Separation of PEGylated Gold Nanoparticles by Micellar Enhanced

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Separation of PEGylated Gold Nanoparticles by Micellar Enhanced Electrospun Fiber Based Ultrathin Layer Chromatography Yanhui Wang, and Susan V Olesik Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04442 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Analytical Chemistry

Separation of PEGylated Gold Nanoparticles by Micellar Enhanced Electrospun Fiber Based Ultrathin Layer Chromatography Yanhui Wang and Susan V. Olesik* Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, United States of America *Corresponding author: Phone: (614) 292-0733, Fax: (614) 292-1685, E-mail: [email protected] ABSTRACT: Gold nanoparticles (AuNPs) are of great interest in many fields, especially in biomedical applications. Thiol terminated polyethylene glycol (PEG) is the most widely used polymer to increase the biocompatibility of nanoparticle therapeutics. Herein, a rapid method for separation and characterization of PEGylated AuNPs on an ultrathin layer chromatographic (UTLC) plate using electrospun polyacrylonitrile (PAN) nanofibers as the stationary phase is described. AuNPs with sizes ranging from 10 to 80 nm and 30 nm AuNPs coated with various molecular weight of PEG (2, 5, 10 and 20 kDa) were all successfully separated by UTLC using optimized conditions. The fabrication of electrospun UTLC is simple, fast and inexpensive. The UTLC, with much thinner sorbent layer (10 times thinner than traditional TLC) and small fiber size (~300 nm), requires minimal mobile phase solvent and provides faster separation and higher resolution compared to other separation methods for AuNPs. AuNPs with different sizes and different PEG molecular weights were well separated within 5 min with lowest plate height < 2 µm and resolution value > 1.5. As an example of this method, the size transformation of AuNPs in serum protein was determined quantitatively.

Due to their extraordinary physicochemical properties, gold nanoparticles (AuNPs) are increasingly used in many fields including electronics,1 food industry,2 cosmetics,3 and especially biomedical applications, such as bioimaging,4 targeted drug and gene delivery,5 cancer diagnostics and immunotherapy.6 For biomedical applications, one of most crucial factors is the surface functionality of AuNPs.7 Unfunctionalized AuNPs are unstable and prone to agglomerate quickly in biological environments, which in turn will exhibit unpredictable effects that can enormously complicate their interactions with biological molecules and cell organelles.8 Thiol-containing polyethylene glycol (PEG) is one of the most widely used biocompatible capping agents to stabilize AuNPs.9,10,11 PEGylated AuNPs have increased stability in vivo, reduced non-specific binding to proteins, decreased cellular uptake and enhanced systemic circulation times compared to unfunctionalized AuNPs or AuNPs with other capping agents, like citrate and hexadecyltrimethylammonium bromide (CTAB).12,13 The size of the AuNPs is the other most critical factor that can largely affect their performance in biomedical applications. Both cellular uptake and cytotoxicity of AuNPs are greatly dependent on nanoparticle size.14,15 On the other hand, most synthetic methods of producing AuNPs result in a distribution of sizes. Furthermore, the length of the end capping compound can effectively change the hydrodynamic radius of AuNPs. Therefore, attention is turning towards developing efficient methods for size characterization. Traditionally, the size of AuNPs is evaluated by microscopic or spectroscopic techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS).

However, electron microscopic methods may be time consuming. Moreover, artifacts can be generated during sample preparation.16 Most importantly, these techniques do not involve a separation process.17 Although DLS offers an obvious advantage over microscopic methods as it measures the hydrodynamic radius, which includes the size of soft coatings on AuNPs, it is still susceptible to skewed results when a sample is polydisperse.18 In recent years, separation techniques such as size exclusion chromatography (SEC),17,19 hydrodynamic chroPmatography (HDC),20 field-flow fractionation (FFF),21,22 capillary electrophoresis (CE)23,24 and gel electrophoresis (GE)25,26 was applied to fractionate AuNPs by size. In SEC, sample degradation or irreversible adsorption of NPs onto the stationary phase may occur, although this effect can be minimized by adding surfactants into the mobile phase.17 HDC separates NPs based on their hydrodynamic radius and the separation is independent on the coating or surface charge of NPs.16 FFF provides continuous and high-resolution separation for NPs in the size range of 1 nm to 1 µm.16 Hansen et al. showed that asymmetric-flow FFF (AF4) is a useful tool for the separation of 30 nm AuNPs coated with different molecular weights of polyethylene glycol.22 However, FFF usually suffers overloading, incomplete sample recovery, and requires expensive instrumentation.16 CE is commonly-used for the separation of AuNPs based on their size and charge, but it provides low sensitivity to UV detection as only very small volumes of sample can be loaded into CE capillaries.16 PEGylated Au NPs were extensively studied by gel electrophoresis due to the ease of detection via their surface plasmon resonance (SPR).25,26 Nevertheless, this technique is time consuming (30 min to

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several hours) to achieve good separations and requires large sample volume.25 Recently, Yan et al. reported the separation of AuNPs using commercial silica gel high performance thin layer chromatography (HPTLC).27,28 Differently sized citratefunctionalized AuNPs were separated with sufficient resolving power, however, the development time was 20 min. Consequently, there is still demand for other techniques for the separation of AuNPs. Ultrathin layer chromatography (UTLC) was first introduced in 2001 using monolithic silica as the stationary phase. Our group reported electrospun nanofibers as the stationary phases for UTLC. Electrospun UTLC offers several distinct advantages: 1) This method has minimum instrumentation requirements. The fabrication of electrospun UTLC plates is simple, fast and low cost. Moreover, a wide range of polymers or materials can be electrospun into nanofibers;29 2) With almost 10 times thinner sorbent layer compared to commercial HPTLC plates and in the absence of a binder, the reported electrospun UTLC plates have showed enhanced separation efficiency, reduced sample and solvent consumption and increased speed of analysis;30 3) Crude samples can be directly applied onto the UTLC plates with minimum sample preparation.27 Previously, electrospun polyacrylonitrile (PAN) and composite nanofibers were successfully applied for separations of laser dyes,30 polycyclic aromatic hydrocarbons (PAHs),31 amino acids and proteins.32 In this work, for the first time, UTLC was employed to separate PEGylated AuNPs with different sizes and AuNPs coated with various molecular weights of PEG.

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potentials of AuNPs were measured using NanoBrook ZetaPALS Potential Analyzer (Brookhaven Instruments Corporation). Absorption spectra of AuNPs were recorded using an Agilent Cary 5000 UV-Vis-NIR spectrometer; water was used as the reference solvent. Electrospinning. A syringe pump (Harvard Apparatus, Holliston, MA), a high voltage power supply (Spellman, Hauppauge, NY), a stainless-steel collector covered with aluminum foil (Reynold Super Strength) and a Plexiglas enclosure were used as the electrospinning apparatus as previously described.30 A nitrogen purge and a VWR Traceable® humidity sensor was used to control and monitor the relative humidity in the closed chamber while electrospinning. Nanofibers were electrospun at room temperature with relative humidity below 20%. The distance between the collector and the tip of the needle was kept at 15 cm. The voltage was kept at 16 kV, and the flow rate was kept at 0.3 mL/h. Each nanofiber mat was electrospun for 10 min. Ultrathin Layer Chromatography. After the electrospun nanofiber mats were prepared, they were cut into 3 cm × 6 cm UTLC plates. Hamilton® micro syringe (10 µL) were used for spotting AuNPs onto the origin line drawn at 1 cm from the bottom of the plate. All the AuNPs samples have mass concentration of 0.05 mg/mL. The volume of each AuNP solution spotted on UTLC was controlled at 0.25 µL. UTLC plates were then placed in a 250 mL developing chamber containing 5 mL of mobile phase. Prior to each development, the mobile phase was allowed to equilibrate for 10 min. PEGylation of AuNPs. AuNPs with different molecular weight of PEG were prepared according to the method reported by Smith et al.33 Briefly, PEG powder was dissolved in Milli-Q water with the concentration of 6, 15, 30 and 60 mg/mL for the 2, 5, 10 and 20 kDa PEG, respectively. The 1.2 mL of each PEG solution was added to 10 mL of 30 nm citrate stabilized AuNPs. The mixtures were then magnetically stirred at room temperature for 24 h to allow complete exchange of PEG with citrate. Each PEGylated AuNP solution was then centrifuged at 14,000 rpm for 60 min. After centrifugation, the supernatant containing excess PEG was removed and the AuNPs were redispersed in Milli-Q water by vortex mixing.

EXPERIMENTAL SECTION Materials. 10, 30, 50 and 80 nm AuNPs (PEGylated, 5 kDa) and 10 and 30 nm citrated stabilized AuNPs were purchased from NanoComposix Inc. (San Diego, CA). Methoxy-poly (ethylene glycol)-thiol (mPEG-SH) was purchased from Laysan Bio Inc. with different lengths: 2, 5, 10 and 20 kDa. PAN, average Mw 150,000 g mol-1, 3-(cyclohexylamino)-1propanesulfonic acid (CAPS, ≥ 98%), sodium dodecyl sulfate (SDS, BioReagent, ≥98.5), hexadecyltrimethylammonium bromide (CTAB, ≥ 98%), polyoxyethylene (23) lauryl ether (Brij-35) and bovine serum albumin (BSA, lyophilized powder, ≥ 96%) were purchased from Sigma-Aldrich (St. Louis, MO). N, N-Dimethylformamide (DMF) (99.8%), HPLC grade 2propanol (99.9%) and tris(hydroxymethyl)aminomethane (Tris, molecular biology grade) were acquired from Fisher Scientific (Fair Lawn, NJ). Milli-Q water was purified to 18.1 MΩ by a Barnstead Nanopure Infinity System from Thermal Scientific Inc. (Odessa, TX). Instrumentation. The morphology of electrospun nanofibers was characterized using a FEI Nova NanoSEM 400 (FEI Corporate, North America NanoPort, Hillsboro, OR) scanning electron microscope (SEM). Each sample was sputter coated with AuPd for 90 s at 17 mA using a sputter coater (Cressington Scientific Inst., Watford, UK) to create a conductive surface before SEM analysis. Fiber diameters were measured on SEM images using ImageJ software (Available at http://www.rsbweb.nih.gov/ij/index.html). A Canon A650IS 12.1 MP digital camera was used for acquisition and documentation of UTLC images. Hydrodynamic diameters of AuNPs were measured using dynamic light scattering instrument (Brookhaven Instruments Corporation, BI-200SM). Zeta

RESULTS AND DISCUSSION Electrospun fibers. Electrospun PAN nanofibers were characterized by SEM (Figure 1). PAN nanofibers exhibited uniform morphology, which is highly desirable in the application of UTLC. The average fiber diameter was 296 ± 26 nm. The electrospun PAN UTLC possesses microscale interstitial spaces between the nanofibers, which are large enough for AuNPs to enter the spaces and be retained on the surface of nanofibers.

Figure 1. (A) SEM image and (B) the fiber diameter distribution of electrospun PAN nanofibers.


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Analytical Chemistry Mobile Phase Conditions. Mobile phase constituents that may affect the retardation factor (Rf) and efficiency of the sample spots were evaluated. Effect of Surfactants. SDS, CTAB and Brij-35 represents surfactants that differ according to the charge of their polar head: anionic, cationic and nonionic, respectively. These surfactants were used to investigate the effect of surfactant type on the retardation factors of PEGylated AuNPs with size of 10, 30, 50 and 80 nm. Micelles form in the mobile phase when the concentration of the surfactant is above its critical micelle concentration (CMC). A concentration range including below the CMC to concentrations well above CMC was studied for each surfactant. As shown in Figure 2, the selectivity between all AuNPs improved with an increase of SDS concentration above its CMC; on the contrary, all the AuNPs remained strongly retained regardless the concentration change of CTAB in the mobile phase, which indicated that there was minimal association between the PEGylated AuNPs and CTAB. Previous studies noted that surfactants may bind cooperatively to nonionic water-soluble polymers such as PEG to form micelle-polymer complexes,34, 35 but these interactions are largely restricted to anionic surfactants (e.g. SDS).36,37 Manna et al. also found that there was no direct or indirect associative interaction between CTAB micelles and PEG.38 Interactions between PEGylated AuNPs and SDS could be mainly due to the electrostatic interaction between PEG and the headgroup of the SDS and the hydrophobic interaction between the hydrophobic sections of these two species. The Rf values of all AuNPs increased with increasing Brij-35 concentration; however, the selectivity among the AuNPs did not increase for Brij-35 as much as that observed for SDS. This may be a result of the identical structures of PEG and the polar head of Brij-35, therefore, the strong eluent strength of the Brij-35 mobile phase hindered its selectivity. As a consequence, the 3% SDS concentration was adopted for the subsequent studies unless otherwise noted.

fore, the volume percentage of 2-propanol added to the micellar mobile phase was maintained below 25%. Tsianou et al. illustrated that the addition of 2-propanol caused increased clustering of PEG when both SDS and PEG were in an aqueous solution and the SDS micelle shape changed from ellipsoidal to a spherical. Furthermore, the surface area of a SDS micelle decreases from 45 to 16 nm2. However, this decrease in micelle size would also increase the total amount of SDS micelles that would be attached to the PEG.40 Increasing amount of SDS micelles on the Au-PEG can essentially increase the hydrodynamic diameter of AuNPs. As shown in Figure S-1, the hydrodynamic diameter of AuNPs increases with the addition of 2-propanol in the aqueous mobile phase with 3% of SDS. This increase in the hydrodynamic diameter of the AuNPs would cause the observed increase in retention with addition of 2-propanol. Figure 3A shows clearly that less than 10% of 2-propanol, four differently sized AuNPs could be well separated from each other. On the other hand, when aqueous-2-propanol mobile phase system was used, 40-60% of 2-propanol was needed to separate these AuNPs with relatively poor selectivity. In this regard, micellar enhanced UTLC offers not only satisfactory separation for PEGylated AuNPs, but also an eco-friendly chromatographic system. For the subsequent studies, 7.5% (v/v) of 2-propanol/ in water was used.

Figure 3. Effect of (A) in micellar mobile phase, (B) 2-Propanol in aqueous phase, (C) pH on retardation factors of 10 nm, 30 nm, 50 nm and 80 nm Au-PEG.

Effect of pH. For PEGylated AuNPs, changes in pH alters their surface. Specifically, the lone electron pair on the ether oxygen atom of PEG binds to hydrogen ions in aqueous solution making the PEG layer on AuNPs positively charged, which favors the adsorption of the anionic SDS micelles.37,41 As a result, the electrostatic interaction between SDS and PEG depends on the concentration of hydrogen ions in the solution.37 Therefore, it is necessary to optimize the pH of the mobile phase. The effect of pH on the retardation factors of AuNPs was investigated over the pH range of 3-11. As shown in Figure 3C, all the AuNPs were more retained and better separated for pH values ≥ 5. Since the positive charge on PEG is reduced as the pH increases, the increased retention of the AuNPs likely results from decreased electrostatic attraction between the PEGylated AuNPs and the negatively charged SDS micelles in the mobile phase. Basic condition near pH 8-9 provided the highest selectivity. Therefore, pH value of 8.5 was used to

Figure 2. Effect of (A) SDS, (B) CTAB, (C) Brij-35 on retardation factors of 10 nm, 30 nm, 50 nm and 80 nm Au-PEG.

Effect of Alcohol in the Mobile Phase. The effect of the addition of 2-propanol as the organic modifier into the micellar mobile phase was studied. Since hydrophobic interaction is the driving force for the micelle formation, large proportions of modifiers can totally disrupt the micelle structure.39 There-



and indicates that the surface of AuNPs was PEGylated.43 To separate these AuNPs

separate differently sized PEGylated AuNPs. Table 1. Core diameter, concentration, surface area and hydrodynamic diameters of differently sized AuNP.

Au Nanoparticles

Au-PEG (5 kDa) 10 nm

30 nm

50 nm

80 nm

Core Diameter (nm)a

11 ± 1

33 ± 4

51 ± 7

82 ± 11

Mass Concentration (mg/mL)

0.051

0.053

0.052

0.054

Surface Area (nm2)

314

2830

7850

20100

Hydrodynamic Diameter (nm)

43.2

57.4

78.5

111.0

a

Values obtained from nanoComposix.

Separation of AuNPs with Different Sizes and Molecular Weights of PEG. Under the optimized condition, PEGylated AuNPs with size of 10, 30, 50 and 80 nm were well separated from each other (Figure 4A). The results of this work clearly demonstrated that the smaller the size of AuNPs, the faster the migration on the UTLC. This may be attributed to the difference in the surface area of these AuNPs. As listed in Table 1, smaller AuNPs have a smaller surface area, thus, their chance to interact with the PAN stationary phase is smaller than larger AuNPs. Moreover, for the same length of PEG, the number of PEG capped on a smaller AuNP is less than the one on a larger particle,42 therefore, smaller AuNPs can be eluted more easily in the micellar mobile phase. It is worth noting that there were stains remaining at the origin during the separation of AuNPs, and they were most possibly from the impurities in the solution of AuNPs, such as excess Au ions or PEG molecules. To confirm our assumption, standard ionic Au and pure 5 kDa PEG were spotted separately on the PAN UTLC. As shown in Figure S-2, after the micellar mobile phase development, Au ions remained at the origin, while 5 kDa PEG migrated to Rf = 0.4. Yan et al. also observed the strong retention of Au ions at the starting point on silica HPTLC under micellar mobile phase condition.27 Therefore, the stains at the origin are ascribed to the excess ionic gold species in the AuNP solution. The chain length of PEG can also influence the biocompatibility of AuNPs.33 Hence, it is important to separate AuNPs capped with different molecular weight of PEG. In this work, we coated 30 nm AuNPs with 2, 5, 10 and 20 kDa PEG and separated them on electrospun PAN UTLC. After coating and prior to separation, the PEGylated AuNPs and the citrated stabilized AuNPs (starting material) were both characterized by UV-vis spectroscopy. After PEGylation, a slight red shift (2 nm) in the plasmon absorption was observed in Figure S-3, which is in accordance with the results of other studies.42 This shift is due to the increase in the dielectric constant of AuNPs

Figure 4. Separation of (A) differently sized PEGylated AuNPs using 3% SDS in Tris buffer (pH 8.5, 10 mM) with 7.5% 2Propanol as the mobile phase: lane 1, 10 nm; lane 2, 30 nm; lane 3, 50 nm; lane 4, 80 nm; lane 5, mixture of 10 nm and 50 nm; (B) Au NPs with different MW of PEG using 2% SDS in CAPS buffer (pH 10, 10 Mm) with 7.5 % 2-Propanol as the mobile phase: lane 1, 2 kDa; lane 2, 5 kDa; lane 3, 10 kDa; lane 4, 20 kDa; lane 5, mixture of 2 kDa and 20 kDa.

with different length of PEG, the composition of a suitable micellar mobile phase was examined, and the optimization procedure is detailed in SI. As shown in Figure 4B, using 2% SDS in CAPS buffer (pH 10, 10 mM) with 7.5 % 2-propanol, four AuNPs coated with different molecular weight of PEG were sufficiently separated from each other with very compact spots. Figure 4B demonstrates that increasing the PEG molecular weight leads to a slower migration of AuNPs on UTLC. Longer PEG chains can essentially increase the hydrodynamic diameter of the AuNPs. Although all these AuNPs have the same core size of 30 nm, the hydrodynamic diameters of 2, 5, 10 and 20 kDa PEGylated AuNPs measured by DLS are 38, 48, 64 and 96 nm, respectively. Therefore, the trend observed in the separation of differently sized AuNPs holds true for the AuNPs with different length of PEG: the smaller sized AuNPs migrated faster. In addition, the selectivity between these four PEGylated AuNPs can be ascribed to the difference in their hydrophobicity. Increasing the polymer length of PEG substantially enhances its hydrophobicity because the ethylene repeat unit is extended.44


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Analytical Chemistry Table 2. Summary of Flory radius (RF), layer thickness (L), PEG-PEG distance (D), Area per molecule (A), density (d) and effective number of PEG (Neff) for different sized PEGylated AuNPs. Au-PEG (nm)

RF (nm)

L (nm)

D (nm)

A (nm2)

d (nm-2)

10

6.0

16.6

1.3

1.3

0.76

59.3

30

6.0

13.7

1.7

2.3

0.43

301.1

50

6.0

14.3

1.6

2.1

0.48

941.7

80

6.0

15.5

1.4

1.6

0.62

3103.2

Neff

layer (L), which can be determined by DLS in a pure solvent.47 The calculated RF, L and D values for PEG on different sized AuNP are listed in Table 2. When D is greater than RF, the PEG adopts “mushroom” regime with the chains folding back over the AuNP surface resulting a thin PEG layer (small L). When D is smaller than RF, the PEG exists in “brush” regime with the chains stretching out from the AuNP resulting a thick PEG layer (large L).48 By comparing the calculated D and RF for each sized Au-PEG in Table 2, the results suggested that PEG chains on all AuNPs used in this study existed in “brush” regime coating.

PEGylated AuNPs in SDS Micellar UTLC. Upon addition of SDS into the mobile phase, the cyano bonded PAN stationary phase was modified by the adsorption of SDS monomers with their charged heads oriented inward and the hydrophobic tails outward.45 The modified PAN stationary phase acted like a pseudo-alkyl bonded phase, which bound to PEG through hydrophobic interaction. When the concentration of SDS exceeded the CMC, micelles were formed in the mobile phase and they would bind to PEG polymer chains around AuNPs. As the SDS concentration further increased, the number of micelles in the mobile phase increased, whereas the number of free monomers in the mobile phase and the bounded monomers on the PAN remained constant to give a stable modified stationary phase. The presence of SDS micelles can essentially change the conformation of PEG chains. Studies using smallangle neutron scattering illustrate that PEG exhibits a random coil like conformation in diluted aqueous solution without SDS micelles.46 When SDS micelles are present in the solution, they bind along the PEG polymer chain to give an extended and “beaded necklace” structure.40 For instance, in the presence of 3% (w/v) SDS in the mobile phase, the hydrodynamic diameter of a 10 nm Au-PEG measured by DLS changed from 43.2 nm to 63.7 nm (Table S1). The zeta potential of Au-PEG became more negative after the addition of SDS above the CMC, which also confirms the association of negative SDS micelles with the PEG polymers (Table S2). The extended polymer conformations increased the likelihood of their interactions with the stationary phase. While Au-PEG were well separated using the optimized condition on electrospun UTLC (polyacrylonitrile nanofibers), they were all strongly retained on commercial Silica Gel HPTLC plates. This is most likely due to the different surface functionality on each stationary phase (cyano groups on nanofibers, silica on commercial HPTLC) (Figure S-7). SDS in the mobile phase can modify cyano groups to generate pseudoalkyl bonded phase, whereas hydrophilic surface is generated when using SDS on the negatively charged silanol group.45 The completely opposite chromatography mode gave distinct retention of Au-PEG. Retention Order and PEG Density. To understand the retention order, the density of PEG molecules attached to the AuNP must first be confirmed. There are two essential parameters that used to understanding the grafting density of PEG: Flory radius (RF) and the distance between the individual grafted PEG chains (D).47,48 RF can be calculated by knowing the number of monomers (N) and the length of one monomer (a): RF = aN3/5 (1) For 5 kDa of PEG, N=113.5 and a= 0.35 nm. As shown in equation 2, the D value is correlated to the thickness of PEG

D=

⁄

(2)

Knowing the distance between grafted PEG molecules, the area (A) occupied by each PEG chain (nm2) on a spherical AuNP can be calculated using the following equation:

A = π + h = π , h 1.5 and up to 3.1. As previously stated, band broadening was observed at 25 mm migration distance or longer for all AuNPs. Consequently, 15 mm solvent migration distance was used for all the separations in this work. The H and Rs for AuNPs capped with different molecular weight of PEG were also measured under optimized condition and listed in Table S4. Electrospun PAN UTLC exhibited comparable chromatographic performance for PEGylated AuNPs from both categories (size and molecular weight).

Figure 5. Plot of (A) Retardation factor vs. Log (Neff) for different sized Au-PEG with R2 =0.9961 (B) Retardation factor vs. Log (MWeff) for AuNPs capped with different molecular weight of PEG R2 = 0.9983.

Chromatographic Performance of Electrospun UTLC. The chromatographic performance of electropsun ULTC was evaluated in terms of plate height (H), resolution (Rs) and development time (t). The plate number (N) describe the separation efficiency of TLC as it is related to the migration distance (Zs) and the spot width (w) of the sample:

N = 16

(6)

H can be obtained by using the equation H = Zs/N. Therefore, H is inversely proportional to N and directly proportional to w. The goal of efforts to minimize band broadening in TLC is to obtain small H values.49 In this work, the changes in H of each PEGylated AuNPs were examined as a function of solvent migration distance on electrospun PAN UTLC. As shown in Figure 6A, the H values remained approximately the same with increasing distance from 5 to 25 mm for all AuNPs except 80 nm AuNP. The H values of all AuNPs increased significantly at longer migration distance, which were mainly attributed to the longitudinal diffusion, because the mobile phase velocity gradually slowed down as it migrated up on the plate due to the capillary driven force in TLC.49 Based on Figure 6A, the smallest H value can be achieved was ~1.2 µm for 10 nm AuNP, and H for all AuNPs were below 10 µm when the migration distance was within 20 mm. Migration distance exceeding 25 mm could result in band broadening to some extent. However, H itself cannot adequately tell the appropriate migration distance for a good separation to occur. The resolution, Rs, which defines how well two neighboring substances could be differentiated, should also be taken into consideration: R" = 2

$% %&

Figure 6. Separation efficiency: (A) Plate height of 10 nm, 30 nm, 50 nm and 80 nm Au-PEG as a function of migration distance; (B) resolution of 10 nm/30 nm, 30 nm/50 nm and 50 nm/80 nm Au-PEG as a function of migration distance; (C) Mobile phase velocity on electrospun PAN UTLC with R2 = 0.997.

To evaluate the speed of the separation, mobile phase velocity was studied using the following equation: Z = κt (8) where, Zf is the total distance traveled by the mobile phase from the origin, к is the velocity constant and t is the development time.50 By plotting Zf2 versus t (Figure 6C), a straight line was obtained and the mobile phase velocity can be determined as the slope. Deviations from the straight line suggest abnormal flow the mobile phase due to experimental errors or the heterogeneity of the stationary phase.51 Figure 6C shows linearity (R2 > 0.99) between Zf2 and t, which demonstrated the homogeneity of the electrospun PAN stationary phase. On the electrospun PAN UTLC, solvent migration of 15 mm required less than 5 min (285 s) to achieve. In current literature, SEC and CE required at least 8-10 min and HPTLC took 20 min to separate AuNPs with two or three different sizes;19,23,27

(7)


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Analytical Chemistry FFF required 40 min and GE took 4 h to separate AuNPs coated with different length of PEG.22,25 In comparison, this developed method using electrospun UTLC offers much faster separations with excellent chromatographic performance. Monitoring the Transformation of AuNPs in Serum Protein. When AuNPs are introduced into bloodstream, proteins rapidly bind to the surface of AuNPs to form a biological coating, known as protein corona (PC). The formation of protein corona can affect the biological identity of the AuNPs and change the structure of adsorbed proteins, which may further eliminate their physiological functions.52,53 In recent years, a large number of studies have devoted to characterizing AuNPPC complexes in order to further investigate their impact on physiological systems.15,26,54 In this work, we applied the developed method using electrospun UTLC to track the transformation of AuNPs in serum protein. Bovine serum albumin (BSA) is the most abundant protein in bovine plasma and highly homologous to its human counterpart (HSA). To monitor the protein adsorption on nanoparticles, 10 nm PEGlyated AuNPs (Au-PEG) and 10 nm citrated stabilized AuNPs (Aucitrate) were incubated with 5 mg/mL bovine serum albumin (BSA) at 37 °C for 1 h. The 5 mg/mL of BSA was prepared in phosphate buffer saline (PBS) to mimic the physiological condition. After incubation, without washing, Au-PEG and Aucitrate were spotted on the UTLC plates, and non-incubated AuNPs were also spotted as standard references. As shown in Figure 7A (spot a, b, d, e) significant decay in migration distance was observed for both Au-PEG and Au-citrate after incubation, which indicates the increase in their hydrodynamic sizes and BSA adsorption. To confirm the stability of the protein corona, incubated Au-PEG and Au-citrated were washed three times with Milli-Q water. After washing, according to Figure 6A (spot a, c, d, f), Au-PEG migrated the same distance for both incubated and non-incubated NPs, whereas Aucitrated still migrated to a shorter distance than its nonincubated counterpart. This observation can be illustrated using the two different types of protein corona: hard corona and soft corona. Proteins that tightly bounded on the surface of NPs are hard corona, while proteins loosely bounded on the surface are soft corona.55 Soft corona is secondary outer layer and has weak interaction with hard corona, therefore, it is dynamic and can be rinsed off or replaced by proteins with higher affinity to AuNPs over time.56 For Au-citrate, it is believed that there were both hard and soft corona on the surface, and the stable hard corona still presented after washing to give an increased hydrodynamic size of AuNPs. For Au-PEG, the particle size remained stable even when the incubation period was extended to one week (Figure 7B). It is well accepted that there is only soft corona covering the surface of PEGylated AuNPs, and no hard corona is observed because thiol terminated PEG creates a protective barrier on AuNPs to resist protein adsorption.25,57 Hence, PEGylation is now the most widely applied strategy to block nonspecific protein adsorption on AuNPs and increase their biocompatibility.58 Using the developed method, electrospun PAN UTLC can be applied to monitor the size transformation of AuNPs in a biological environment.

ent sizes in the range of 10-80 nm were well separated from each other. It demonstrated that the Rf value increases with decreasing size of AuNPs. This method also permits the separation of AuNPs capped with different molecular weight of PEG in the range of 2-20 kDa. The results indicated that the Rf value increases with decreasing length of the PEG coating. Micellar mobile phases were adopted to provide a highly biodegradable chromatographic system. This novel method exhibits excellent separation performance with the smallest plate heights < 2 µm and resolution of each pair of AuNPs > 1.5. Electrospun PAN UTLC shows uniform morphology and allows fast mobile phase migration velocity. Separations for all PEGylated AuNPs could be achieved within 5 min. This method was applied to monitor the transformation of AuNPs in serum protein, and provided clear evidence of size changes for Au-citrate and the stability of Au-PEG.

Figure 7. A. UTLC of a) nonincubated Au-PEG, b) incubated AuPEG before washing, c) incubated Au-PEG after washing, d) nonincubated Au-citrate, e) incubated Au-citrated before washing, d) non-incubated Au-citrate after washing. B. Rf values of incubated Au-PEG before and after washing at different incubation period.

ASSOCIATED CONTENT Supporting Information DLS measurements of AuNPs; Zeta potential of Au-PEG without and with SDS in the mobile phase; the effect of micellar mobile phase on the separation of AuNPs with different PEG length; Au ions and PEG in the chromatographic system; UV spectra of PEGylated AuNP; different concentration of AuNPs on UTLC; Capability of the method for separation of AgNP and AuNP with PVP. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

CONCLUSION

*Phone: +1-614-292-0733. Fax: +1-614-688-5402. E-mail: [email protected]

To the best of our knowledge, this is the first use of UTLC for the separation of nanoparticles. PEGylated AuNPs with differ-



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Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This material is based upon work partially supported by the National Science Foundation under Grant No. CHE-1610254. Also support was provided by the Ohio State University.

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