Accepted Manuscript Increased cellular uptake of peptide-modified PEGylated gold nanoparticles Bo He, Yuan Zhang, Qiang Zhang, Bing He, Wenbing Dai, Xueqing Wang, Dan Yang, Mengmeng Qin, Hua Zhang, Changcheng Yin PII:

S0006-291X(17)31988-5

DOI:

10.1016/j.bbrc.2017.10.026

Reference:

YBBRC 38643

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 26 September 2017 Accepted Date: 5 October 2017

Please cite this article as: B. He, Y. Zhang, Q. Zhang, B. He, W. Dai, X. Wang, D. Yang, M. Qin, H. Zhang, C. Yin, Increased cellular uptake of peptide-modified PEGylated gold nanoparticles, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Increased cellular uptake of peptide-modified PEGylated gold nanoparticles Bo Hea, Yuan Zhangb, Qiang Zhangc,d, Bing Hec,d, Wenbing Daic,d,

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Xueqing Wangc,d, Dan Yangc,d, Mengmeng Qinc,d, Hua Zhangc,d,

a

Department of Biophysics, School of Basic Medical Sciences, Peking University

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Health Science Center, Beijing, China b

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Changcheng Yina

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,

University of Rhode Island, Kingston, RI, USA c

Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China

d

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing,

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China

Corresponding author:

Hua Zhang, State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China. Laboratory of Natural and Biomimetic Drugs, Peking University,

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Beijing, China. E-mail address: [email protected]

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Changcheng Yin, Department of Biophysics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. E-mail address: [email protected]

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Abstract: Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface.

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However, the instability and low bioavailability of gold nanoparticles compromise clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their

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stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible

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spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively

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coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon

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resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of

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gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles.

ACCEPTED MANUSCRIPT Keywords: Gold nanoparticles; cell uptake; CPP; SPR; TEM

Introduction

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Functionalized gold nanoparticles（GNPs） have broad application in various fields, due to their excellent biocompatibility and versatile surface modification

capability. GNPs have been widely used as sensors for different chemically and

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biologically important molecules [1] and drug delivery carriers [2,3]. Unmodified

GNPs are unstable and will gradually undergo aggregation in biological medium. To

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improve stability, protective agents were used to coat GNPs through covalent or non-covalent conjugation, such as peptides, protein, and polymers [4-6]. Polymers are most frequently used to stabilize GNPs by acting as a scaffold and preventing them

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from aggregation. However, polymers coated on GNPs prevent their interaction with cell membrane and lower their bioavailability. As cell-penetrating peptides (CPPs) have been used for improved intracellular delivery, we hypothesize that cell

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penetrating peptide (CPP) and PEG modification on GNPs could greatly enhance their

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cellular uptake and biostability in physiological conditions. CPP, also known as protein transduction domain (PTD), is a short amino acid

peptide fragment that can direct nanoparticles, macromolecules or virions to pass through the cell membrane for sensing and drug delivery applications [7,8]. The most well-known CPP was HIV-1 TAT derived peptide (YGRKKRRQRRR). CPP derived from Drosophila Antennapedia homodomain (RQIKWFQNRRMKWKK) has a similar function [9-11]. Many peptide fragments with similar characteristics to

ACCEPTED MANUSCRIPT penetratin were also found, such as transpartan (GWTLNSAGYLLGKINLKALAALAKKIL), polyarginine peptide, and polylysine peptide of different lengths [12,13]. In addition to cationic CPPs that have been

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widely investigated, other types of CPPs also enhance cellular uptake. Recently, hydrophobic CPPs, including Kaposi’s fibroblast growth factor

(AAVALLPAVLLALLAP) [14], Pep-7(SLWEMMMVSLACQY) [15], FGF

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(PIEVCMYREP) [16] and β3(VTVLALGALAGVGVG) [17] have been of great

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interest. Our lab has investigated many highly effective CPP-conjugated drug delivery systems. We found that compared with unmodified liposome, PFVYLI (PFV), a hydrophobic CPP modified liposome could significantly enhance the intracellular delivery of DOX [8]. The intracellular deliver of a pro-apoptotic peptide (PAD) could

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also be enhanced by conjugating it with a cyclic peptide cyclosporin A (CsA) [18]. As a proof of concept, in this work we choose a cationic CPP CALRRRRRRRR (R8), and a hydrophobic CPP CALNNPFVYLI (PFV), to enhance cellular uptake of

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GNPs. Both peptides have a cysteine residue which can bind to GNPs by forming

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Au-S bond. Arginine-rich CPPs have attracted much attention as a promising carrier for intracellular delivery of therapeutic molecules [19]. It is believed that cationic CPPs, such as octa-argine (R8) and Tat peptides, bind to membrane-associated fusion proteins followed by different manners of endocytosis, such as clathrin-mediate endocytosis [20], micropinocytosis [21] and caveolae-mediated endocytosis [22]. CPPs-containing cargoes could enter cells not only by endocytosis, but also by direct cell penetration through the plasma membrane [23]. The efficient intracellular drug

ACCEPTED MANUSCRIPT transport to cytosol by direct penetration or endosome escape is important for drug delivery application. PFVYLI peptide, derived from Kaposi Fibroblast Growth Factor, is considered as an extremely hydrophobic CPP without positive charge [24]. The

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internalization of PFV-cargoes is either through an endocytosis pathway or directly across the plasma membrane [8]. It is known that the internalization patterns of

CPP-cargoes can be strongly affected by variable factors, such as the physicochemical

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nature of peptides, cell types, and the cargoes to be delivered. To the best of our

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knowledge, there is no report on utilizing PFV or R8 to enhance the cell uptake of GNPs. We studied two representative CPPs with different properties and investigated the membrane interaction mechanisms and cell uptake pathways of CPP -modified GNPs.

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The mechanism of energy-dependent endocytosis has been widely studied in nanoparticles with size ranging from 10 to 100nm [25-27]. It is recently found that GNPs coating with multiple ligands, with the size of 2-20nm, were capable of fusion

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and penetration through lipid bilayers without membrane disruption [28]. We choose

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to investigate the GNPs with core size of 6.1 nm, due to the fact that they have small particle size, high surface area, efficient escape from RES system [29], and efficient cellular uptake and potential biomedical applications of energy-independent pathway [30].

In our study, we selected A549 and B16 cell lines as the cancer cell models. CPP-modified GNPs were taken up by B16 and A549 cells more significantly than that of PEG. The inhibitor experiment indicated that the majority of CPP-modified

ACCEPTED MANUSCRIPT GNPs were taken up by cells in clathrin-mediated endocytosis and macropinocytosis pathways. Transmission electron microscopy (TEM) further confirmed the cellular uptake data. To elucidate the mechanism of interaction between CPP-modified

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nanoparticles and the cell membrane, liposomes composing of DOPC (1, 2-dioleoyl-sn-glycero-3-phosphocholine) were used as a surrogate of the cell

membrane and incubated with nanoparticles. The interaction between liposomes and

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nanoparticles was observed under TEM. In addition, the interactions between

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phospholipids and nanoparticles were measured by Surface plasmon resonance (SPR)

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analysis assay.

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Materials and methods Experimental materials

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Peptides CALNNPFVYLI and CALRRRRRRRR were purchased from Hong Jiang Biotechnology Ltd. (Jiangsu, China). DOPC was purchased from Avanti Polar lipids (Alabaster, USA). GNPs were synthesized in-house. All other chemicals were

purchased from Sigma-Aldrich (St. Louis, MO.), and all solvents were reagent grade.

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Cell culture reagents were purchased from Macgene Biotech Co. Ltd, (Beijing,

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China).

Synthesis of citrate-coated GNP

GNPs (6.1± 1.1 nm) were prepared by citrate and tannins reduction of HAuCl4 according to a published method [31,32]. An aqueous solution of HAuCl4 (80 mL,

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0.32 mM) was refluxed for 5-10 min, and a pre-warmed (50-60°C) citrate solution (10 mL, 20.8 mM) and tannins solution (10 mL, 0.59 mM) were added immediately. The

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reflux was continued for another 30 min until a dark red solution was obtained. GNP was stored at room temperature until use.

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Functionalization of GNPs

ACCEPTED MANUSCRIPT PEGylated GNPs were prepared by mixing citrate-coated GNP (100 mL, 50 µg/mL) with thiollated-PEG2000 (2 mL, 1 mg/mL). Peptides and PEG2000 coated GNPs were prepared by mixing citrate-coated GNP (100 mL, 50 µg/mL) with thiolated-PEG2000 (2 mL, 1 mg/mL) for 1 h before adding 2 mg peptides to the

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mixtures. The reaction continued for at least 7 days, and unreacted molecules were removed via ultrafiltration (Millipore, 15 mL, 100 kD). GNP coated with

thiollated-PEG2000 is GNP@PEG, GNP coated with thillated-PEG2000 and PFV

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peptides is GNP@PFV, GNP coated with thillated-PEG2000 and R8 peptides is

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GNP@R8.

UV-visible spectrometry

UV-vis absorption spectra were recorded at room temperature (400-800 nm) in

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500 µL disposable polystyrene micro cuvettes using a Thermo Electron Corporation Evolution 500 spectrometer (Beckman, CA,).

TEM micrograph

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Particle images were acquired using a F20 TEM operated at 200 KV. To prepare

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TEM samples, 7 µL particle solution was dropped on a glow-discharged continuous carbon film coated 200-mesh grid (Quantifoil Micro Tools GmbH, Jena, Germany) for 2 min before draining the mesh grid with absorbent paper. Hydrodynamic size and zeta potential were measured by dynamic light scattering (DLS) using a Malvern Zetasizer nano ZS (Malvern, UK), and each sample was measured in triplicate.

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Cell culture B16 (mouse melanoma) and A549 (human lung adenocarcinoma) cell lines were provided by the Institute of Basic Medical Science, Chinese Academy of Medical

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Science (Beijing, China). Both cell lines were cultured in RPMI-1640 (Macgene,

Beijing, China) medium supplemented with 10% FCS (Gibco), 100 U/mL penicillin

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and 100 µg/mL streptomycin (Macgene, Beijing, China). Both cell lines were

Cellular uptake

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maintained at 37°C in a 5% CO2 humidified incubator.

B16 and A549 cells were incubated with 200 nM GNPs solution (0.5 mL of solution of 400 nM GNPs added to 0.5 mL of medium) for 10, 30, 60, and 720 min,

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and washed 5 times with PBS buffer. Samples were digested with aqua regia, and gold was measured using ICP-AES. The protein amount in cells was measured with a

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Bicinchoninic Acid Kit (Sigma).

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Liposome synthesis First, 5 mg of DOPC were dissolved in 10 mL of chloroform in a round-bottomed

flask and dried under a vacuum using a rotary evaporator. Then, 1 mL PBS was added to the flask and the flask was vortexed for ~1 min. The suspension was transferred to a cryogen vial and frozen with liquid nitrogen. This freeze-thaw cycle was repeated 5 times, followed by extrusion through a polycarbonate filter with a pore size of 100 nm (Avestin, Germany) to obtain 100 nm unilamellar vesicles.

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Surface plasmon resonance (SPR) analysis To demonstrate the interactions between CPP-functionalized GNPs and liposome composed of DOPC as well as to determine the mechanism of interaction, an SPR

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assay was performed at 25°C using a Biacore 3000 instrument (GE) with L1 chip and

Cryo- Electron Microscopy

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HPA chip.

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Liposomes (4 mg/mL) were incubated with GNPs (400 nM) at 37°C for 4 h. A 2µl aliquot of the above-prepared samples was applied on a glow-discharged continuous carbon film coated on a 200-mesh R1.2/1.3 Quantifoil holy grid (Quantifoil Micro Tools GmbH, Jena, Germany). Specimens were prepared by Vitro

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Blot (FEI, Netherlands) with a blot time of 3 s, a blot force of 0 and a wait time of 30 s. Images were obtained using F30 TEM (FEI) at 120 KV.

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Results and discussion Preparation and characterization of GNPs

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The GNPs were synthesized by the reduction of gold salt with citrate and tannins, and further functionalized by thiol-PEG and CPPs. The GNPs were spherical in shape and monodisperse, as shown in the TEM micrograph (Figure 1A). We measured at least 500 GNPs in TEM micrographs and the average core size of GNPs was calculated to be 6.1±1.1nm((Figure 1C). UV-spectra of these GNPs present maximum absorption peak at 515nm for GNP@PEG, 520nm for GNP@PFV and 525nm for GNP@R8 (Figure 1B). The plasmon resonance showed a bathochromic shift of

ACCEPTED MANUSCRIPT CPP-functionalized NP. Since the plasmon resonance affected by the interactions on the gold surface (e.g., adsorption of polymers or protein), the shift of GNPs absorption peak indicated the modification of CPPs on GNPs. To further confirm

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ligands have conjugated to GNPs, XPS was used to analyze element content in three types of nanoparticles (Figure 1D). GNP@PFV and GNP@R8 contained 1.1% and

2.4% of nitrogen atom respectively, indicating that nanoparticles were functionalized

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by peptides. Nanoparticles could be stably stored at 4°C for more than 1 month

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without aggregation.

Cellular uptake of functionalized GNP Nanoparticles were incubated with A549 and B16 cell lines for 10min, 30min, 1h and 12h at 37℃, ICP-AES was used to measure the cellular uptake of GNPs and the

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total protein in cells was measured by a Bicinchoninic Acid Kit (Figure 2A). Our result showed that GNP@PEG can be hardly taken up by cells, and both CPPs significantly increased the endocytosis of GNPs. PEG may prevent GNPs interacting

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with cell membrane, and both CPPs increased the adherence of GNPs to the cell

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membrane, thus promoting their intracellular delivery. However, the endocytic pathways of two CPP modified GNPs were different. Uptake of GNP@PFV was rapid during the initial hour and declined over time before reaching a plateau. GNP@R8 uptake was slower than that of GNP@PFV in the first hour but the total cellular uptake was greater. The result indicated that GNP@PFV and GNP@R8 shared different endocytic pathways.

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Investigation of endocytosis pathway of GNPs Low incubation temperature or a metabolic inhibitor that can deplete ATP decreased energy-dependent endocytosis. As shown in Figure 2B, GNPs incubate

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with cells for 1h at 4°C and 37°C, GNP@R8 was endocytosed in both cell types and

energy-independent endocytosis of GNP@PFV was not obvious. TEM confirmed that

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GNP@R8 entered the cytoplasm through direct transmembrane mechanism (Figure 2 C, D, E). It is reported that R8 could bind with cell membranes, and gradually

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aggregate and diffuse into cells. The internalization of R8 is affected by the membrane and CPP concentration. At low concentration, the cellular uptake of R8 depended on endocytosis; while, at high concentration, a direct transmembrane effect occurred [33,34]. However, this R8 transmembrane concentration threshold varies in

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different cell lines and nanoparticles.

Cellular uptake data at 4°C indicated that nanoparticles entered cells via

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energy-dependent endocytosis. Inhibitors (chlorpromazine, filipin, genistein, cytochalasin D, 5-N-ethyl-N-isopropylamiloride [EIPA], and dynasore) were used to

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verify endocytosis pathways (Figure 3A). Chlorpromazine resulted in a 60% decrease in the cellular uptake of GNP@PFV and a 62% decrease in the cellular uptake of GNP@R8 comparing to the control. As chlorpromazine could inhibit clathrin-mediated endocytosis by removing clathrin and AP2 adaptor proteins on the cell membrane, both CPP modified GNPs were taken up through clathrin-mediated endocytosis. In addition, EIPA can inhibit macropinocytosis by the blockage of sodium-proton exchange [35]. The addition of EIPA resulted in a 56% decrease in the

ACCEPTED MANUSCRIPT cellular uptake of GNP@PFV and a 72% decrease in the cellular uptake of GNP@R8, suggesting that micropinocytosis plays a more important role in the cellular uptake of GNP@PFV than that of GNP@R8. After the addition of cytochalasin D, the

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endocytosis of GNP@PFV and GNP@R8 were decreased to 64% and 69%, respectively. Thus, both CPPs-modified GNPs enter cells mainly through clathrin

mediated endocytosis and micropinocytosis. Macropinocytosis was the major uptake

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pathway for GNP@PFV and GNP@R8 enter cell mainly through clathrin mediated

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endocytosis. TEM data showed that endocytosed vesicles were 100-1,000 nm (Figure 3B), suggesting that uptake of CPP-modified GNP was mainly through CvME-mediated endocytosis and macropinocytosis.

Interaction between membrane and GNP

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Both energy-dependent endocytosis and energy-independent endocytosis require the binding of nanoparticles onto the surface of cell membrane as the first step. To elucidate the mechanism of interaction between CPP-modified nanoparticles and the

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cell membrane, liposome was used as an alternative for the cell membrane and

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nanoparticles were added to the liposome solution. Cationic CPP binding to negatively charged cell membrane through the electrostatic interaction has been confirmed by many researches [13,33]. However, some studies found that negatively charged phospholipid at high concentration could decrease the amount of CPP inserted into the lipid bilayer [36], indicating the electrostatic interaction may not be the only way leading to the interaction between cationic CPPs and the lipid membrane. Although the zeta potential of GNP@R8 was higher than that of GNP@PFV, the

ACCEPTED MANUSCRIPT value was still negative. In order to investigate the interaction between CPPs and lipid material, a neutral phospholipid DOPC, was used to make liposomes of 100 nm in diameter. GNPs (200 nM) were incubated with liposomes for 4 h, and negative

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staining samples were prepared for TEM imaging to observe the interaction between liposome and GNP (Figure 4A). The TEM images showed that GNP@PEG did not interact with plasma membrane, and was homogenously distributed in the

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microscopic field. A lot of GNP@PFV and GNP@R8 were adherent to the surface of

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liposome, and damage of the membrane could be observed. In order to study their interaction in water, we used cryo-electron microscopy to observe the undehydrated liposome and GNP (Figure 4B). The cryo-electron images illustrated that GNP@PEG was homogenously distributed in the solution, and there was significant steric

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hindrance between liposome and GNP@PEG. The average distance between GNP@PEG and liposome was 6.5 nm, which was consistent with the hydration layer outside GNP@PEG. GNP@PFV and GNP@R8 mainly located around the liposome,

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and tightly adhered to the surface of liposome. This result suggested that CPP on the

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GNP had already inserted into the lipid bilayer, helping GNP to bind to the surface of the lipid bilayer. Thus, in addition to the potential interaction between CPP and the cell membrane, the outer phospholipid also played a very important role in the interaction.

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SPR analysis L1 chips were used to analyze interactions between lipid bilayer and GNPs (Figure 4C). The L1 chip data showed that GNP@PFV could bind with liposomes

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effectively and this may be because GNP@PFV on the liposome surface may expose the hydrophobic lipid tail and nanoparticles could bind with the exposed hydrophobic

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site. GNP@PFV bound with the liposome did not dissociate after washing with PBS, suggesting a strong hydrophobic binding capacity of GNP@PFV. GNP@R8’s affinity

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for liposomes was greater than that of GNP@PFV at low concentrations, and as concentration increased, more nanoparticles come off from the liposome. About 20-30% of chip nanoparticles were washed away with PBS. Molecular simulation showed that positively charged R8 peptide repulses positively charged choline groups

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and ions and attracts negatively charged phosphate groups. R8 peptides are adsorbed at the bottom of lipid head groups and not on the lipid surface [37]. 60-90% of the

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GNP@PEG could be washed away due to the weak binding. A lipid monolayer was applied to the HPA chip and all nanoparticles tested had a similar binding curve,

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suggesting that the interaction between CPP-modified nanoparticles and lipid materials was dependent on the lipid bilayer of membrane..

Conflict and interest statement None of the authors of this manuscript had financial or personal relationships with other people or organizations that could inappropriately influence the work presented. None of the funding sources for this study played any role in study design, in the collection, analysis and interpretation of data; in the writing of the manuscript;

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Acknowledgments This work was supported by the National Natural Science Foundation of China

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(No. 81690264 and No.31570732) and the National Basic Research Program of China (973 program, 2015CB932100).

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Figure legends: Figure 1: Characterization of GNPs. (A) TEM images for A1 (GNP@PEG)，A2(GNP@PFV) and A3(GNP@R8) (B) UV-spectra peaks of GNPs. (C) Core size distribution of GNPs in TEM images. (D)

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Elemental analysis of GNP@PFV and GNP@R8 (carbon and nitrogen) determined by XPS. Figure 2 Cellular uptake of GNPs by A549, uptake of nanoparticles was evaluated by ICP-AES and

TEM. (A)Cellular uptake of GNPs by (A1) A549 cell line and (A2) B16 cell line with time. (B) Uptake

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of GNPs@PFV and GNP@R8 in A549 cell line at 4°C. (C) The distribution of GNP@R8 in cytoplasm

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of A549 cell. (D) Aggregation of GNP@R8 on A549 cell membrane. (E) Direct translocation of GNP@R8 on A549 cell. Insets are magnified on the right of each image highlighted with respective red box.

Figure 3 Effect of various endocytosis inhibitor treatments on the uptake of GNPs and TEM images of

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GNPs take up by energy dependent pathway.(A) Cellular uptake of GNP@PFV and GNP@R8 in A549 cell line (n=3, * p