Protein Corona Formed from Different Blood Plasma Proteins Affects

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Protein Corona Formed from Different Blood Plasma Proteins Affects the Colloidal Stability of Nanoparticles Differently Yan Teck Ho, Nurul 'Ain Azman, Fion Wen Yee Loh, Gabriella Kai Teng Ong, Gokce Engudar, Seth Allan Kriz, and James Chen Yong Kah Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00743 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

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Bioconjugate Chemistry

Protein Corona Formed from Different Blood Plasma Proteins Affects the Colloidal Stability of Nanoparticles Differently Yan Teck Ho1, Nurul ‘Ain Azman2, Fion Wen Yee Loh2, Gabriella Kai Teng Ong2, Gokce Engudar2†, Seth Allan Kriz3, James Chen Yong Kah1,2 *

1NUS

Graduate School for Integrative Sciences and Engineering, National University of

Singapore, Centre for Life Sciences (CeLS), 28 Medical Drive, #05-01, Singapore 117456. 2Department

of Biomedical Engineering, National University of Singapore, 4 Engineering Drive

3, Engineering Block 4, #04-08, Singapore 117583. 3Department

of Chemical Engineering, Michigan Technological University, Building 203, 1400

Townsend Drive, Houghton, Michigan 49931, United States of America. †Current

address: Department of Chemistry, Technical University of Denmark, 2800 Lyngby,

Denmark. CORRESPONDING AUTHOR *[email protected]

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

ABSTRACT Significant progress in the characterization of protein corona has been made. However, insights on how the corona affects the aggregation of nanoparticles (NPs) and consequent biological identity are still lacking. Here, we examined how the corona formed from four major serum proteins: immunoglobulin G (IgG), fibrinogen (FBG), apolipoprotein A1 (ApoA1), and human serum albumin (HSA) over a range of concentrations affect the aggregation of gold NPs (AuNPs). We found that at physiological pH of 7.4, all four proteins aggregated the AuNPs at low concentrations but conferred colloidal stability at high concentrations due to the complete “corona coat” around individual AuNP. Due to their immune-related functions, IgG and FBG aggregated the AuNPs to a greater extent compared to HSA and ApoA1 which were mostly involved in transport of small molecules. We then introduced the AuNP-corona formed from each protein into an acidic solution at pH 6.2 with high ionic concentration for up to 24 h as a model of the tumor microenvironment to examine for changes in their aggregation. We observed that protein corona formation sterically stabilized the AuNP-corona for all four proteins, resulting in a smaller increase in aggregation and size compared to citrate-capped AuNPs. This was especially true for corona formed at high protein:AuNP ratios. Our study therefore showed that the formation of a complete “corona coat” around NPs at sufficiently high protein:NP ratio was required for colloidal stability of designed NP systems in both physiological and cancer microenvironment to maintain efficiency and efficacy in cancer drug delivery.


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INTRODUCTION In recent years, we have gained a better understanding of how different physical properties of nanoparticles (NPs) such as size, shape, composition, surface charge, hydrophobicity and functional groups, and external environments e.g. cell culture media and temperature, affect the nano-bio interactions, leading to differences in protein corona formation, composition and evolution 1. These in turn adversely affect downstream biological responses such as cell uptake 25,

targeting efficiency

6-8,

biodistribution

7, 9,

cytotoxicity

10-13,

and immune response

7, 14-17.

At

the individual protein level, we have also achieved a better understanding of how these NPs properties affect protein immobilization and unfolding 18-21 to express possible epitopes that may likewise elicit undesirable biological responses. However, the converse of how different proteins affect the physical behavior of NPs, and their downstream biological response is still not as well studied and understood. Aggregation is one such behavior that can occur in NPs during protein adsorption and corona formation on their surface

22, 23.

While some groups have shown that the coat of protein corona conferred colloidal

stability to NPs

24-26,

others have shown that the formation of protein corona induced clustering

or aggregation of NPs 7, 26, 27. Colloidal stability and aggregation of NPs by protein adsorption poses downstream biological consequences in their delivery efficiency since they affect the size of NPs to extravasate across endothelial barrier, and diffuse towards tumor core

28-30.

Although tumor dependent

studies reported endothelial permeability size limits of ~200 nm for NPs

32-34,

31,

most

with smaller NPs

tending to penetrate better in tumors 35. Therefore, a better understanding of the aggregation of NPs by protein adsorption could be imperative in predicting their effectiveness in cancer drug delivery.


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The differences in colloidal stability of NPs with protein adsorption could be dependent on pH, protein concentration, and the types of proteins used in different studies. Different plasma proteins play different roles in the blood, and may thus have different propensity to aggregate NPs. Unfortunately, an understanding of the link between protein adsorption and colloidal stability or aggregation of NPs is still lacking with conflicting results and a lack of consensus being reported across different studies. Therefore, there have been no general guidelines to date for us to predict the colloidal stability and aggregation behavior of NPs with different proteins. In this study, we aim to elucidate how the spontaneous protein corona formed from each of the four most abundant plasma proteins: human serum albumin (HSA, 66.5 kDa); apolipoprotein A1 (ApoA1, 28.3 kDa); fibrinogen (FBG, 340 kDa); immunoglobulin G (IgG, 150 kDa) (physical properties summarized in Table 1); and their concentrations, affect the colloidal stability of gold NPs (AuNPs) differently. We used AuNPs as our model NP of interest since its strong optical absorption is sensitive to aggregation, and can be easily quantified by UV-Vis absorption spectroscopy. These four proteins are classified either as transport-related proteins (HSA and ApoA1) or immune-related proteins (FBG and IgG). While the transport-related proteins are known to play a role in the transport of small molecules in the blood

36, 37,

the

immune-related proteins are known to interact, opsonize, and hence aggregate certain entities in the blood to trigger an immune response 38, 39. We examined the concentration and time-dependent change in hydrodynamic diameter (DH) and aggregation level of NPs induced by these proteins at physiological and slightly acidic pH with elevated levels of Na+ to mimic the acidic tumor microenvironment with high ionic concentrations

40-42.

Studies have shown that increased levels of Na+ induced Warburg-like

metabolism in cancer cells, aiding their unregulated proliferation


41, 43.

The outcomes provided


insights not just on how different proteins affect NP stability, but also how the cancer microenvironment could further modulate this colloidal stability.


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Table 1. Physical properties of the four common plasma proteins used in this study. HSA and ApoA1 were grouped as transport-related proteins with functions linked to small molecule transport, while IgG and Fibrinogen were grouped as immune-related proteins with functions in the human immune system. IgG44-47

Fibrinogen48-50

ApoAI51-55

HSA56, 57

Physiological concentration

8-17 mg/ml (83 µM)

1.8-3.5 mg/ml (7.8 µM)

0.8-1.7 mg/ml (44 µM)

35-50 mg/ml (630 µM)

Molecular weight

150 kDa

340 kDa

28.3 kDa

67 kDa

Dimensions

14.5 X 8.5 X 4 nm

5 X 45 nm (elongated)

2.5 X 8 nm (globular)

4 X 4 X 14 nm (triangular prism shape)

Protein structure Description

Tetrameric quaternary structure with 2 γ heavy chains, and 2 light chains

Dimer arrangement α-, β-, γ-chains

Single polypeptide chain with 245 amino acids and a high content of α-helixes.

Globular Single polypeptide chains with 585 amino acids, held in 3 homologous domains

PI

5.9-6.1

5.5-5.8

5.0-5.5

4.7

RESULTS AND DISCUSSION Synthesis and characterization of AuNPs In this study, we used 20 nm citrate-capped spherical AuNPs as our model NPs to examine aggregation behavior with different proteins due to their popularity in biomedical applications and aggregation-sensitive optical properties that allowed us to easily characterize their colloidal stability with absorbance measurements. The TEM image and DLS measurement showed the spherical AuNPs had a mean DH of 20.9 ± 0.7 nm (Figure 1A and 1B) and zeta potential, ζ of 7 ACS Paragon Plus Environment


28.7 ± 1.0 mV (mean ± standard error). The AuNPs in its monodispersed state showed a peak absorbance at 520 nm that was sensitive to aggregation (Figure 1C).

Figure 1. Characterization of citrate-capped AuNPs synthesized using the Turkevich’s method. (A) TEM images of the spherical AuNPs whose size agreed with the mean DH value measured by DLS. (B) Representative DH distribution of the AuNPs and (C) UV-Vis absorbance spectrum of the synthesized 20-nm citrate-capped AuNPs.

Effect of common plasma proteins on aggregation of AuNPs We examined how the non-specific adsorption of the four most common plasma proteins affects the aggregation of citrate-capped AuNPs by incubating them in increasing concentrations of transport-related proteins (ApoA1 and HSA) and immune-related opsonin proteins (IgG and FBG) separately for 24 h at 37°C in physiological pH of 7.4 before measuring their Aggregation 8 ACS Paragon Plus Environment

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Index (AI) to ensure the formation of a “hard” corona so the proteins are stably bound on the NPs

58, 59.

We observed that all four proteins induced aggregation of AuNPs in a similar

concentration-dependent manner where at low protein:AuNP incubation ratios, the aggregation increased with protein:AuNP ratio towards a maximum aggregation before becoming more colloidally stable again as the ratio continued to increase further. Here, the maximum aggregation occurred at different protein:AuNP ratios for different plasma proteins (Figure 2).

Figure 2. Aggregation index (AI) of AuNPs at different protein:AuNP ratios for all four proteins: (A) IgG; (B) FBG; (C) ApoA1; and (D) HSA, after incubating the AuNPs with these proteins for 24 h at 37 °C in physiological pH of 7.4. The y-axis was drawn to the same scale to allow comparison of AI between the two types of proteins.



Between the two groups of proteins, we also observed that the immune-related proteins IgG and FBG aggregated AuNPs to a greater degree as evidenced from their higher peak AI values, and over a wider range of protein:AuNP incubation ratios compared to transport-related proteins ApoA1 and HSA. Here, IgG aggregated the AuNPs to a peak AI of 1.10 ± 0.02 at 8 IgG molecules per AuNP, before decreasing towards a constant AI of 0.52 ± 0.03 as the ratio of IgG:AuNP increased beyond 70 (Figure 2A). Similarly, at low FBG:AuNP ratios, AuNP aggregation increased with FBG:AuNP ratios towards a peak AI of 0.99 ± 0.04 with 8 FBG molecules per AuNP, and remained high until 25 FBG molecules per AuNP before decreasing towards a constant AI of 0.44 ± 0.01 as the ratio of FBG:AuNP increased beyond 30 (Figure 2B). In contrast, AuNPs incubated with ApoA1 and HSA aggregated to a peak AI of only 0.58 ± 0.02 at 8.5 ApoA1 molecules per AuNP (Figure 2C) and 0.70 ± 0.04 at 3 HSA molecules per AuNP (Figure 2D) respectively. For both proteins, further increase in the ratio beyond the peak saw the AuNPs regaining colloidal stability at a constant AI of 0.43 ± 0.02 and 0.43 ± 0.01 at high protein: AuNP ratios for ApoA1 and HSA respectively. Similar aggregation characteristics were also observed by Deng et al. on poly(acrylic acid) (PAA) passivated AuNPs (AuNP-PAA) with FBG 60, and Cukalevski et al. on comparably sized 27 nm carboxylated polystyrene NPs (pNPs), sulphonated pNPs, and poly(methyl methacrylate) (PMMA) NPs with IgG and FBG 26. In the study by Deng et al., the aggregation of AuNP-PAA at low FBG:AuNP-PAA ratios was eliminated when FBG was enzymatically digested by plasmin, suggesting that the intact FBG molecule was mediating the aggregation between the AuNPs 60. Similarly, the increased aggregation of carboxylated pNPs, sulphonated pNPs, and PMMA NPs observed by Cukalevski et al. when these NPs were incubated in bovine serum with low


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concentrations of IgG was diminished when the NPs were incubated in IgG-depleted bovine serum and fetal calf serum (which contained low levels of IgG) 26. Furthermore, when the NPs were pre-coated with human serum albumin to pre-form a corona before they were incubated in low concentrations of IgG, aggregation was also not observed in the NPs 26. These observations together suggest that the NP aggregation present at low FBG and IgG concentrations was mediated by the proteins which formed bridges between the NPs at low concentration, and not a result of direct NP-to-NP aggregation

26.

In fact, Cukalevski et al. found that the aggregation

induced by IgG molecules on carboxylated pNPs was mediated by the Fc domain of the IgG molecules 26. In general, the adsorption of proteins on NPs is a thermodynamically driven process due to the intrinsically high surface energy of the NPs 61, 62. The results from our study further validate the proposed mechanism suggested in previous studies by both Deng et al.60 and Cukalevski et al.26, where at low protein concentrations, the proteins served as bridges that cross-linked NPs together, allowing them to overcome their charge repulsion and come into proximity with each other to result in aggregation. As protein concentration increased, the clusters grew in size until sufficient proteins were subsequently able to completely “coat” a single NP to form a corona that provided steric stabilization between adjacent NPs, hence reducing their aggregation. While it would be useful to further validate the aggregation mechanism, doing so using conventional imaging methods such as transmission electron microscopy (TEM) and negative staining would be non-trivial, given that it would be difficult to differentiate between the adsorbed proteins of the AuNP-corona complexes and the citrate cap on the AuNPs used in this study 63. It would also be difficult to differentiate the aggregation induced by the proteins from the aggregation of AuNPs when dried on the TEM grid during the sample preparation process.



Such aggregation would occur not just on the plane of the grid, but also out of the plane i.e. stacking of NPs, and this would further add to the complexity of TEM analysis. However, it is also interesting to note that the trend of aggregation observed above could also be dependent on the size of the protein relative to the NPs. Liu et al. observed that incubating 25 nm diameter gold nanorods (AuNRs) of different aspect ratios with fixed concentrations of bovine serum albumin (BSA) and HSA resulted in the formation of AuNR-albumin aggregates, whereas incubating the AuNRs with identical concentrations of IgG and immunoglobulin A (IgA) led to the formation of a complete corona coating each AuNR, colloidally stabilizing the AuNRimmunoglobulin complexes

64.

This was likely a result of the albumins being much smaller in

size as compared to the immunoglobulins (Table 1), which at the same molar concentrations tested, were insufficient to form a complete protein coat around the AuNRs 64. . To further probe this relative size dependency, we incubated much larger 60 nm spherical citrate-capped AuNPs (zeta potential, ζ of -33.9 ± 0.4 mV) with HSA at varying HSA:AuNP ratios. Unlike the smaller 20 nm citrate-capped AuNPs, we observed no significant change to the AI or DH of the 60 nm AuNPs over a wide range of incubation ratios (Figure S1A and B, Supporting Information). While the proteins were still likely to have adsorbed on the surface of individual 60 nm AuNPs, the significantly smaller size of the HSA (~14 nm in diameter, Table 1) relative to the 60 nm AuNPs meant that the inter-particle distance between adjacent NPs bridged or cross-linked by HSA at low HSA:AuNP ratios would result in greater repulsion between the AuNPs (Figure S1C, Supporting Information), compared to smaller 20 nm AuNPs where the inter-particle distance bridged by HSA resulted in energetically more favorable aggregated state due to the lower repulsion experienced between the AuNPs (Figure S1D, Supporting Information). Similar observations were also made with 60 nm AuNPs and FBG (data not


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shown). Hence, the 60 nm AuNPs that were much larger than the proteins were also more likely to remain isolated even at low protein:AuNP ratios. Therefore, the general aggregation trend observed in this study could be likely confined to NPs of sizes comparable to the proteins with which the corona was formed. This holds important implications since there are a plethora of studies extoling the benefits of smaller NPs (< 50 nm) in biological applications due to their advantageous delivery characteristics compared to larger NPs 28, 65-67. The outcomes of our study would provide insights to the colloidal stability of many of these smaller NPs as they contact the plasma. More importantly, we demonstrated that this aggregation behavior was not exclusive to immune-related proteins, but was observed in other proteins in general, although we noted that aggregation by immune-related proteins was more severe compared to transport-related proteins as evidenced by their higher AI and larger DH of the aggregates. Furthermore, we also observed that the immune-related proteins aggregated AuNPs over a wider range of protein:AuNP ratios than transport-related proteins. The AI of AuNPs with adsorbed proteins was significantly higher (p < 0.05) than that of citrate-capped AuNPs over a range of 6.3 to 10.6 ApoA1 per AuNP and 3.0 to 6.0 HSA per AuNP for transport-related proteins. This range was much smaller than that of 2.0 to 50.0 IgG per AuNP and 4.0 to 20.0 FBG per AuNP for immune-related proteins, thus showing that the immune-related proteins required a much higher incubation ratio to achieve colloidal stability of AuNPs with the corona compared to transport-related proteins. We had previously characterized the binding kinetics and affinity of the four proteins: HSA, ApoA1, FBG, and IgG to the same citrate-capped AuNPs using surface plasmon resonance (SPR) biosensing

68.

From the SPR measurements, we observed significantly different binding

affinities between the four proteins to AuNPs (in descending order of binding affinity based on



KD value): ApoA1 (KD,ApoA1 = 00.12 ± 0.07 µM), FBG (KD,FBG = 00.53 ± 0.08 µM), HSA (KD,HSA = 4.93 ± 2.41 µM) and IgG (KD,IgG = 10.13 ± 3.28 µM). Here, ApoA1 bound most strongly while IgG bound most weakly to AuNPs. Comparing the binding affinities with our observations on aggregation of NPs, the binding affinities between the proteins and AuNPs were likely mediators of the aggregation behavior of the NP-protein complexes, as the incubation ratios necessary for ApoA1 and IgG to stabilize the AuNPs agreed well with their respective binding affinities. However, it was likely that other factors, such as the size difference between the two groups of proteins could also play a role in affecting the AuNP-protein aggregation characteristics since the smaller ApoA1 (28.3 kDa) and HSA proteins (67 kDa) (Table 1) generally induced lower levels of AuNPs aggregation compared to the larger IgG (150 kDa) and FBG (340 kDa). This could be due to the smaller proteins packing better on the surface of AuNPs whereas the larger proteins presenting poorer conformation and packing on the AuNPs surface, or that the smaller proteins present smaller inter-particle distance between adjacent NPs bridged by the proteins to result in higher and hence less favorable energy state to be maintained in aggregated form as discussed above. Furthermore, the intrinsic agglutinative or “sticky” property of IgG and FBG in ‘trapping’ foreign entities, were also contributors to the observed differences in aggregation behavior of NPs by the proteins, given that HSA required a lower incubation ratio to achieve colloidal stability than FBG despite having a lower binding affinity 68. Besides being an opsonin, IgG is known to agglutinate foreign entities which resulted in their clumping and immobilization, thus facilitating subsequent phagocytic clearance 69. While FBG is more widely known for its role in blood coagulation and hemostasis through the generation of a gelatinous fibrin scaffold for


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trapping and agglutinating red blood cells and platelets in a blood clot 70, FBG is also involved in trapping pathogens and foreign entities as part of the innate immunity 71, 72. In contrast, HSA and ApoA1 are much smaller proteins involved in transport of small molecules in blood. ApoA1, a flexible protein in its lipid-free form, is a major constituent in high density lipoproteins involved in the binding of lipids

55, 73, 74,

while HSA, being the most

abundant protein found in human serum are mostly involved in the transport of many molecules, including hormones, fatty acids, bilirubin, drugs, ions, and toxic substances

36, 75, 76.

Therefore,

the larger surface area of immune-related proteins due to their size which allowed for more surface interactions with AuNPs coupled with their intrinsic agglutination behavior promoted a higher degree of AuNPs aggregation with FBG and IgG compared to HSA and ApoA1. While it may be interesting to determine the specific protein domains or sequence features responsible for the “stickiness” or aggregation of AuNPs, doing so would require domain mapping for all four proteins. This conventionally involves repeating the experiments after introducing mutations to the different domains of each protein 77, which would be non-trivial and beyond the scope of this study.

Effect of common plasma protein on size of AuNP-protein agglomerates The aggregation of AuNPs induced by proteins could have resulted in the formation of large AuNP-protein agglomerates comprising clusters of AuNPs entrapped in a protein matrix

78.

While the AI provided a quantitative indication of the aggregation state of AuNPs from UV-Vis spectral measurements, it was limited in quantifying the size of the AuNP-protein agglomerates which also included the proteins. It was this size that determined their physiological behavior and consequent downstream biological response in cells and tissues 79-81.



We therefore measured the DH of the AuNP-protein agglomerates formed from all four proteins at different protein:AuNP incubation ratios and observed that DH varied with protein:AuNP ratio in a similar manner as the AI of AuNPs. The DH of AuNP-protein increased with protein:AuNP ratio towards a peak DH before decreasing back to a constant DH at high protein:AuNP ratios where a stable protein corona was formed (Figure 3).

Figure 3. DH of AuNP-protein agglomerates at different protein:AuNP ratios for all four proteins: (A) IgG; (B) FBG; (C) ApoA1; and (D) HSA, after incubating the AuNPs with these proteins for 24 h at 37°C in physiological pH of 7.4.

Amongst the four proteins, AuNP-IgG showed the largest increase in DH after formation of the IgG corona on AuNPs at high IgG:AuNP ratios. Here, the DH of 20.9 nm citrate-capped AuNPs increased to 138.8 ± 45.2 nm (ΔDH = 117.9 nm) and 215.7 nm ± 165.9 nm (ΔDH = 194.8 16 ACS Paragon Plus Environment

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nm) for IgG:AuNP incubation ratios of 70 and 90 respectively (Figure 3A). Since the AI of AuNP-IgG at these IgG:AuNP ratios indicated low levels of aggregation of AuNPs (Figure 2A) and IgG had a mean DH of ~10.6 nm 82, the increase in DH thus suggested more than a monolayer thickness of IgG molecules forming a corona around each AuNP. Additional measurements showed that free IgG of concentrations 0.04 mg/mL, 0.08 mg/mL, and 1 mg/mL in 10 mM sodium phosphate buffer (1x PhB) had mean DH values of 391.1 ± 17.4 nm, 373.5 ± 8.87 nm, and 266.9 ± 86.9 nm respectively (Figure S2A, Supporting Information). The DH histogram distributions for free IgG at all three concentrations showed a peak population at ~11.7 nm, likely attributable to the individual IgG molecules present in the solution (Figure S2B, Supporting Information). However, larger sized populations with peaks at ~70 nm and ~396 nm were also observed in the free IgG at all three concentrations, with IgG at 1 mg/mL displaying an additional peak at ~5,560 nm. The histogram distributions also showed broadening of the peaks at higher concentrations, suggesting increasing heterogeneity in the free IgG population due to clustering or agglomeration of IgG molecules prior to incubation with AuNPs. Such agglomeration or clustering of antibodies including IgG both physiologically and in vitro have also been previously reported by others, although the mechanism remained unclear 83-85. With 0.08 mg/mL of free IgG being comparable to the concentration of IgG present at an incubation ratio of IgG:AuNP = 90, these additional DLS measurements suggested that the large increase in DH observed at high IgG:AuNP ratios could be attributed to the adsorption of IgG agglomerates to form IgG multilayers on AuNPs. By repeating the incubation with filtered IgG (IgG filtered with 200 nm pore sized syringe filters to remove agglomerated IgG molecules) at high IgG:AuNP ratios of 90 and 150, we observed an average ΔDH of ~34 nm in the AuNPs, which was comparable to the DH of the filtered free IgG molecules (Figure S3, Supporting



Information), thus suggesting the formation of an IgG monolayer instead of multilayer on the 20 nm citrate-capped AuNPs. Similarly, for ApoA1, HSA and FBG, the increase in DH at high protein:AuNP ratio was equivalent to the DH of the individual protein (Figures 3B – D). The DH of AuNP-ApoA1 at high ApoA1:AuNP ratio of 25.3 was 28.3 ± 5.2 nm. This was an increase of ΔDH = 5.4 nm over its uncoated AuNP and corresponded well to the DH of ApoA1 molecule of ~1.4 nm 86. Similarly, the increase in DH following the formation of AuNP-HSA and AuNP-FBG corona at high protein:AuNP incubation ratios of 90 for HSA and 30 for FBG were 8.3 nm and 35.4 nm respectively (Figure 3B and 3D). These increases were also consistent with the reported size of ~7 nm and ~22 nm of HSA and FBG molecules respectively 82, and suggested the formation of a monolayer of ApoA1, HSA and FBG corona on each AuNP at high protein:AuNP ratios, unlike the unfiltered IgG. However, the DH of the AuNP-protein agglomerates was large for all four proteins at lower protein:AuNP ratios, and was likely attributed to aggregation as observed in our AI measurements. Similar to AI, we also observed that immune-related proteins IgG and FBG aggregated the AuNPs to a larger DH of 511 nm and 1,199 nm respectively compared to the smaller DH of 410 nm and 68 nm induced by ApoA1 and HSA respectively at low protein:AuNP ratios. This was likely due to the immune-related proteins being larger and more “sticky” than the transport-related proteins as discussed previously. Furthermore, the increase in DH correspond well to increases in AI of all four AuNP-proteins (Figure S4, Supporting Information), and suggested that while the protein layer(s) influence the final DH of the AuNPcorona complex, the aggregation state of AuNPs was also a large determinant of their final DH.


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Effect of cancer-mimicking microenvironment on aggregation of AuNP-proteins Studies have shown that the tumor microenvironment is mostly acidic, with pH ranging from 5.8 to 7.6

87,

to promote tumor cell proliferation and survival

42, 88.

A significant source of this

acidic environment is attributed to the Warburg effect arising from increased lactate secretions 42. The tumor microenvironment also exhibits increased Na+ concentration which promotes this Warburg effect in tumor cells 41. Unfortunately, both pH and high ionic concentrations are also modulators of NPs 89 and protein aggregation 90, 91. Here, we gradually introduced AuNPs with ApoA1, HSA, IgG, and FBG non-specifically adsorbed at four selected protein:AuNP incubation ratios into a 10x PhB solution at pH 6.2 via dialysis to simulate their transition from physiological pH in the blood to a low pH and high ionic tumor microenvironment, and examined the change in aggregation and size of AuNPprotein complex. After dialyzing the AuNP-protein complex over time, we observed that protein corona formed at high protein:AuNP ratios from all four proteins generally prevented AuNPs from further aggregation as they entered a low pH and high ionic strength environment. This was evidenced by a smaller increase in AI (∆AI) compared to citrate-capped AuNPs (Figure 4, black line), especially at the highest protein:AuNP ratio for all proteins.



Figure 4. Fold changes in the AI of AuNPs over time at different protein:AuNP incubation ratios as the AuNPs traversed from physiological pH 7.4 to a low pH 6.2 and high ionic microenvironment that modelled that of tumors. AuNPs were pre-incubated with (A) ApoA1; (B) HSA; (C) IgG; or (D) FBG for 24 h at four selected protein:AuNP ratios, and dialyzed for up to 24 h at 37 °C in 10x PhB at pH 6.2. The time-dependent fold change in AI for each protein:AuNP ratio from the initial at time t = 0 h showed that the non-specific adsorption of proteins at a sufficiently high protein:AuNP incubation ratio minimized further aggregation of AuNPs induced by the low pH and high ionic concentrations microenvironment.

We observed citrate-capped AuNPs aggregating strongly at lower pH and higher ionic concentration due to charge screening, with the AI increasing over time from 0.48 at 0 h to 1.09 (fold change in AI, ∆AI = 2.27) after 2.5 h of dialysis, and remaining relatively unchanged 20 ACS Paragon Plus Environment

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thereafter (Figure 4A – D). An increase in AuNPs aggregation was also observed at the lowest protein:AuNP ratio of 2.1 for AuNP-ApoA1 where inadequate steric stabilization conferred by the small sized Apo-A1 (Table 1) caused significant increase in AI (∆AI = 2.03) within 2.5 h (Figure 4A). However, aggregation was much less pronounced at higher ApoA1:AuNP ratios of more than 4.2 (∆AI ≈ 1.2) due to increasing steric stabilization conferred by the ApoA1 corona (Figure 4A). These smaller increases in AI were also observed for all of the AuNP-protein complexes formed from HSA, IgG, and FBG proteins (Figure 4B-D). The larger molecular weights of these proteins (Table 1) likely conferred greater steric stabilization to the AuNP-protein complexes, resulting in little or no further change in the aggregation state of AuNPs over time as they traversed into the cancer microenvironment, even at low protein:AuNP ratios.

Effect of a cancer-mimicking microenvironment on DH of AuNP-proteins Apart from AI, we also examined the change in DH of the AuNP-protein complex at the same protein:AuNP ratios and time points during dialysis as the pH was gradually lowered and ionic concentration gradually increased during dialysis. At the highest protein:AuNP ratio where the proteins formed a well-defined coat of protein corona to confer steric and charge stabilization, we observed a significantly smaller fold change in DH (∆DH) of the AuNP-protein complex for all four proteins over the 24 h course of dialysis into the new low pH and high ionic strength environment compared to citrate-capped AuNPs (average ∆DH ~ 82.1) (Figure 5). This agreed well with our observation on the change in AI (Figure 4).



Figure 5. Changes in the DH of AuNPs over time at different protein:AuNP incubation ratios as the AuNPs traversed from physiological pH 7.4 to a low pH 6.2 and high ionic strength microenvironment that modelled that of tumors. AuNPs were pre-incubated with (A) ApoA1; (B) HSA; (C) IgG; or (D) FBG for 24 h at four selected protein:AuNP ratios, and dialyzed for up to 24 h at 37 °C in 10x PhB at pH 6.2. The time-dependent fold change in DH (∆DH) for each protein:AuNP ratio over the initial at time t = 0 h showed that the non-specific adsorption of proteins at a sufficiently high protein:AuNP incubation ratio minimized further aggregation of AuNPs induced by the low pH and high ionic concentrations microenvironment.

However, the AuNP-protein complexes formed at low protein:AuNP ratios still showed signs of further agglomeration in the tumor microenvironment as they experienced a larger increase in DH (∆DH = 74.1 for ApoA1:AuNP = 2.1, ∆DH = 27.8 at IgG:AuNP = 4, ∆DH = 37.5 at 22 ACS Paragon Plus Environment

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FBG:AuNP = 8) over 24 h compared to AuNP-protein complexes at the highest protein:AuNP ratios owing to insufficient stabilization by the proteins, with the exception of HSA. Nonetheless, the ∆DH was still significantly lower than that of citrate-capped AuNPs. By observing the changes in both the AI and DH of the AuNP-protein complex at different protein:AuNP incubation ratios for all four proteins, it was evident that the envelopment of AuNP aggregates or single AuNP by the protein corona formed from each of the four proteins at a sufficiently high ratio, regardless of whether they were transport or immune-related, served to sterically stabilize the AuNPs from further aggregation, which would otherwise occur in an acidic and high ionic concentration microenvironment. Here, we also noted that such a “sufficiently high ratio” was different for different proteins due to their differences in size and functionality, which dictated their interaction with the AuNPs 25.

Such a ratio could be empirically determined using simple salt-induced aggregation

techniques widely reported to determine the minimum protein amount (MPA) of proteins needed to colloidally stability the NPs 25.

CONCLUSION The non-specific adsorption of proteins did not simply coat the surface of NPs, but also changed their colloidal stability and altered the effective size of NPs that biological systems ‘see’ and interact with, in both protein type- and concentration-dependent manner. This poses several implications in their downstream biological behavior such as vascular extravasation, tumor targeting and cell uptake in vivo 92. It was clear from our study that a sufficiently high protein:NP ratio was required for nonspecific adsorption to form a corona coat on NPs to protect them from further aggregation as the NPs traverse from vascular flow into the tumor interstitium of low pH and high ionic strength. A 23 ACS Paragon Plus Environment


lower concentration of proteins would otherwise result in aggregation in physiological environment, and the aggregation could possibly be exacerbated as the NPs traverse into the tumor environment. With a tightly controlled physiological concentration of major proteins in the plasma, this placed constraints on the concentration of NPs to be administered into the circulation to achieve the required protein:NP ratio that minimized aggregation and formed a corona to confer colloidal stability during tumor delivery. This would also help to ensure that the designed NP system remained as close to their designed size as possible to maintain efficiency and efficacy in delivery 93. Although most of the protein corona on NPs were formed from the full suite of plasma proteins in actual biological systems, our study on the aggregation behavior of NPs by individual type of protein nonetheless presented its merit. Apart from a simpler system to understand the aggregation characteristics of NPs upon introduction into a physiological environment, more importantly, we also wanted to study how the pre-formed protein corona constituted from different single plasma protein would affect the aggregation of the AuNPs as they were introduced into a cancer microenvironment. This is important in understanding the biological behaviour of a single protein coating system on NPs given the widespread use of ApoA1 94, HSA 95,

IgG 96, 97, and FBG 98, 99 coated NPs in a myriad of biomedical and nanomedicine applications,

including cancer drug delivery. These applications demanded a proper understanding of the colloidal stability of NPs at a particular protein concentration and microenvironment. While our study might have employed an idealized in vitro system to model and examine tumor microenvironment-induced aggregation, this system has shown the ability of the protein corona to confer NP stability for cancer drug delivery. This lays the foundation for further studies characterizing the aggregation profiles of different NP-protein complexes within the


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microenvironments of different tumors, which studies have shown, could be significantly different for different cancers 100. Furthermore, the unique aggregation profile of different plasma proteins used in our study also suggested possible use of NP aggregation as a technique to identify and characterize corona composition. Many downstream applications exploiting these aggregation traits for the sensing of proteins related to diagnosis and prediction of diseases could potentially be possible. Therefore, our study provided some fundamental insights on how different proteins could affect the biological identity of NPs through changing their size, which could present us with new opportunities in improving tumor delivery and disease diagnostics.

MATERIALS AND METHODS Synthesis and characterization of Colloidal AuNPs Spherical AuNPs of 20 nm were synthesized using Turkevich’s method 101, and purified with repeated centrifugation at 7,000 rcf for 20 min before being re-suspended in Milli-Q water and stored at 4 °C until further use. The washed citrate-capped AuNPs were characterized for their absorbance using UV-Vis spectrometry (Cary 60, Agilent Technologies, U.S.A); DH with dynamic light scattering (DLS) at 25 °C with back-scattering detector (173°) (Nano ZS, Malvern, UK); and surface charge with a Zetasizer (Nano ZS, Malvern, UK). Measurements were carried out in triplicates with each set being the average of five measurements of the sample measured. Transmission electron microscopy (TEM) (Jem 3010, JEOL, Japan) images of reference AuNP solutions were also taken to verify the size, shape, and morphology of the synthesized AuNPs. The concentration of the citrate-capped AuNPs were calculated from the absorbance spectra using both empirical and theoretical coefficients from a previously described method

102.

Spherical 60 nm citrate-capped AuNPs was synthesized based on the protocol previously 25 ACS Paragon Plus Environment


Page 26 of 43

described by Leng et al.103 The larger AuNPs were also purified and characterized using the same techniques as the smaller AuNPs.

Quantifying aggregation of AuNPs Monodispersed AuNPs colloid typically exhibit a peak surface plasmon resonance (SPR) absorbance at ~520 nm with its intensity correlated to particle concentration

104-106

and sensitive

to aggregation of AuNPs. Particle aggregation typically resulted in a red shift and broadening of the peak SPR absorbance

106-115,

thus causing the absorbance at 580 nm to increase with

aggregation. This allowed us to quantify aggregation of AuNPs in a concentration-independent manner by normalizing the absorbance at 580 nm to that at 520 nm

116

to give an Aggregation

Index (AI) from the absorbance spectra: 𝐴𝑔𝑔𝑟𝑒𝑔𝑎𝑡𝑖𝑜𝑛 𝐼𝑛𝑑𝑒𝑥 (𝐴𝐼) = Here, A580

nm

and A520

nm

𝐴580 𝑛𝑚

(1)

𝐴520 𝑛𝑚

referred to the absorbance at 580 nm and 520 nm respectively.

Normalizing the absorbance value at 580 nm to the peak at 520 nm gave a dimensionless parameter associated with NP aggregation that is independent of particle concentration. Here, a higher AI value thus corresponded to a higher degree of aggregation of AuNPs.

Non-specific adsorption of common plasma proteins on AuNPs All four proteins used in our study were purchased commercially. Stock concentrations of each of the four proteins: human ApoA1 (Abcam, U.K.), HSA (Sigma-Aldrich, U.S.A.), IgG (Athens research and technology, U.S.A), and FBG (Sigma-Aldrich), were reconstituted and serially diluted in 10 mM sodium phosphate buffer (1x PhB) over a range of protein concentrations that covered physiological concentration reported in human plasma 26 ACS Paragon Plus Environment

117.

Serial

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dilutions of each protein were then added in 1:1 volume ratio to the washed citrate-capped AuNPs at physiological pH of 7.4 and different protein:AuNP incubation ratios from 0 to 120 (summarized in Table S1, Supporting Information). The AuNP-protein mixture was incubated under constant rotation for 24 h at 37 °C to allow spontaneous non-specific adsorption of each protein around the AuNPs and their accompanying aggregation to form the AuNP-protein agglomerates, before their DH and UV-Vis absorption spectrum were measured with DLS (Nano ZS, Malvern, UK) and UV-Vis spectrophotometry (Multiskan GO microplate spectrophotometer, Thermo Fisher Scientific, U.S.A) respectively. The AI of the AuNPs at each protein:AuNP ratio was then derived from the absorbance measurements.

Aggregation of AuNPs in tumor-mimicking extracellular microenvironment The AuNPs with each of the four proteins non-specifically adsorbed at physiological pH and at selected ratios were then transferred into a low pH environment with elevated concentration of Na+ to probe for changes in the aggregation behavior of AuNPs as they traversed from a physiological to tumor-mimicking microenvironment. This was performed by transferring the AuNP-protein complexes to dialysis tubings (12 kDa molecular weight cutoff, Sigma-Aldrich, U.S.A), sealed tightly, and introduced into a 10x PhB solution at pH 6.2 with [Na+] = 100 mM in a 37 °C water bath for dialysis to occur. The pH 6.2 acidic reservoir was obtained by adjusting the pH of 100 mM (10x) PhB (1st Base Biochemicals, Singapore) with L-(+)-lactic acid (Sigma-Aldrich, U.S.A) to model the acidity contributed by lactate arising from the Warburg effect in the tumor microenvironment, while the 10x PhB simulated the high ionic environment due to high Na+ concentration present in the tumor extracellular space 40, 41.



The pH of the AuNP-protein complex in the dialysis bag gradually decreased from pH 7.4 to 6.2 within 1.5 h of incubation in the acidic reservoir (data not shown), and the AuNP-protein colloid were sampled at 2.5, 6, and 24 h of dialysis for their DH and AI to be measured. At these time points, the pH of the environment of AuNPs in the dialysis tubing had already reached 6.2 The fold change in AI (∆AI) and DH (∆DH) over the reference at t = 0 h (physiological condition at pH 7.4) were tracked over time for all AuNP-protein complexes at different protein:AuNP ratios by normalizing the measured AI and DH at the selected time points to the initial AI and DH values at t = 0 h.

AUTHOR INFORMATION Corresponding Author *[email protected] Author contribution JCY Kah and YT Ho conceptualized the study. YT Ho, NA Azman, FWY Loh, GKT Ong, and SA Kriz conducted the experiments and performed the analysis for this study. G Engudar contributed in the optimization of the study and provided input on the characterization of the nanoparticles. All authors were involved in the drafting and reviewing of this manuscript. We also declare that none of the authors possess competing financial interest in this study. Supporting Information Supporting Information on the protein:AuNP incubation ratios used for each of the four proteins in this study, the change in AI and DH of larger 60 nm AuNPs after protein incubation and data


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on aggregation of IgGs are available. One figure juxtaposing Figures 2 and 3 in the main article is also provided as a supplementary figure. Acknowledgment YT Ho and NA Azman would like to acknowledge the scholarship support from National University of Singapore Graduate School of Integrative Sciences and Engineering (NGS) and National University of Singapore Biomedical Engineering department respectively. SA Kriz would like to acknowledge the support from NSF IRES programme. The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 1 Grant. Conflict of Interest The authors declare no competing financial interest.

ABBREVIATIONS ∆AI: Fold change in aggregation index ∆DH: Fold change in hydrodynamic diameter AI: Aggregation index ApoA1: Apolipoprotein A1 AuNP: Gold nanoparticle AuNR: Gold nanorods BSA: Bovine serum albumin DH: Hydrodynamic diameter FBG: Fibrinogen HSA: Human serum albumin



IgA: Immunoglobulin A IgG: Immunoglobulin G KD: dissociation constant NP: Nanoparticle PMMA: poly(methyl methacrylate) pNP: Polystyrene nanoparticle SPR: Surface plasmon resonance TEM: Transmission electron microscopy

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Lynch, I., and Dawson, K. A. (2008) Protein-nanoparticle interactions. Nano today 3, 40-47. Lesniak, A., Fenaroli, F., Monopoli, M. P., Aberg, C., Dawson, K. A., and Salvati, A. (2012) Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. ACS nano 6, 5845-5857. Lesniak, A., Campbell, A., Monopoli, M. P., Lynch, I., Salvati, A., and Dawson, K. A. (2010) Serum heat inactivation affects protein corona composition and nanoparticle uptake. Biomaterials 31, 9511-9518. Gunawan, C., Lim, M., Marquis, C. P., and Amal, R. (2014) Nanoparticle-protein corona complexes govern the biological fates and functions of nanoparticles. Journal of Materials Chemistry B 2, 2060-2083. Walkey, C. D., Olsen, J. B., Song, F., Liu, R., Guo, H., Olsen, D. W., Cohen, Y., Emili, A., and Chan, W. C. (2014) Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS nano 8, 2439-2455. Mirshafiee, V., Mahmoudi, M., Lou, K., Cheng, J., and Kraft, M. L. (2013) Protein Corona Significantly Reduces Active Targeting Yield. Chemical communications (Cambridge, England) 49, 2557-2559. Bargheer, D., Nielsen, J., Gebel, G., Heine, M., Salmen, S. C., Stauber, R., Weller, H., Heeren, J., and Nielsen, P. (2015) The fate of a designed protein corona on nanoparticles in vitro and in vivo. Beilstein journal of nanotechnology 6, 36-46. Khan, S., Gupta, A., and Nandi, C. K. (2013) Controlling the Fate of Protein Corona by Tuning Surface Properties of Nanoparticles. The Journal of Physical Chemistry Letters 4, 3747-3752. Aggarwal, P., Hall, J. B., McLeland, C. B., Dobrovolskaia, M. A., and McNeil, S. E. (2009) Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Advanced drug delivery reviews 61, 428-437. Rivera-Gil, P., Jimenez De Aberasturi, D., Wulf, V., Pelaz, B., Del Pino, P., Zhao, Y., De La Fuente, J. M., Ruiz De Larramendi, I., Rojo, T. f., and Liang, X.-J. (2012) The challenge to relate the physicochemical properties of colloidal nanoparticles to their cytotoxicity. Accounts of chemical research 46, 743-749. 30 ACS Paragon Plus Environment

Page 30 of 43

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


(11) (12) (13) (14)

(15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27)

Soenen, S. J., Rivera-Gil, P., Montenegro, J.-M., Parak, W. J., De Smedt, S. C., and Braeckmans, K. (2011) Cellular toxicity of inorganic nanoparticles: common aspects and guidelines for improved nanotoxicity evaluation. Nano today 6, 446-465. Alkilany, A. M., and Murphy, C. J. (2010) Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? Journal of nanoparticle research 12, 2313-2333. Alkilany, A. M., Nagaria, P. K., Hexel, C. R., Shaw, T. J., Murphy, C. J., and Wyatt, M. D. (2009) Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects. Small 5, 701-708. Shannahan, J. H., Podila, R., Aldossari, A. A., Emerson, H., Powell, B. A., Ke, P. C., Rao, A. M., and Brown, J. M. (2015) Formation of a protein corona on silver nanoparticles mediates cellular toxicity via scavenger receptors. Toxicological sciences : an official journal of the Society of Toxicology 143, 136-146. Foldbjerg, R., Jespersen, L. V., Wang, J., Sutherland, D., and Autrup, H. (2010) in Nanotoxicology 2010, Edinburgh Napier University. Sakulkhu, U., Mahmoudi, M., Maurizi, L., Salaklang, J., and Hofmann, H. (2014) Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings. Sci. Rep. 4. Walkey, C. D., and Chan, W. C. (2012) Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chemical Society reviews 41, 27802799. Wang, A. L., Vangala, K., Vo, T., Zhang, D. M., and Fitzkee, N. C. (2014) A Three-Step Model for Protein-Gold Nanoparticle Adsorption. J Phys Chem C 118, 8134-8142. Zhang, D., Neumann, O., Wang, H., Yuwono, V. M., Barhoumi, A., Perham, M., Hartgerink, J. D., Wittung-Stafshede, P., and Halas, N. J. (2009) Gold nanoparticles can induce the formation of protein-based aggregates at physiological pH. Nano letters 9, 666-671. Fei, L., and Perrett, S. (2009) Effect of nanoparticles on protein folding and fibrillogenesis. International journal of molecular sciences 10, 646-655. Deng, Z. J., Liang, M., Monteiro, M., Toth, I., and Minchin, R. F. (2011) Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nature nanotechnology 6, 39-44. Bharti, B., Meissner, J., and Findenegg, G. H. (2011) Aggregation of silica nanoparticles directed by adsorption of lysozyme. Langmuir : the ACS journal of surfaces and colloids 27, 9823-9833. Gebauer, J. S., Malissek, M., Simon, S., Knauer, S. K., Maskos, M., Stauber, R. H., Peukert, W., and Treuel, L. (2012) Impact of the nanoparticle-protein corona on colloidal stability and protein structure. Langmuir : the ACS journal of surfaces and colloids 28, 9673-9679. Dominguez-Medina, S., Blankenburg, J., Olson, J., Landes, C. F., and Link, S. (2013) Adsorption of a Protein Monolayer via Hydrophobic Interactions Prevents Nanoparticle Aggregation under Harsh Environmental Conditions. ACS Sustain Chem Eng 1, 833-842. Yeo, E. L. L., Chua, A. J. S., Parthasarathy, K., Yeo, H. Y., Ng, M. L., and Kah, J. C. Y. (2015) Understanding aggregation-based assays: nature of protein corona and number of epitopes on antigen matters. RSC Advances 5, 14982-14993. Cukalevski, R., Ferreira, S. A., Dunning, C. J., Berggård, T., and Cedervall, T. (2015) IgG and fibrinogen driven nanoparticle aggregation. Nano Research 8, 2733-2743. Kah, J. C., Grabinski, C., Untener, E., Garrett, C., Chen, J., Zhu, D., Hussain, S. M., and HamadSchifferli, K. (2014) Protein coronas on gold nanorods passivated with amphiphilic ligands affect cytotoxicity and cellular response to penicillin/streptomycin. ACS nano 8, 4608-4620.



(28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46)

Nakamura, Y., Mochida, A., Choyke, P. L., and Kobayashi, H. (2016) Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer? Bioconjugate Chemistry 27, 2225-2238. Liu, L. J., Brown, S. L., Ewing, J. R., Ala, B. D., Schneider, K. M., and Schlesinger, M. (2016) Estimation of Tumor Interstitial Fluid Pressure (TIFP) Noninvasively. PloS one 11, e0140892. Wu, M., Frieboes, H. B., Chaplain, M. A., McDougall, S. R., Cristini, V., and Lowengrub, J. S. (2014) The effect of interstitial pressure on therapeutic agent transport: coupling with the tumor blood and lymphatic vascular systems. Journal of theoretical biology 355, 194-207. Hashizume, H., Baluk, P., Morikawa, S., McLean, J. W., Thurston, G., Roberge, S., Jain, R. K., and McDonald, D. M. (2000) Openings between defective endothelial cells explain tumor vessel leakiness. The American journal of pathology 156, 1363-1380. Kobayashi, H., Watanabe, R., and Choyke, P. L. (2013) Improving conventional enhanced permeability and retention (EPR) effects; what is the appropriate target? Theranostics 4, 81-89. Maeda, H. (2001) The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Advances in enzyme regulation 41, 189-207. Miao, L., and Huang, L. (2015) Exploring the tumor microenvironment with nanoparticles. Cancer treatment and research 166, 193-226. Jiang, W., Huang, Y., An, Y., and Kim, B. Y. (2015) Remodeling Tumor Vasculature to Enhance Delivery of Intermediate-Sized Nanoparticles. ACS nano 9, 8689-8696. Curry, F. E., and Clough, G. F. (2002) Flow-dependent changes in microvascular permeability -an important adaptive phenomenon. The Journal of physiology 543, 729. Shah, P. K., Kaul, S., Nilsson, J., and Cercek, B. (2001) Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part I. Circulation 104, 2376-2383. Koval, M., Preiter, K., Adles, C., Stahl, P. D., and Steinberg, T. H. (1998) Size of IgG-opsonized particles determines macrophage response during internalization. Experimental cell research 242, 265-273. Whitnack, E., and Beachey, E. H. (1985) Inhibition of complement-mediated opsonization and phagocytosis of Streptococcus pyogenes by D fragments of fibrinogen and fibrin bound to cell surface M protein. The Journal of experimental medicine 162, 1983-1997. Amara, S., Ivy, M. T., Myles, E. L., and Tiriveedhi, V. (2016) Sodium channel gammaENaC mediates IL-17 synergized high salt induced inflammatory stress in breast cancer cells. Cellular immunology 302, 1-10. Amara, S., and Tiriveedhi, V. (2017) Inflammatory role of high salt level in tumor microenvironment (Review). International journal of oncology 50, 1477-1481. Kato, Y., Ozawa, S., Miyamoto, C., Maehata, Y., Suzuki, A., Maeda, T., and Baba, Y. (2013) Acidic extracellular microenvironment and cancer. Cancer cell international 13, 89. Epstein, T., Xu, L., Gillies, R. J., and Gatenby, R. A. (2014) Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane. Cancer & metabolism 2, 7. Van de Winkel, J., and Anderson, C. L. (1991) Biology of human immunoglobulin G Fc receptors. Journal of leukocyte biology 49, 511-524. van de Winkel, J. G., and Capel, P. J. (1993) Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunology today 14, 215-221. Saphire, E. O., Parren, P. W., Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M., Dwek, R. A., Stanfield, R. L., Burton, D. R., and Wilson, I. A. (2001) Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 293, 1155-1159. 32 ACS Paragon Plus Environment

Page 32 of 43

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


(47) (48) (49) (50) (51) (52)

(53) (54)

(55) (56) (57) (58) (59) (60) (61) (62) (63) (64) (65)

Janeway Jr, C. A., Travers, P., Walport, M., and Shlomchik, M. J. (2001) The structure of a typical antibody molecule, in Immunobiology: The Immune System in Health and Disease, Garland Science, New York. Kollman, J. M., Pandi, L., Sawaya, M. R., Riley, M., and Doolittle, R. F. (2009) Crystal structure of human fibrinogen. Biochemistry 48, 3877-3886. Marder, V. J., Shulman, N. R., and Carroll, W. R. (1969) High molecular weight derivatives of human fibrinogen produced by plasmin I. Physicochemical and immunological characterization. Journal of Biological Chemistry 244, 2111-2119. Mustard, J., Packham, M., Kinlough-Rathbone, R., Perry, D., and Regoeczi, E. (1978) Fibrinogen and ADP-induced platelet aggregation. Blood 52, 453-466. Borhani, D. W., Rogers, D. P., Engler, J. A., and Brouillette, C. G. (1997) Crystal structure of truncated human apolipoprotein AI suggests a lipid-bound conformation. Proceedings of the National Academy of Sciences 94, 12291-12296. DiDonato, J. A., Huang, Y., Aulak, K., Even-Or, O., Gerstenecker, G., Gogonea, V., Wu, Y., Fox, P. L., Tang, W. W., and Plow, E. F. (2013) The function and distribution of apolipoprotein A1 in the artery wall are markedly distinct from those in plasma. Circulation, CIRCULATIONAHA. 113.002624. Ishikawa, T., Fidge, N., Thelle, D., Førde, O., and Miller, N. (1978) The Tromsø Heart Study: Serum apolipoprotein Al concentration in relation to future coronary heart disease. European journal of clinical investigation 8, 179-182. Wu, Z., Gogonea, V., Lee, X., May, R. P., Pipich, V., Wagner, M. A., Undurti, A., Tallant, T. C., Baleanu-Gogonea, C., and Charlton, F. (2011) The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering. Journal of Biological Chemistry 286, 12495-12508. Frank, P. G., and Marcel, Y. L. (2000) Apolipoprotein A-I: structure-function relationships. Journal of lipid research 41, 853-872. Sugio, S., Kashima, A., Mochizuki, S., Noda, M., and Kobayashi, K. (1999) Crystal structure of human serum albumin at 2.5 Å resolution. Protein engineering 12, 439-446. Soltys, B. J., and Hsia, J. C. (1978) Human serum albumin. Biol Chem 253, 3029-3034. Miclăuş, T., Beer, C., Chevallier, J., Scavenius, C., Bochenkov, V. E., Enghild, J. J., and Sutherland, D. S. (2016) Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro. Nature communications 7. Winzen, S., Schoettler, S., Baier, G., Rosenauer, C., Mailaender, V., Landfester, K., and Mohr, K. (2015) Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition. Nanoscale 7, 2992-3001. Deng, Z. J., Liang, M., Toth, I., Monteiro, M. J., and Minchin, R. F. (2012) Molecular interaction of poly(acrylic acid) gold nanoparticles with human fibrinogen. ACS nano 6, 8962-8969. Rahman, M., Laurent, S., Tawil, N., Yahia, L. H., and Mahmoudi, M. (2013) Nanoparticle and protein corona, in Protein-nanoparticle interactions pp 21-44, Springer, Berlin, Heidelberg. Tran, R., Xu, Z., Radhakrishnan, B., Winston, D., Sun, W., Persson, K. A., and Ong, S. P. (2016) Surface energies of elemental crystals. Scientific data 3, 160080. Uchimiya, M., Pignatello, J. J., White, J. C., Hu, S. L., and Ferreira, P. J. (2017) Surface Interactions between Gold Nanoparticles and Biochar. Scientific reports 7, 5027. Liu, H., Pierre-Pierre, N., and Huo, Q. (2012) Dynamic light scattering for gold nanorod size characterization and study of nanorod–protein interactions. Gold bulletin 45, 187-195. Ho, Y. T., Kamm, R. D., and Kah, J. C. Y. (2018) Influence of protein corona and caveolaemediated endocytosis on nanoparticle uptake and transcytosis. Nanoscale 10, 12386-12397.



(66) (67) (68)

(69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) (84)

Blanco, E., Shen, H., and Ferrari, M. (2015) Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nature biotechnology 33, 941-951. Zhang, B., Hu, Y., and Pang, Z. (2017) Modulating the Tumor Microenvironment to Enhance Tumor Nanomedicine Delivery. Frontiers in pharmacology 8, 952. Patra, A., Ding, T., Engudar, G., Wang, Y., Dykas, M. M., Liedberg, B., Kah, J. C. Y., Venkatesan, T., and Drum, C. L. (2016) Component‐Specific Analysis of Plasma Protein Corona Formation on Gold Nanoparticles Using Multiplexed Surface Plasmon Resonance. Small 12, 1174-1182. Reed, W. P., Stromquist, D. L., and Williams, R. C., Jr. (1983) Agglutination and phagocytosis of pneumococci by immunoglobulin G antibodies of restricted heterogeneity. The Journal of laboratory and clinical medicine 101, 847-856. Lord, S. T. (2007) Fibrinogen and fibrin: scaffold proteins in hemostasis. Current opinion in hematology 14, 236-241. Esmon, C. T., Xu, J., and Lupu, F. (2011) Innate immunity and coagulation. Journal of thrombosis and haemostasis : JTH 9 Suppl 1, 182-188. Pahlman, L. I., Morgelin, M., Kasetty, G., Olin, A. I., Schmidtchen, A., and Herwald, H. (2013) Antimicrobial activity of fibrinogen and fibrinogen-derived peptides--a novel link between coagulation and innate immunity. Thrombosis and haemostasis 109, 930-939. Marcel, Y. L., and Kiss, R. S. (2003) Structure-function relationships of apolipoprotein A-I: a flexible protein with dynamic lipid associations. Current opinion in lipidology 14, 151-157. Sorci-Thomas, M., Kearns, M. W., and Lee, J. P. (1993) Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships. The Journal of biological chemistry 268, 21403-21409. Carter, D. C., He, X. M., Munson, S. H., Twigg, P. D., Gernert, K. M., Broom, M. B., and Miller, T. Y. (1989) Three-dimensional structure of human serum albumin. Science 244, 1195-1198. Rosenoer, V. M., Oratz, M., and Rothschild, M. A. (2014) Albumin: Structure, function and uses, Elsevier, Oxford, United Kingdom. Albertson, R., Chabu, C., Sheehan, A., and Doe, C. Q. (2004) Scribble protein domain mapping reveals a multistep localization mechanism and domains necessary for establishing cortical polarity. Journal of cell science 117, 6061-6070. Kah, J. C., Chen, J., Zubieta, A., and Hamad-Schifferli, K. (2012) Exploiting the protein corona around gold nanorods for loading and triggered release. ACS nano 6, 6730-6740. Chikaura, H., Nakashima, Y., Fujiwara, Y., Komohara, Y., Takeya, M., and Nakanishi, Y. (2016) Effect of particle size on biological response by human monocyte-derived macrophages. Biosurface and Biotribology 2, 18-25. Albanese, A., Tang, P. S., and Chan, W. C. (2012) The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annual review of biomedical engineering 14, 1-16. Zellner, R. (2015) Biological responses to nanoscale particles. Beilstein journal of nanotechnology 6, 380-382. Armstrong, J. K., Wenby, R. B., Meiselman, H. J., and Fisher, T. C. (2004) The Hydrodynamic Radii of Macromolecules and Their Effect on Red Blood Cell Aggregation. Biophysical Journal 87, 42594270. Amin, S., Barnett, G. V., Pathak, J. A., Roberts, C. J., and Sarangapani, P. S. (2014) Protein aggregation, particle formation, characterization & rheology. Current Opinion in Colloid & Interface Science 19, 438-449. Stefani, M. (2004) Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1739, 5-25. 34 ACS Paragon Plus Environment

Page 34 of 43

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


(85) (86) (87) (88) (89)

(90) (91) (92) (93) (94) (95) (96)

(97) (98) (99) (100) (101) (102)

Vazquez-Rey, M., and Lang, D. A. (2011) Aggregates in monoclonal antibody manufacturing processes. Biotechnology and bioengineering 108, 1494-1508. Huang, R., Silva, R. A., Jerome, W. G., Kontush, A., Chapman, M. J., Curtiss, L. K., Hodges, T. J., and Davidson, W. S. (2011) Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma. Nature structural & molecular biology 18, 416-422. Tannock, I. F., and Rotin, D. (1989) Acid pH in tumors and its potential for therapeutic exploitation. Cancer research 49, 4373-4384. Justus, C. R., Dong, L., and Yang, L. V. (2013) Acidic tumor microenvironment and pH-sensing G protein-coupled receptors. Frontiers in physiology 4, 354. Carneiro-da-Cunha, M. G., Cerqueira, M. A., Souza, B. W., Teixeira, J. A., and Vicente, A. A. (2011) Influence of concentration, ionic strength and pH on zeta potential and mean hydrodynamic diameter of edible polysaccharide solutions envisaged for multinanolayered films production. Carbohydrate Polymers 85, 522-528. Kastelic, M., Kalyuzhnyi, Y. V., Hribar-Lee, B., Dill, K. A., and Vlachy, V. (2015) Protein aggregation in salt solutions. Proceedings of the National Academy of Sciences of the United States of America 112, 6766-6770. Sahin, E., Grillo, A. O., Perkins, M. D., and Roberts, C. J. (2010) Comparative effects of pH and ionic strength on protein-protein interactions, unfolding, and aggregation for IgG1 antibodies. Journal of pharmaceutical sciences 99, 4830-4848. Monopoli, M. P., Walczyk, D., Campbell, A., Elia, G., Lynch, I., Bombelli, F. B., and Dawson, K. A. (2011) Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. Journal of the American Chemical Society 133, 2525-2534. Maiolo, D., Del Pino, P., Metrangolo, P., Parak, W. J., and Baldelli Bombelli, F. (2015) Nanomedicine delivery: does protein corona route to the target or off road? Nanomedicine : nanotechnology, biology, and medicine 10, 3231-3247. Li, Y., Rui, M., Tang, H., and Xu, Y. (2013) The distribution and cell uptake of ApoA1 modified lipid carriers of siRNA in mouse liver in vivo. Asian Journal of Pharmaceutical Sciences 8, 228-233. Quan, Q., Xie, J., Gao, H., Yang, M., Zhang, F., Liu, G., Lin, X., Wang, A., Eden, H. S., Lee, S., et al. (2011) HSA Coated Iron Oxide Nanoparticles as Drug Delivery Vehicles for Cancer Therapy. Molecular pharmaceutics 8, 1669-1676. Kolhar, P., Anselmo, A. C., Gupta, V., Pant, K., Prabhakarpandian, B., Ruoslahti, E., and Mitragotri, S. (2013) Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium. Proceedings of the National Academy of Sciences of the United States of America 110, 10753-10758. Arruebo, M., Valladares, M., and González-Fernández, Á. (2009) Antibody-conjugated nanoparticles for biomedical applications. Journal of Nanomaterials 2009, 37. Roy, S., and Dasgupta, A. K. (2007) Controllable self-assembly from fibrinogen–gold (fibrinogen– Au) and thrombin–silver (thrombin–Ag) nanoparticle interaction. FEBS Letters 581, 5533-5542. Krystofiak, E. S. (2013) Fibrinogen-Conjugated Gold-coated Magnetite Nanoparticles for Antiplatelet Therapy. University of Wisconsin Milwaukee Theses and Dissertations Paper 717. Oliver, A. J., Lau, P. K., Unsworth, A. S., Loi, S., Darcy, P. K., Kershaw, M. H., and Slaney, C. Y. (2018) Tissue-Dependent Tumor Microenvironments and Their impact on immunotherapy Responses. Frontiers in immunology 9, 70. Kimling, J., Maier, M., Okenve, B., Kotaidis, V., Ballot, H., and Plech, A. (2006) Turkevich method for gold nanoparticle synthesis revisited. The journal of physical chemistry. B 110, 15700-15707. Haiss, W., Thanh, N. T., Aveyard, J., and Fernig, D. G. (2007) Determination of size and concentration of gold nanoparticles from UV-vis spectra. Analytical chemistry 79, 4215-4221.



(103) (104) (105) (106) (107) (108) (109) (110) (111) (112) (113) (114) (115) (116) (117)

Leng, W., Pati, P., and Vikesland, P. J. (2015) Room temperature seed mediated growth of gold nanoparticles: mechanistic investigations and life cycle assesment. Environmental Science: Nano 2, 440-453. Xiulan, S., Xiaolian, Z., Jian, T., Zhou, J., and Chu, F. S. (2005) Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1. International Journal of Food Microbiology 99, 185-194. De Roe, C., Courtoy, P. J., and Baudhuin, P. (1987) A model of protein-colloidal gold interactions. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 35, 1191-1198. Ho, Y. T., Poinard, B., Yeo, E. L., and Kah, J. C. (2015) An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles. The Analyst 140, 1026-1036. Bohren, C. F., and Huffman, D. R. (2008) Absorption and scattering of light by small particles, John Wiley & Sons, Weinheim, Germany. Chah, S., Hammond, M. R., and Zare, R. N. (2005) Gold Nanoparticles as a Colorimetric Sensor for Protein Conformational Changes. Chemistry & Biology 12, 323-328. Jain, P. K., Huang, X., El-Sayed, I. H., and El-Sayed, M. A. (2007) Review of some interesting surface plasmon resonance-enhanced properties of noble metal nanoparticles and their applications to biosystems. Plasmonics 2, 107-118. Kah, J. C., Zubieta, A., Saavedra, R. A., and Hamad-Schifferli, K. (2012) Stability of gold nanorods passivated with amphiphilic ligands. Langmuir : the ACS journal of surfaces and colloids 28, 8834-8844. Kreibig, U., and Vollmer, M. (1995) Optical properties of metal clusters, Vol. 25, Springer Berlin. Link, S., and El-Sayed, M. A. (1999) Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. The Journal of Physical Chemistry B 103, 8410-8426. Link, S., and El-Sayed, M. A. (2000) Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals. International Reviews in Physical Chemistry 19, 409-453. Link, S., and El-Sayed, M. A. (2003) Optical properties and ultrafast dynamics of metallic nanocrystals. Annual review of physical chemistry 54, 331-366. Papavassiliou, G. C. (1979) Optical properties of small inorganic and organic metal particles. Progress in Solid State Chemistry 12, 185-271. Kah, J. C. Y. (2013) Stability and aggregation assays of nanoparticles in biological media. Nanomaterial Interfaces in Biology: Methods and Protocols, 119-126. Pagana, K. D. (2013) Mosby's manual of diagnostic and laboratory tests, Elsevier Health Sciences, Canada.

TABLE OF CONTENTS GRAPHIC


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Transport Related Proteins

Physiological Microenvironment pH 7.4 Low Ionic Concentration

Immune Related Proteins

Increasing Protein Concentration

Tumor Microenvironment pH 6.2 High Ionic Concentration

Time

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


The corona formed from immune and transport-related proteins aggregate AuNPs in a concentration and protein-type dependent manner. At low protein concentrations, the proteins formed “bridges” between AuNPs, causing aggregation that could exacerbate in a tumormimicking microenvironment, unlike the colloidal stability conferred by the corona at higher protein concentrations.



Figure 1. Characterization of citrate-capped AuNPs synthesized using the Turkevich’s method. (A) TEM images of the spherical AuNPs whose size agreed with the mean DH value measured by DLS. (B) Representative DH distribution of the AuNPs and (C) UV-Vis absorbance spectrum of the synthesized 20-nm citrate-capped AuNPs. 289x202mm (300 x 300 DPI)

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Figure 2. AI of AuNPs at different protein:AuNP ratios for all four proteins: (A) IgG; (B) FBG; (C) ApoA1; and (D) HSA, after incubating the AuNPs with these proteins for 24 h at 37 °C in physiological pH of 7.4. The yaxis was drawn to the same scale to allow comparison between the two types of proteins. 289x202mm (300 x 300 DPI)



Figure 3. DH of AuNP-protein complex at different protein:AuNP ratios for all four proteins: (A) IgG; (B) FBG; (C) ApoA1; and (D) HSA, after incubating the AuNPs with these proteins for 24 h at 37°C in physiological pH of 7.4. 289x202mm (300 x 300 DPI)


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Figure 4. Fold changes in the AI of AuNPs over time at different protein:AuNP incubation ratios as the AuNPs traversed from physiological pH 7.4 to a low pH 6.2 and high ionic microenvironment that modelled that of tumors. AuNPs were pre-incubated with (A) ApoA1; (B) HSA; (C) IgG; or (D) FBG for 24 h at four selected protein:AuNP ratios, and dialyzed for up to 24 h at 37 °C in 10x PhB at pH 6.2. The time-dependent fold change in AI for each protein:AuNP ratio from the initial at time t = 0 h showed that the non-specific adsorption of proteins at a sufficiently high protein:AuNP incubation ratio minimized further aggregation of AuNPs induced by the low pH and high ionic concentrations microenvironment. 289x202mm (300 x 300 DPI)



Figure 5. Changes in the DH of AuNPs over time at different protein:AuNP incubation ratios as the AuNPs traversed from physiological pH 7.4 to a low pH 6.2 and high ionic strength microenvironment that modelled that of tumors. AuNPs were pre-incubated with (A) ApoA1; (B) HSA; (C) IgG; or (D) FBG for 24 h at four selected protein:AuNP ratios, and dialyzed for up to 24 h at 37 °C in 10x PhB at pH 6.2. The time-dependent fold change in DH (∆DH) for each protein:AuNP ratio over the initial at time t = 0 h showed that the nonspecific adsorption of proteins at a sufficiently high protein:AuNP incubation ratio minimized further aggregation of AuNPs induced by the low pH and high ionic concentrations microenvironment. 289x202mm (300 x 300 DPI)


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The corona formed from immune and transport related proteins aggregate AuNPs in a concentration and protein dependent manner. At low protein concentrations, the proteins formed “bridges” between AuNPs, causing aggregation that could exacerbate in a tumor-mimicking microenvironment, unlike the colloidal stability conferred by the corona at higher protein concentrations. 592x465mm (120 x 120 DPI)


Protein Corona Formed from Different Blood Plasma Proteins Affects

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