HIV Peptide-Mediated Binding Behaviors of Nanoparticles on a Lipid

Subscriber access provided by University Libraries, University of Memphis

Article

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on Lipid Membrane Minh Dinh Phan, Heesuk Kim, Songyi Lee, Chung-Jong Yu, Bongjin Moon, and Kwanwoo Shin Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04234 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 12

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

Langmuir

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on Lipid Membrane Minh Dinh Phan,

†

Heesuk Kim,

†

Songyi Lee,

†

Chung-Jong Yu,

and Kwanwoo Shin

‡

Bongjin Moon,

†

∗,†

†Department of Chemistry, Sogang University, Seoul, Korea ‡Beamline Division, Pohang Accelerator Laboratory, Pohang, Korea E-mail: [email protected]

Abstract

state to the closely packed state upon the increase of the charged density of the membrane. The present study also provides insights on the binding mechanisms of the cellpenetrating peptide-coated nanoparticles to the lipid membranes, which is a common theme of delivery systems in pharmaceutical research.

Bioinspired design of ligands for nanoparticle coating with remarkable precision in controlling anisotropic connectivity and with universal binding efficiency to the membrane have made a great impact on nanoparticle self-assembly. We utilize HIV-1 derived trans-activator of transcription peptide (TAT), a member of the cell-penetrating peptides, as a soft shell coating on gold nanoparticles (GNPs), and characterize TAT pepidemediated binding behaviors of GNPs on the lipid membrane. Whereas the peptides enable GNPs to firmly attach to the membrane, the binding structures are driven by two electrostatic forces: the interparticle peptiderepulsion and the peptide-membrane attraction. Although transmission electron microscopic images showed that the densities of membrane-embedded GNPs were almost equal, X-ray reflectivity revealed significant difference in binding structures of GNPs along the surface normal upon the increase of charged densities (φ) of the membrane. Especially, GNPs densely suspended at φ = 70%, while they adopted an additional welldefined layer underneath the membrane at φ = 100%, in addition to a translocation of the initially-bound particles into the membrane. The observed behaviors of GNPs manifest a 3D to 2D transformation of the self-assembled structures from the diffused

Introduction Ensembles of nanoparticles (NPs) can display new electronic, magnetic, and optical properties that are different to those displayed by individual NPs and bulk samples. 1,2 Organizing NPs into microscopically well-defined two- or three-dimensional (2D or 3D) structures has been approached by a variety of methods, in which self-assembly of NPs in solutions 3 and at interfaces 4–8 are the two conventional strategies. Assembled-structures of particles can be vastly varied upon different derivatives coated onto the particles’ surface. For examples, the formation of 3D bodycentred-cubic crystalline assemblies of gold NPs in solution is mediated by interactions between complementary DNA derivative, 3 while 2D hexagonal closed packed assemblies at air-water interfaces are induced by the repulsive forces between the negatively charged non-complementary DNA molecules attached to the NPs’ surface. 4 Therefore, the

ACS Paragon Plus Environment

1

Langmuir

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

elaborate design of ligands is a prerequisite for directing self-assembly of NPs. Especially, ligand-mediated interactions can be dominant in the case of soft-corona coated NPs with the thickness of the corona layer comparable to the size of the particle core because the dominant interparticle ligand-ligand interactions could outweigh the van der Waals interactions between the NP cores. 9 Bioinspired ligand designs have attracted a great deal of attention because of their remarkable precision in controlling anisotropic connectivity (for example, artificial DNA) 3,10 and their universal efficiency in membrane binding (for example, viruses and multimeric proteins). 11–13 Among the most frequently used cell-penetrating peptides (CPPs) for drug delivery is human immunodeficiency virus type 1 (HIV-1) derived trans-activator of transcription (TAT) peptide. In this report, we investigate the TAT-mediated binding behaviors of gold NPs (GNPs) on the biological membrane upon various densities of negatively charged lipids. TAT molecules impart highly positive charge on the surface of GNP due to a large portion of lysine (K) and arginine (R) residues in TAT’s sequence (YGRKKRRQRRRGGGC). Langmuir monolayer of phospholipids is used as a model biological membrane, to which TAT-capped GNPs are expected to bind strongly. 14 Besides the fundamental and technological aspects of NP self-assembly at the lipid interface, understanding NP-membrane interaction is crucial in solving problem in nano-toxicology. 15 In addition, since CPPs are known for their capability of entering the body in a noninvasive manner, 12,13 incorporating TATs, a member of CPP family, into versatile cargo-carrying platforms leads to creation of novel drug-delivery systems that ensures improved coated-drug uptake, as well as their targeted recognition and controlled release via stimulus-response. 16 TAT peptides are used to cap 10% of soft corona composed of 11-mercaptoundecylpenta(ethylene glycol) disulfide and maleimideterminated 11-mercaptoundecyl-hexa(ethylene glycol) disulfide on GNPs’ surface (see

Scheme S1). Since the length of ligands, ∼ 26 − 49 Å in respect of without and with capped-TAT, is comparable to the core radius of the GNP, ∼ 34 ± 5 Å (see Figure S1), one can speculate that the macroscopic structures of GNPs are governed by two electrostatic interactions: interparticle ligand-ligand repulsion, and attraction between the positively charged TAT-capped ligands and negatively charged lipids on membrane. The membrane monolayer is composed of zwitterionic phosphocholine 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), and phosphoserine with negatively charged headgroup 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS). The overall negatively charged density of membrane can be varied by changing the DPPS’s fraction (φ), which has been performed in this study at four different compositions, ranging from 0% to 100%. Experimentally, the adsorption isotherms and transmission electron microscopic (TEM) images showed that the initial binding amounts of GNPs on lipid monolayers were almost equal regardless of φ. X-ray reflectivity (XR) results, however, revealed the difference in macroscopic binding structures of NPs along the surface normal. Especially, from φ = 70% to 100%, the binding structure transformed from a highly diffuse within a confined volume to a well-defined compact layer.

Experimental Section Materials Hydrogen tetrachloroaurate (III) hydrate (HAuCl4 ·3H2 O, 99.999%), tetraoctylammonium bromide (TOAB, 98%), sodium borohydride (NaBH4 , 99%), 4-(N,N-dimethylamino) pyridine (DMAP, 99%), anhydrous sodium sulfate (Na2 SO4 , ≥99.0%), methylene chloride (CH2 Cl2 , ≥99.8%), chloroform (CHCl3 , ≥99.8%) were purchased from Aldrich and used without further purification. Toluene (C6 H5 CH3 , 99.5%), sulfuric acid (H2 SO4 , 95.0%), ethanol (CH3 CH2 OH,


2

Page 2 of 12

Page 3 of 12

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

Langmuir

99.9%), methanol (CH3 OH, 99.5%), sodium hydroxide (NaOH, 99.9%) and acetic acid (CH3 COOH, 99.0%) were purchased from Jin Chemical Co., Ltd (Kyunggi-do, South Korea) and used without further purification. The maleimide-terminated linker (linker 1) and penta(etlyene glycol)-terminated linker (linker 11) were synthesized by a previously reported method. 17 TAT peptides (YGRKKRRQRRRGGGC) were purchased from AnyGen (Gwangju, South Korea). 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dipalmitoylsn-glycero-3-phosphoserine (DPPS) were purchased from Avanti Polar Lipids and their stock solutions at the concentration of 0.5 mg/ml were prepared in chloroform and chloroform-methanol co-solvent (4:1 v/v), respectively. Deionized water (DI water) was obtained at 18.2 mΩ·m by using an ultrafiltration system (PURELAB Classic, ELGALabWater).

added to 10 ml of TOAB-GNP solution. The gold nanoparticles were immediately transferred to water phase including DMAP from toluene phase. The mixture was left at room temperature without stirring or shaking for 1 hour. Finally, aqueous DMAP-GNP solution was isolated from the mixture and stored at 4 ◦ C.

Synthesis of TAT-Conjugated Gold Nanoparticles (TAT-GNPs) TAT modified GNPs were prepared by twophase method via ligand exchange (see Scheme S1). Firstly, gold nanoparticles were synthesis with TOAB as stabilizing agent in toluene phase. When DMAP solution of 0.1 M was added into TOAB stabilized GNP solution, DMAP molecules were moved from water phase to water/toluene interface. At this concentration of DMAP, they are present mostly in a form of free base. Then, DMAP molecules were strongly bound on gold surface and exchanged with TOAB molecules in water phase. 18 Secondly, the thiol solution (maleimide-terminated linker) was added into the DMAP-GNP solution. The linkers were also bounded on gold surface by ligand-exchange process. Then linker-GNPs were precipitated into CH2 Cl2 . All ligandexchange process was carried out in mild condition, without stirring or shaking. Finally, TAT peptides (YGRKKRRQRRRGGGC) solution was added into linker-GNP solution. The terminal thiol groups of TAT peptides were covalently bound to maleimide groups of linkers 1. Thus, TAT peptides were conjugated with linker-GNPs via the stable carbonsulfur bonds. The conjugation of the linkers (linker 1: linker 11 = 1:9 in molar ratio) on the surface of gold nanoparticle was carried out by ligandexchange method. 9 ml of the linker solution (0.45 × 10−3 M in 95% ethanol) was added into 6 ml of aqueous DMAP-GNP solution. Subsequently, 6 ml of CH2 Cl2 was added into this mixture. By the addition of acetic acid (20%), the pH of mixture was adjusted to 5.

Synthesis of DMAP protected gold nanoparticles via two-phase reduction DMAP protected GNPs were synthesized by Gittins and Caruso’s method. 18 First, HAuCl4 ·3H2 O was dissolved in deionized water at concentration of 30 mM. 30 ml of this solution was added to 80 ml of tetraoctylammonium bromide solution (25 mM) in toluene. After vigorous stirring of the mixture, the gold salt was transferred to organic phase from water phase. Subsequently, 25 ml of ice NaBH4 solution (0.4 M) was dropped in the mixture. TOAB stabilized GNPs were immediately formed by the reduction of HAuCl4 . 19 The particle solution was kept stirring for 30 min. Then, the toluene phase including TOAB-GNPs was separated from the mixture and washed with 0.1 M H2 SO4 , 0.1 M NaOH, and deionized water, respectively. The toluene phase was dried over anhydrous Na2 SO4 . Second, TOAB-GNPs were changed to DMAP-GNPs by phase transfer. 10 ml of aqueous DMAP solution (0.1 M) was gently


3

Langmuir

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 functionalities on gold nanoparticle were immediately changed from DMAPs to linkers by place-exchange reaction. In the meantime, the gold nanoparticles were transferred from aqueous phase to organic phase. TAT peptides were conjugated to linker coated GNPs (linker-GNPs) via phase transfer from organic phase to aqueous phase. 1 ml of TAT solution (0.0001M) in DI water was added into 1 ml of linker-GNP solution in CH2 Cl2 . The upper TAT solution was separated from the bottom linker-GNP solution. After gentle shaking of it, the linkerGNPs were moved to aqueous phase including TAT peptides by the conjugation of TAT peptides and linker-GNPs. This solution was kept without stirring for additional 12 hours. Subsequently, the upper solution (TAT-GNPs) was carefully taken from water/organic interface and centrifuged twice at 4 ◦ C with DI water to remove the excess TAT peptides. Finally, the precipitate was re-dispersed in the DI water and stored at 4 ◦ C.

Page 4 of 12

filled to a positive menicus was used to minimize the amount of TAT-GNPs injected, as well as optimize the uniform dispersion and adsorption of particles from the underneath of membranes. Surface pressure was monitored using a Nima Technology surface pressure sensor. Lipids were deposited to a measured surface pressure of ∼25 mN/m and allowed to relax 30 min before X-ray scanning. Subsequently, ∼300 µl TAT-GNP at ∼ 30 mM was injected through a side port on the trough to a final concentration of ∼0.36 mM. The entire system was enclosed in a Plexiglas box with Kapton covered windows for in/out X-ray beams, and was placed on an antivibration table. XR data were refined by using the effective-density model to provide the electron density (ED) profiles perpendicular to the film layer. 20,21 The detailed information on X-ray setup and interpretation are described in literature elsewhere. 22 The quantitative fitting parameters for all XR profiles are summarized (see Supporting Information, Table I-II).

TEM measurements

Results and discussion

TEM images were taken from High Resolution TEM (JEM-2100F, JEOL) to capture the morphology of the nanoparticles and their binding behaviors to the lipid membranes. Several carbon coated copper grids were attached to the silicon wafer which had been dipped inside the bulk water from the beginning. The monolayers were compressed to desired Π which represents for each kinetics section, before being transferred onto the grids by lifting up the wafer as a typical LB deposition at a dipping speed of 1 mm/min.

The synthesis scheme for GNPs and their structures are summarized in Scheme 1, Supporting Information. The presence of linkers

X-ray Reflectivity Measurements XR measurements were performed at a wavelength of λ = 1.542 Å from Cu Kα source using D8 Advance (Bruker AXS, Karlsruhe, Germany) with a vertical goniometer, which allows the liquid’s surface to be studied without disturbing the Langmuir trough during the measurements. A custom mini Teflon trough with a total volume of ∼25 ml when

Figure 1: The dynamic adsorption isotherms of GNPs on different membrane’s compositions, at initial surface pressure of 25 mN/m. and TAT peptides on particles’ surface was


4

Page 5 of 12

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

Langmuir

characterized and confirmed by ultravioletvisible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy (see Figure S2 and S3). The electrostatic properties of particle’s surface were characterized by zeta potential technique (see Figure S4), showing that the charge was switched from negative to positive upon the conjugation of TAT. Figure 1 shows the surface pressure (Π) changes by the adsorption of GNPs on the lipid membranes. Since the aim of study is to characterize the TAT-mediated binding behaviors of GNPs on the membrane, the adsorption isotherms were taken by fixing the trough area, not to allow the membrane area expanding or contracting, and monitoring the change of Π as function of time. The steep increase of Π up to 35 mN/m in the initial binding regime marked in Figure 1 exhibits almost equally in the magnitude and slope for all compositions. After the initial binding processes are saturated, the adsorption isotherms show no significant change for all compositions, except for pure DPPS. Those observations, thus, feature two points: first, the NP adsorption is driven biologically by the binding efficiency of TAT into the films during the initial binding regime; second, the number of GNPs whose TAT-capped linkers incorporated into the lipid membrane is almost equal for φ = 0 − 70%, and is maintained constantly within the course of experiments. Note that TAT peptides themselves can barely cause any increase in Π, 23 but they enable the strong binding of linkers to the membrane and facilitate the interaction between GNPs and the membrane. For φ = 100% Π further increases up to 45 mN/m, indicative of an additional adsorption of NPs due to the exceeding negative charge on the membrane. TEM were used to confirm the presence and visualize the distribution of GNPs binding to membranes (Figure 2). The samples were deposited by the mean of Langmuir Blodgett (LB) technique at the equilibrium states, 35 mN/m for all compositions, plus at 37 and 44 mN/m for pure DPPS layer in accordance with the adsorption isotherms (Figure 1). As a result, the densities of GNPs at the equilib-

rium states at 35 mN/m are of 24.2%, 31.0%, 32.4%, and 33.9% (estimated by Image J program 24 ) for φ = 0%, 30%, 70%, and 100%, respectively. The TEM images of DPPS at fur-

Figure 2: TEM images of GNPs’ distribution on the films at different compositions and surface pressures. The films are transferred on carbon coated copper grids, which were attached on silicon wafer prior to spreading the lipids, by Langmuir Blodgett (LB) technique at desired pressures. Only GNPs physically binding to the films are transferred on the substrate and observed. Scale bars for all subimages are 50 nm. ther increased Πs, 37 and 44 mN/m, clearly display local aggregation or multilayered-like structure of NPs. Next, we employed XR to assess structural details along the surface normal that were not directly provided by the adsorption isotherms and TEM images. There are several questions that are possibly resolved by XR


5

Langmuir

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

Page 6 of 12

Figure 3: XR profiles for φ = 0% - 70% (a) taken at equilibrium states after the initial binding are shown on the adsorption isotherms (Figure 1), and their corresponding ED profiles (b, b’). The ED profiles of sub-layers deconvoluted from the overall ED profiles are presented as dash-dotdot, dash, and dot lines for tail, head regions, and initially-bound GNPs, respectively, for both panels (b) and (b’). The suspended GNP layer is presented by dash-dot line in panel (b’). Profile of bare DPPC monolayer without the introduction of GNPs is added for comparison. The zero position in each ED profile is assigned at position of headgroup.


6

Page 7 of 12

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

Langmuir

measurements, such as the binding structures of GNPs, the effect of TAT binding and possible GNP translocation into the membrane, and how membrane structures changing to accommodate those processes. XR profiles in Figure 3a were taken at the equilibrium states after the initial binding (Figure 1). The reflectivity signal from interfacial surfaces, R, is plotted on a semilog scale as Rq4z where qz is the surface-normal scattering wave vector. Compared to the XR profile of the bare DPPC interface, the data for the GNP binding interfaces show higher overall reflectivity and shift of modulation at qz ∼ 0.23 Å−1 to lower q (Figure 3a). These changes suggest the increases of overall thickness and ED of the film, which are obviously an indication of GNPs binding to the membrane. Furthermore, the reflectivity intensity also increase upon the increase of negatively charged density of membrane, suggesting the increase of density of GNPs. However, the adsorption isotherms (Figure 1) and TEM images (Figure 2) did not show any significant difference in the amount of GNPs incorporated into membrane. Therefore, the increase of GNPs’ density should be within the bulk solution close to the membrane and not physically incorporated into the membrane. Consequently, the 3-layer model, including 2 layers accounted for the lipid membrane (head and tail regions) and 1 layer accounted for the initially-bound GNPs, was applied for fitting all GNP binding profiles, while the 2layer model, including head and tail regions, was used for the bare DPPC monolayer. The corresponding ED profiles for φ = 0% and 30% (Figure 3b) show the increase of ED values for GNP layer, from 0.41 to 0.45 e− /Å3 , while the thickness of GNP layer in both cases were almost identical, 5.4 and 5.9 Å, respectively. 25 These values do not reflect the actual particle size, but rather may arise from the highly diffusive state of the particles bound to the membrane by TAT-capped ligands. Interestingly, for φ = 70%, there are two distinct features shown in XR profile; first, the reflectivity intensity at low qz regime is dramatically higher than that of φ = 0% and 30%; sec-

ond, there is a shift of a critical angle to higher qz , from 0.021 (of water) to 0.031 Å−1 (Figure 3a). These features, in principle, should be attributed to a dramatic increase in ED either of the subphase or of the overall film with a thick adsorbed GNP layer. The former presumption may lead to an unphysical model where the number of injected GNPs would be very large to partially displace the bulk water in entire volume within a penetration range of X-ray beam. Therefore, in this case, the XR profile for φ = 70% is refined based on the latter presumption by vastly varying the ED and thickness of adsorbed layer, together with the roughness of its boundary along water subphase. As a result, a dense suspension of GNPs, with ED value of 0.49 e− /Å3 (identical to that of φ = 30%), ∼ 120 Åin thickness, and a diffuse boundary (∼ 60 Å) along the subphase, 25 is obtained in corresponding ED profile (Figure 3b’). The thickness of this suspended layer is approximately 1.5-time of the size of individual GNP particle. Although for different φs the membrane structures can also be different, the headgroup thicknesses obtained from the XR fitting for GNP binding interfaces are quite consistent, 4.9 to 5.1 Å, at about a half of bare DPPC’s headgroup thickness, 9.5 Å. The other half of headgroup thickness is quite matching with the thicknesses of initially-bound GNP layers, 5.4 to 5.9 Å, thus, suggesting that the initially-bound GNPs have inserted about a half-way through the headgroup region. These initially-bound particles are expected to repel particles arriving at the membrane later, which results in no change in the adsorption isotherms for φ = 0 − 70% after the initial binding regime (Figure 1). Such the interparticle repulsion also prevents the un-bound GNPs to be transferred onto the substrate, resulting in the equal densities of GNPs shown in TEM images at the equilibrium states for all compositions (Figure 2). The in situ measurements to characterize the binding progress of GNPs on pure DPPS monolayer were inspired by the progressive increase of Π shown in the adsorption isotherm (Figure 1) and the presence of multi-


7

Langmuir

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

Page 8 of 12

(Figure 4a). Accordingly, the fittings were solved by 6layer model, including 3 layers accounted for the additional GNP interface (linker facing the bulk, core GNP, linker facing the membrane), 1 layer accounted for the initiallybound GNPs, and 2 layers accounted for the lipid membrane (head and tail regions). The ED profiles show a progressive increment of ED values, from 0.36 to 0.44 e− /Å3 , indicating the growth in the density of the additionally adsorbed GNP layer upon time (Figure 4b). The total thickness of all GNP-related layers is ∼ 78 ± 36 Å, which is approximately same size as of an individual GNP. Besides, the ED values of head regions also increase dramatically, from 0.43 to 0.46 e− /Å3 for 5hr and 20hr, respectively, compared to 0.40 e− /Å3 of bare DPPS, 25 clearly suggesting that GNPs have incorporated into membrane. The roughness values of GNP-incorporating lipid headgroups, 10.8 − 16.5Å, are also much greater than the bare DPPS membrane, 3Å, along with the decreases of the headgroup thicknesses and ED of the lipid tails, suggesting that the incorporation of GNPs into the membrane significantly disrupt the packing state of the headgroup layer. The total thickness of the rough headgroup surface, however, is still less than an approximated diameter of the core gold NPs, possibly leading to an expectation of shape distortion of the particles upon incorporating into the membrane. This phenomenon of shape distortion of NPs was observed before, 26 where the loss of uniformity of ligand coverage on the particle surface after spreading is believed to lead a shape changing from spherical to oblate. In our case, the linkers on entire particle incorporated into the membrane are expected to either orient toward the air or interact with the lipid tails, which possibly leave the other side facing the bulk less covered or even naked, hence become less stable. As a result, they will tend to either locally aggregate with surrounding particles, which is observed from TEM images (Figure 2), or adopt a new shape to minimize the surface exposure to the bulk and maximize the interaction with

layer in TEM images (Figure 2). Compared to the XR profile of bare DPPS, the data for GNP binding interfaces show additional modulation at qz = 0.06 Å−1 ; and the progressive increase in the amplitude of the modulation, from 0hr to 20hr, is related to the density of the adsorbed GNP layer. These changes ba-

Figure 4: In situ XR characterization for binding progress of GNPs on DPPS (φ = 100%) (a) and their corresponding ED profiles (b). The inserted cartoon illustrates the location and lengths of sub-layers on the ED profiles. The zero position in each ED profile is assigned at position of headgroup. The intensity scale in (a) is offset for clarity. “Before” denotes bare DPPS, while “hr” denotes at time (in hour) of in situ measurements after injection of TATGNPs underneath the membrane. sically indicate the evolution of an additional GNP interface toward a denser layer, as we apply in our fitting motifs and discuss below


8

Page 9 of 12

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

Langmuir

Supporting Information Available

the lipid membrane. One of the important features, which highlights the difference in the binding behaviors of GNPs on the membrane between φ = 70% and 100%, is that the critical angle shifting in XR profile for φ = 70% (Figure 3a) is not observed for all in situ profiles for φ = 100%, indicating that GNPs are no longer suspended in the bulk but rather have reached close to the interface and formed a dense layer due to a dominant attraction between GNPs and membrane. In other word, along the surface normal (1D), the binding structures of GNPs are transformed from the diffused state to the closely packed state upon the increase of the charged density of the membrane, from 70% to 100%, respectively.

The following files are available free of charge. Schematic of TAT-GNP synthesis, additional characterization for the structures and properties of TAT-GNPs, X-ray fitting Table 1-2, and additional reference.

References (1) Hodes, G. When small is different: some recent advances in concepts and applications of nanoscale phenomena. Adv. Mater. 2007, 19, 639–655. (2) Whitesides, G. M. Nanoscience, nanotechnology, and chemistry. Small 2005, 1, 172–179.

Conclusion In summary, our study demonstrates how TAT-capped GNPs, after strongly binding to the biological membrane due to the interactions between TAT peptides and the membrane, direct the distribution of other particles in the bulk. The balance of the GNPmembrane attraction and the interparticle repulsion determines the macroscopic structure of GNP self-assembly. This results in varying degrees of ordering structures, ranging from particles highly scattered in the bulk solution at φ ≤ 30%, to a dense GNP suspension within a confined volume close to the membrane at φ = 70%, and to a well-defined GNP layer underneath the membrane at φ = 100%.

(3) Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-guided crystallization of colloidal nanoparticles. Nature 2008, 451, 549–552. (4) Srivastava, S.; Nykypanchuk, D.; Fukuto, M.; Gang, O. Tunable Nanoparticle Arrays at Charged Interfaces. ACS Nano 2014, 8, 9857–9866. (5) Heath, J. R.; Knobler, C. M.; Leff, D. V. Pressure/temperature phase diagrams and superlattices of organically functionalized metal nanocrystal monolayers: the influence of particle size, size distribution, and surface passivant. J. Phys. Chem. B 1997, 101, 189–197.

Acknowledgement We thank Dr. Sushil Satija for valuable comments on the manuscript. This work was supported by the Leading Foreign Institute Recruitment Program (2013K1A4A3055268), the Mid-career Researcher Program (2016R1A2B3015239), the Advanced Research Center for Nuclear Excellence and Basic Science Research Program (2013R1A1A2010265), through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning, Korea.

(6) Fukuto, M.; Heilmann, R. K.; Pershan, P. S.; Badia, A.; Lennox, R. B. Monolayer/bilayer transition in Langmuir films of derivatized gold nanoparticles at the gas/water interface: An xray scattering study. J. Chem. Phys. 2004, 120, 3446–3459. (7) Duan, H.; Wang, D.; Kurth, D. G.; Möhwald, H. Directing self-assembly of nanoparticles at water/oil interfaces.


9

Langmuir

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

Angew. Chem. Int. Ed. 2004, 43, 5639– 5642.

Page 10 of 12

(17) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Maleimide-functionalized selfassembled monolayers for the preparation of peptide and carbohydrate biochips. Langmuir 2003, 19, 1522–1531.

(8) Rabani, E.; Reichman, D. R.; Geissler, P. L.; Brus, L. E. Dryingmediated self-assembly of nanoparticles. Nature 2003, 426, 271–274.

(18) Gittins, D. I.; Caruso, F. Spontaneous phase transfer of nanoparticulate metals from organic to aqueous media. Angew. Chem. Int. Ed. 2001, 40, 3001–3004.

(9) Garbin, V.; Jenkins, I.; Sinno, T.; Crocker, J. C.; Stebe, K. J. Interactions and Stress Relaxation in Monolayers of Soft Nanoparticles at Fluid-Fluid Interfaces. Phys. Rev. Lett. 2015, 114, 108301.

(19) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. Synthesis of thiol-derivatised gold nanoparticles in a two-phase liquid–liquid system. J. Chem. Soc., Chem. Commun. 1994, 801–802.

(10) Liu, J.; Lu, Y. A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. 2003, 125, 6642–6643.

(20) Tolan, M. X-ray scattering from soft-matter thin films; Springer, 1999.

(11) Fonseca, S. B.; Pereira, M. P.; Kelley, S. O. Recent advances in the use of cellpenetrating peptides for medical and biological applications. Adv. Drug Deliver. Rev. 2009, 61, 953–964.

(21) Parratt, L. G. Surface studies of solids by total reflection of X-rays. Phys. Rev. 1954, 95, 359. (22) Phan, M. D.; Shin, K. Effects of Cardiolipin on Membrane Morphology: A Langmuir Monolayer Study. Biophys. J. 2015, 108, 1977–1986.

(12) Gupta, B.; Levchenko, T. S.; Torchilin, V. P. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv. Drug. Deliver. Rev. 2005, 57, 637–651.

(23) Peetla, C.; Rao, K. S.; Labhasetwar, V. Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles. Mol. Pharmacol. 2009, 6, 1311–1320.

(13) Nakase, I.; Akita, H.; Kogure, K.; Graslund, A.; Langel, U.; Harashima, H.; Futaki, S. Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides. Accounts Chem. Res. 2011, 45, 1132–1139.

(24) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9, 671–675.

(14) Herce, H. D.; Garcia, A. E. Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes. Proc. Natl. Acad. Sci. 2007, 104, 20805–20810.

(25) The ED values displayed in ED profiles tend to be smaller than the absolute values obtained directly from the XR analyses due to the contribution of interfacial roughness. For the details of absolute ED values, see Table 1 and 2 in Supporting Information.

(15) Elsaesser, A.; Howard, C. V. Toxicology of nanoparticles. Adv. Drug Deliver. Ref. 2012, 64, 129–137. (16) Koren, E.; Torchilin, V. P. Cellpenetrating peptides: breaking through to the other side. Trends Mol. Med. 2012, 18, 385–393.

(26) Sun, Y.; Frenkel, A. I.; Isseroff, R.; Shonbrun, C.; Forman, M.; Shin, K.; Koga, T.; White, H.; Zhang, L.; Zhu, Y.;


10

Page 11 of 12

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

Langmuir

Rafailovich, M. H.; Sokolov, J. C. Characterization of palladium nanoparticles by using X-ray reflectivity, EXAFS, and electron microscopy. Langmuir 2006, 22, 807– 816.


11

Langmuir

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

Graphical TOC Entry


12

Page 12 of 12

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on a Lipid

Recommend Documents