Communication pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Complement Activation by PEGylated Gold Nanoparticles Quang Huy Quach,† Roxanne Li Xian Kong,† and James Chen Yong Kah*,†,‡ †
Department of Biomedical Engineering and ‡NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 115783 Singapore S Supporting Information *
ABSTRACT: Gold nanoparticles (AuNPs) are widely used in biomedical applications, but much less is known about their immunological properties, particularly their interaction with the complement system, a key component of innate immunity serving as an indicator of their biocompatibility. Using a library of different-sized AuNPs (10, 20, 40, and 80 nm) passivated with polyethylene glycol (PEG) of different molecular weight (Mw = 1, 2, 5, and 10 kDa), we demonstrated that citrate-capped AuNPs activated the whole complement system in a size-dependent manner, characterized by the formation of the end-point activation product, SC5b-9, in human serum. Although PEGylation of AuNPs mitigated, but did not abolish, the activation level, complement activation by PEGylated AuNPs was independent of both the core size of AuNPs and the molecular weight of PEG. The cellular uptake of both citrate-capped and PEGylated AuNPs by human U937 promonocytic cells which expresses complement receptors were highly correlated to the level of complement activation. Taken together, our results provided new insights on the innate complement activation by PEGylated AuNPs that are widely considered to be inert biocompatible nanomaterials. he complement system is a collection of ∼40 serum and membrane-bound proteins that exerts its effector functions on both innate and adaptive immunity upon being activated.1,2 Although the system is activated through three pathways: classical, lectin, and alternative pathway, all lead to the formation of SC5b-9 complex as the final activation product.3 While complement activation has been exploited to enhance the adaptive immune response in the development of subunit vaccines, 4−6 it also serves as an indicator of biocompatibility,7−9 as it is associated with hypersensitivity, inflammation, cancer, and Alzheimer’s disease.3,10 We have previously reported on complement activation by gold nanomaterials of different shapes11 that were widely used for the diagnosis and treatment of diseases in vivo,12,13 which resulted in the formation of SC5b-9 complex at comparable levels between the shapes. Others have also found that complement proteins including C1q, C3, and C4 constituted a significant proportion of protein corona around citratecapped gold nanoparticles (AuNPs) of 30 and 50 nm, although no significant difference in the amount of complement activation products was induced by these AuNP.14,15 Apart from complement activation, many complement proteins (C1q, C3b, and C4b) also serve as opsonins which marked engineered nanoparticles (NPs) for rapid clearance from the circulation.16 Such opsonization is often mitigated through surface functionalization of NPs with polyethylene glycol (PEG) which suppresses the nonspecific interaction between NPs and opsonins to prolong blood residence.17,18 Despite the perceived biocompatibility associated with
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© XXXX American Chemical Society
PEGylation, recent studies have reported its adverse biological effects, including causing cell cycle arrest and DNA damage,19 reducing cellular uptake of NPs,20 and activating the complement system, leading to hypersensitivity reactions.21 Furthermore, the presence of anti-PEG IgM which binds to PEG moieties has been reported in human serum to activate the classical pathway of the complement system,22−24 leading to accelerated blood clearance of PEGylated NPs.25 More recently, other studies have shown that PEGylated AuNPs induced gene modifications in the human cell line at low doses. These suggested that PEG may not be as biologically benign as commonly thought.26 PEGylated AuNPs have also been shown to adsorb complement proteins (C1q, C3, C4, C4a, and Ficolin-2),15,27 leading to an elevated level of Bb protein, an activation product associated with the alternative pathway.27 These complement proteins, which absorbed on the surface of AuNPs, have been found to mediate their cellular uptake in macrophages.28 While these findings suggest an adverse biological impact of PEGylated AuNPs, their ability to activate the complement system has not been fully characterized to date. We have previously found that PEGylation of AuNPs with 2 kDa PEG did not minimize the complement activation level of AuNPs.11 Here, we further examined the effects of AuNPs size and molecular weight of PEG on complement activation by Received: December 13, 2017 Revised: January 28, 2018 Published: February 12, 2018 A
DOI: 10.1021/acs.bioconjchem.7b00793 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 1. Synthesis and PEGylation of AuNPs. The citrate-capped AuNPs (dotted lines in A−D) were incubated with PEG of different Mw (denoted as 1K, 2K, 5K, and 10K in the graphs for 1, 2, 5, and 10 kDa PEG, respectively). PEGylated Au10 (A), Au20 (B), Au40 (C), and Au80 (D) remained stable under salt-induced aggregation (solid lines) while citrate-capped AuNPs aggregated (dash lines). The insets in A−D show TEM images of citrate-capped Au10, Au20, Au40, and Au80, respectively. (E) Hydrodynamic diameter, Dh, of PEGylated AuNPs increased as Mw of PEG increased, while (F) all PEGylated AuNPs had similar zeta potentials, ζ, regardless of the Mw of PEG. Each data point represents the mean ± standard deviation (SD) of triplicate experiments.
Figure 2. (A) PEG density, (B) PEG footprint grafted on the surface of AuNPs, (C) partition coefficient of PEGylated AuNPs as determined by its LogP value, which was a measure of its hydrophilicity, with a negative value being more hydrophilic. Each data point represents the mean ± standard deviation (SD) of triplicate experiments.
colloidally stable at higher PEG:AuNPs ratios for each size, likely due to sufficient passivation conferred by PEG (Figure S2−S5, A−H, SI). The aggregation was confirmed by a significant increase in their Dh (Figure S2−S5, I, SI), and higher polydispersity indexes (Figure S2−S5, J, SI) at the lower ratios. We found that a minimum PEG:AuNP incubation ratio of 1000 was needed to maintain colloidal stability of PEGylated Au10 for all Mw of PEG, as determined by no further change in its Dh, polydispersity index, and ζ as the ratio was increased further (Figure S2, I−K, SI). In a similar manner, the minimum PEG:AuNP ratio of 5000, 25,000, and 125,000 was needed to maintain colloidal stability of Au20, Au40, and Au80, respectively for all Mw of PEG (Figure S3−S5, I−K, SI) after repeated centrifugation to remove excess PEG. We repeated PEGylating the AuNPs of all sizes and with all Mw of PEG basing on these minimum PEG:AuNPs ratios to confer colloidal stability for each AuNP size. Consistently, the resulting PEGylated AuNPs showed a greater absorbance and minor red-shift in UV−vis spectrum (Figure 1A−D, solid lines) compared to citrate-capped AuNPs (Figure 1A−D, dot lines), and an increase in the Dh of PEGylated AuNPs over the citratecapped AuNPs (Figure 1E), likely due to the presence of PEG layer on the surface of AuNPs.19,20,32 By subtracting the Dh of citrate-capped AuNPs, we determined the thickness of PEG
passivating AuNPs of different sizes (10, 20, 40, and 80 nm) with PEG of different molecular weights (1, 2, 5, and 10 kDa). The UV−vis absorption spectrum of AuNPs showed a redshift in the surface plasmon resonance (SPR) peaks from 517, 524, 530, to 549 nm as our expected size of AuNPs increased from 10, 20, 40, to 80 nm, respectively (dotted lines in Figure 1A−D), which was consistent with the theoretical account.29−31 The TEM images showed that the synthesized AuNPs were relatively spherical and monodispersed with average diameters of 10.58 ± 1.28 (Au10), 18.74 ± 1.86 (Au20), 41.66 ± 3.31 (Au40), and 81.13 ± 7.58 (Au80) (insets of Figure 1A−D and Figure S1A−D in Supporting Information, SI). The hydrodynamic diameters (Dh) of the synthesized Au10, Au20, Au40, and Au80 dispersed in water were 13.39 ± 0.36, 23.35 ± 0.88, 39.66 ± 4.35, and 78.09 ± 1.46, respectively (Figure 1E), which agreed well with the TEM. All citrate-capped AuNPs had similar zeta potentials (ζ) of around −30 mV (Figure 1F). Unlike previous studies,15,19 we varied the PEG:AuNP incubation ratio to determine the minimum amount of PEG that saturates the surface of AuNP, instead of varying the PEG density on the surface of AuNPs. The PEGylated AuNPs aggregated at low PEG:AuNP ratios after repeated centrifugation to remove any excess PEG, as characterized by a significant SPR peak broadening, while PEGylated AuNPs remained B
DOI: 10.1021/acs.bioconjchem.7b00793 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
cascade proceeds to generate its final end-point nonlytic activation product marker, SC5b-9. Apart from SC5b-9, other activation products generated along the complement cascade such as C3a, C5b, or pathway-specific activation products, such as C4d and Bb could also be used as markers for complement activation.4−6,38−40 Here, we focused on measuring the overall level of complement activation; therefore, we used SC5b-9 as a marker for the activation of complement system by our AuNP library. We quantified the amount of SC5b-9, an end-point product of complement activation, in commercially available normal human serum after incubation with both citrate-capped AuNPs and PEGylated AuNPs (Figure 3) using established calibration
layer to be ∼5, 7, 12, and 20 nm for PEG of 1, 2, 5, and 10 kDa, respectively (Table S1, SI). More importantly, all the PEGylated AuNPs were resistant to salt-induced aggregation while citrate-capped AuNPs aggregated (Figure 1A−D, dash lines). All PEGylated AuNPs, regardless of their sizes or Mw of PEG were consistently less negative in their zeta potentials (Figure 1F). The presence of PEG on the surface of AuNPs was further confirmed from negative staining TEM in which PEG layer was observed as white coating around AuNPs (the insets of Figure S1, E,F, SI). Taken together, these results confirmed the successful PEGylation of AuNPs. The PEG density grafted on the surface of AuNPs was indirectly determined by quantifying the excess unbound PEG in the supernatant after conjugation based on the preestablished calibration curves using Ellman’s reagent (Figure S6, SI).33 We found that the density of PEG grafted on the surface of AuNPs decreased from 6.46 to 2.91 nm−2 with the increase in Mw of PEG (Figure 2A), resulting in the increase of the footprint of each PEG molecule with the Mw of PEG, with average footprint ranging from 0.15 to 0.35 nm2, depending on the size of AuNPs and the Mw of PEG grafted (Figure 2B). These grafting densities resulted in an average interdistance between grafted PEG chains ranging from 0.0084 to 0.04 nm (See Supporting Information for details on the calculation). These distances were much lower than Flory radius of PEG (Table S2), suggesting that PEG of all Mw adopted the brush conformation on the surface of AuNPs across all sizes.34 Within a fixed Mw of PEG, its density increased (hence, the PEG footprint on surface decreased) with size of AuNPs (Figure 2A,B). These values also agreed well with that reported by others, with slight differences due to different technique in quantifying the amount of PEG.32,35 Interestingly, the PEG density increased with size of AuNPs and could be attributed to a reduced curvature with larger AuNPs that enabled more PEG molecules to be packed together. The partition coefficient of PEGylated AuNP as determined by its LogP value, which was a measure of its hydrophilicity, showed that the LogP of PEGylated AuNPs was generally more negative compared to citrate-capped AuNPs (Figure 2C). This agreed with the general understanding that PEG conferred the hydrophilicity to AuNPs, which were otherwise known to have a more hydrophobic surface.36,37 Furthermore, an increase in the PEG chain length generally resulted in a corresponding increase in the hydrophilicity of PEGylated AuNPs (Figure 2C), theoretically due to the increase in the amount of water molecules trapped between increasing Mw of PEG molecules.17,18,22 The above was true except for PEGylated Au80 whose LogP was about −1.0 regardless of PEG Mw (Figure 2C). This was likely due to a lower curvature of Au80 compared to smaller sizes, which made the methoxy-terminated ends of grafted PEG on Au80 very near to each other. Coupled with a higher PEG density and a larger size as discussed earlier, the access of water molecules into the spaces between PEG chains would be limited. This would be true regardless of the Mw of PEG. Hence, the LogP of PEGylated Au80 was generally less hydrophilic and independent of the Mw of PEG. In fact, LogP of PEGylated Au80 was similar to that of AuNPs of other sizes PEGylated with 1 kDa PEG (Figure 2C). Although the three pathways of the complement system can be activated separately or simultaneously, their termination phase is the same for all pathways.3 After converging at the central step of generating C3 convertase, the proteolytic
Figure 3. Detection of end-point complement activation product, SC5b-9, by ELISA kit. Elevated levels of SC5b-9 were found in commercially available human serum. 1× PBS and zymosan (a wellknown complement activator derived from yeast cell wall, 10 mg/mL) were used as negative control (C, “-”) and positive control (C, “+”), respectively. Each data point represents the mean ± standard deviation (SD) of triplicate experiments.
curve for determination of SC5b-9 (Figure S7, SI). The citratecapped AuNPs activated the complement system in a sizedependent manner, with SC5b-9 amount detected in increasing amount of 0.51, 0.68, 0.88, and 0.99 μg/mL for Au10, Au20, Au40, and Au80, respectively (Figure 3). This could best be explained by the increase in surface area of AuNPs available for complement proteins to nonspecifically adsorb and trigger the complement cascade. Although PEGylation of AuNPs led to significant decreases in the level of complement activation compared to citratecapped AuNPs, it failed to prevent PEGylated AuNPs from activating the complement system as SC5b-9 amount was still significant compared to our negative control of serum incubated with saline only (Figure 3). Interestingly, the sizedependent complement activation observed in citrate-capped AuNPs was negated upon PEGylation, indicating that the PEGylation process made the complement activation by AuNPs independent of its size. Among the different PEG Mw, we observed that 2 kDa PEG had the least complement activation in smaller Au10 and Au20, while the 5 kDa PEG was most effective in reducing the complement activation in the larger Au40 and Au80. We also noted that 10 kDa PEG generally activated the complement system at higher levels across all sizes compared to PEG of lower molecular weights. Although the reason behind this was not entirely clear to us, we suspected it could be due to its lower PEG density or larger number of chemical groups responsible for activation in the longer chain. A deeper study on the contribution of anti-PEG IgM on overall complement activation by PEGylated AuNPs may provide further interesting insights. Nonetheless, the differences in SC5b-9 level between different molecular weight PEGs were much smaller and not C
DOI: 10.1021/acs.bioconjchem.7b00793 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry statistically significant in almost all cases compared to the difference with citrate-capped AuNPs. Previous study by Hamad et al. has shown that the change in configuration of poly(ethylene oxide) (PEO) on nanoparticles from mushroom to brush switched complement activation from classical to lectin pathway.40 Here, we found that PEG of all Mw adopted the brush conformation on the surface of AuNPs; hence, we inferred that PEGylated AuNPs activated the complement system mainly by lectin pathway. However, with a high occurrence (4% to 25%) of anti-PEG antibodies in the healthy blood donor population,41,42 we did not exclude the contribution of these antibodies to the overall activation level via classical pathway, although this contribution may vary depending on blood source. Equal amounts of complement proteins, including C1q, C3, and C4, have been found to absorbed on the surface of PEGylated AuNPs even as the Mw of PEG increased from 2 to 20 kDa.27 We reasoned that similar level of complement proteins adsorption also occurred on our PEGylated AuNPs, leading to similar activation levels observed in our study. This Mw-independence of complement activation also agreed with previously published result which reported that complement activation by PEGylated carbon nanotubes was independent of both Mw and surface density of PEG.38 Regardless of the Mw of PEG, the increase in hydrophilicity (Figure 2C) and reduction in the level of complement activation conferred by PEGylation suggested a relationship between hydrophilicity and complement activation, which could be explained by a reduction in the adsorption of complement proteins in the serum on the surface of AuNPs as it became more hydrophilic from the PEGylation. This would consequently lead to a reduction in triggering the downstream complement activation cascade and hence the amount of intermediate and final activation products formed. Complement proteins deposit on the surface of complement activators in a process known as opsonization, priming the activators for recognition by phagocytic immune cells. The human macrophage-like U937 promonocytic cells expressed complement receptors and could recognize complement protein-bound activators to enhance their cellular uptakes. The uptake of AuNPs by U937 cells was examined by dosing the cells with 0.5 nM citrate-capped and PEGylated AuNPs for 3 h. The concentration used was noncytotoxic as the cells maintained high cell viability >90% with the alamarBlue assay even at 1 nM of the AuNPs for all sizes and Mw of PEG (Figure S8, SI). We also confirmed that all AuNPs were colloidally stable under the same incubation condition (in RPMI medium for 3 h at 37 °C), although both hydrodynamic diameters and zeta potential of AuNPs changed significantly after incubation. This was likely due to the adsorption of serum proteins instead of aggregation of AuNPs resulting in precipitation (Figure S9, SI). Hence, the measured cellular uptake was not affected by precipitation of AuNPs. From a quantitative analysis of the AuNPs cell uptake using ICP-MS, we observed significant reduction ranging from 29% to 93% drop in the cellular uptake of AuNPs by U937 cells after PEGylating AuNPs with increasing Mw of PEG compared to citrate-capped AuNPs as our normalized 100% control (Figure 4A). This could be correlated to their increased hydrophilicity associated with the longer PEG and hence reduced complement proteins adsorption as discussed earlier which minimized macrophage recognition. The reduction in the cell uptake by PEGylation was dependent on the size of AuNPs with the
Figure 4. Cellular uptake of PEGylated AuNPs by human macrophage-like U937 promonocytic cells. (A) Relative cellular uptake of PEGylated AuNPs of different sizes by U937 cells (106 cell/well in 24well plate) in RPMI medium containing active serum. Amount of citrate-capped AuNPs of each size taken up by cells was normalized to 100%, and relative cellular uptake of PEGylated AuNPs was compared to that of citrate-capped AuNPs. (B) Intracellular gold (Au) mass as quantified by ICP-MS in 106 U937 cells cultured in RPMI media supplemented with active serum and heat-deactivated serum after incubating the cells with citrate-capped and PEGylated Au20 of different Mw. Each data point represents the mean ± standard deviation (SD) of triplicate experiments.
smaller Au10 experienced the least reduction of ∼50% on average, while the larger Au20, Au40, and Au80 experienced >70% reduction in their cell uptake (Figure 4A). This showed that PEGylation was more effective in shielding larger AuNPs from phagocytosis compared to smaller ones. To further explore the mediating role of complement activation in cellular uptake, we deactivated by heat complement proteins and repeated the U937 cell uptake of AuNPs with cell culture medium supplemented with heat-deactivated serum. The intracellular gold (Au) mass from citrate-capped Au20 in U937 cells decreased by 43% in medium containing deactivated serum compared to medium containing active serum. A similar significant reduction in cell uptake (36%) was also observed for Au20 PEGylated with 1 kDa PEG (Figure 4B). This could be due to the lost in proteolytic activity of complement proteins after heating; hence it failed to amplify complement signal, leading to the decrease in U937 cellular uptake. On the other hand, no significant reduction in the intracellular Au mass in U937 cells was observed with deactivated serum compared to active serum for Au20 PEGylated with 2, 5, and 10 kDa (Figure 4B), suggesting that complement protein in deactivated serum still bound to the surface of AuNPs. Together, these observations confirmed the mediating role of complement protein in U937 cellular uptake of AuNPs.28 In summary, we reported on complement activation by PEGylated AuNPs, and established the link between hydrophilicity of AuNPs by PEGylation and the consequent reduction in phagocytosis that was mediated through a reduced adsorption of complement proteins on AuNPs and their downstream complement activation. We also examined the effect of both AuNPs size and Mw of PEG in mediating the process, and concluded that PEGylation of spherical AuNPs, while being able to reduce the level of complement activation over citrate-capped AuNPs, was not able to totally avoid it. These findings thus shed new insights on the interactions between PEGylated AuNPs and the complement system, and therefore helped explain the failure of PEGylated nanoparticles in escaping the reticuloendothelial system (RES), and their subsequent high accumulation in the major RES organs. With this elucidated mechanism, one could make use of the D
DOI: 10.1021/acs.bioconjchem.7b00793 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Lambris, J. D. (2008) Modulation of the antitumor immune response by complement. Nat. Immunol. 9, 1225−1235. (11) Quach, Q. H., and Chen Yong Kah, J. (2017) Non-Specific Adsorption of Complement Proteins Affects Complement Activation Pathways of Gold Nanomaterials. Nanotoxicology 11, 382. (12) Hu, M., Chen, J., Li, Z.-Y., Au, L., Hartland, G. V., Li, X., Marquez, M., and Xia, Y. (2006) Gold nanostructures: engineering their plasmonic properties for biomedical applications. Chem. Soc. Rev. 35, 1084−1094. (13) Huang, X., Jain, P. K., El-Sayed, I. H., and El-Sayed, M. A. (2007) Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine 2, 681. (14) Dobrovolskaia, M. A., Patri, A. K., Zheng, J., Clogston, J. D., Ayub, N., Aggarwal, P., Neun, B. W., Hall, J. B., and McNeil, S. E. (2009) Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles. Nanomedicine 5, 106−17. (15) Walkey, C. D., Olsen, J. B., Guo, H., Emili, A., and Chan, W. C. (2012) Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J. Am. Chem. Soc. 134, 2139−2147. (16) Bertrand, N., and Leroux, J.-C. (2012) The journey of a drugcarrier in the body: an anatomo-physiological perspective. J. Controlled Release 161, 152−163. (17) Harris, J. M., and Chess, R. B. (2003) Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2, 214−221. (18) Otsuka, H., Nagasaki, Y., and Kataoka, K. (2003) PEGylated nanoparticles for biological and pharmaceutical applications. Adv. Drug Delivery Rev. 55, 403−419. (19) Uz, M., Bulmus, V., and Alsoy Altinkaya, S. (2016) Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle−Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage. Langmuir 32, 5997−6009. (20) Del Pino, P., Yang, F., Pelaz, B., Zhang, Q., Kantner, K., Hartmann, R., Martinez de Baroja, N., Gallego, M., Möller, M., and Manshian, B. B. (2016) Basic physicochemical properties of polyethylene glycol coated gold nanoparticles that determine their interaction with cells. Angew. Chem., Int. Ed. 55, 5483−5487. (21) Khan, A. A. C., Szebeni, J., Savay, S., Liebes, L., Rafique, N., Alving, C., and Muggia, F. (2003) Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil®). Annals of Oncology 14, 1430−1437. (22) Knop, K., Hoogenboom, R., Fischer, D., and Schubert, U. S. (2010) Poly (ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem., Int. Ed. 49, 6288−6308. (23) Verhoef, J. J., Carpenter, J. F., Anchordoquy, T. J., and Schellekens, H. (2014) Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics. Drug Discovery Today 19, 1945−1952. (24) Yang, Q., and Lai, S. K. (2015) Anti-PEG immunity: emergence, characteristics, and unaddressed questions. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 7, 655−677. (25) Lila, A. S. A., Kiwada, H., and Ishida, T. (2013) The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J. Controlled Release 172, 38−47. (26) Falagan-Lotsch, P., Grzincic, E. M., and Murphy, C. J. (2016) One low-dose exposure of gold nanoparticles induces long-term changes in human cells. Proc. Natl. Acad. Sci. U. S. A. 113, 13318. (27) Dobrovolskaia, M. A., Neun, B. W., Man, S., Ye, X., Hansen, M., Patri, A. K., Crist, R. M., and McNeil, S. E. (2014) Protein corona composition does not accurately predict hematocompatibility of colloidal gold nanoparticles. Nanomedicine 10, 1453−63. (28) Saha, K., Rahimi, M., Yazdani, M., Kim, S. T., Moyano, D. F., Hou, S., Das, R., Mout, R., Rezaee, F., and Mahmoudi, M. (2016) Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona. ACS Nano 10, 4421−4430. (29) Slot, J., and Geuze, H. (1984) A method to prepare isodisperse colloidal gold sols in the size range 3−17 nm. Ultramicroscopy 15, 383.
interaction between complement proteins and nanoparticles to predict immune evasion to facilitate rational and efficient design of surface passivation strategies to avoid RES clearance.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00793. Experimental sections and additional data on the size distribution of AuNPs, characterization, concentration calibration curves for determination of mPEG-SH, SC5b9, cell viability, and colloidal stability of PEGylated AuNPs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
James Chen Yong Kah: 0000-0002-2247-6929 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Funding was from the MOE AcRF Tier 1 Grant. The authors thank Dr. Laurent Berkale for helpful discussions. REFERENCES
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DOI: 10.1021/acs.bioconjchem.7b00793 Bioconjugate Chem. XXXX, XXX, XXX−XXX
