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Ligand Size and Conformation Affect the Behavior of Nanoparticles Coated with In Vitro and In Vivo Protein Corona Huajin Zhang, Tianmu Wu, Wenqi Yu, Shaobo Ruan, Qin He, and Huile Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16096 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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ACS Applied Materials & Interfaces

Ligand Size and Conformation Affect the Behavior of Nanoparticles Coated with In Vitro and In Vivo Protein Corona Huajin Zhang1, Tianmu Wu2, Wenqi Yu1, Shaobo Ruan1, Qin He1, Huile Gao1,* 1

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of

Pharmacy, Sichuan University, Chengdu 610041, China. 2

State Key Laboratory of Oral Diseases and West China Hospital of Stomatology,

Sichuan University, Chengdu 610041, China.

Abstract Protein corona is immediately established on the surface of nanoparticles upon their introduction into biological milieu. Several studies have shown that the targeting efficiency of ligand-modified nanoparticles is attenuated or abolished, owing to the protein

adsorption.

Here,

transferrin

receptor–targeting

ligands,

including

LT7

(CHAIYPRH), DT7 (hrpyiahc, all D-form amino acids), and transferrin, were used to identify the influence of the ligand size and conformation on protein corona formation. The results showed that the targeting capacity of ligand-modified nanoparticles was lost after incubation with plasma in vitro, while it was partially retained after in vivo corona formation. Results from sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry revealed the difference in the composition of

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in vitro and in vivo corona, wherein the ligand size and conformation played a critical role. Differences were observed in cellular internalization and exocytosis profiles based on the ligand and corona source. Keywords: protein corona, transferrin, T7 peptide, active targeting nanoparticle, cellular uptake, exocytosis, proteomics.

Introduction Nanoparticles are versatile platforms that are extensively used for disease diagnosis and delivery of chemicals drugs, peptides, proteins, and nucleic acids1,

2

. Several

nanoparticles have been approved for clinical applications, such as doxorubicin liposomes (Doxil)3, paclitaxel-albumin–bound nanoparticles (Abraxane)4, iron oxide nanoparticles (Feridex)5, and paclitaxel-encapsulated poly lactic acid micelles (Genexol)6. Although these nanoparticles are passively distributed into the tumor tissue, their therapeutic efficiencies rely on the phenomenon of enhanced permeability and retention effect7. These particles have been modified with several ligands that specifically interact with receptors overexpressed on target cells to improve their delivery efficiency and expand their applications from the treatment of tumors to other diseases8. At present, several ligand-modified nanoparticles are under clinical evaluation9; however, none of these combinations have been clinically approved. Aside from the safety concern, the in


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vivo efficacy of ligand-modified nanoparticles is still controversial. However, some studies suggest that the ligand-based modification may fail to elevate the accumulation of nanoparticles at the target site10, 11, other reports claim contrasting results9, 12. Therefore, it is important to elucidate the reason underlying these controversial data and identify the factors influencing the in vivo performance of nanoparticles. Once introduced in the blood, nanoparticles are immediately covered with a protein corona on their surfaces13, 14. Protein corona considerably affects the biodistribution of nanoparticles and their interactions with receptors and cells15. The composition of protein corona is influenced by the particle size, shape, and surface properties such as zeta potential, hydrophobicity, and functional groups14,

16-19

. The targeting ligands may be

proteins, peptides, small molecules, and oligonucleotides; protein and peptide ligands are widely studied20. Transferrin receptor (TfR) is known to be overexpressed on several malignant cells, and many ligands have been developed to bind TfR, including transferrin (Tf) and LT7 (CHAIYPRH) peptide21, 22. Tf has been widely used as the model ligand to evaluate the targeting mechanism and functions of protein corona, whereas LT7 is a small peptide selected by phage display and exhibits a high binding affinity to TfR22, 23. In addition, D-peptides constructed to overcome the poor stability of L-peptides in blood have been known to show promising outcomes24. Although there are contrasting reports on the inhibitory effect of the protein corona on targeted delivery of nanoparticles19, 23, 25-



27

, no systemic study has elucidated the influence of ligand size and conformation on

protein corona formation and targeting capacity. Here, we hypothesize that ligand properties such as size and confirmation may affect the composition of protein corona and subsequent interaction between the ligand and target cells and that these characteristics may provide an important reference for constructing drug delivery systems. In this study, LT7, DT7, and Tf were selected as model ligands and coupled to polystyrene nanoparticles (PNs) to investigate the influence of ligand size and conformation on protein corona composition and PN behavior. The corona composition was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and label-free proteomic analysis using nanoliquid chromatography tandem mass spectrometry (Nano-LC-ESI-MS/MS). In addition, the influence of the protein corona on the interaction between ligand-modified PNs and HepG2 cells overexpressing TfR was investigated with cellular uptake and exocytosis studies. Our study results may serve as a foundation for the evaluation of the in vivo efficacy of ligand-modified nanoparticles and provide guidelines for the construction of nanoparticles for in vivo active targeted delivery. Results and discussion Preparation and characterization of nanoparticles. Amino-functionalized PNs were synthesized by microemulsion polymerization28 and reacted with N-hydroxysuccinimide–


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and maleimide-functionalized polyethylene glycol (Mal-PEG5000-NHS, Seebio, Shanghai, China). The thiolated human Tf-SH, LT7, and DT7 were coupled to PNs through the reaction between maleimide and thiol groups23. The particle size of PEG-modified PN (PEG-PN) was 80 nm (Table 1). After the attachment of 144 LT7, 135 DT7, and 190 Tf (Supplementary Table S1) to a single PN, the particle size of LT7-modified PNs (LT7-PN), DT7-modified PNs (DT7-PN), and Tf-modified PNs (Tf-PN) increased to 92, 89, and 84 nm, respectively, with a small polydispersity index (Table 1 and Supplementary Figs. S1, S2). Transmission electron microscopy (TEM, JEM 100CX, JEOL, Japan) revealed the round shape of the particles (Fig. 1a-d). Circular dichroism (CD) spectroscopy demonstrated that all ligands retained their unique conformations after their conjugation onto PNs (Supplementary Fig. S3). We evaluated the binding affinity of various PNs to TfR with label-free surface plasmon resonance (SPR) assay (Biacore 200, GE, USA) (Supplementary Fig. S4). The binding affinity of LT7-PN and DT7-PN to TfR was considerably higher than that of PEG-PN (1.6-fold and 2.2-fold, respectively), indicating that LT7 and DT7 may specifically target TfR, which is consistent with the results of previous studies24, 29. The binding affinity of Tf-PN to TfR was significantly higher than that of PEG-PN (2.7-fold), LT7-PN, and DT7-PN; thus, Tf-PN reserved its specific interaction with TfR, as observed with CD result.



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Figure 1. Characterization of various PNs before and after in vitro human protein corona formation. a-d. TEM images of PEG-PN (a), LT7-PN (b), DT7-PN (c), Tf-PN (d). e-h. TEM images of PNs with in vitro coronas. Scale bars represent 100 nm. Table 1. Characterization of particle size and zeta potential of bare PNs and PNs with in vitro corona by dynamic light scattering (n=3).

bare PNs

PNs with in vitro corona

Particle size (nm)

PDI

Zeta potential (mV)

PEG-PN LT7-PN DT7-PN

80.77±1.73 89.50±5.87 83.61±8.96

0.15±0.01 0.12±0.01 0.15±0.02

2.11±0.08 16.2±0.53 17.1±0.40

Tf-PN PEG-PN LT7-PN DT7-PN

92.47±8.88 138.7±3.60 136.5±0.80 142.0±2.60

0.15±0.03 0.18±0.02 0.15±0.02 0.14±0.02

1.25±0.20 -7.46±0.27 -10.1±0.44 -14.5±0.17

Tf-PN

151.8±11.3

0.19±0.05

-15.0±0.71

PNs were purified using molecular-exclusion chromatography following in vitro incubation with 100% human plasma (male, type B, Rh negative) for 1 h at 37 °C (Supplementary


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Fig. S5)27. The size of PEG-PN, LT7-PN, DT7-PN, and Tf-PN with in vitro protein corona significantly increased to 139, 137, 142, and 152 nm, respectively (Table 1). The zeta potential of all PNs with in vitro corona changed from positive to slightly negative, indicative of the adsorption of the electronegative proteins around PNs. The amount of in vitro protein corona adsorbed on PEG-PN, LT7-PN, DT7-PN, and Tf-PN was 45, 47, 100, and 53 µg per milligram of PN, respectively. Factors such as the size increase, zeta potential change, and corona concentration suggest that DT7-PN may absorb more proteins than other PNs. We examined the particle size of PNs with/without in vitro protein corona in phosphate-buffered saline (PBS, pH 7.4) for 24 h and found that the particles were stable during incubation (Supplementary Table S2). Furthermore, we evaluated the formation of in vivo corona on PNs by collecting plasma samples 10 min after the intravenous injection of various PNs into mice27. The size of PNs coated with in vivo corona was larger than that of the uncoated PNs (Supplementary Fig. S6). Influence of ligand properties on in vitro/in vivo corona composition. Although LT7, DT7, and Tf showed binding affinity toward TfR, their properties differed. Tf is a large sized-protein (molecular weight [MW] = 77 kDa) and may pose difficulty for the construction of drug delivery systems30. In addition, Tf-receptor combination would be competitively inhibited by the endogenous Tf31. Previous experiments have shown that peptides interacting with TfR bind to a unique binding site and show no interference with



Tf binding. Although the accurate binding sites are unknown, at least 15 cavities with no overlapping binding site with holo-Tf are known29. With respect to LT7, its binding site for TfR is different from that for Tf. The peptide DT7 was artificially constructed and its amino acid sequence is retro-inverso (Supplementary Figs. S3, S7-S10). Retro-inverso Dpeptides exhibit the ability to overcome disadvantages of L-peptides32. We performed SDS-PAGE followed by Coomassie blue staining to elucidate the composition of protein corona (Fig. 2a, c). Higher protein concentration was observed in the corona from LT7-PN, DT7-PN, and Tf-PN as compared with that from PEG-PN. Thus, the ligand modification resulted in elevated protein adsorption, owing to the alteration in the surface property14. It is interesting that the composition of the protein coronas from different PNs was different, particularly for proteins with MW of 90, 70, 55, and 45 kDa. Furthermore, in vivo corona showed more complex composition as compared with in vitro corona (Fig. 2a, c and Supplementary Tables S3, S4) and may cause different cellular response. Nano-LC-ESI-MS/MS was used to elucidate the protein abundance in the corona. Bioanalytical tools33 were further employed to classify the identified proteins in accordance with their functions in biological processes. After analyzing 170 types of proteins from in vitro or in vivo corona, the most abundant proteins were found to be apolipoproteins, which may be influenced by the hydrophobicity of PNs34. In addition, the


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species of proteins were significantly different between in vivo and in vitro coronas, as previously reported27. Aside from apolipoproteins, fibrinogen was frequently identified in in vitro corona on PNs (fibrinogen α 94.6 kDa and fibrinogen γ 52.3 kDa), consistent with the findings of a previous study35 (Fig. 2b and Supplementary Table S3). As fibrinogen chains are highly associated with protein polymerization, fibrinogen may induce aggregation of protein coronas on PN surface and form a compact corona, thereby hindering the binding of ligands to TfR (Supplementary Fig. S11 and Supplementary Table S5). In addition, fibrinogen may induce the signal of non-specific transduction (Supplementary Fig. S11 and Supplementary Table S5) and further up-regulate the release of inflammatory cytokines36, which may disturb the interaction between ligands and TfR. In this study, different properties of ligands significantly influenced the composition of the protein corona. Low concentration of fibrinogen γ chain was observed in the in vitro corona of LT7-PN, while low concentration of apolipoprotein A-I and high concentration of fibrinogen γ chain and plasma protein were observed in the in vitro corona of DT7-PN. In the in vitro corona of Tf-PN, the concentration of apolipoprotein A-I and fibrinogen γ chain slightly increased and that of plasma protein decreased. All the above mentioned factors of in vitro protein corona may disturb the interaction between the ligand-modified PNs and TfR.



Figure 2. Proteomics analysis of in vitro and in vivo corona. a. SDS-PAGE of in vitro corona. The gel was stained with Coomassie blue (Supplementary Fig. S12). Lanes 1-4 indicate PEG-PN, LT7-PN, DT7-PN, and Tf-PN with in vitro corona (in order). b. NanoLC-ESI-MS/MS label-free proteomic analysis heat map of the abundant proteins (>1%) in in vitro protein corona. c. SDS-PAGE of in vivo corona with sliver staining (Supplementary Fig. S13). Lanes 1-4 indicate PEG-PN, LT7-PN, DT7-PN, and Tf-PN with


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in vivo corona (in order). d. Mass spectrometric analysis heat map of the abundant proteins (>1%) in in vivo protein corona.

Different high abundance proteins were observed with in vivo corona; the species and amount of apolipoproteins were more abundant that others (Fig. 2d, Supplementary Table S4). Different from in vitro corona, the adsorption of the in vivo fibrinogen was less than 1%. The amount of albumin and clusterin were significantly higher than that observed in in vitro corona. Clusterin (51.6 kDa) is an important corona component and reduces the non-specific cellular uptake34. In addition, complement proteins were significantly abundant in the in vivo corona of LT7-PN and DT7-PN. Several studies have demonstrated the conclusive roles played by complement proteins from protein corona in the process of complement activation37-39 either through the classical or alternative pathway (Supplementary Fig. S14). Complement proteins were sparse in plasma, suggesting that LT7/DT7 functionalization may attract a mass of complement proteins and cause complement activation to further induce macrophage phagocytic activity40. Tf-functionalized PNs lacked complement proteins adsorption, suggestive of their better biocompatibility than LT7-PN and DT7-PN.

TfR-binding affinity and cellular uptake of ligand-modified PNs with/without corona.



Localized surface plasmon resonance (LSPR) assay was used to evaluate the influence of corona on the binding affinity of ligand-modified PNs to TfR. Although bare PNs retained the initial binding affinity to TfR (Fig. 3a), the binding affinity considerably reduced to the level of PEG-PN following corona formation on LT7-PN, DT7-PN, and TfPN (Fig. 3b). Thus, the protein corona may hinder the specific interaction and ligands may be completely masked by in vitro protein corona. Similar observations were reported in previous studies, wherein the active targeting capacity was lost after protein corona formation23, 41.

Figure 3. Targeting capacity of various PNs. a. Binding affinity of PEG-PN, LT7-PN, DT7PN, and Tf-PN to TfR without protein corona, as determined by LSPR assay in triplicates. b. Binding affinity of PNs to TfR after in vitro corona formation, as determined by LSPR assay in triplicates. c. Flow cytometry (FCM) evaluation of HepG2 cells 1 h after the


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uptake of PNs functionalized with LT7, DT7, or Tf with/without in vitro corona; PEG-PN served as a control; error bars represent the variability of the data; statistically significant differences (two-tailed t-test) were marked with *P PEG-PN. As per the results of the proteomic study, the lower adsorption of fibrinogen and higher adsorption of clusterin may reserve the specific uptake capacity of PNs with in vivo corona as compared with PNs with in vitro corona. Several reports have shown that the corona composition was influenced by the protein source27,

43-45

. In

addition, studies have been performed for the analysis of in vivo corona27, 38; however, no study has explored the quantitative difference in the targeting capacity of particles with in vivo and in vitro corona. In this direction, we evaluated the difference between in vitro and in vivo protein corona. Confocal microscopy was used to verify the cellular internalization and study the localization of PNs. We performed semi-quantitative analysis to evaluate the percentage of various PNs that diffused out of lysosomes at three time points (0.5, 1, and 1.5 h) (Fig. 3e). The distribution of Tf-PN in the cytoplasm was higher than that of other particles,



indicating that the higher cellular uptake of Tf-PN was attributed to its faster transport from the lysosomes to the cytoplasm. The distribution of LT7-PN and DT7-PN in the cytoplasm was similar to that of PEG-PN, suggesting that the relatively low binding affinity of DT7 and LT7 to TfR may hamper their transport from lysosomes to the cytoplasm. The coating of PNs with in vitro hard corona resulted in a significant reduction in the uptake of PNs to the level observed with the shield of hard corona reported in previous studies25,

46

. Although in vivo corona interfered with the binding between the

receptor and ligand bound PNs, the cellular uptake of ligand-modified PNs with in vivo corona was much higher than that of PEG-PN, suggestive of the retention of the specificity of ligand-modified PNs. The distribution ratio in the cytoplasm was significantly higher for ligand-modified PNs with in vivo corona than PEG-PNs (Fig. 3f). In addition, PNs functionalized with Tf—a representative protein ligand—showed a strong intracellular transportation capacity, resulting in the prolonged retention of PNs inside the cells. The internalization of T7 via TfR is thought to be assisted by endogenous Tf. Although the endocytosis of T7 was faster than that of Tf, the amount of the internalized T7 was lower than that of Tf29. The results of the cellular uptake analysis by confocal microscopy were similar to those of the quantitative analysis (Fig. 4). These preliminarily results shed light on the differences between in vitro and in vivo corona. The bicinchoninic acid assay showed that the total amount of in vivo protein corona on PNs


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was nearly similar to that of in vitro protein corona (Supplementary Table S6), suggesting that the difference in the corona composition, not the amount of corona, influenced the performance of ligand-modified PNs with in vitro and in vivo corona.

Exocytosis of PNs. To understand the role of ligands in the exocytosis of PNs with or without protein corona, the exocytosis liquid discharged from cells was collected and the amount of PNs expelled from the cells was evaluated41. To ensure similar internalization level for all cells, the concentration was elevated to a level much higher than the normal level (1.5 mg/mL for PEG-PN and LT7-PN, 1 mg/mL for DT7-PN and Tf-PN, and 2 mg/mL for PNs with in vitro corona) (Supplementary Figs. S17, S18). PEG-PN, LT7-PN, DT7-PN, and Tf-PN were barely expelled from HepG2 cells, owing to the strong binding affinity between PNs without corona and cell membrane (Fig. 5a). As ligand-modified PNs had a high binding affinity to TfR, the clearance of TfR was hindered in the endosomes and cytoplasm. As a consequence, the recycling of TfR to the cell surface was reduced47, leading to decreased exocytosis48. In addition, the high potential for the re-uptake of ligand-modified PNs by cells further attenuated the exocytosis rate; thus, the ligandmodified PNs without corona may be retained in the cells for a longer duration. However, the amount of PNs extracted from the cells significantly increased following coating with in vitro protein corona, indicating that in vitro corona may hinder the interaction between



particles and cells, as observed in the plasma binding assay (Fig. 5a). The retention of the corona on PN surface during the complete intracellular transportation and exocytosis process would result in similar exocytosis rate for LT7-PN, DT7-PN, as well as Tf-PN and PEG-PN, as demonstrated by the cellular uptake study. For PNs with in vitro corona, the exocytosis rate was in the following order: PEG-PN > DT7-PN > LT7-PN > Tf-PN. Thus, ligand modification significantly inhibited the exocytosis of PNs. These results, in combination with the cellular uptake and exocytosis analysis, suggest that more Tf-PNs may be reserved in cells than LT7-PNs and DT7-PNs and that Tf-PNs were suitable for intracellular drug delivery.

Figure 5. Exocytosis process of PN. The ratio of PNs retained and expelled from the HepG2 cells at 100 min. P values represent statistically significant difference in two-tailed t-test for PN with its corresponding corona at 100 min time point.


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Figure 6. Statistical analysis of the amount of proteins involved in the cellular component from in vitro (a) and in vivo (b) corona, counted according to the top 10 related proteins.

The proteomic analysis of the cellular component (Fig. 6a, b) showed that many of the proteins adsorbed onto different ligand-modified PNs were functional in the extracellular region (Supplementary Table S7) and may have accelerated the removal of PNs with corona from the cells before PN decomposition49. PEG modification is suggested to reduce the interaction between nanoparticles and intracellular proteins; this phenomenon may explain the highest exocytosis rate observed for PEG-PN with in vitro corona as compared to other ligand-modified PNs41.



Conclusion The difference in the ligand size and conformation altered the composition of in vitro and in vivo corona, resulting in variations in their cellular uptake and exocytosis. As the exact functions of each protein in the protein coronas remain unclear, further studies should evaluate the effects of different protein combinations during the processes of cell cycle.

Materials and methods Synthesis of amino functionalized fluorescent polystyrene nanoparticles Aqueous solution composed of 0.3 g AEMH, 1.0 g Lutensol AT50 (BASF, Germany) and 24 g deionized water was transferred into a 50 mL round-bottom flask, magnetic stirring at 500 rpm and 72 oC in oil bath. The oil phase consisted of 6.0 g styrene monomer, 1.0 mg V59 (2,2′-Azobis(2-methylbutyronitrile)) and 2.5 g cetane were added drop-wisely into the flask, and stirred at a rate of 500 rpm for 1 h of pre-emulsification. The mixture was then well dispersed under probe ultrasonication (400 W, 1 s for 20 times), and reacted at 300 rpm, 72 oC for another 20 h. Conjugation of LT7, DT7 and Tf onto PEG-PN Firstly, in order to obtain sodium thioglycollate modified Tf (Sigma-Aldrich), Tf was reacted with truant’s reagent 2-iminothiolane (molar: molar = 1:40), and 0.15 mol/L PBS with 0.1 mmol/L EDTA (pH 8.5) for 1 h at room temperature, then purified with molecular


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exclusion chromatography (Sephadex G-100, Sigma-Aldrich). Afterwards, LT7 (PHTD PEPTIDE CO., LTD, Zhengzhou, China), DT7 (PHTD PEPTIDE CO., LTD, Zhengzhou, China), and Tf were reacted with PEG conjugated PNs (molar:molar = 1:1, the estimate density of ligand density 100 per nanoparticle) mildly at 500 rpm, room temperature over night. The particles were then ultrafiltrated to remove unconjugated ligands. The particles were purified using ultrafiltration tube (100 kDa, Millipore) at 10000 rpm for 15 min and then the particles were resuspended in 0.5 mL PBS (pH 7.4). The operation was repeated 3 times to remove unloaded compounds. The ligation efficiency was measured by BCA assays for unconjugated ligands (Supplementary Table S1). Transmission electron microscope PNs with/without in vitro protein corona were negatively stained by phosphotungstic acid and then images were taken under 75 kV operating voltage by transmission electron microscope. Localized surface plasmon resonance assay Firstly, the COOH sensor chip (Nicoya lifesciences, Canada) was activated by EDC/NHS reaction, and then 200 µL TfR (10 µg/mL) were injected for covalent conjugation. After loading 200 µL blocking solution, 200 µL of all the PN samples (0.5 mg/mL) were loaded in turn to interact with the TfR on the sensor chip. And all the sample injection had been repeated 3 times.



Preparation of in vivo corona coated PNs 100 µL of various PNs (100 mg/mL) was intravenously administrated in male Kunming mice. After 10 min, blood was sampled by eyeball removal method. We collected about 1 mL whole blood into the tubes pre-coated with 1% heparin sodium, and centrifuged at 3000 rpm, 4 oC for 10 min. After centrifugation, we collected about 0.4 mL supernatant (plasma with PNs). Then we loaded the plasma with PNs on to the Sepharose CL-4B (Sigma-Aldrich) column (16x1.0 cm) and eluted with PBS (pH 7.4) buffer and get about 1.5 mL PNs with corona liquids with the concentration of 2.5-3 mg/mL. The turbidimetry of each PN sample was determined, and the concentration of PNs was calculated by the line equation of the standard curve. Before adding the PNs with in vivo corona to the cells, the serum-free DMEM medium was added to the PNs samples to adjust all the concentration to 0. 5 mg/mL. Male Kunming mice (30 ± 2 g) were all purchased from Dashuo Biotechnology Co., Ltd, (Chengdu, China) and maintained under standard conditions. All animal experiments were performed under the guidelines of the ethics committee of Sichuan University. Adsorbed proteins isolation (identification) by SDS-PAGE Ligand-modified PNs coated with hard corona were all adjust to the concentration of 0.15 mg/mL, and the liquid was boiled with loading buffer (250 mM Tris-HCl , 10% SDS, 0.05% Bromophenol Blue, 50% Glycerol and 5% β- mercaptoethanol, 3:1) at 100 oC for 8


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min to collect and denature proteins. All the in vitro/in vivo PNs samples with an equal volume of 15 µL were loaded onto the polyacrylamide gels, followed by running the gel at 90 V for 1 h and then 120 A for 2 h. The gels were stained by silver staining kit (Beyotime, Haimen, China) and coomasside blue. Stained gels were further scanned using GelDocTM XR scanner (Bio-Rad, America). Proteomics Firstly, proteins from PNs with in vitro/in vivo corona samples were separated from PNs with SDS-PAGE50. Then in-gel-digestion was performed followed by capillary-high performance liquid chromatography (Easy nLC1000, Thermo scientific) separation and proteomic mass spectrometry (Q-Exactive HF, Thermo scientific, America). Mscot and Proteome Discoverer (Thermo Scientific, America) were used to qualitatively and quantitatively match the proteins to the library. Exocytosis of PNs from HepG2 cells HepG2 cells were incubated in 24-well plate with different ligand functionalized PNs for 1 h. Then the culture medium was removed, and washed quickly by PBS (pH 7.2) for 3 times. Then cells were treated with 2 mL phenol red-free and serum-free DMEM medium to collect 100 µL discharged liquid each time from every well at the following time points: 10, 20, 30, 40, 45, 60, 90 and 100 min. And plate reader (Thermo Scientific, America) was used to detect the fluorescence intensity of the coumarin-6 stained PNs under 466



nm excitation and 504 nm emission. SDS-PAGE for protein corona of excreted PNs After exocytosis for 2 h, 500 µL upper liquid was collected from each well. To enrich the loading sample collected from the wells, the liquid was all freeze-dried for 48 h and redissolved with 100 µL deionized water. And the protein samples were treated and analyzed in the above-mentioned method.

Author contributions H.G. supervised the study. H.Z. and H.G. designed the experiments. H.Z. performed all experiments with assistance by T.W., W.Y., S.R. and Q.H.. H.Z. and H.G. analyzed the data and wrote the paper.

Supporting information Supporting information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxx. Synthesis of amino functionalized fluorescent polystyrene nanoparticles. Conjugation of PNs with NHS-PEG5000-Mal linker. Characterization of PEG-PN, LT7-PN, DT7-PN and TfPN. Preparation of in vitro protein corona coated PNs. In vitro and in vivo cellular uptake. Adsorbed proteins isolation (identification) by SDS-PAGE. Supplementary figures and


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Tables mentioned in the manuscript.

Corresponding author *E-mail: [email protected]; [email protected] ORCID Huile Gao: 0000-0002-5355-7238 Notes The authors declare no competing financial interest.

Acknowledgements The work was granted by National Natural Science Foundation of China (31571016, 81402866, 81402642). Authors thank Dr. Zhonglian Cao (School of Pharmacy, Fudan University, Shanghai, China) for the assistance of SPR assay. Authors thank Dr. Jingyuang Xiong (West China School of Public Health, Sichuan University) for the English editing. Authors acknowledge the support of West China School of Public Health, research center for public health and preventive medicine, Sichuan University.

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Graphic for manuscript.


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Figure 1. Characterization of various PNs before and after in vitro human protein corona formation. a-d. TEM images of PEG-PN (a), LT7-PN (b), DT7-PN (c), Tf-PN (d). e-h. TEM images of PNs with in vitro coronas. Scale bars represent 100 nm. 94x72mm (300 x 300 DPI)



Figure 2. Proteomics analysis of in vitro and in vivo corona. a. SDS-PAGE of in vitro corona. The gel was stained with Coomassie blue (Supplementary Fig. S12). Lanes 1-4 indicate PEG-PN, LT7-PN, DT7-PN, and Tf-PN with in vitro corona (in order). b. Nano-LC-ESI-MS/MS label-free proteomic analysis heat map of the abundant proteins (>1%) in in vitro protein corona. c. SDS-PAGE of in vivo corona with sliver staining (Supplementary Fig. S13). Lanes 1-4 indicate PEG-PN, LT7-PN, DT7-PN, and Tf-PN with in vivo corona (in order). d. Mass spectrometric analysis heat map of the abundant proteins (>1%) in in vivo protein corona. 199x228mm (300 x 300 DPI)


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Figure 3. Targeting capacity of various PNs. a. Binding affinity of PEG-PN, LT7-PN, DT7-PN, and Tf-PN to TfR without protein corona, as determined by LSPR assay in triplicates. b. Binding affinity of PNs to TfR after in vitro corona formation, as determined by LSPR assay in triplicates. c. Flow cytometry (FCM) evaluation of HepG2 cells 1 h after the uptake of PNs functionalized with LT7, DT7, or Tf with/without in vitro corona; PEG-PN served as a control; error bars represent the variability of the data; statistically significant differences (two-tailed t-test) were marked with *P

Ligand Size and Conformation Affect the Behavior ... - ACS Publications

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