Size-Dependent Facilitation of Cancer Cell Targeting by Proteins

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Size-Dependent Facilitation of Cancer Cell Targeting by Proteins Adsorbed on Nanoparticles Gaoxing Su, Xiaofei Zhou, Hongyu Zhou, Ye Li, Xianren Zhang, Yin Liu, Dapeng Cao, and Bing Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10967 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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ACS Applied Materials & Interfaces

Size-Dependent Facilitation of Cancer Cell Targeting by Proteins Adsorbed on Nanoparticles

Gaoxing Su†, ‡, #, Xiaofei Zhou†, #, Hongyu Zhou*, §, Ye Li⊥, Xianren Zhang‖, Yin Liu†, Dapeng Cao‖, and Bing Yan*, §

†

School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China

‡

School of Pharmacy, Key Laboratory of Inflammation and Molecular Drug Targets of Jiangsu

Province, Nantong University, Nantong 226001, China §

School of Environment, Guangzhou Key Laboratory of Environmental Exposure and health and

Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University, Guangzhou 510632, China ‖

Division of Molecular and Materials Simulation, State Key Laboratory of Organic-Inorganic

Composites, Beijing University of Chemical Technology, Beijing 100029, China ⊥

College of Biological Sciences and Biotechnology, Beijing Forest University, Beijing 100083,

China

ACS Paragon Plus Environment

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KEYWORDS: cell targeting, protein adsorption, nanomedicine, delivery, cell recognition

ABSTRACT

Understandings of how biomolecules modify nanoparticles in a biological context and how these exchanges impact nano-bio interactions are fundamental to nanomedicine and nanotoxicology research. In this work, cancer-targeting gold nanoparticles (TGNPs) with different sizes (5, 15, and 40 nm) were designed and synthesized. These nanoparticles spontaneously adsorbed proteins in complete cell culture medium (DMEM with 10% human serum). Although the targeting ligands on the surface of nanoparticles were likely to be shielded by adsorbed proteins, the targeting capability of nanoparticles was maintained due to the highly dynamic nature of protein adsorption. By regulating the size and surface curvature of nanoparticles, we found that smaller TGNPs (5 nm, large surface curvature) recognize folate receptors on HeLa cells mainly through one-on-one bindings, and adsorbed proteins partially interfered with their binding, inducing a reduction of cell uptake by ~30%. Larger TGNPs (40 nm, small surface curvature) bound to cell surface receptors through multivalent interactions, and their binding affinity was, in contrast, enhanced by adsorbed proteins, resulting in an increased cell uptake by ~13%. Computational modeling further corroborated our experimental findings. The compelling findings from this work demonstrated how nanoparticle’s size controlled its biological activity and provided key design principles for nanomedicine agents.

Introduction


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Nanoparticles serve as excellent carriers for drugs, genes and imaging agents for cancer therapy and diagnosis.1-5 An increasing number of nanotheranostic formulations are either in clinical trials5-8 or have already been approved by the FDA5,8,9, even though these nanoparticles are not cancer cell-selective. Cancer-targeting nanocarriers and related therapy and diagnoses will immensely improve the general issue of selectivity in cancer treatment. Cancer-specific targeting is achieved by functionalizing nanoparticles with targeting antibodies,10-12 aptamers,13,14 peptides,15,16 or small molecules16,17 to specifically bind to overexpressed receptors on the cancer cell surface. However, when nanoparticles enter the bloodstream, serum proteins are spontaneously adsorbed to nanoparticles due to their large surface areas and high surface reactivity.18-21 Protein adsorption immediately changes the physicochemical properties of nanoparticles, such as size,22 surface properties,23 zeta potential,24 and aggregation status,25 and probably the ways nanoparticles interact with cells.22,26-33 A critical question is whether protein adsorption on nanoparticles prohibits the targeting capability of nanoparticles. We have conjugated GD2-targeting antibody molecules to drug-carrying gold nanoparticles (GNPs) and found that even with protein adsorption, these nanoparticles target neuroblastoma cells with a 62fold higher binding affinity than that of normal cell models.12 Reports also show enhanced cell uptake in cell culture medium when nanoparticles adsorbed serum proteins22,28,34 and a higher tumor accumulation compared to non-targeting nanoparticles when they are administered by intravenous injection in vivo.3,5,35 These studies seem to indicate that the targeting capability of nanoparticles is mostly maintained after protein adsorption on nanoparticles. However, contradictory results have also been reported.36-39 In one case, the adsorbed proteins block the cell recognition of the transferrin-functionalized nanoparticles.36 Therefore, the question remains on how the adsorbed proteins impact cell recognition and targeting.40


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Before we explore the answers to the above question, we must point out two important factors: first, the dimensions of nanoparticles may play a significant role because size defines the curvature of the particle surface and how surface ligands are projected during their binding with cell surface receptors41; second, the adsorbed proteins might be in a dynamic equilibrium18,42 with free proteins in solution. This dynamic exchange process may give targeting ligands enough time and space to interact with cell surface receptors. With these questions and hypothesis in mind, we designed and synthesized folate-targeting GNPs with three different diameters (5, 15, and 40 nm) and analyzed the dynamic adsorption of serum proteins and their effects on cancer cell targeting. Our results revealed an astonishing fact: the adsorbed serum proteins only partially reduced the cell targeting of smaller GNPs (5 nm), while enhanced the targeting of larger GNPs (40 nm) compared to no protein adsorption with a cut-off diameter around 15 nm.

Results and Discussion Cancer-targeting and non-targeting GNPs of three sizes. To reveal the effects of adsorbed proteins on cell targeting by nanoparticles and to address the roles that particle size and protein binding dynamics play in cell recognition, we first assembled targeting GNPs of various sizes and corresponding non-targeting GNPs. In many cancers, cells overexpress folate receptors (FR) on their surfaces. Therefore, we synthesized FR-targeting GNPs with diameters of 5, 15, and 40 nm (TGNP-5, TGNP-15 and TGNP-40) by reduction of hydrogen tetrachloroaurate(III) trihydrate with NaBH4 or sodium citrate. Folic acid (FA)-terminated thiolated PEG5000 ligands were bound to the surface of GNPs through strong S-Au bonding to form various targeting nanoparticles (Figure 1A). As controls, non-targeting GNPs (NGNP-5, NGNP-15 and NGNP-40)


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with the same diameters were also synthesized by the conjugation of thiolated methoxyterminated PEG5000 to GNPs (Figure 1A). The TEM images of both targeting and non-targeting GNPs showed that these nanoparticles were all spherical particles (Figure 1B) with narrow size distributions. We also characterized their aggregation status in solution by measuring their dynamic light scattering (DLS) in water and serum suspensions (Table 1). After surface ligand conjugation, the hydrodynamic diameters of targeting GNPs were 16 nm, 30 nm, and 52 nm, respectively. When these GNPs were dispersed in cell culture medium with 10% human serum, the hydrodynamic diameters of GNPs were 57 nm, 89 nm, and 124 nm, indicating strong protein adsorption. DLS results (PDI98%) and trisodium citrate dehydrate were purchased from Sigma-Aldrich. All reagents were used as received. Glassware used for GNP synthesis was immersed in aqua regia overnight, then washed with ultrapure water several times. Ultrapure water (18.2 MΩ•cm, Milli-Q) was used in all experiments. Synthesis of GNPs. GNPs (diameter: 5 nm) were prepared according previous work with minor modification.51 Briefly, HAuCl4•3H2O solution (10 mM, 2.54 mL) were added to a flask with 88 mL of water. Next, trisodium citrate solution (38.8 mM, 2 mL) was mixed and stirred for one minute. Fresh NaBH4 solution (0.075%, 1 mL) was mixed with vigorous stirring for 5 min. Obtained GNPs were approximately 5 nm. GNPs (diameter: 15 nm) were prepared as shown in our previous work.12 Using a seed-mediated growth method, GNPs (diameter :40) nm were prepared.52 Synthesized 15 nm GNPs were used as seeds. The stock solution of 15 nm GNPs was diluted with water (118 mL) and trisodium citrate solution (60 mM, 2 mL). The mixture was heated to 90 °C with stirring. Then, HAuCl4•3H2O solution (1 mL, 25 mM) was added. After 30 min, this process (adding 1 mL 25 mM HAuCl4•3H2O solution) was repeated three times. The first generation of GNPs was obtained. After that, 37.5 mL of the first generation of GNP solution was diluted with water (112 mL) and sodium citrate solution (60 mM, 2 mL). The mixture was heated to 90 °C with stirring. Then, HAuCl4•3H2O solution (25 mM, 1 mL) was added. After 30


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min, this process (adding 1 mL 25 mM HAuCl4•3H2O solution) was duplicated three times. GNPs with a diameter of 40 nm were obtained. Functionalized with Targeting Molecules. Before the conjugation of PEG, 50 mL of GNP stock solution was concentrated to 10 mL by centrifugation. Then, thiolated and FA-terminated PEG or thiolated methoxy-terminated PEG were mixed and stirred at 60 °C for 1 h. After that, PEG-conjugated GNPs were purified by ultrafiltration (MWCO: 10 kDa) and washed with water three times. The supernatant was kept and the absorbance at 280 nm was measured to calculate amounts of unbound PEG. The PEG grafting density was obtained by subtracting unbound PEG from the total used PEG in the reactions. Characterization of GNPs. The size and shape of as-synthesized GNPs were characterized employing TEM (JEOL, 1200 EX, operating at 80 kV). The hydrodynamic diameters and zeta potentials of PEG-conjugated GNPs were measured using dynamic light scattering (DLS) (Malvern Nano Z Zetasizer, Malvern Instruments Ltd.) in water. Characterization of Bound Proteins with SDS-PAGE. Each PEG-conjugated GNP (a total surface area of 50 cm2) was dispersed into Dulbecco's Modified Eagle’s Medium (DMEM) with 10% human serum (500 μL). After incubation for 2 h, particles absored proteins were collected by centrifugation (20000 g for 1 h at 4 °C) and washed several times with PBS to remove unbound and loose bound proteins. The bound proteins on GNPs were detached using cleavage solution (PBS with LDS loading buffer and mercaptoethanol) at 70 °C for 10 min. After that, the mixture was centrifuged, the supernatant containing isolated proteins was loaded to SDS-PAGE and stained with silver. Dynamic Protein Exchange. GNPs were incubated with 1 mg/mL BSA-FITC solution at 37 °C for 1 h. Then, the solution was centrifuged at 20000 g (4 °C, 1 h). The pellets were


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redispersed with 1 mg/mL BSA solution. The fluorescence intensity of the solution was determined with a fluorospectrophotometer at different time points. Quantitation of Cell Uptake of TGNPs and NTGNPs. HeLa and A549 cells were cultured in complete medium (DMEM with 10% fetal bovine serum (FBS) ). In a 12-well plate, cells were seeded with the density of 150,000 cells per well. After growing 24 h, each well was washed once with PBS, and TGNPs or NTGNPs in complete medium (DEME with 10% human serum) (25 µg/mL) or serum-free DMEM were added to the well. After 4 h incubation, each well was washed three times with PBS and detached by trypsin solution (0.25 %). Cells in each well were counted and digested with

aqua regia overnight for ICP-MS measurements. Each

experiment was done three times. Gold Concentration Measurements. The gold content of each cell was measured by ICPMS. The procedure was same as reported in our previous work.24 TEM Images of Cells. HeLa cells were seeded in a 6-well plate and incubated with TGNPs for 4 h at 25 µg/mL in complete DMEM or serum-free DMEM and washed once with PBS. After that, cells were fixed with glutaraldehyde (2.5%) for 30 min at room temperature and then washed twice with PBS. The cells were gathered, sectioned, and observed with TEM (JEOL 1200) Cytotoxicity Measurements. The XTT assay was employed to evaluate the cell viability after incubation with TGNPs or NTGNPs. After cells were seeded for 24 h in a 96-well plate, complete DMEM containing TGNPs or NTGNPs (50 µg/mL) was added. Cells were cultured for 24 h. Then, XTT solution (50 μL, 1 mg/mL) and medium (100 μL) were added to each well and cultured at 37 °C for 4 h. The absorbance of each well was determined at 480 nm and 650 nm. The cell viabilities were calculated following the standard procedures.


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Models and Simulation Methods. In this work, the N-varied dissipative particle dynamics (DPD) was performed to investigate the mechanism for the endocytosis of protein adsorbed nanoparticle. The framework of the N-varied DPD method uses beads i and j that interact with each other via a pairwise additive force consisting of a conservative force FijC , a dissipative force FijD , and a random force FijR . Thus, the total force exerted on bead i can be expressed as Fi   (FijC  FijD  FijR ) (Detail methods are in the Supporting Information). In our simulation i j

system, the lipid molecule53 has a hydrophilic headgroup (denoted as H) and two hydrophobic tails (denoted as T). The model of the lipid membrane can show typical phase behaviors.54,55 Besides, 1/4 of lipid molecules in the membrane were set as receptors (R). The nanoparticles (N) were modeled as sphere coated with ligands (L).56 The coarse-grained proteins (P) were represented by chain models to simplify the question. Solvent molecules (W) were modeled as single beads.

ASSOCIATED CONTENT Supporting Information. Calibration curve used to calculate the concentration of free PEG and cytotoxicity of nanoparticles were included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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E-mail: [email protected] (B. Yan); [email protected] (H. Zhou) Author Contributions #

Gaoxing Su and Xiaofei Zhou contributed equally to this work. All authors have given approval

to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (2016YFA0203103), the National Natural Science Foundation of China (21137002, 91543204, 21405084 and 91334203), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030401). REFERENCES (1)

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Figure 1. (A) Synthesis of TGNPs and NGNPs. (B) TEM images of gold nanoparticles. (a) 5 nm; (b) 15 nm; (c) 40 nm.


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Table 1. Characterization of targeting and non-targeting GNPs. c)

Diameters

a)

b) DH

PDI (in water)

(nm)

(in water) (nm)

Zeta Potential (in water) (mV)

DH

PDI (in Complete Medium)

(in Complete Medium)

(nm)

Zeta Potential (in Complete Medium)

Ligand Density -2 (nm )

(mV)

NGNP-5

5±2

15±3

0.28±0.02

-8.6±1.7

52±10

0.26±0.05

-12.4±0.9

1.06

d)

NGNP-15

15±2

25±5

0.26±0.02

10.9±2.2

86±15

0.28±0.06

-13.6±1.2

1.06

d)

NGNP-40

40±5

50±4

0.27±0.02

13.4±0.5

110±17

0.25±0.01

-15.9±1.0

1.06

d)

TGNP-5

5±2

16±4

0.23±0.03

27.6±2.6

57±11

0.21±0.03

-18.3±1.3

1.08

TGNP-15

15±2

30±4

0.20±0.04

20.7±0.3

89±12

0.22±0.02

-23.2±2.8

0.97

TGNP-40

40±5

52±5

0.20±0.01

26.3±5.4

124±13

0.24±0.02

-15.7±0.8

1.12

a)

Obtained from the statistics analysis of 50 GNPs in TEM images; b)Hydrodynamic diameters of

GNPs dispersed in water measured by dynamic light scattering (DLS);

c)

Hydrodynamic

diameters of GNPs dispersed in cell culture medium with 10% human serum measured by DLS; d)

assume that PEG densities are the same between targeting GNPs and non-targeting GNPs. The

values were the average PEG densities of TGNP-5, TGNP-15 and TGNP-40


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Figure 2. Protein adsorption on NGNPs and TGNPs. (A) Tight-binding proteins on NGNPs and TGNPs separated by SDS-PAGE and stained with silver. (B) Amounts of adsorbed proteins on GNPs were determined by the bicinchoninic acid (BCA) assay.


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Figure 3. Dynamic protein exchange on NGNPs and TGNPs. (A) Fluorescence spectra of the mixture containing BSA-FITC bound TGNP-40 and free non-fluorescent BSA, which were recorded at 0, 1, 2, 4, 6, 12 h since they were mixed. (B) Time course of fluorescence intensity of dynamic protein exchanges for TGNP-40 and NGNP-40. (C) Schematic illustration the dynamic protein adsorption on the surface of GNPs.


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Figure 4. Specific binding of targeting nanoparticles in protein-rich media. (A-F) TEM images of cells after incubation with TGNP-5 (A, D), TGNP-15(B, E) or TGNP-40 (C, F) in complete DMEM (A-C) or serum-free DMEM (D-F) at 25 µg/mL for 4 hrs. (G) Cellular uptake of


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targeting and non-targeting nanoparticles (25 μg/mL) with diameters of 5 nm, 15 nm, 40 nm in complete cell culture medium for 4 hrs. (H) Scheme to illustrate how the specific targeting occurs in the presence of protein corona.


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Figure 5. Effects of adsorbed proteins on the small TGNPs. (A) Cellular internalization of TGNP-5 was analyzed in serum-free cell culture medium or complete cell culture medium at GNP doses of 10, 25, and 50 μg/mL for 4 hrs. (B) Competitive cell uptake of targeting


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nanoparticles with free folic acids in serum-free and -complete cell culture medium of GNPs with diameters of 5 nm. The cells were pre-treated with folic acid with different concentrations (0, 30, 60, and 120 μg/mL) for 1 hrs and then incubated with TGNP-5 for 4 hrs. TGNP-5 dose was 25 μg/mL. (C) The amount of bound proteins decreases with a serum concentration decreasing. Bound proteins were separated by SDS-PAGE and stained with silver. (D) Cell uptake of TGNP-5 in culture media with 0%, 0.5%, 1%, 2%, 5%, 10% human serum at 25μg/mL for 4 hrs. (E) Illustration the one-on-one bindings between a small TGNP and a cell and the interactions between a protein bound small TGNP and a cell.


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Figure 6. Simulation of Cell uptake of protein bound small nanoparticles. After Protein binding, small nanoparticles (diameter: 4.5 nm) fail to enter the cells. In the snapshot, the lipid head is shown in green, lipid tail in yellow, receptor head in pink, receptor tail in orange, protein in pink, nanoparticle in purple, ligand in white. Water molecules are not shown in the snapshots for clarity.


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Figure 7. Effects of adsorbed proteins on the targeting of larger GNPs. (A) Cellular internalization of TGNP-40 in serum-free cell culture medium or complete cell culture medium at GNP doses of 10, 25, and 50 μg/mL for 4 hrs. (B) Competitive cell uptake of targeting nanoparticles with free folic acids in serum-free and -complete cell culture medium of TGNP-40. The cells were pre-treated with folic acid with different concentrations (0 and 120 μg/mL) for 1 hrs. GNP dose was 25 μg/mL. (C) Cartoons showing the multivalent binding between a large TGNP and a cell.


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Figure 8. Cell uptake was enhanced by protein recognition of the adsorbed proteins on GNPs with a diameter of 40 nm. (A) The adsorbed proteins enhanced cell uptake of non-targeting nanoparticles in HeLa cells, 25 μg/mL, 4 hrs. (B) Targeting nanoparticle were uptaken by FAnegative A549 cells in Serum-free DMEM and complete DMEM.


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Figure 9. Simulation of Cell uptake of protein bound large particles. Protein binding cannot prevent cell uptake of large nanoparticles (diameter: 9.7 nm).


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Cell Uptake (Х107 nm2/Cell)

Page 35 of 36

A)

40

60 Complete Serum-free

B)

50

30

40

20

30 20

10 0

10 0

TGNP-5

TGNP-15 TGNP-40

10

25 Dose (µg/mL)

50

Figure 10. (A) Cell uptake of TGNP-5, TGNP-15, and TGNP-40 at 25 μg/mL in serum-free cell culture medium or complete cell culture medium for 4 hrs. (B) Cellular internalization of TGNP15 in serum-free cell culture medium or complete cell culture medium at TGNP-15 doses of 10, 25, and 50 μg/mL for 4 hrs.


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Table of Content (TOC)


36

Size-Dependent Facilitation of Cancer Cell Targeting by Proteins

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