25 mM gold(III) chloride trihydrate (HAuCl4·3H2O, Sigma). After becoming orange red in about 15 min, the solution was cooled down to 90 °C and maintained at this temperature. Next, 1 mL of 25 mM Au3+ was injected twice at 30 min intervals. The solution was diluted by replacing 55 mL of the solution with 53 mL of Nanopure water (Thermo Scientific) and 2 mL of 60 mM sodium citrate. Addition of 1 mL of the 25 mM Au3+ solution was repeated 3 times every 30 min. The solution was used for subsequent NP growth. The process of dilution, addition of sodium citrate solution, and three doses of Au3+ solution was repeated again until the AuNPs measure roughly 25 nm in diameter, as evidenced by their surface plasmon resonance peak at 521 nm.97 To prepare alkyl-terminated, PEG-coated AuNPs (alkylx%-PEG-AuNPs) for ICP-MS and TEM studies, an aqueous suspension of AuNP solution was mixed with x mol % of HS-PEG5000CnH2n+1 and (100 − x) mol % of HS-PEG5000-OCH3 (JenKem Technology) by keeping the total PEG concentration at 20 PEG molecules per nm2 of AuNP surface to be coated. All PEGylation reactions lasted for 1 h with sonication, followed by purification by 5 rounds of centrifugation at 13,000 rpm for 15 min and resuspension in deionized water. To prepare Cyanine 5 (Cy5)-containing dodecyl4%PEG-AuNPs for confocal imaging, 0.13 mol % of HS-PEG5000-Cy5 (Nanocs), 4 mol % of HS-PEG5000-C12H25, and 94.7% of HS-PEG5000OCH3 were added to the AuNP solution while keeping the total number of HS-PEG5000-dodecyl strands per NP unchanged. Characterization of Alkylx%-PEG-AuNPs. The concentration of NPs was determined by UV−vis-NIR spectrophotometry (Agilent Cary 5000) based on the Beer−Lambert’s law and the molar extinction coefficient of AuNPs of 25 nm in diameter (1.1 × 109 M−1 cm−1).97 The hydrodynamic diameters and ζ-potentials of NPs were measured by dynamic light scattering (Zetasizer Nano ZS90, Malvern). All reported hydrodynamic sizes represent the “Z-average” values from three independent measurements. Additional DLS size profiles and size distribution data measured in intensity (di), number (dn), and volume (dv) are provided in the Supporting Information. To reveal the presence of the PEG corona on the AuNP surface by TEM, 10 μL of 2 nM NP solution was drop-cast onto a TEM copper grid (200 mesh; Beijing Zhongjingkeyi Technology) and left for 30 min. After removing the NP solution, 10 μL of 2% phosphotungstic acid (Sigma) was added onto the TEM grid for another 1 min. After removing the PTA solution, the stained grid was allowed to dry at RT for at least 4 h before visualization under TEM at a beam voltage of 100 kV (Hitachi H7700). Quantification of the Loading Density of PEG Strands. The density of PEG grafted onto AuNPs was determined by thiol depletion via the Ellman’s assay.34,35 One mL of 2 nM AuNPs was mixed with different mole ratios of HS-PEG5000-OCH3 and HS-PEG5000-CnH2n+1 at a total PEG concentration of 20 PEG molecules per nm2 of AuNP surface for 1 h under sonication. After centrifuging the NP solution at 15,000 rpm for 15 min, the supernatant was lyophilized and resuspended in 60 μL of deionized water. Twenty μL of the concentrated PEG sample was mixed with 100 μL of Ellman’s assay buffer [1 mM EDTA (Sigma) in 0.1 mM Na2HPO4; pH = 8]. The resultant PEG sample was subsequently mixed with 50 μL of Ellman’s detection buffer [0.5 mg/mL of Ellman’s reagent (5,5-dithio-bis(2nitrobenzoic acid)) (JenKem Technology) in the assay buffer]. AuNPs not functionalized with PEG strands were included as negative control. After 10 min of reaction, the absorbance of the reaction mixture was

CONCLUSIONS In this work, we have systematically investigated the effect of alkylation on the cellular uptake of densely PEGylated AuNPs, which are acknowledged for their limited uptake by mammalian cells. We have demonstrated that inclusion of an extremely modest fraction of alkyl chains to the dense PEG shell (