Protein Immobilization in Hollow Nanostructures and Investigation of

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Protein Immobilization in Hollow Nanostructures and Investigation of the Adsorbed Protein Behavior Xi Qian,†,‡,§ Alex Levenstein,†,‡ Jennifer E. Gagner,†,‡,§,⊥ Jonathan S. Dordick,*,†,‡,§,∥ and Richard W. Siegel*,†,‡,§ †

Department of Materials Science and Engineering, ‡Rensselaer Nanotechnology Center, §Center for Biotechnology and Interdisciplinary Studies, and ∥Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ABSTRACT: Understanding nanomaterial-biomolecule interactions is critical to develop broad applications in sensors, devices, and therapeutics. During the past decade, in-depth studies have been performed on the effect of nanoscale surface topography on adsorbed protein structure and function. However, a fundamental understanding of nanobio interactions at concave surfaces is limited; the greatest challenge is to create a nanostructure that allows such interactions to occur and to be characterized. We have synthesized hollow nanocages (AuNG) through careful control of morphology and surface chemistry. Lysozyme was used as a model to probe interactions between a protein and these nanostructures. Solid Au nanoparticles with a similar morphology and surface chemistry were also used as a reference. Through a series of quantitative analyses of protein adsorption profiles and enzymatic activity assays of both nanobioconjugates, we discovered that a significant amount of protein could be delivered into the core of AuNG, while maintaining a substantial fraction of native activity.

1. INTRODUCTION The interface between biotechnology and nanotechnology has led to significant advances in protein, drug, and gene delivery,1,2 cancer therapy,3,4 bioimaging,5,6 and the preparation of antimicrobial surfaces,7,8 among myriad other applications. The interaction of biomolecules with nanomaterials profoundly influences bound proteins, as well as cells and tissues.9,10 In most cases, the nanomaterials used have been simple, albeit highly controlled structures. Significant information has been obtained on biomolecule-nanomaterial interactions via the formation of protein−nanoparticle (NP) conjugates.11,12 Such conjugates have been studied extensively, including the influence of surface chemistry,13 topography,14,15 and crystallinity16 on protein structure and function. One of the most significant influences is the size of the NP and, hence, its associated surface curvature,17−19 which can induce conformational changes of adsorbed proteins and ultimately affect protein function.12 To date, the vast majority of protein−nanomaterial interactions have been studied on nanomaterials with convex (positive curvature) or flat surfaces. Our current understanding of protein−nanomaterial interactions on concave (negative curvature) surfaces, however, is essentially limited to mesoporous silica materials,20−23 which possess tubular pores of 5−15 nm in diameter. This lack of understanding is primarily due to the great difficulty in reproducibly synthesizing hollow monodispersed nanoparticles exhibiting negative curvature. This is unfortunate, as the internal surfaces in hollow nanostructures may provide fundamentally different concave © 2014 American Chemical Society

and macromolecular-crowding environments and, hence, distinct protein−nanomaterial interactions. An interesting nanostructure possessing negatively curved internal surfaces that could potentially adsorb proteins is the gold−silver alloy nanocage (AuNG)24 that is synthesized by galvanization. In contrast to mesoporous silica, AuNGs possess a near-spherical hollow core of 20−50 nm in diameter. As the surface area of AuNG cavities is much larger than a protein, this hollow core provides a surface that can potentially adsorb a significant amount of protein. Moreover, AuNGs are distinguished by their high surface area, low bulk density, tunable surface plasmon resonance (SPR), and low cytotoxicity,25 rendering them promising for biomedical applications such as imaging and photothermal tumor ablation.26 Their hollow cores could also facilitate use as materials for drug delivery27 and protein delivery.28 Considering these promising features, a major challenge beyond monodispersity in studying or utilizing protein-AuNG conjugates, however, is the verification of successful protein internalization within the nanocage core. Although a variety of characterization methods can be applied to determine the amount of adsorbed protein onto AuNGs,29,30 due to the nature of their hollow structures, there are relatively few ways to distinguish whether the proteins are adsorbed onto the internal or the external surfaces of the AuNG. Unfortunately, Received: December 17, 2013 Revised: January 21, 2014 Published: January 22, 2014 1295

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removed. Both SEM and XRD samples were prepared by drop-drying aqueous nanoparticle suspensions on silicon wafers. Inductively coupled plasma mass spectroscopy (ICP-MS) was performed via Bruker Varian 820-MS ICP-MS (Billerica, MA), and the samples were prepared by dissolving nanoparticle suspensions into aqua regia and then diluted in 5% nitric acid. 2.4. Determination of Protein Loading. Quantification of adsorbed protein was made using standard μBCA and BCA assays. Briefly, protein stock solutions were prepared in PBS solution (2 mM, pH 7.4). Protein solution was then added in concentrations ranging from 10 to 350 μg/mL to aliquots of nanoparticle solution (2 mM PBS, pH 7.4). Samples were shaken at 4 °C and 125 rpm for 72 h to allow AuNP−Lyz solutions to reach equilibrium. To measure protein loading, the nanobioconjugates were centrifuged at 12 000 rpm and 4 °C for 10 min, then redispersed in buffer via vortex mixing, and centrifuged again to ensure that all free protein was removed. Supernatants from centrifugations were collected to measure free protein concentration in μBCA and BCA assays. Both assays were measured in a Molecular Devices (Sunnyvale, California) SpectraMax M5 microplate reader. Protein uptake pseudoisotherms were fit using the Langmuir equation eq 2

direct microscopic observation is not presently capable of visualizing such adsorbed species. Nevertheless, in order to analyze protein-nanocage interactions on internal surfaces, protein internalization must be experimentally demonstrated by other methods.31 In the current work, we used the hydrolytic enzyme lysozyme (Lyz) to probe protein internalization within nanocages. Taking advantage of our group’s previous in-depth study of Lyz−gold nanocube (Lyz−AuNC) conjugates,16 we performed a comparative study between Lyz−AuNC and Lyz−AuNG conjugates. Enzymatic activity of the conjugates was assayed using two substrates with different accessibilities to the interior space of AuNGs. By combining activity data with protein adsorption profiles, a quantitative understanding of protein internalization and functionality in a hollow nanostructure has been achieved.

2. METHODS 2.1. Materials. Gold(III) chloride trihydrate (99.9%), silver nitrate ACS reagent (99+%), 11-mercaptoundecanoic acid (11-MUA) (90%), sodium sulfide nonahydrous, 1,5-pentanediol (96%) (PD), and polyvinylpyrrolidone (Mw ≈ 55 000) (PVP) were purchased from Sigma-Aldrich (St. Louis, MO). Ethylene glycol was purchased from J. T. Baker (Center Valley, PA). Lysozyme (Lyz) from chicken egg white, Micrococcus lysodeikticus, and 4-methylumbelliferyl β-DN,N′,N″-triacetylchitotrioside (4-MU-β(GluNAc)3) were purchased from Sigma-Aldrich as dry powders and used without further purification. Bicinchoinic acid (BCA) and microbicinchoninic acid (μBCA) assay reagents were purchased from Pierce Biotechnology, Inc. (Rockford, IL). 2.2. Nanoparticle Synthesis. Silver nanocubes (AgNCs) and gold−silver alloy nanocages (AuNGs) were synthesized based on the method reported by Skrabalak et al.32 A reduction of Ag+ salt into Ag was achieved by using AgNO3 as the silver substrate, ethylene glycol as both the solvent and reducing agent, PVP as the capping agent, and Na2S as the catalyst. The concentration of Na2S and reaction times were carefully controlled to ensure a good yield of distinct AgNCs with uniformed structure. Subsequently, the AgNC suspension was used as a sacrificial template in the galvanization reaction (eq 1) to obtain AuNGs.33

3Ag(s) + AuCl4 −(aq) → Au(s) + 3Ag +(aq) + 4Cl(aq)

[P]ads N[P] = app free [NP] Kd + [P]free

(2)

where [NP] is the concentration of AuNPs, [P]ads is the concentration of adsorbed protein, [P]free is the concentration of unbound protein, N is the number of protein molecules per AuNP at saturation, and Kapp d is the apparent dissociation constant for the respective sites. Curves were fit using a nonlinear regression method in Matlab r2013b (The Mathworks, Inc., Natick, MA) by maximizing the correlation coefficient. Conservative estimates of particle surface coverage were determined using a method similar to that used by Cedervall et al.36 The published radius of gyration for Lyz (1.5 nm37) was used to determine the cross-sectional area of an individual protein molecule, and then the available surface area on the nanoparticle was estimated by determining the total surface area at one protein radius-length away from the nanoparticle surface. 2.5. Enzymatic Activity Assays. According to our previous work,14,16 Lyz−AuNP may form microscale aggregates at high concentrations, which results in a significant loss in enzymatic activity. As a result, samples were prepared with low AuNP concentrations and saturated surface protein coverage. To that end, Lyz−AuNP conjugates were incubated for 72 h (2 mM PBS buffer, pH 7.4 at 4 °C) to allow sufficient time for protein internalization into Lyz− AuNGs; the amount of added Lyz was carefully controlled to 300% of the saturation amount, approximately 1 μM, to make sure that the amount of adsorbed Lyz is adequate for both activity assays.38,39 After incubation, nanobioconjugates were spun down gently to remove free protein. The amount of adsorbed protein was determined by measuring the protein concentration of the spun supernatant via the μBCA assay. Lyz activity was assayed by two methods, the first of which being turbidity measurements of M. lysodeikticus monitored at 450 nm for 300 s. Briefly, M. lysodeikticus solution was prepared in 30 mM potassium phosphate buffer (pH 7.4) and diluted to an absorbance of 0.6 AU. Then 100 μL of washed Lyz−AuNP conjugate was added to 900 μL of M. lysodeikticus solution. Incubation of M. lysodeikticus with active Lyz results in decreased solution turbidity as the cells are lysed. Specific activity was normalized against the activity of the same concentration of free Lyz. The enzymatic activity of Lyz−NP conjugates was also measured in a fluorescence assay by measuring the fluorescent intensity of 7hydroxy-4-methylcoumarin (4-MU) that was cleaved from Lyzcatalyzed hydrolysis of 4-MU-β-(GluNAc)3. Washed Lyz−AuNP conjugates were centrifuged to remove free Lyz and incubated at 37 °C with 2 mL of 0.09 mg/mL 4-MU-β-(GluNAc)3 prepared in sodium phosphate buffer (0.1 M, pH 6.5). During incubation, 300 μL aliquots were taken at 0, 2, 4, 8, and 20 h, respectively, and immediately mixed

(1)

In the galvanization reaction, aqueous HAuCl4 solution was added to a boiled AgNC solution with a syringe pump, while maintaining careful control of the concentration of both solutions. A dramatic color change from pale yellow to grayish blue was observed during galvanization. Multiple galvanization experiments were performed to obtain the optimum amount of HAuCl4, so that the highest Au/Ag ratio could be achieved without collapsing the cage structure.34 The synthesis of AuNCs followed the protocol of Seo et al.35 Briefly, HAuCl4 and PVP were added into heated 1,5-pentanediol with refluxing, and AgNO3 was carefully added to control AuNC morphology. A variety of characterization methods, including SEM and XRD, were used to determine the morphology and uniformity of the nanoparticles. After their morphologies had been confirmed, the nanoparticles underwent a ligand exchange process. The ligand exchange and subsequent characterizations followed a protocol previously reported by Gagner et al.16 Specifically, gold nanoparticles were incubated in ethanol with 10 mg/mL 11-MUA to replace the nanoparticle surface ligand from PVP to 11-MUA. After the ligand exchange, AuNGs were stored in 2 mM phosphate-buffered saline (PBS, pH 7.4) and ready for subsequent protein experiments. 2.3. Nanoparticle Characterization. Scanning electron microscopy (SEM) was performed via a Carl Zeiss Supra-55 VP field emission SEM (Jena, Germany). X-ray diffraction (XRD) patterns were collected via a Panalytical X’Pert diffractometer (Westborough, MA) using Cu Kα radiation (λ = 0.1542 nm), and the background was 1296

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Figure 1. Characterization of AgNC and AuNC: Representative scanning electron micrographs of AgNC (A) and AuNC (C); X-ray diffraction pattern of AuNC and AgNC (B). Scale bars = 100 nm. with the same amount of glycerin buffer (0.8 M, pH 10.4) to quench the reaction. The mixed aliquots were then centrifuged again to remove the AuNPs, which could potentially interfere with the fluorescence measurement. After centrifugation, supernatants that contain 4-MU residue were transferred to a 96-well plate, and the concentration of 4-MU was measured in triplicate using fluorescence spectroscopy (λex = 355 nm, λem = 460 nm). A free Lyz sample was also involved in this assay as a control. The specific activity of each nanobioconjugate sample was normalized with respect to the same concentration of free Lyz.

3. RESULTS AND DISCUSSION 3.1. Nanoparticle Characterization. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to characterize the structures of AgNCs and AuNCs (Figure 1). For AgNCs, as their morphology plays a critical role in the subsequent galvanization synthesis of AuNGs, its dominant crystal facet must be {100} to ensure a near-perfect cubic structure of the nanocrystals. Similarly, for AuNCs, their nearperfect cubic morphology is also important in the quantitative analysis of protein adsorption. As shown in Figure 1B, the dominant diffraction peak of AgNCs is at 2θ = 44°, corresponding to the {200} facet of a face-centered cubic (FCC) silver crystal, which demonstrates that the silver template is dominated by perfect or near-perfect nanocubes. The XRD of AuNC shows that the dominant peak is the {200} facet as well, demonstrating that AuNCs are also dominated by near-perfect nanocubes. SEM micrographs of AgNC and AuNC (Figure 1A and C) further supported the XRD result. SEM was used to determine the exact morphologies and sizes of the nanoparticles, for which average data were used to calculate the available surface areas for protein adsorption. According to the size distribution histograms of the three nanoparticles (Figure 2), the mean edge lengths of AgNCs, AuNGs, and AuNCs were 52 ± 7, 54 ± 10, and 74 ± 14 nm, respectively, indicating good monodispersities. AuNGs are slightly larger than their AgNC templates, which is consistent with the reported galvanization mechanism.34 Meanwhile for AuNGs (Figure 3A,B), the reaction parameters were carefully controlled according to the mechanistic studies of Ag-HAuCl4 galvanization of Sun et al.34,40 Galvanization reactions were quenched at the dealloying step so that the interior and exterior surfaces of AuNGs are chemically similar. According to inductively coupled plasma mass spectroscopy (ICP-MS) measurements, these AuNG have a Au:Ag ratio of ∼3:1, indicating a significant replacement of Ag atoms. Mahmoud et al.41 demonstrated that such AuNG’s external surface is dominated by Au atoms, and electron diffraction characterization34 further demonstrated that both the AuNG walls and

Figure 2. Size distributions of AgNC (green), AuNC (red), and AuNG (blue) measured from SEM micrographs. The average edge lengths of each are shown.

AuNC surfaces are {100} faceted. Such similarities ensure that the AuNG and AuNC possess similar surface chemistry after 11-MUA ligand exchange. For AuNGs, it is difficult to calculate their total surface area accurately, as the interior structure has not been precisely determined. Therefore, AuNG geometry was estimated using a cubic box model with a cylindrical pore on each facet. Furthermore, according to earlier characterizations of AuNGs (Figure 3C,D),42,43 their hollow cores approximate a rounded cube, as schematically illustrated in Figure 3F,G. Gagner et al.16 demonstrated that the external surface area of rounded-cubic nanoparticles could be calculated by summing the flat facets and rounded edges and corners. However, in the present work, the internal edge lengths and corner radii can only be roughly and separately estimated, and thus cannot provide a good estimation of overall internal surface area. However, by assuming the interior hollow as an “effective perfect cube”, and hence, neglecting the exact sizes of truncated edges and corners (as demonstrated by the blue dotted line in Figure 3E), it is possible to obtain a reliable range for the internal surface area. The volume of the hollow core can be estimated. As both Au and Ag crystals have an FCC structure and their lattice constants are very close (4.08 Å for Au and 4.09 Å for Ag), one can assume that, during galvanization, the volume of an Ag nanocube template is decreased according to eq 3. 108

[Ag]nanocage + 197 [Au]nanocage VAuNG = VAgNC [Ag]template

(3)

According to ICP-MS, the volume is decreased by approximately 50−60% after galvanization. Due to the standard 1297

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Figure 3. Characterization and geometric models of AuNG. (A) and (B) SEM micrographs of AuNG; (C) AuNG cross section measured by X-ray tomography; (D) AuNG cross section observed by SEM in backscattered mode; (E) 2-D schematic model of the cross section of AuNG, where the dashed line is indicating the “effective cube” in the calculation of eq 2; (F) 3-D model of AuNG; (G) 3-D model of the interior hollow of AuNG. Scale bars = 50 nm. Panel C reprinted with permission from ref 42. Copyright 2010 American Chemical Society. Panel D reprinted with permission from ref 40. Copyright 2007 American Chemical Society.

Table 1. Geometric Parameters of AuNG and AuNC

a

average exterior edge length (nm)

estimated average interior hollow edge length (nm)

exterior surface area per NP (nm2)

estimated interior surface area per NP (nm2)

# of lysozyme per NP at 100% coveragea

AuNG

54 ± 10

42−45

18200

10 600−12 200

AuNC

74 ± 14

ext: 2600 int: 1500−1700 4700

32900

Lyz are considered as spheres 1.5 nm in gyration radius.

deviations of the size measurements from AuNC and AgNC, only a range, instead of an exact value, can be obtained from eq 3. Since a spherical or concave hollow core contains more volume than a cubic or polyhedral hollow core with the same surface area, the upper limit of the hollow core surface area can be calculated by assuming that the hollow core is a perfect cube. As shown in Figure.3E, with a pore radius of r, the nanocage wall thickness of d, and the AuNG edge length of L, and assuming one pore at each cubic surface, the exterior surface area can be expressed by eq 4 SAE = 6(L2 − πr 2)

VH = (L − 2d)3

(8)

VAuNG = L3 − VH − VP

(9)

From the SEM micrographs, it is possible to estimate that L = 55 nm and r ≈ 10 nm. Also, according to the literature, d < 15 nm.42 At this scale, VP represents less than 1% of the total hollow core volume, and hence, can be neglected numerically. Thus, eq 9 can be reduced to eq 10

(4)

VAuNG = L3 − VH

(10)

.By combining eq 6 and 8, an estimated d value is calculated to be 4 nm, and the surface area of the associated effective cube can thus be calculated, providing an upper limit of the hollow core surface area. To estimate the lower limit of the hollow core surface area, we employed a geometric model developed by Buchin et al.44 In this so-called “inflated cube” model, the authors demonstrated that by truncating the corners and edges of a perfect

(5)

and the interior hollow surface area can be expressed by eq 6 SAH = 6(L − 2d)2 − 6πr 2

(7)

.

the pore surface area can expressed by eq 5

SAP = 12πrd

Vp = 6dπr 2

(6)

The hollow core volume of an AuNG is comprised of two parts; the six cylinders of the pores (VP) and the center cubic hollow core (VH). These volumes can be calculated via eqs 5−7 1298

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3.2. Protein Uptake and Immobilized Protein Activities. Following nanoparticle synthesis and characterization, Lyz−AuNP conjugates were prepared via physical adsorption of the protein onto the AuNP surfaces. Negatively charged AuNPs coated with 11-MUA (pKa ≈ 4.8) were expected to interact with the positively charged Lyz (pI ≈ 11) primarily through electrostatic interactions. According to previous studies on mesoporous silica,22 Lyz can access a pore that is only several nanometers in radius, and thus, it is possible to immobilize Lyz on the nanocage’s internal surfaces simply by diffusion. Protein adsorption was quantified using the microbicinchoninic acid (μBCA) and bicinchoninic acid (BCA) assays to determine the amount of protein in solution after two rounds of centrifugation. Adsorption curves were fitted to the classical Langmuir isotherm (eq 18),45 where applicable, to determine the number of binding sites on the AuNP, as well as the apparent dissociation constants (Kapp d ) for each conjugate. The data points and fitted adsorption pseudoisotherms are shown in Figure 4.

cube, while maintaining its surface area, a cube’s volume would increase by 25%. Thus, since a perfect cube can increase its volume by 25% without changing its surface area, the smallest possible surface area of the hollow core is equivalent to an effective cube that has a volume of VH/1.25. As a result, the range of the hollow core surface area can be determined and, as shown in Table 1. The possible surface area is confined to a relatively small range, about 70% of that compared to the exterior surface area. Moreover, with these calculated geometrical parameters, the pore surface area is only ∼5% of the total surface area; hence, the pores can be neglected in calculating protein loading. For the same reason, the circumferential surface areas of the pores were also neglected during subsequent calculations. By combining the morphological parameters of each nanoparticle and the element concentration measured by ICP-MS, one can calculate nanoparticle concentrations and determine the total available surface area per unit volume for protein adsorption. As shown by the outlines of AuNC in the SEM micrographs (Figure 1), most nanoparticles are nearperfect cubes; hence, the AuNC concentrations can be calculated via eq 11 [NP] =

[Au] lAu 3ρAu

(11)

where [NP] is the nanoparticle concentration, [Au] is the element concentration of Au in mass, l is the average cube edge-length of an AuNC, and ρ is density. Moreover, the total available AuNC surface area can be calculated via eq 12 [A] = 6l 2[NP]

(12)

,where [A] is the total available surface area per unit volume. These calculations result in an AuNC concentration of 7.7 × 1010 mL−1, with an available surface area of 3.0 × 1015 nm2/mL, as summarized in Table 2.

Figure 4. Protein uptake curves of Lyz−AuNG nanobioconjugates and (inset) the protein uptake curve of AuNC. In both graphs, dashed lines indicate 100% protein coverage with respect to the exterior surface, while the solid lines indicate the fitted Langmuir pseudoisotherms.46 The error bars shown are the standard deviation of measurements.

Table 2. ICP-MS and Protein Loading Data of AuNG and AuNCa AuNC AuNG a

ICP-MS: Au (μg/mL)

ICP-MS: Ag (μg/mL)

Particles/ml

605 115

0 30

7.7 × 1010 3.0 × 1011

The protein uptake curve of AuNG shows two distinct regions (Figure 4), as is commonly observed for mesoporous silica when proteins adsorb to both exterior and interior surfaces,47 first saturating at low concentrations ([free Lyz] < 1 μM), then dramatically increasing at [free Lyz] = 2−3 μM, and finally approaching saturation at higher free protein concentrations ([free Lyz] > 10 μM). At even higher free Lyz concentrations, severe aggregation was observed in the solution and further measurements were prevented. The first region ([free Lyz] < 3 μM) of the AuNG−Lyz uptake curve can be fitted by a Langmuir isotherm, resulting in Lyz uptake of 3500 ± 400 molecules/AuNG, which yields ca. 137% surface coverage, and Kapp d = 0.038 ± 0.03 μM. For comparison, the Langmuir fit for Lyz−AuNC conjugates (Figure 5 inset) estimates a Lyz uptake of 4800 ± 500 molecules/AuNC, a Kapp d = 0.036 ± 0.02 μM, and a theoretical saturation coverage of ca. 100%. The second step of the protein uptake curve ([free Lyz] > 3 μM) was not fit to a particular isotherm. The observed strong affinity is likely due to strong electrostatic interactions between the highly basic protein and the 11-MUA-coated surfaces. The fitted saturation coverage is consistent with our previous study of Lyz−AuNC conjugates.16

Standard deviations of measurements are below 5%.

For AuNGs, again, their concentrations cannot be calculated directly via eq 9 due to their hollow structure. However, considering that during the galvanization process, three Ag atoms were substituted by one Au atom, then one can calculate the actual concentration of Ag in the Ag precursor template using eq 13. 108 [Ag]template = [Ag]nanocage + 3 [Au]nanocage (13) 197 Since the nanoparticle concentrations of AgNC templates can be calculated as described by eq 9, and by assuming that a majority of AgNC are able to be entirely converted into AuNG, one can obtain a relatively good estimate of the AuNG concentration to be 1.50 × 1011 mL−1 (Table 2). All geometrical parameters of AuNCs and AuNGs are summarized in Table 1, and the concentration parameters are summarized in Table 2. 1299

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D 9 = 1 + λ ln λ − 1.5603λ + 0.528155λ 2 + 1.91521λ 3 D0 8 − 2.81903λ 4 + 0.270788λ 5 + 1.10115λ 6 − 0.435933λ 7

(14)

where λ is the ratio between the protein size and the pore diameter. This equation is valid only when 0 < λ < 0.95; D is the through-pore diffusivity of proteins and D0 is the free solution diffusivity determined by the Stokes−Einstein equation31 (eq 15) D0 =

KBT 6πμR 0

(15)

where KB is Boltzmann’s constant, T is the temperature in Kelvin, μ is the solution viscosity, and R0 is the gyration radius of the protein. In the present study, the average pore radius is about 5 nm and the protein gyration radius is about 1.5 nm; hence according to eq 14, D ≈ 0.2D0. Thus, Lyz internalization in AuNG is theoretically possible. Protein internalization can also be interpreted with the help of AuNG’s Lyz uptake curve, as shown in Figure 4, where almost 300% coverage would be concluded, if only the external surfaces are considered. However, according to our earlier studies, 11-MUA capped AuNP−Lyz nanobioconjugates are very likely to aggregate beyond 100% exterior surface coverage, and hence prevent multilayered protein uptake. For example, according to our previous studies at similar free-protein concentrations, AuNC-Lyz nanobioconjugates saturated at ∼110% exterior surface protein coverage, and Au nanooctahedra-lysozyme (AuNO-Lyz) nanobioconjugates saturated at ∼100% coverage16 (in both cases nanoparticles were also capped by 11-MUA and in the same ionic environment). As a result, we conclude that the relatively high protein uptake observed in the present work indicates that the interior surfaces of AuNG are accessible for protein internalization. Moreover, the two distinct regions of protein uptake of AuNG−Lyz are further clarified by the likelihood of protein internalization. Specifically, when the equilibrium free protein concentration is below 3 μM, AuNG and AuNC behave similarly in terms of protein uptake, and both uptake curves can be described by Langmuir isotherms. Furthermore, their fitted protein uptake pseudoisotherms yield similar protein−surface affinity values, which is consistent with the fact that the AuNG

Figure 5. Enzymatic activity of Lyz−AuNP nanobioconjugates in different assays. The relative activity values are normalized to the same amount of free Lyz. The error bars shown are the standard deviation of measurements.

The enzymatic activity of Lyz−AuNP conjugates was measured in two assays, the cell wall assay, with Micrococcus lysodeikticus as the substrate, and a fluorescence assay with 4MU-β-(GluNAC)3 as a fluorogenic glycanase substrate (Figure 5). Compared with free Lyz, the Lyz−AuNCs retained ∼28% of their native activity in both assays, consistent with our previous results. 16 Interestingly, for Lyz−AuNG conjugates, the fluorogenic substrate activity, vs free enzyme, resulted in a nearly identical activity as that with AuNCs (ca. 27% activity retention), yet indicated only ca. 15% of native activity in the cell wall assay. 3.3. Verification of Protein Internalization in AuNG. Immobilizing proteins at the concave interior surfaces of a hollow nanostructure is an issue that must be addressed to understand associated nanobio interactions. The delivery of proteins into the nanocage hollow core is a diffusion-controlled process in which the major challenge is the diffusion hindrance caused by the AuNG pores. As the pore size is close to that of the protein size, protein molecules may not be able to migrate easily through the pores. Assuming proteins are hard spheres, then their diffusivity in a cylindrical pore can be described by the Higdon-Muldowney equation,48 as shown in eq 14

Figure 6. Schematics that illustrate the accessibility into AuNG: (A) The flows of Lyz (blue dots) into and out from AuNG and (B) the accessibility of fluorogenic glycanase and M. Lysodeikticus substrates to AuNG, respectively. The M. lysodeikticus is too big to enter an AuNG and thus cannot access Lyz inside AuNG. The 4-MU-β-(GluNAc)3 can enter an AuNG, be digested by Lyz inside the AuNG, and release 4-MU fluorescent residue, which can finally diffuse to the outside of the AuNG. 1300

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As shown schematically in Figure 6B, M. lysodeikticus is ∼1 μm in size and thus cannot enter the interior space of the AuNG. Because the cell wall assay measures the rate of digestion of M. lysodeikticus cells, the lysozyme adsorbed inside the AuNG is unable to access the cells. Thus, to Lyz−AuNG nanobioconjugates, the cell wall assay only measures the activity of Lyz at the external surfaces of AuNG, which is similar to the surface of AuNC, and should yield similar specific activity as the Lyz−AuNC nanobioconjugates (∼28%). In fact, the low measured specific activity of Lyz−AuNG (∼15%) is the average of the effective external Lyz and noneffective internal Lyz. Therefore, from these relative activity values, one can quantitatively estimate the ratio between the amount of internally and externally adsorbed Lyz, yielding that ∼47% (1 − (15%/28%)) of the adsorbed Lyz is internalized. On the other hand, the fluorogenic glycanase substrate, 4MU-β-(GluNAC)3, is easily able to enter AuNG cores and release the cleaved fluorescent residue, 4-MU, to the solution. Thus, the fluorescence assay for Lyz−AuNG measures the overall activity of both internally and externally adsorbed Lyz. Since the internal and external Lyz may have different activity, the following relationship should exist for Lyz−AuNG in the fluorescent assay

surfaces, both interior and exterior, are materially the same as the surface of AuNC after 11-MUA ligand exchange. As for the protein coverage, at low free protein concentrations ( 3 μM, yet a more quantitative model would require precise consideration of the combined effects of protein−surface attraction, free protein concentration, and nanobioconjugate aggregation and is hence beyond the scope of the present research. 3.4. Effects of Concave Surfaces on Adsorbed Lysozyme Activity. The Lyz−AuNG and Lyz−AuNG nanobioconjugates show similar activities in the fluorescence assay, while showing different activities in the cell wall assay (Figure 5). Considering the fact that these two assays are based on similar enzyme mechanisms, the most probable explanation of such a difference is the different accessibility of the two assay substrates.

Figure 7. Adsorbed lysozyme activities renormalized after accounting for substrate accessibility. The relative activity values are normalized to the same amount of free Lyz. The error bars of AuNG-Ext and AuNGInt are the accumulated error from the calculation using eq 18, and the error bar of AuNC is the standard deviation of the measurement of AuNC-Lyz activity in the M. Lysodeikticus assay.

The deconvolution of adsorbed protein activities reveals that Lyz inside the AuNG also retains a substantial activity similar to that for Lyz adsorbed externally on both AuNC and AuNG, indicating that the protein remains functional inside AuNG cores as well. Previous studies indicate that when adsorbed onto surfaces, proteins may exhibit perturbations in their secondary and/or tertiary structure resulting in loss of their native activity.18,19 Yet, quantitatively, such perturbations depend on the particular nanobio interactions between the surface and immobilized proteins, as well as the adsorbed protein−protein interactions. 1301

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4. SUMMARY AND CONCLUSIONS In this study, we have demonstrated that Lyz adsorbs to the internal core of hollow AuNG. Via carefully controlled galvanization synthesis, AuNG with small surface pores and concave hollow interior spaces were obtained. The delivery of proteins into the interior space of AuNG was not only demonstrated to be theoretically possible, but also experimentally proven. Comparing with Lyz−AuNC nanobioconjugates, which have already been thoroughly studied by our research group,16 Lyz−AuNG nanobioconjugates show almost 100% higher protein uptake amounts than the AuNC investigated in the present or previous studies, even though the AuNG are significantly smaller in size than the AuNC. This indicates that an AuNG has more total surface area accessible for protein uptake per unit volume than AuNC. Moreover, the observed two-step protein uptake curve of AuNG indicates that the protein internalization of AuNG is affected by the electrostatic attraction between surface and proteins, and by the increase of free protein concentration. In enzymatic activity assays, using two different substrates with very different accessibilities into AuNG, and using AuNC as a reference, the activity of internalized Lyz in AuNG could be estimated, showing that Lyz is substantially active inside AuNG cores. This study provides insight into nanostructure−biomolecule interactions of a novel type of nanostructure. The ability of AuNG to internalize a significant amount of proteins allows further study for their biomedical applications. In particular, when combined with their novel electromagnetic and chemical properties, AuNG can serve as a powerful tool in drug delivery, bioimaging, photothermal therapy of tumors, and a number of other applications.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ⊥

Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02115.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Nanoscale Science and Engineering Initiative of the National Science Foundation under Grant No. DMR-0642573.

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REFERENCES

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