Unexpected Changes in Functionality and Surface Coverage for Au

Jun 26, 2015 - drug delivery and an effective probe for interrogating NP−cell interactions. ..... resulting from heterogeneous coverage of AuNP surf...
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Unexpected Changes in Functionality and Surface Coverage for Au Nanoparticle PEI Conjugates: Implications for Stability and Efficacy in Biological Systems Tae Joon Cho, John M. Pettibone, Justin M. Gorham, Thao M. Nguyen, Robert I. MacCuspie, Julien Gigault, and Vincent A. Hackley* Materials Measurement Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States S Supporting Information *

ABSTRACT: Cationic polyethylenimine conjugated gold nanoparticles (AuNP-PEI) are a widely studied vector for drug delivery and an effective probe for interrogating NP−cell interactions. However, an inconsistent body of literature currently exists regarding the reproducibility of physicochemical properties, colloidal stability, and efficacy for these species. To address this gap, we systematically examined the preparation, stability, and formation mechanism of PEI conjugates produced from citrate-capped AuNPs. We considered the dependence on relative molar mass, Mr, backbone conformation, and material source. The conjugation mechanism of Au-PEI was probed using attenuated total reflectance FTIR and X-ray photoelectron spectroscopy, revealing distinct fates for citrate when interacting with different PEI species. The differences in residual citrate, PEI properties, and sample preparation resulted in distinct products with differentiated stability. Overall, branched PEI (25 kDa) conjugates exhibited the greatest colloidal stability in all media tested. By contrast, linear PEI (25 kDa) induced agglomeration. Colloidal stability of the products was also observed to correlate with displaced citrate, which supports a glaring knowledge gap that has emerged regarding the role of this commonly used carboxylate species as a “place holder” for conjugation with ligands of broad functionalities. We observed an unexpected and previously unreported conversion of amine functional groups to quaternary ammonium species for 10 kDa branched conjugates. Results suggest that the AuNP surface catalyzes this conversion. The product is known to manifest distinct processes and uptake in biological systems compared to amines and may lead to unintentional toxicological consequences or decreased efficacy as delivery vectors. Overall, comprehensive physicochemical characterization (tandem spectroscopy methods combined with physical measurements) of the conjugation process provides a methodology for elucidating the contributing factors of colloidal stability and chemical functionality that likely influence the previously reported variations in conjugate properties and biological response models.

1. INTRODUCTION On the basis of their lack of toxicity,1,2 unique physicochemical properties,3 and facile surface modification,4 gold nanoparticles (AuNPs) remain the most promising nanomaterial for many biomedical applications.5,6 Beyond this conceptual statement, in recent years, remarkable results regarding biomedical applications of AuNPs have been demonstrated for sensing,7−10 imaging,11−15 and therapeutics.16−21 The fate and efficacy of AuNP-based technologies in biological systems is driven largely by interfacial interactions between the local environment and the NP surface (functionality), where surface functionality contributions of NPs have been recently reviewed.5 For therapeutics, in particular, cationic (positively charged) AuNPs are of interest in view of biochemical delivery related to their cellular uptake22−24 and gene transfection20,25−30 efficacies. These properties are related to the cationic nature of positively charged AuNPs, which drive electrostatic approaches © XXXX American Chemical Society

to negatively charged biological entities (such as cells). Among the many cationic coating materials, polyethylenimine (PEI) is one of the most studied polycations due to its attractive intrinsic characteristics including water solubility, complex formation with metal ions31 and/or anionic polyelectrolytes,32 and, most interestingly for biological applications, gene transfection/delivery efficiency.33 Hence, a significant body of work exists on PEI-conjugated AuNPs (Au-PEIs).29,30,34−43 Although the results of Au-PEIs with respect to their preparation,34−37 including layer-by-layer (LBL) assembly,38−40 properties,44−47 and biological activities,29,30,34,39−43 have been discussed at length in the literature, a knowledge gap still exists regarding the role of PEI structure, size (relative molar mass, Received: May 4, 2015 Revised: June 19, 2015

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Figure 1. Characterization of Au-(branched)PEIs: (a) batch-mode DLS size distribution of citrate-stabilized AuNPs (solid line), Au-PEI2kB-mod (circle), Au-PEI10kB (square), and Au-PEI25kB (triangle). (b) SPR bands by UV−vis for citrate-stabilized AuNPs (solid line), Au-PEI2kB-mod (dashed−dotted line), Au-PEI10kB (dotted line), and Au-PEI25kB (dashed line). (c) Particle core size and height measured by TEM and AFM, respectively. Error bars in (a) and (c) represent one standard deviation for replicate means and population means, respectively; the particle count is given in Table 1.

Mr) and preparation methods with respect to the response and stability in different media. Furthermore, anecdotal evidence from attempts in our laboratory to replicate published procedures for generating Au-PEI conjugates as well as other types of biologically active cationic AuNPs has yielded results that are frequently inconsistent with the published reports in terms of product uniformity, stability, and lack of agglomerated byproducts, complicating the correlation of material properties with the biological response or application efficacy. To avoid conflating contributions from the NP and PEI characteristics (source, structure, Mr,), manufactured citratecapped AuNPs are commonly used as the initial platform for conjugation. However, systematic evaluations examining the role of citrate in the conjugation process have not been conducted, and the complete removal of citrate during ligand exchange processes or persistence in LBL assemblies is generally assumed. Recently, contributions of citrate in other biologically relevant applications have suggested less than passive or spectator roles in ligand exchange reactions, which will be dependent on the preparation conditions and procedures. For example, Park and Shumaker-Parry recently examined the thiol displacement of citrate on metal NPs and unexpectedly identified persistent heterogeneous surfaces containing coadsorbed thiol and residual citrate, contrary to widely held assumptions regarding thiol displacement of the weakly bound anion.48 Zhang et al. reported that different biomolecules introduced to citrate-capped AuNPs resulted in distinct modes based on size and aggregation state.49 The authors suggested that the role of citrate as a place holder or active participant in the NP attachment can change in systems of similar functionality. Although they did not use molecular spectroscopy to examine specific processes, the study highlights the complexity of preparation and processing in the development of stable, effective NP probes or delivery vectors. A growing body of literature exists that suggest more thorough investigations of attachment and displacement mechanisms are necessary for developing robust, reliable nanomaterial-based biomedical products. Therefore, the current study systematically evaluated the influence that PEI parameters have on the formation of Au-PEI and its corresponding physicochemical properties and more specifically on the conjugation mechanisms of different PEIs

and the connection to colloidal stability. Key parameters of PEI for this work were (A) conformation, comparing linear and branched PEIs (chain and backbone), and (B) Mr. Monodisperse citrate-stabilized AuNPs were used as a platform for functionalization to elucidate any PEI−citrate interactions that could contribute to the previously reported product variations. Additionally, stability studies were conducted covering a broad range of biologically relevant conditions through the systematic use of well-defined protocols.50,51 Together, an improved understanding of specific aspects of the preparation process on the material’s molecular properties can be better correlated with stability and response.

2. EXPERIMENTAL SECTION Specific reagents used in this study are identified in the Supporting Information (SI). All chemicals were used without further purification. Details regarding instrumentation and methodology are also provided in the Supporting Information (SI). 2.1. General Method for the Formation of Au-PEI Conjugates (Au-PEI2kB, Au-PEI10kB, Au-PEI25kB, and AuPEI25kL). One milliliter of each PEI solution (1.0% mass fraction in DI water) was added dropwise to 10 mL of a citrate-stabilized gold colloid suspension, which was then stirred at room temperature for 5 h and purified using centrifugal filtration (Amicon Ultra, Millipore; regenerated cellulose membrane, MWCO = 100 kDa). The filtrate was removed and replenished with filtered DI water, and then purified AuPEIs were passed through a 0.2 μm nylon filter to remove any large impurities such as dust. It is important to note that the purification step was not applied to Au-PEI2kB and Au-PEI25kL because of their rapid aggregation during the conjugation process. 2.2. Modified Method for Au-PEI2kB-mod. To 1 mL of a 10% mass fraction of PEI2kB solution was added dropwise 10 mL of citratestabilized AuNPs, and then the suspension was stirred at room temperature for 5 h. The purification process was the same as the general method. This method ensures continual oversaturation of PEI during the conjugation process.

3. RESULTS AND DISCUSSION 3.1. Preparation of Au-PEIs. Two preparation methods for the Au-PEI conjugates (Au-PEI2kB, 2kB-mod, 10kB, 25kB, and 25kL) were examined in this study. The first procedure (general method) involved the addition of each PEI solution (1.0% mass fraction in DI water) to a suspension of citratestabilized AuNPs (10 nm diameter, nominal); this resulted in B

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Langmuir varying rates of color change relative to the original AuNP suspension, a clear indication of instability in the suspensions that was inconsistent across PEI samples. The addition of PEI2kB and PEI25kL to the AuNP suspensions resulted in immediate color changes and/or precipitation within 1 h. In contrast, the introduction of PEI10kB and PEI25kB to the AuNP suspension yielded a very slight color change (to purplish red) after stirring for 5 h, which indicates improved colloidal stability for the Au-PEI conjugate product. Furthermore, the positive charge of the Au-PEIs hindered the purification process because there was considerable loss of particles onto the filter (regenerated cellulose) during centrifugal filtration. In an effort to improve suspension stability during the conjugation step, the citrate-AuNP suspension (10 mL) was added dropwise to excess PEI2kB and PEI25kL solutions, which is referred to herein as the modified method. Dropwise addition of the citrate-stabilized AuNPs to a 10% mass fraction solution of PEI2kB (1 mL) afforded better suspension stability (the suspension was a slight purple-red color after stirring for 5 h, hereafter Au-PEI2kB-mod) than the product produced by the general method. However, Au-PEI25kL did not display improved stability using the modified method (1.0% mass fraction solution of PEI25kL; the solubility of PEI25kL does not allow the dissolved concentration to reach 10% mass fraction in DI water) and resulted in observable precipitation over 1 h that was similar to the material obtained by the general method. 3.2. Characterization and Physicochemical Properties. To elucidate the contributing factors to reported variability in stability and the response of Au-PEIs, a combination of complementary and orthogonal measurement techniques were used to examine physical properties and mechanistic aspects. The z-average diameter (dz) and zeta potential (ZP) of Au-PEIs were measured in batch mode, and the uncertainties were expressed as one standard deviation of five and three measurements, respectively. As shown in Figure 1a, the purified Au-PEI10kB and Au-PEI25kB prepared by the general method resulted in monomodal size distributions with dz = (62.9 ± 0.3) nm and (78.8 ± 0.6) nm, respectively. The mean polydispersity index (PI) obtained for these measurements was 0.21 and 0.14, respectively. Au-PEI2kB-mod obtained by the modified method exhibited a smaller dz, (21.8 ± 0.73) nm; however, a bimodal size distribution with broader peak widths (PI = 0.38) was observed, indicating that some agglomeration was occurring during the conjugation process. PI values associated with each Au-PEI conjugate provide evidence that higher-Mr PEI more effectively preserves the colloidal stability of the Au-PEI conjugates. The measured ZP of Au-PEIs (at pH ∼8 to 9, dispersed in DI water) was (+24.6 ± 3.0) mV for Au-PEI2kB-mod, (+25.8 ± 0.7) mV for Au-PEI10kB, and (+29.4 ± 1.3) mV for AuPEI25kB, which confirmed the presence of positively charged coronas surrounding the gold core in all cases. The surface plasmon resonance (SPR) band maxima of Au-PEIs were centered at 524, 530, and 534 nm for Au-PEI2kB-mod, 10kB, and 25kB, respectively (Figure 1b), which are consistent with AuNPs in this size range. Au-PEIs were visualized by two microscopy techniques (Figure 1c and Table 1). The average diameter of the gold core by TEM was found to remain essentially constant for asreceived citrate-stabilized AuNPs and conjugated Au-PEI samples (Figure 1c). In contrast to the core size, the mean

Table 1. Summary of AFM and TEM Measurements of Au(Branched) PEIs Showing the Mean Size (Particle Height or Core Diameter, Respectively) with One Standard Deviation and the Number, N, of Particles Evaluated AFM sample citrate AuNPs Au-PEI2kB-mod Au-PEI10kB Au-PEI25kB

TEM

size (nm)

N

± ± ± ±

103 118 114 114

9.1 10.0 11.1 12.1

2.9 1.6 3.2 3.3

size (nm)

N

± ± ± ±

550 221 240 415

8.3 8.3 8.4 8.4

1.3 0.8 0.7 0.9

AFM height was found to increase from ∼9 to ∼12 nm with increasing Mr of the PEI conjugated to AuNPs. Representative TEM and AFM images are presented in the SI (Figures S1 and S2). A4F was used to fractionate and characterize the size distribution of the Au-PEI conjugates utilizing nominal conditions that we previously developed for positively charged nanomaterials.50,52 A4F fractionations of Au-PEI10kBs and AuPEI25kBs exhibit different retention times, tR (SI, Figure S3a,c, solid vertical arrows; note that Au-PEI2kB was not analyzed by A4F because of its relatively high level of polydispersity based on DLS results). Au-PEI10kB eluted faster (tR = 15.4 min) compared to Au-PEI25kB (tR = 17.0 min), consistent with an increase in the observed hydrodynamic size from online light scattering measurements of (61.0 ± 22.6) nm to (76.0 ± 22.0) nm, averaged across the peak full width at half-maximum (fwhm, selected area). The online DLS measurements for the two samples were consistent with the batch-mode DLS results (Figure S3b,d; dashed arrows), showing that the size increases continually across the selected peak area. This broad distribution of products in the observed conjugates is likely resulting from heterogeneous coverage of AuNP surfaces by the PEI but could also indicate that agglomeration is occurring in the channel during the focusing and/or elution steps. The size range observed by online mode DLS is too broad and reaches sizes too large to be accounted for solely by a heterogeneous PEI coating. The distribution of sizes is distinct from the previously reported Au-positively charged dendron conjugates (Au-PCD),50 which exhibited a narrow distribution of z-average sizes across the entire peak. The heterogeneity of the NP surface is further investigated in the subsequent sections to provide a correlation between coverage, composition and colloidal stability. 3.3. Conjugation Mechanism Study. To examine the mechanism of conjugation for PEI as a function of Mr, ATRFTIR and XPS measurements were employed. To examine the interaction between PEI and the citrate moiety on AuNPs, we conducted flow cell experiments with ATR-FTIR (Figure 2). Figure 2 displays the ATR-FTIR spectra for the addition of PEI2kB to the citrate-stabilized AuNP film (spectrum (a)), in aqueous solutions. The spectrum after the addition of three 1 mL aliquots of a 1.0% mass fraction solution of PEI over a similar mixing time for the general method is shown in spectrum (b). The data clearly show the loss of each characteristic citrate vibrational mode after the addition of the PEI2kB. Although the amount of citrate remaining was not quantitatively determined, the resulting spectrum for the PEI2kB-modified AuNP film after flushing and drying predominantly exhibits bands associated with PEI. The removal of citrate is further supported by the comparison of the dried film after rinsing (spectrum c) and the thoroughly washed, wellC

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N stretching modes are observed near 1597 and 1122 cm−1, respectively.53 Characteristic CH2 bending modes and C−C stretching modes are observed at 1454 and 1047 cm−1, respectively.54 Two N−H stretching modes were observed for the primary amine group near 3355 and 3277 cm−1, and an overtone band near 3180 cm−1 was also present. Characteristic C−H stretching modes were observed between 3000 and 2700 cm−1, with the most intense band centered near 2805 cm−1. For the citrate-stabilized AuNPs, the symmetric and asymmetric stretching modes of C−O are observed at 1575 and 1394 cm−1, respectively. The signal intensities for the citrate moiety are significantly depressed in all Au-PEI samples, most clearly depicted by the removal of the band at 1394 cm−1, which suggests that PEI displaces citrate during the conjugation process. However, the resulting conjugation products are not consistent for different Mr PEI samples. For the Au-PEI10kB samples, distinct differences in the IR signatures of the conjugated PEI are observed in the ATR-FITR spectrum (Figure 3, spectrum c). First, the N−H symmetric stretching and C−H stretching bands at 3355 and 2805 cm−1 have been further diminished (Figure 3, spectrum c #) compared to the Au-PEI2kB-mod and Au-PEI25kB samples. Concomitantly, a new band is present that is centered near 988 cm−1, which is consistent with the formation of a quaternary ammonium.55 Although Au-PEI2kB-mod and Au-PEI25kB spectra contain a similar vibrational mode, the relative intensity of the peak is significantly depressed compared to that of the Au-PEI10kB sample. Furthermore, the relative intensity of the C−N stretching mode was diminished relative to that of the C−C stretching modes along with the possible appearance of an additional mode centered near 1335 cm−1. To examine the reproducibility of the observed results, we used three different batches of PEI10kB from two vendors and observed a similar formation of the 988 cm−1 band for each conjugate; we also prepared Au-PEI1.8kB-mod as an analogous conjugate of AuPEI2kB-mod from a different vendor as well to confirm the reproducibility. The ATR-FTIR spectra were consistent for AuPEI2kB-mod and Au-PEI1.8kB-mod (not shown). Investigation of the Au-PEI conjugates was also conducted with XPS to provide further characterization of the surface products and to help with identification of the observed products. In Figure S4 (SI), XPS spectral profiles of the N (1s), C (1s), and Au (4f) regions are shown for one citrate-stabilized AuNP sample, representative purified samples for each branched Au-PEI conjugate, and a representative PEI control because the XPS spectra of each PEI Mr are similar (SI, Figure S5). For the conjugated Au-PEIs, the peak maxima for the Au (4f) region was (83.9 ± 0.1) eV while the C (1s) peak maximum was at (286.5 ± 0.1) eV after energy referencing the N (1s) peak maximum to 400 eV (mean and standard deviation of 18 measurements). The peak-to-peak separation of the C (1s) and N (1s) peak maximums of 113.5 eV was consistent with literature values for PEI.56,57 A qualitative estimation of the relative surface coverage of different Mr PEIs on the AuNP surface was performed by examining the Au/N ratio shown in Figure 4(a). The results demonstrated that the Au/N ratio follows Au-PEI10kB > AuPEI2kB-mod > Au-PEI25kB. Assuming uniform coverage of each PEI size on the AuNPs, the Au/N ratio for a given set of samples would decrease with increasing PEI Mr, but this rationale is inconsistent with the observed trend. Instead, the data suggest that Au-PEI10kB had either a less complete or a thinner polymer layer relative to both the Au-PEI2kB and the

Figure 2. In situ (in the flow cell) ATR-FTIR spectra for (a) citratestabilized AuNPs, (b) loss spectrum after the addition of PEI2kB in the flow cell, (c) dried and washed product after PEI2kB addition, and reference spectrum of (d) dried Au-PEI2kB-mod conjugate (separate batch, purified) and (e) dried PEI2kB for comparison. Absorbance has been normalized and spectra offset on the vertical axis for ease of viewing.

dispersed batch sample (spectrum d) that exhibits signatures very similar to predominantly PEI vibrations. These serial results obtained in situ support the replacement of citrate moieties by PEI chains. Consequently, the ATR spectra provide evidence that conjugation is principally accomplished by a ligand exchange reaction rather than an electrostatically induced layering interaction between the negative surface (citrate) and the positively charged ligand (PEI). Figure 3 shows the ATR-FTIR spectra for Au-PEI2kB-mod, Au-PEI10kB, Au-PEI25kBs, and citrate-stabilized AuNPs. The top spectrum (spectrum a) is representative of a pure PEI25kB sample dried onto the crystal, where the N−H bending and C−

Figure 3. ATR-FTIR spectra for dried AuNP conjugates (a) PEI25kB, (b) Au-PEI25kB, (c) Au-PEI10kB, and (d) Au-PEI2kB-mod, with the native citrate-stabilized AuNPs (Cit. AuNPs). # indicates a substantially diminished stretching band for Au-PEI10kB. Spectra have been normalized on the basis of absorbance and offset on the vertical axis for ease of viewing. D

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the apparent chemical change of the PEI, we examined the solution-phase reaction of equimolar sodium citrate and PEI10kB (10 mmol/L total concentration) using the same experimental conditions, including temperature and mixing, used for the citrate exchange study over more than 1 week. The resulting product of the solution-phase mixture in the absence of the AuNP surface did not result in similar signatures observed for Au-PEI10kB in the ATR-FTIR spectra (SI, Figure S6), which indicates that the reaction is not simply the solutionphase processing of the citrate and PEI, and thus the AuNP likely contributes to the reaction. Combining the conjugation and solution processing data provides a more complete picture regarding the products on the AuNP surfaces as a function of Mr for the branched PEI. First, the vibrational mode centered near 988 cm−1 is consistent with the formation of a quaternary ammonium (R-N(CH2)2R).55 This is also supported by the shift and broadening in the N (1s) region to higher binding energy species that was observed with XPS, which represents more highly oxidized functional groups. The well-known polymeric reactions of carboxylic acids and PEI that result in amide bonds54 would not result in the observed shift of the N 1s region to higher binding energies for Au-PEI10kB observed in the XPS data, which is further corroborating evidence for the quaternary ammonium species; however, the presence of both higher-binding-energy species in the C (1s) region and the preservation of the C−O signatures in the ATR-FTIR data support a reaction between PEI and citrate. Furthermore, because the reaction did not occur in the absence of the AuNP surface, it is possible that the reaction with PEI10kB could be catalyzed by the gold nanoparticle surface, resulting in the formation of a more highly coordinated amine or oxidized amine. The current results outlining a distinct change to the conjugated PEI may have significant implications for both stability and intended efficacy. The observed chemical changes to quaternary ammonium species can affect processing and uptake in biological systems driven by the interfacial reactions of the cells and the nanoparticle surface functionality.58 This unexpected reaction based on either functionality of PEI or undetected impurity in the commercial PEI sample highlights the need for complete characterization methods that include ligand or coating characterization. Furthermore, citrate plays a definitive role in the conjugation process. Relying solely on nanoparticle physical characterization methods would not have distinguished the chemical changes, possibly introducing large uncertainties into subsequent data analysis for stability, reactivity, or biological response. 3.4. Examination of Contributing Factors to Agglomerated Systems. As mentioned in Preparation of Au-PEIs, Au-PEI2kB (prepared by the general method) and Au-PEI25kL (prepared with linear PEI) are agglomerated and consequently precipitated out of solution during the conjugation process using the general method. Again, PEI2kB produced disparate products, either stable conjugates (Au-PEI2kB-mod) or agglomerates (Au-PEI2kB), depending on the preparation method. In this section, we characterize the formation of AuPEI2kB and Au-PEI25kL agglomerates (not Au-PEI2kB-mod conjugates). Interestingly, the resulting agglomerates of the two Au-PEI conjugates exhibited different structures. After storage for 12 h, Au-PEI2kBs were agglomerated and the solution contained large flocculates. Au-PEI25kL samples formed a slightly turbid, purple suspension; the color suggests the formation of agglomerates that are small enough to remain

Figure 4. (a) Au/N ratio for each of the Au-PEI systems plotted vs the PEI Mr. (b) Total carboxyl percentage (COO %) plotted vs ammonium percentage (NR4+%). The line is purely meant as a guide to the eye. Set A was measured at 150 W; set B was measured at 120 W. Each data point represents a single measurement.

Au-PEI25kB. Additionally, it is important to note that there was further variability observed between sets of Au-PEI conjugates created at different times. This was most pronounced when comparing 10 kDa samples between sets (Figure 4a). Peak fitting analysis was performed on the spectra to examine potential variations in the chemistry of the PEI coatings, as seen in Figure S4. By fitting both the citrate-stabilized AuNPs and the PEI controls, the C (1s) line shapes of the Au-PEI samples could be consistently fit with four spectral features. Three of these were the following: a hydrocarbon (CH/CC) feature at (285.3 to 285.4) eV, a carboxyl feature (COO−) at (288.6 to 288.8) eV, and an unknown highly oxidized carbon feature (Cunk) at (289.7 to 290.2) eV, all of which were derived from the citrate-AuNP control. The carbonyl feature (CO) at (286.8 to 286.9) eV from the citrate control was combined into one spectral peak with the amine functionality (C−N) at (286.5 to 286.6) eV from the PEI controls because it was not possible to deconvolute the two features reliably due to proximity. The N (1s) region was fit with a peak representative of the amine feature at ∼400 eV and with an ammonium component (NR4+) which was allowed to span the range from (401 to 401.9) eV. Using the aforementioned fitting procedure, the evaluation of functionalities can be conducted in a consistent manner. For Au-PEI conjugates, the analysis revealed that predominantly CO/CN and COO− functionalities were present in the C (1s) region irrespective of Mr. The surface concentration of the COO− functionality and the ammonium functionality within the N (1s) region appeared to be greatest for the Au-PEI10kB. This can be more clearly observed by plotting the COO− functionality vs NR4+ functionality in atomic percentages, as shown in Figure 4b. Generally speaking, the ammonium and carboxyl functionalities (as measured by XPS) were greatest for Au-PEI10kB and were least for Au-PEI2kB-mod. The source of the COO−-type functionality is likely to be residual citrate not displaced during the conjugation process. The percentage of ammonium species appeared to increase with the COO− functionality following what appeared to be a linear trend. Additionally, this is consistent with FTIR results, where the relative intensity of the symmetric CO stretching mode persisted with the appearance of the 988 cm−1 band in the spectra for Au-PEI specimens. To further investigate the origin of the unique functionality observed for Au-PEI10kB and specifically the role of AuNP in E

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Figure 5. Pictorial representation of disparate agglomerate formation for PEI2kB and PEI25kL conjugation. (a) TEM images of Au-PEI2kB and (b) Au-PEI25kL. Scale bars represent 200 nm, and insets show suspensions Au-PEI2kB (in a) and Au-PEI25kL (in b), 12 h after mixing. (c) Cartoon depiction of conjugation and resulting products.

Figure 6. ATR-FTIR spectra for dried films of (a) Au-PEI2kB and (b) Au-PEI25kL agglomerates. For (a), spectrum (i) shows Au-PEI2kB-mod (well dispersed by modified method), and spectrum (ii) shows washed Au-PEI2kB agglomerates. For (b), spectrum (i) shows PEI25kL only (control), and spectrum (ii) shows washed Au-PEI25kL. Spectra have been scaled on the vertical axis for ease of viewing.

suspended during the experimental time frame (Figure 5(a) Au-PEI2kBs, (b) Au-PEI25kLs; scale bar 200 nm). TEM revealed that Au-PEI2kBs (Figure 5a) form large fractal-like structures that likely result from rapid agglomeration. The AuPEI25kLs formed substantially smaller agglomerate structures, again with a fractal-like appearance (Figure 5b). Furthermore, TEM images suggest that in both cases the gold cores are not fused or in close contact but rather are clearly separated by the PEI coating. This indicates that the PEI surface modification also controls the particle agglomeration mechanism. We hypothesized that this agglomeration process would be induced by bridging effects between particles due to the citrate ions and PEI or would result from incomplete coverage during preparation. To investigate the possible roles that PEI and citrate play in the agglomeration observed in the TEM images, ATR-FTIR

spectra of dried conjugates prepared with different methods were compared for both PEI2kB and PEI25kL (Figure 6). For the Au-PEI2kB (Figure 6a), incomplete removal of the characteristic citrate band at 1394 cm−1 was apparent even after three cycles of centrifugal filtration (spectrum ii), which is clearly differentiated by the blue shift relative to the welldispersed system (spectrum i). Furthermore, a comparison of the ratio of the integrated peak intensities of the bands near 1575 cm−1, which is predominantly citrate, and near 1460 cm−1, which represents the CH2 bending mode of PEI, provides an estimate of the amounts of PEI to citrate present on the surface for all Au-PEI conjugates and is referred to herein as [Cit]/ [PEI]. On the basis of this metric, which is plotted in Figure S7 (SI), for all Au-PEI branched conjugates the [Cit]/[PEI] for spectrum i is greater than that for spectrum ii by more than 50%, indicating that more citrate was retained in the product F

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Figure 7. Stability of Au-PEIs in PBS over time monitored by UV−vis absorbance: (a) Au-PEI2kB-mod, (b) Au-PEI10kB, and (c) Au-PEI25kB.

PEI25kL agglomerates, respectively (as determined by ATRFTIR), are seemingly dependent on the morphological structure of PEI. The rapid and more densely packed structure of Au-PEI2kB agglomerates seems to limit the surface site availability and results in the incomplete removal of citrate. By contrast, the more open fractal structure of Au-PEI25kL presumably allows PEI to more effectively displace the citrate. The relatively good particle dispersion behavior observed for Au-PEI10kB and Au-PEI25kB prepared under the same conditions (by the general method with 1% ligand concentration) is not yet clear, but we speculate that the higher Mr associated with these branched structures could contribute to an enhancement of colloidal stability by providing a thicker barrier less subject to interparticle bridging. 3.5. Stability Studies. Because of the thorough physical and chemical characterization of each conjugate in the previous sections (vide supra), the contributing factors affecting overall stability, including the molecular functionality and coverage that are functions of the preparation methods,51 can be better evaluated and weighted, which is critical to the commercial application of functionalized AuNPs. The best colloidal stability was observed for Au-PEI10kB and Au-PEI25kB, where replicate samples were aged for 6 months under ambient laboratory conditions. The aged samples yielded nearly identical DLS mean sizes and size distributions compared to the initial, freshly prepared and purified products (SI, Figure S9, top). In contrast, the hydrodynamic size distribution for Au-PEI2kB-mod significantly increased within 1 month, indicating reduced colloidal stability compared to that of the higher-Mr PEIs. The associated SPR band intensities and peak positions (UV−vis) provide a more sensitive probe for changes in the near-field environment of the AuNPs (Figure S9, bottom). The substantially red-shifted (Δ ≈ 40 nm) absorbance maximum for Au-PEI2kB-mod suggests a large increase in size via agglomeration. Au-PEI10kB and Au-PEI25kB showed minor red shifts with Δλmax ≈ 5 and 2 nm, respectively. These results suggest that Au-PEIs encapsulated by higher-Mr PEI offer better long-term stability based on the observed changes in the physicochemical properties probed by DLS and UV−vis. For an accurate assessment of the biological activity of AuNPs, an evaluation of their stability in physiologically relevant media is critical. Au-PEIs were tested over a 48 h period in a PBS buffer, consistent with many cell-based exposure assays. Au-PEIs diluted into PBS exhibited improved ligand-dependent stability compared to citrate AuNPs based on UV−vis results. As shown in Figure 7, the trends observed for

that exhibited greater agglomeration both optically and in the TEM images. In contrast to the Au-PEI2kB system, ATR-FTIR spectra of the Au-PEI25kL agglomerates clearly exhibit more efficient removal of citrate through the centrifugal washing step (Figure 6b). The evidence for more efficient removal is based on the observation of no significant band shifts in the washed sample when compared to the pure PEI25kL spectrum toward the characteristic citrate bands at 1394 and 1261 cm−1 following centrifugal purification, as was observed for the Au-PEI2kB. Also, the similar peak heights of N−H bending modes in the region from 1600 to 1700 cm−1 compared to the characteristic C−O stretching mode of citrate at 1575 cm−1 provide further evidence of more efficient citrate removal (SI, Figure S8). Together, the data support a more complete removal of citrate for the linear versus the branched PEI species conjugated to AuNPs examined in the present study, which corresponds to an observed correlation between a higher retention of citrate and more tightly packed agglomerates for these two species (Figure 5). These spectral results provide insight into the mechanisms occurring during the agglomeration process; it is evident that some form of self-assembly of PEI-capped particles was the predominant process controlling the final agglomerate structure, rather than electrostatic interactions (bridging) between PEI and citrate species on the surface. On the basis of TEM and ATR-FTIR experiments, the AuNP agglomeration process in the presence of PEI2kB and PEI25kL is proposed and depicted in Figure 5c. Au-PEI2kB-mod, prepared by the modified method, was relatively well dispersed (Figure 5c bottom); however, Au-PEI2kB prepared by the general method led to the formation of large agglomerates. The difference in the preparation methods exposes the nanoparticles to very different local environments; specifically, the modified method yields AuNPs in a sea of PEI that allows the NPs to remain dispersed during the ligand exchange process. Conversely, the addition of the PEI to the AuNP solution induces the assembly (agglomeration) of unsaturated PEI coatings due to the dilution of PEI in the case of PEI2kB (Figure 5c, top). The data suggest that the Au-PEI25kL agglomerates were formed by a similar self-assembly process, but the final morphology is significantly impacted by the linear structure of PEI25kL (Figure 5c, middle). PEI25kL seemingly has the ability to trap AuNPs, resulting in smaller, more elongated fractal-like agglomerates aligned with the linear chain of the PEI (thin coating on the Au core). The presence and absence of observable citrate in the Au-PEI2kB and AuG

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beyond the scope of the present study, the addition of 10% fetal bovine serum to DMEM, a common medium for growing mammalian cells, may impact the colloidal stability as a result of the interaction with serum proteins, principally bovine serum albumin. An investigation of protein interactions with PEIconjugated AuNPs would be relevant to therapeutic applications and worthy of considerable attention in a future study. Next, we examined the effect of pH on Au-PEI behavior, which is a critical metric for evaluating test materials for biomedical applications because of the wide range of pH environments potentially encountered in physiological systems (ranging from highly acidic to mildly alkaline). The pHdependent stability of Au-PEIs was evaluated over a 12 h period. Notably, all Au-PEIs exhibit very stable behavior in either strongly acidic (50 mmol/L HCl, pH ∼1.3) or highly basic solutions (50 mmol/L NaOH, pH ∼12.7) as shown in Figure S10 (SI). In general, the resistance against acid destabilization of Au-PEIs is greatly improved relative to that of citrate-stabilized AuNPs, which precipitate immediately in strong acid. The Mr of the PEI surface coating on the Au core was not a determining factor for pH resistance. The improved stability of Au-PEIs under acidic conditions can be attributed to the amine functionality acting as a proton sponge to induce increased particle repulsion, unlike the citrate-stabilized AuNPs that quickly agglomerate and precipitate due to neutralization of the carboxylate functional groups. Resistance to acidic conditions may have significant implications for therapeutic applications, particularly for oral delivery and uptake by cytoplasmic lysosomes. At pH 7.05, Au-PEI10kB and AuPEI25kB are stable but notably, and Au-PEI2kB-mod shows instability over time. The behavior of Au-PEI2kB-mod at pH 7.05 was similar to that in PBS, suggesting that a 50 mmol/L salt concentration (NaCl) obtained from mixing HCl and NaOH was sufficient to induce the observed instability. However, this same ionic strength did not induce agglomeration for Au-PEI10kB and Au-PEI25kB. Overall, the series of stability tests performed in aqueous media (shelf life), PBS, DMEM, and over a wide range of pH values reveals a general order for Au-PEI stability in physiologically relevant media (AuPEI25kB ≫ Au-PEI10kB > Au-PEI2kB-mod) and suggests that, in general, higher Mr increases stability. The thermal stability for each Au-PEI conjugate was evaluated by UV−vis absorbance measurements over the range from (20 to 60) °C, which covers the relevant range for most biomedical applications or biological assays. Samples were incubated for 30 min at each temperature before measurements were conducted. The constancy of the SPR band (from UV−vis spectra) confirms that all Au-PEIs are stable with respect to temperature variations over the tested range (SI, Figure S11). As an improved approach to the long-term storage of AuNPs, we assessed the response of Au-PEIs to a lyophilization− reconstitution cycle. Samples were chosen on the basis of the long-term aqueous colloidal stability that has been demonstrated by monitoring particle size and UV−vis absorbance spectra over a 6 month period (Au-PEI10kB and Au-PEI25kB, Figure S9). It has been shown previously that citrate-stabilized AuNPs cannot be reconstituted following lyophilization,51 but AuNPs with other surface modifications have been reported to have a more successful reconstitution capacity.59−61 As shown in Figure S12 (SI), an analysis of reconstituted AuPEIs lyophilized (without the aid of excipients) revealed relatively poor recovery of the primary NP size, resulting in large shifts in the z-average size for both Au-PEI10kB and Au-

the Au-PEI samples in PBS buffer resulted in distinct end points for each sample, which differed from the results obtained for DI water. For Au-PEI2kB-mod (Figure 7a), the red-shifted λmax along with decreased intensity (∼40% reduction over 12 h, where the spectral changes appeared to plateau) and increased scattering signatures (near 700 nm) were observed over 12 h, similar to behavior in DI water. The UV−vis spectrum for AuPEI10kB (Figure 7b) exhibited an initial red shift and increase in absorbance at 700 nm only during the first 4 h. Additionally, the optical density for Au-PEI10kB continuously decreased over 48 h (Figure 7b, up to about 80% loss in λmax), which was the most significant loss in SPR intensity observed among all Au-PEI conjugates studied in the PBS buffer. The loss of signal intensity is consistent with agglomeration products settling (sedimentation process), which differs from the stability in the other media (e.g., DI water) that displayed a relatively constant absorbance peak maxima over 6 months. In stark contrast, AuPEI25kB (Figure 7c) displayed no notable shift in λmax and only an ∼30% reduction in optical density after 48 h in PBS, where the spectral changes appeared to plateau. Finally, there was no observed increase in the baseline near 700 nm, providing further evidence for the absence of a significant agglomerate population in the solution. Overall, the Au-PEI25kB sample showed the greatest stability in the PBS buffer, consistent with stability studies in other media. The stability of the Au-PEI2kB-mod and Au-PEI10kB samples showed distinctly different behavior in the PBS buffer. On the basis of the UV−vis data, the Au-PEI2kB-mod samples form agglomerates that remain suspended in solution, but the Au-PEI10kB samples do not form similar agglomerate types (with respect to either packing density or size) that result in sedimentation. Qualitatively, the degree of loss in SPR absorbance with respect to the period of PBS storage time was consistent with the degree of surface coverage measured by XPS as observed in Figure 4a. On the basis of the FTIR and XPS studies, it is reasonable to surmise that the susceptibility of the Au-PEI10kB in the PBS buffer is due to the unique chemical nature (quaternary ammonium functionality) outlined in previous sections. Furthermore, the relative coverage of PEI or the chemically modified PEI is lowest for the Au-PEI10kB sample, which could also be contributing to the observed decreased stability compared to that of Au-PEI25kB. DLS results in PBS showed trends consistent with UV−vis. The size of Au-PEI2kB-mod and Au-PEI10kB increased to a few hundred nanometers (data omitted, numerical accuracy is not reliable) over time, but Au-PEI25kB data exhibited a nearly constant mean size (∼80 nm) under identical conditions. Compared to the citrate-AuNPs, which are immediately precipitated in PBS,51 all Au-PEIs showed a marked improvement in stability, which is attributed to the larger molecular size of the PEI chains and their hyperbranched structure as an electrosteric protective coating that substantially reduces salt sensitivity (screening of electrostatic charge). DMEM is another common biological test medium that is often used for cell cultures. All Au-PEIs in this study exhibited less stability in DMEM than in PBS, based on UV−vis and DLS (data not shown). This result suggests more complicated interactions between the Au-PEIs and the DMEM components that lead to instability and induced particle agglomeration (e.g., DMEM contains amino acids, salts, and glucose as well as vitamins). Overall, the Au-PEIs demonstrate a notable improvement in stability relative to citrate-stabilized AuNPs, and the degree of stability depends primarily on Mr. Although H

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sequences. The likelihood that the Au core plays a catalytic role in this chemical modification is strongly suggested by the results. Additionally, we demonstrated that spectroscopic analysis of the ligand chemical speciation and state can shed light on the physical properties and mechanisms of conjugation beyond that which can be determined from measurements of stability, zeta potential, and conjugate size alone. In general, the outcome of prepared conjugates and their colloidal stability improve with branching and increased Mr. The preparatory steps for conjugation with the branched and linear PEI lead to variable results, where the amount of citrate retained (on the AuNP core) is impacted by the conjugation processing steps. For branched PEI, a general trend of improving colloidal stability was observed with increasing Mr in all media examined, indicating that the surface coating thickness is a key parameter in determining stability.

PEI25kB samples (Figure S12a,c). Additionally, there was a significant difference in the SPR band shifts (red shift, Figure S12b,d) between Au-PEI10kB and Au-PEI25kB (Δλmax = 32 and 7 nm, respectively), and these corresponded to visibly distinguishable color changes (Figure S12 bottom; left vial, AuPEI10kB; right vial, Au-PEI25kB) before and after lyophilization−reconstitution. The data further supports the general trend observed above for the solution experiments that the effect of ligand Mr on overall stability is obvious and is fairly remarkable when compared to the citrate-stabilized AuNPs, which when subjected to the same process result in complete agglomeration.51,59 Presumably, the PEI coating acts as a barrier to prevent gold core-to-core fusion (or close approach), providing a marked improvement in stability. It is noteworthy that even though DLS results clearly show the presence of substantial agglomeration following lyophilization−reconstitution, the UV−vis spectra for Au-PEI25kB conjugates indicates only a slight red shift and optical density reduction. This suggests that the Au cores, though trapped in larger assemblies, are sufficiently separated to avoid perturbing the SPR absorption due to the thickness of the PEI25k coating. Now, through a series of stability studies we can conclude that Au-PEI25kB, the branched and highest-Mr PEI gold conjugates, exhibited the most stable behavior, in general, and has improved stability compared to that of citrate-stabilized AuNPs in PBS, DMEM, and acidic solution and after the lyophilization−reconstitution cycle. A comparison of the stability of the Au-PEI25kB to our previously developed positively charged dendron-stabilized AuNP (Au-PCD),50 which contained a disulfide linker and three branched polyethylene glycol chains with trimethylammonium termini (Mr ≈ 2.6kD), demonstrates that the PEI conjugate is relatively less stable in physiological salt media (PBS and DMEM) and the lyophilization−reconstitution cycle. However, the PEI conjugate demonstrated better stability than Au-PCD under strongly acidic conditions. This result demonstrates that the metal−ligand bonding character is more effective for salts and phase-transform resistance whereas surface chemical functionality is a dominant effect against acidic attack due at least in part to the buffering (proton-scavenging) capacity of the amine functional groups on the Au-PEI surface.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details including reagents, instrumentation, and measurement methodologies. TEM and AFM images. Supporting data from A4F, XPS, and (ATR)-FTIR measurements and stability tests. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.5b01634.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 301-975-5790. Fax: 301-975-5334. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity 1. Small 2005, 1, 325−327. (2) Nelson, B. C.; Petersen, E. J.; Marquis, B. J.; Atha, D. H.; Elliott, J. T.; Cleveland, D.; Watson, S. S.; Tseng, I. H.; Dillon, A.; Theodore, M.; Jackman, J. NIST gold nanoparticle reference materials do not induce oxidative DNA damage. Nanotoxicology 2013, 7, 21−29. (3) Eustis, S.; El-Sayed, M. A. Why gold nanoparticles are more precious than pretty gold: Noble metal surface plasmon resonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes. Chem. Soc. Rev. 2006, 35, 209−217. (4) Daniel, M. C.; Astruc, D. Gold nanoparticles: Assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem. Rev. 2004, 104, 293−346. (5) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Gold Nanoparticles for Biology and Medicine. Angew. Chem., Int. Ed. 2010, 49, 3280−3294. (6) Yeh, Y. C.; Creran, B.; Rotello, V. M. Gold nanoparticles: preparation, properties, and applications in bionanotechnology. Nanoscale 2012, 4, 1871−1880. (7) Bunz, U. H. F.; Rotello, V. M. Gold Nanoparticle-Fluorophore Complexes: Sensitive and Discerning ″Noses″ for Biosystems Sensing. Angew. Chem., Int. Ed. 2010, 49, 3268−3279. (8) Jiang, Y.; Zhao, H.; Lin, Y. Q.; Zhu, N. N.; Ma, Y. R.; Mao, L. Q. Colorimetric Detection of Glucose in Rat Brain Using Gold Nanoparticles. Angew. Chem., Int. Ed. 2010, 49, 4800−4804. (9) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. H. Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells. Anal. Chem. 2008, 80, 1067−1072.

4. CONCLUSIONS Through a systematic approach, the stability and physicochemical properties of synthesized PEI-AuNP conjugates were thoroughly investigated, and the roles of preparation methodology, PEI structure, Mr, and material source were elucidated, advancing the pursuit of the rational design of these materials in biomedical applications. Using a combination of spectroscopic measurements (ATR-FTIR and XPS), insight into the reaction mechanism of conjugation was achieved, including a distinct role of citrate in both stability and ligand-specific chemical changes. The products of the Au-PEI conjugation were dissimilar for different branched PEI samples and highlight an important observation for applied systems relying on the design of the ligand-system interface for transport or functionality: complete physicochemical characterization of the precursors and resulting products is necessary to developing property− response relationships for these widely used systems. Specifically, we observed the formation of quaternary ammonium species from the amine functionality for AuPEI10kB, which may alter the interaction between cells and conjugated NPs and thereby result in unanticipated conI

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Article

Langmuir (10) Rosi, N. L.; Mirkin, C. A. Nanostructures in biodiagnostics. Chem. Rev. 2005, 105, 1547−1562. (11) Peng, C.; Zheng, L. F.; Chen, Q.; Shen, M. W.; Guo, R.; Wang, H.; Cao, X. Y.; Zhang, G. X.; Shi, X. Y. PEGylated dendrimerentrapped gold nanoparticles for in vivo blood pool and tumor imaging by computed tomography. Biomaterials 2012, 33, 1107−1119. (12) Leduc, C.; Jung, J. M.; Carney, R. R.; Stellacci, F.; Lounis, B. Direct Investigation of Intracellular Presence of Gold Nanoparticles via Photothermal Heterodyne Imaging. ACS Nano 2011, 5, 2587−2592. (13) Kim, D.; Jeong, Y. Y.; Jon, S. A Drug-Loaded Aptamer-Gold Nanoparticle Bioconjugate for Combined CT Imaging and Therapy of Prostate Cancer. ACS Nano 2010, 4, 3689−3696. (14) El-Sayed, I. H.; Huang, X. H.; El-Sayed, M. A. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer. Nano Lett. 2005, 5, 829−834. (15) Popovtzer, R.; Agrawal, A.; Kotov, N. A.; Popovtzer, A.; Balter, J.; Carey, T. E.; Kopelman, R. Targeted Gold Nanoparticles Enable Molecular CT Imaging of Cancer. Nano Lett. 2008, 8, 4593−4596. (16) Chou, L. Y. T.; Ming, K.; Chan, W. C. W. Strategies for the intracellular delivery of nanoparticles. Chem. Soc. Rev. 2011, 40, 233− 245. (17) Van de Broek, B.; Devoogdt, N.; D’Hollander, A.; Gijs, H. L.; Jans, K.; Lagae, L.; Muyldermans, S.; Maes, G.; Borghs, G. Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy. ACS Nano 2011, 5, 4319−4328. (18) Choi, C. H. J.; Alabi, C. A.; Webster, P.; Davis, M. E. Mechanism of active targeting in solid tumors with transferrincontaining gold nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1235−1240. (19) Chompoosor, A.; Saha, K.; Ghosh, P. S.; Macarthy, D. J.; Miranda, O. R.; Zhu, Z. J.; Arcaro, K. F.; Rotello, V. M. The Role of Surface Functionality on Acute Cytotoxicity, ROS Generation and DNA Damage by Cationic Gold Nanoparticles. Small 2010, 6, 2246− 2249. (20) Ghosh, P. S.; Kim, C. K.; Han, G.; Forbes, N. S.; Rotello, V. M. Efficient Gene Delivery Vectors by Tuning the Surface Charge Density of Amino Acid-Functionalized Gold Nanoparticles. ACS Nano 2008, 2, 2213−2218. (21) Cheng, Y.; Meyers, J. D.; Broome, A. M.; Kenney, M. E.; Basilion, J. P.; Burda, C. Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates. J. Am. Chem. Soc. 2011, 133, 2583−2591. (22) Cho, E. C.; Xie, J. W.; Wurm, P. A.; Xia, Y. N. Understanding the Role of Surface Charges in Cellular Adsorption versus Internalization by Selectively Removing Gold Nanoparticles on the Cell Surface with a I-2/KI Etchant 1. Nano Lett. 2009, 9, 1080−1084. (23) Taylor, U.; Klein, S.; Petersen, S.; Kues, W.; Barcikowski, S.; Rath, D. Nonendosomal Cellular Uptake of Ligand-Free, Positively Charged Gold Nanoparticles 3. Cytometry, Part A 2010, 77A, 439− 446. (24) Arvizo, R. R.; Miranda, O. R.; Thompson, M. A.; Pabelick, C. M.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.; Prakash, Y. S.; Mukherjee, P. Effect of Nanoparticle Surface Charge at the Plasma Membrane and Beyond. Nano Lett. 2010, 10, 2543−2548. (25) Lee, S. H.; Bae, K. H.; Kim, S. H.; Lee, K. R.; Park, T. G. Aminefunctionalized gold nanoparticles as non-cytotoxic and efficient intracellular siRNA delivery carriers 1. Int. J. Pharm. 2008, 364, 94− 101. (26) Niidome, T.; Nakashima, K.; Takahashi, H.; Niidome, Y. Preparation of primary amine-modified gold nanoparticles and their transfection ability into cultivated cells 1. Chem. Commun. 2004, 1978−1979. (27) Noh, S. M.; Kim, W. K.; Kim, S. J.; Kim, J. M.; Baek, K. H.; Oh, Y. K. Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles 2. Biochim. Biophys. Acta, Gen. Subj. 2007, 1770, 747−752.

(28) Sandhu, K. K.; McIntosh, C. M.; Simard, J. M.; Smith, S. W.; Rotello, V. M. Gold nanoparticle-mediated Transfection of mammalian cells 1. Bioconjugate Chem. 2002, 13, 3−6. (29) Song, W. J.; Du, J. Z.; Sun, T. M.; Zhang, P. Z.; Wang, J. Gold Nanoparticles Capped with Polyethyleneimine for Enhanced siRNA Delivery. Small 2010, 6, 239−246. (30) Thomas, M.; Klibanov, A. M. Conjugation to gold nanoparticles enhances polyethylenimine’s transfer of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9138−9143. (31) Bekturov, E. A.; Mamutbekov, G. K. Syntheses and properties of films from polymer gels based on polymer-metal complexes. Macromol. Chem. Phys. 1997, 198, 81−88. (32) Kramer, G.; Buchhammer, H. M.; Lunkwitz, K. Investigation of the stability of surface modification by polyelectrolyte complexes influence of polyelectrolyte complex components and of substrates and media. Colloids Surf., A 1998, 137, 45−56. (33) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and In-vivo Polyethyleneimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (34) Lee, Y.; Lee, S. H.; Kim, J. S.; Maruyama, A.; Chen, X. S.; Park, T. G. Controlled synthesis of PEI-coated gold nanoparticles using reductive catechol chemistry for siRNA delivery. J. Controlled Release 2011, 155, 3−10. (35) Note, C.; Kosmella, S.; Koetz, J. Poly(ethyleneimine) as reducing and stabilizing agent for the formation of gold nanoparticles in w/o microemulsions. Colloids Surf., A 2006, 290, 150−156. (36) Sun, X. P.; Dong, S. J.; Wang, E. K. One-step synthesis and characterization of polyelectrolyte-protected gold nanoparticles through a thermal process. Polymer 2004, 45, 2181−2184. (37) Sun, X. P.; Dong, S. J.; Wang, E. K. One-step preparation of highly concentrated well-stable gold colloids by direct mix of polyelectrolyte and HAuCl4 aqueous solutions at room temperature. J. Colloid Interface Sci. 2005, 288, 301−303. (38) Boyer, C.; Bousquet, A.; Rondolo, J.; Whittaker, M. R.; Stenzel, M. H.; Davis, T. P. Glycopolymer Decoration of Gold Nanoparticles Using a LbL Approach. Macromolecules 2010, 43, 3775−3784. (39) Elbakry, A.; Zaky, A.; Liebkl, R.; Rachel, R.; Goepferich, A.; Breunig, M. Layer-by-Layer Assembled Gold Nanoparticles for siRNA Delivery. Nano Lett. 2009, 9, 2059−2064. (40) Lee, M. Y.; Park, S. J.; Park, K.; Kim, K. S.; Lee, H.; Hahn, S. K. Target-Specific Gene Silencing of Layer-by-Layer Assembled GoldCysteamine/siRNA/PEI/HA Nanocomplex. ACS Nano 2011, 5, 6138−6147. (41) Cebrian, V.; Martin-Saavedra, F.; Yague, C.; Arruebo, M.; Santamaria, J.; Vilaboa, N. Size-dependent transfection efficiency of PEI-coated gold nanoparticles. Acta Biomater. 2011, 7, 3645−3655. (42) Sullivan, M. M. O.; Green, J. J.; Przybycien, T. M. Development of a novel gene delivery scaffold utilizing colloidal gold-polyethylenimine conjugates for DNA condensation. Gene Ther. 2003, 10, 1882− 1890. (43) Hu, C.; Peng, Q.; Chen, F. J.; Zhong, Z. L.; Zhuo, R. X. Low Molecular Weight Polyethylenimine Conjugated Gold Nanoparticles as Efficient Gene Vectors. Bioconjugate Chem. 2010, 21, 836−843. (44) Kim, K.; Lee, H. B.; Lee, J. W.; Park, H. K.; Shin, K. S. Selfassembly of poly(ethylenimine)-capped Au nanoparticles at a toluenewater interface for efficient surface-enhanced Raman scattering. Langmuir 2008, 24, 7178−7183. (45) Chen, C. C.; Hsu, C. H.; Kuo, P. L. Effects of alkylated polyethylenimines on the formation of gold nanoplates. Langmuir 2007, 23, 6801−6806. (46) Wang, S. T.; Yan, J. C.; Chen, L. Formation of gold nanoparticles and self-assembly into dimer and trimer aggregates. Mater. Lett. 2005, 59, 1383−1386. (47) Kuo, P. L.; Chen, C. C.; Jao, M. W. Effects of polymer micelles of alkylated polyethylenimines on generation of gold nanoparticles. J. Phys. Chem. B 2005, 109, 9445−9450. J

DOI: 10.1021/acs.langmuir.5b01634 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir (48) Park, J.-W.; Shumaker-Parry, J. S. Strong Resistance of Citrate Anions on Metal Nanoparticles to Desorption under Thiol Functionalization. ACS Nano 2015, 9, 1665−1682. (49) Zhang, S.; Moustafa, Y.; Huo, Q. Different Interaction Modes of Biomolecules with Citrate-Capped Gold Nanoparticles. ACS Appl. Mater. Interfaces 2014, 6, 21184−21192. (50) Cho, T. J.; MacCuspie, R. I.; Gigault, J.; Gorham, J. M.; Elliott, J. T.; Hackley, V. A. Highly Stable Positively Charged DendronEncapsulated Gold Nanoparticles. Langmuir 2014, 30, 3883−3893. (51) Cho, T. J.; Zangmeister, R. A.; MacCuspie, R. I.; Patri, A. K.; Hackley, V. A. Newkome-Type Dendron-Stabilized Gold Nanoparticles: Synthesis, Reactivity, and Stability. Chem. Mater. 2011, 23, 2665−2676. (52) Gigault, J.; Cho, T. J.; MacCuspie, R. I.; Hackley, V. A. Gold nanorod separation and characterization by asymmetric-flow field flow fractionation with UV-Vis detection. Anal. Bioanal. Chem. 2013, 405, 1191−1202. (53) Yang, T.; Hussain, A.; Bai, S.; Khalil, I. A.; Harashima, H.; Ahsan, F. Positively charged polyethylenimines enhance nasal absorption of the negatively charged drug, low molecular weight heparin. J. Controlled Release 2006, 115, 289−297. (54) Jager, M.; Schubert, S.; Ochrimenko, S.; Fischer, D.; Schubert, U. S. Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application. Chem. Soc. Rev. 2012, 41, 4755−4767. (55) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, 1991. (56) Suriyanarayanan, S.; Lee, H. H.; Liedberg, B.; Aastrup, T.; Nicholls, I. A. Protein-resistant hyperbranched polyethyleneimine brush surfaces. J. Colloid Interface Sci. 2013, 396, 307−315. (57) Louette, P.; Bodino, F.; Pireaux, J.-J. Poly(ethylene imine) (PEI) XPS Reference Core Level and Energy Loss Spectra. Surf. Sci. Spectra 2005, 12, 54−58. (58) Yang, T.; Hussain, A.; Bai, S.; Khalil, I. A.; Harashima, H.; Ahsan, F. Positively charged polyethylenimines enhance nasal absorption of the negatively charged drug, low molecular weight heparin. J. Controlled Release 2006, 115, 289−297. (59) Daniel, M. C.; Grow, M. E.; Pan, H. M.; Bednarek, M.; Ghann, W. E.; Zabetakis, K.; Cornish, J. Gold nanoparticle-cored poly(propyleneimine) dendrimers as a new platform for multifunctional drug delivery systems. New J. Chem. 2011, 35, 2366−2374. (60) Rahme, K.; Vicendo, P.; Ayela, C.; Gaillard, C.; Payre, B.; Mingotaud, C.; Gauffre, F. A Simple Protocol to Stabilize Gold Nanoparticles using Amphiphilic Block Copolymers: Stability Studies and Viable Cellular Uptake. Chem. - Eur. J. 2009, 15, 11151−11159. (61) Zhang, L.; Li, P.; Li, D.; Guo, S.; Wang, E. Effect of freezethawing on lipid bilayer-protected gold nanoparticles. Langmuir 2008, 24, 3407−3411.

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DOI: 10.1021/acs.langmuir.5b01634 Langmuir XXXX, XXX, XXX−XXX