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Coating Metal Nanoparticle Surfaces With Small Organic Molecules Can Reduce Non-Specific Cell Uptake Desiree Van Haute, Alice T. Liu, and Jacob M. Berlin ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b03025 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017
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Coating Metal Nanoparticle Surfaces With Small Organic Molecules Can Reduce Non-Specific Cell Uptake Desiree Van Haute, Alice T. Liu, Jacob M. Berlin* Department of Molecular Medicine, Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute at City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010, United States. *Corresponding Author Jacob M. Berlin, Ph.D Associate Professor, Division of Molecular Medicine City of Hope, 1500 East Duarte Rd, Duarte, CA 91010 Phone [626/256-4673] Email:
[email protected] KEYWORDS: Cell Uptake, gold nanoparticles, cyanide, surface coverage, protein opsonization, polyethylene glycol
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Abstract
Elucidation of mechanisms of uptake of nanoparticles by cells and methods to prevent this uptake is essential for many applications of nanoparticles. Most recent studies have focused on the role of proteins that coat nanoparticles and have employed PEGylation, particularly dense coatings of PEG, to reduce protein opsonization and cell uptake. Here we show that small molecule coatings on metallic nanoparticles can markedly reduce cell uptake for very sparsely PEGylated nanoparticles. Similar results were obtained in media with and without proteins, suggesting that protein opsonization is not the primary driver of this phenomenon. The reduction in cell uptake is proportional to the degree of surface coverage by the small molecules. Probing cell uptake pathways using inhibitors suggested that the primary role of increased surface coverage is to reduce nanoparticles’ interactions with the scavenger receptors. This work highlights an under-investigated mechanism of cell uptake that may have played a role in many other studies and also suggests that a wide variety of molecules can be used alongside PEGylation to stably passivate nanoparticle surfaces for low cell uptake.
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A major challenge in using nanoparticles for targeted delivery is that NPs administered intravenously (IV) primarily accumulate and persist in the liver and spleen.1-4 Considering the Kupffer cell population in the liver and the large macrophage population in the spleen, this accumulation is generally attributed to the phagocytosis of nanoparticles by macrophages in the body, though recent work has implicated other immune cells as well.3, 5,6 Decreasing uptake of nanoparticles by macrophages in vitro has been an active area of research in the hunt for longcirculating, liver-avoiding nanoparticles that could improve targeted delivery for diseases such as cancer. Many different passivating agents — polyethylene glycol (PEG),7-9 chitosan,10 hyaluronic acid,11 polyoxazoline,12 among others — have been investigated for their ability to shield the nanomaterials from phagocytic cells in vitro. PEG is by far the most commonly used passivating agent. PEG acts as a barrier layer that prevents the direct interaction between nanoparticles and their environment. PEG functionalized nanoparticles are endocytosed by macrophages less than their unfunctionalized counterparts.13 In general, it has been reported that as the amount of PEG on the surface of the nanoparticle increases, the cellular uptake of these particles decreases. It is commonly thought that this phenomenon is due to PEG’s ability to reduce protein opsonization since it has been demonstrated that by increasing the density of PEG13 or the length of PEG,14,15 there is a decrease – but never an elimination – of protein opsonization. Most reports focus on increasing the density of the PEG on the surface of the nanoparticle either through backfilling,16 using branched PEGs,9 using a hydrophobic core as a spacer,17 or by incubating the particles in an excess of PEG.13 It has also been demonstrated that the conformation of the PEG coating (brush vs mushroom) can play a significant role in NP uptake and, as a result, increasing PEGylation is not always beneficial.18 For all PEGylation strategies, fully protecting the surface of the NPs appears to be quite challenging as, for example,
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even with “dense” coatings it was found that cysteine residues on peptides or proteins can penetrate the PEG layer and attach to the surface of the particle.19 Recent reports have suggested that the protein corona hypothesis is more complex than simply the amount of protein adsorbed onto the surface of a nanoparticles.20 The identity and abundance of proteins composing the corona have been observed to influence nanoparticles ability to avoid cell uptake.21,22 Recognizing that composition may be more important than amount, protein corona fingerprinting has been undertaken with metallic nanoparticles in order to predict in vitro cell uptake based on the composition of the protein corona.22 It has also been demonstrated that for liposomes the identity of the proteins forming the corona depends on PEG length and this change in composition correlates with a change in cell uptake.14 Interestingly, this report focused on using apolipoproteins in the protein corona to enhance uptake by cancer cells expressing a high level of scavenger receptor class B type 1, while another study suggested that apolipoprotein J may be the active mediator of the low cell uptake achieved by PEGylation.15 Additionally, reports have investigated the role of the conformation of the proteins with evidence that suggests that denatured albumin on the surface of a nanoparticles could trigger its cell uptake in a phagocytic cell.23 Overall, the protein corona is widely believed to be a key mediator of cell uptake but there remains a debate on how the corona mediates the uptake process. In our previous publication on the controlled synthesis of gold nanoparticle aggregates using a small molecule crosslinker, pentaerythritol tetrakis-(3-mercaptopropionate) (PTMP), we noticed what appeared to be unusually low cell uptake in a human macrophage-like cell line (THP-1).24 In transmission electron micrographs, most cells were void of nanoparticle aggregates and the cells that contained nanoparticle aggregates generally had one aggregate per cell. This result was in contrast to publications that showed endosomes full of gold nanoparticle
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aggregates25-27 or solid gold spheres of similar size (60 nm).28,29 In further work, we found that these aggregates have an extremely high level of surface coverage as measured by rate of dissolution in potassium cyanide (KCN) (Figure S1A) and hypothesized that this is responsible for their extremely low uptake (Figure S1B).
In order to study the effect of surface coverage in a controlled manner, we prepared 50 nm solid gold spheres with similar levels of PEGylation but varying surface coverage by PTMP. We found that uptake by three different cell lines correlated with the degree of surface coverage with the highest level of surface coverage closely matching the behavior of the aggregates. Interestingly, the degree of uptake could be decreased significantly below that observed for 50 nm solid gold spheres with a higher amount of PEGylation but no small molecule surface coverage. While amount of cell uptake was inversely proportional to surface coverage on the gold nanoparticles, there was no correlation with amount of protein opsonization and similar results were obtained in serum-free media. In particular, the fact that there was no change in the uptake trends when serum-free media was used strongly suggests that the inhibition of uptake by the small molecule coating is not driven by alterations in the composition of the protein corona. An inhibitor study identified the scavenger receptors as the predominant mechanism of uptake for the nanoparticles with incomplete surface coverage, suggesting that more complete surface coverage suppresses this mechanism – likely by inhibiting direct interaction between the scavenger receptors and the NP surface. Overall, we show that, for gold nanoparticles, in vitro uptake can be markedly suppressed by coating their surface with a small molecule and this likely occurs by suppressing uptake via the scavenger receptors and is independent of protein opsonization.
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Results and Discussion Previously synthesized24 biocompatible nanoparticle aggregates had remarkably low cellular uptake in vitro. Electron micrographs of THP-1 cells differentiated into a macrophage phenotype and exposed to gold nanoparticle aggregates for 24 hours showed that the majority of cells contained no aggregates and those that did had only one or two aggregates. This low cell uptake was in contrast to other studies in our lab15a, 17 and in the literature25-29 which showed high levels of uptake with multiple vesicles per cell containing numerous particles. We were interested in determining why these aggregates showed such low uptake into a phagocytic cell line. We found that these aggregates have an extremely high level of surface coverage as measured by rate of dissolution in potassium cyanide (KCN) (Figure S1A) and hypothesized that this is responsible for their extremely low uptake. Since the aggregates possess different morphology than standard spherical NPs and the degree of surface coverage cannot be readily tuned for the aggregates, we decided to investigate the influence of surface coverage with a small molecule in the context of solid spherical gold nanoparticles.
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We developed a library of nanoparticles with variable surface coverage, characterized their physical properties, and measured their stability in media, salt, and cyanide before determining their cellular uptake. Our library of nanoparticles was based on 50 nm citratestabilized gold nanoparticles functionalized with variable amounts of a tetravalent thiol small molecule, pentaerythritol tetrakis (3-mercaptoproprionate) (PTMP). While PTMP has previously been used to assemble 5- 15 nm gold nanoparticles into aggregates, 50 nm gold nanoparticles are too large to create aggregates.24 We were able to modulate surface coverage of solid 50 nm particles by varying the amount of PTMP in the reaction mixture (Scheme 1). Particles are referred to as high, medium, or low surface coverage particles depending on the amount of PTMP measured on the surface of the nanoparticles. Unfortunately, NPs only coated with PTMP are unstable in high salt solutions (like 1x PBS and media) and require PEGylation for stability Scheme 1: Library Synthesis
in these environments. This prevents evaluating the impact of PTMP coating in isolation. Thus, to best study the effect of PTMP coating, high, medium and low surface coverage NPs were
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functionalized with PEG-maleimide which reacts with free thiols on the NP surface. The degree of PEGylation was kept to the amount necessary for stability in solution and was so minimal that we could not directly measure it (described further below and in Table 1). PEGylated 50 nm particles, citrate-stabilized 50 nm particles and 50 nm aggregates assembled from 5 nm particles were used as controls. PEGylated particles were used to understand how coverage with PTMP compared to the most commonly used passivating agent. Of note, the amount of PEG on the PEGylated particles was much higher than for any of the three PTMP-coated NPs as these NPs were not stable with the minimal amount of PEGylation necessary to stabilize the small molecule-coated NPs. Citrate-stabilized particles were used as a positive control since they are highly endocytosed.13 The 50 nm nanoparticle aggregates were used as a control due to their previously noted low cellular uptake and to ensure that the simplified model was able to match the characteristics and behavior of the nanoparticle aggregates. All of the particles were similar in size and charge (Table 1). While the amount of PEG on the PEGylated 50 nm particles was 8% by TGA, the high, medium and low surface coverage nanoparticles did not have enough organic material (PEG-Maleimide plus PTMP) to accurately quantify by TGA with our instrument. Based on the sensitivity of our system and the amount of material we were able to prepare, we know the particles contained 95%, 71% and 29%, respectively (See Figure S8 for assumptions and calculations). Importantly for this discussion, the amount of organic material was much less than that seen with the PEGylated particles. No evidence of aggregation was seen with these particles using TEM or UV measurements (Figure S3).
Table 1: Surface Coverage Library Characterization 50 nm 50 nm Particle Size Citrate PEG Aggregates Coating
50 nm High Coverage
50 nm Medium Coverage
50 nm Low Coverage
Hydrodynamic 52 59 50 66.3 74.6 74.3 Diameter (nm) Zeta Potential -28 -10 -18 -26 -9 -6 (mV) Weight Percent 1 1 1 8% 10%
