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Orthogonal Analysis of Functional Gold Nanoparticles for Biomedical Applications
De-Hao Tsai,1, 2, Yi-Fu Lu,2 Frank DelRio,1 Tae Joon Cho,1 Suvajyoti Guha,3 Michael R. Zachariah,3,4, Fan Zhang,1 Andrew Allen,1 Vincent A. Hackley1,
1Materials Measurement Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
2Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
3Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899, USA
4Departments of Chemical Engineering and Chemistry, University of Maryland, College Park, MD, 20740 USA
Corresponding author. E-mail: HYPERLINK "mailto:dhtsai@mx.nthu.edu.tw" dhtsai@mx.nthu.edu.tw
Abstract
We report a comprehensive strategy based on implementation of orthogonal measurement techniques to provide critical and verifiable material characteristics for functionalized gold nanoparticles (AuNPs) used in biomedical applications. Samples wer e a n a l y z e d b e f o r e a n d a f t e r H" 5 0 m o n t h s o f c o l d s t o r a g e ( H" 4 o C ) . B i o m e d i c a l a p p l i c a t i o n s r e q u i r e l o n g - t e r m s t o r a g e a t c o l d t e m p e r a t u r e s , w h i c h c o u l d h a v e a n i m p a c t o n A u N P t h e r a p e u t i c s . T h i o l a t e d p o l y e t h y l e n e g l y c o l ( S H - P E G ) - c o n j u g a t e d A u N P s w i t h d i f f e r e nt terminal groups (methyl-, carboxylic-, and amine-) were chosen as a model system due to their high relevancy in biomedical applications. Electrospray-differential mobility analysis, asymmetric-flow field flow fractionation, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, inductively coupled plasma mass spectrometry, and small-angle X-ray scattering were employed to provide both complementary and orthogonal information on (1) particle size and size distribution, (2) particle concentrations, (3) molecular conjugation properties (i.e., conformation and surface packing density), and (4) colloidal stability. Results show that SH-PEGs were conjugated on the surface of AuNPs to form a brush-like polymer corona. The surface packing density of SH-PEG was H" 0 . 4 2 n m - 2 f o r t h e m e t h y l - P E G - S H A u N P s , H" 0 . 2 6 n m - 2 f o r t h e a m i n e - S H - P E G A u N P s , a n d H" 0 . 1 8 n m - 2 f o r t h e c a r b o x y l i c - P E G - S H A u N P s b e f o r e c o l d s t o r a g e , a p p r o x i m a t e l y 1 0 % o f i t s t h e o r e t i c a l m a x i m u m v a l u e . T h e c o n f o r m a t i o n o f s u r f a c e - b o u n d S H - P E G s w a s t h e n e s timated to be in an intermediate state between brush-like and random-coiled, based on the measured thicknesses in liquid and in dry states. By analyzing the change in particle size distribution and number concentration in suspension following cold storage, the long term colloidal stability of AuNPs was shown to be significantly improved via functionalization with SH-PEG, especially in the case of methyl-PEG-SH and carboxylic-PEG-SH (i.e., we estimate that > 80 % of SH-PEG5K remained on the surface of AuNPs during storage). The work described here provides a generic strategy to track and analyze the material properties of functional AuNPs intended for biomedical applications, and highlights the importance of a multi-technique analysis. The effects of long term storage on the physical state of the particles, and on the stability of the ligand-AuNP conjugates, are employed to demonstrate the capacity of this approach to address critical issues relevant to clinical applications.
Introduction
Gold nanoparticles (AuNPs), principally in colloidal form, are one of the most promising building blocks for biomedically-relevant nanoparticle enabled products (NEPs) and applications. ADDIN EN.CITE ADDIN EN.CITE.DATA 1-6 AuNPs have the advantageous property of tunable surface plasmon resonance (SPR), ADDIN EN.CITE Eustis200674747417Eustis, S.El-Sayed, M. A.El-Sayed, MA
Georgia Tech, Sch Chem & Biochem, Laser Dynam Lab, Atlanta, GA 30332 USA
Georgia Tech, Sch Chem & Biochem, Laser Dynam Lab, Atlanta, GA 30332 USAWhy 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 shapesChemical Society ReviewsChem Soc Rev
Chem Soc RevChemical Society ReviewsChem Soc Rev209-217353optical-propertiesraman-scatteringnanosphere lithographyquantum dotssizenanostructuresfluorescenceassembliesdependencechemistry20060306-0012WOS:000235989600001<Go to ISI>://WOS:000235989600001English7 an optical phenomenon that has been exploited for applications ranging from imaging and optical-based molecular detection to hyperthermal therapy for cancer treatment, combined with facile synthetic methods ADDIN EN.CITE Daniel200475757517Daniel, M. C.Astruc, D.Astruc, D
Univ Bordeaux 1, LCOO, UMR CNRS 5802, Mol Nanosci & Catalysis Grp, F-33405 Talence, France
Univ Bordeaux 1, LCOO, UMR CNRS 5802, Mol Nanosci & Catalysis Grp, F-33405 Talence, FranceGold nanoparticles: Assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnologyChemical ReviewsChem Rev
Chem RevChemical ReviewsChem Rev293-3461041langmuir-blodgett-filmsmonolayer-protected clustersnonlinear-optical responseshape-controlled synthesisaqueous chloroaurate ionsenhanced raman-scatteringcore-shell nanoparticlessingle-crystal surfacesquantitative colorimetric detectionwater/aot/n-heptane microemulsions2004Jan0009-2665WOS:000188217400009<Go to ISI>://WOS:000188217400009English8 and general biocompatibility. ADDIN EN.CITE ADDIN EN.CITE.DATA 9 Furthermore, thiol-Au chemistry provides an effective route for rational design by surface modification (i.e., attachment of functional molecules to the surface of AuNPs). By conjugation with drug ligands, for example, AuNPs can function as stealthy vectors to improve drug-delivery efficacy. ADDIN EN.CITE ADDIN EN.CITE.DATA 6,10-12 The concept of applying AuNPs in nanomedicine is attractive, because in theory they can provide a therapeutic synergetic effect while avoiding or mitigating harm to healthy tissue. ADDIN EN.CITE ADDIN EN.CITE.DATA 1,6,10
In order to achieve the desired functionality described above, it is necessary to identify and make use of AuNPs with suitable material properties. For this, the concept of materials-by-design offers a powerful and fascinating platform. A major challenge for successful implementation of the design is the accurate and precise specification of material properties for AuNPs in NEPs (defined as AuNP-NEPs), including batch-to-batch uniformity. On this basis, it is of essential importance to understand the correlations between the properties of the materials in design and their formulation chemistry. However, serious questions have been raised about the quality of NEPs, where the uncertainties in the accuracy of material property measurements lead to challenges in assessing and understanding the results of their corresponding efficacy and safety. Specifically, validated measurement methods do not exist to sufficiently assess the purity of NEPs or to compare nanomaterials from different vendors or laboratories. These general, yet crucial concerns, if unaddressed, could adversely affect the eventual implementation of these promising AuNP-NEPs. For example, to preclude potential hazards to human health, regulators may halt or disapprove the use of these AuNP-NEPs for biomedical applications.
From the perspective of product development and quality assurance, it is critical to have suitable AuNPs characterized by traceable characterization methods. The development of standard reference materials has partially satisfied the need for traceability in AuNP size measurements. Similarly, to obtain complete information of material properties for AuNP-NEPs, it is necessary to establish suitable measurement metrology and methodology, the importance of which has been recognized by regulatory agencies and pharmaceutical companies world-wide. ADDIN EN.CITE ADDIN EN.CITE.DATA 1,13,14 Once established, material characterization will help bridge the gap between AuNP-NEP design and performance, and facilitate the screening of AuNP-NEPs for disqualifying properties prior to performing laborious and expensive biological tests. ADDIN EN.CITE ADDIN EN.CITE.DATA 13,14
Physical dimensions (including surface area), surface properties, colloidal stability, and particle concentration are considered among the most important material properties for applications in nanomedicine. Among these properties, physical dimensions, especially the primary and/or secondary particle diameter, have arguably the greatest impact on the AuNP functionality (e.g., transport behavior, circulation half-life, ligand loading, drug dosage, etc.). For example, to avoid renal filtration through the glomerular capillary wall in the kidney, while still being able to target and accumulate at a tumor site without recognition by the reticuloendothelial system (RES), the acceptable primary diameter for sphere-like AuNPs should range from about 10 nm to 100 nm. ADDIN EN.CITE ADDIN EN.CITE.DATA 15,16 Therefore, physical dimensions are always the first consideration during the design and formulation of a NEP.
Similarly, surface properties of AuNPs determine their interactions with ligands, cells, or physiological systems in the human body (e.g., hemolytic properties), affecting the therapeutic performance and biodistribution of AuNP-NEPs. ADDIN EN.CITE ADDIN EN.CITE.DATA 17 Surface engineering of AuNPs with targeting moieties via thiol-Au chemistry has proven advantageous, where transport properties (including circulation time), imaging/diagnostic ability, and drug loadings can be enhanced by judiciously choosing suitable ligands. ADDIN EN.CITE Wang201149494917Wang, J.Byrne, J. D.Napier, M. E.DeSimone, J. M.DeSimone, JM
Univ N Carolina, Dept Chem, Lineberger Comprehens Canc Ctr, Carolina Ctr Canc Nanotechnol Excellence, Chapel Hill, NC 27599 USA
Univ N Carolina, Dept Chem, Lineberger Comprehens Canc Ctr, Carolina Ctr Canc Nanotechnol Excellence, Chapel Hill, NC 27599 USA
Univ N Carolina, Dept Pharmacol, Inst Adv Mat, Inst Nanomed, Chapel Hill, NC 27599 USA
N Carolina State Univ, Dept Chem & Biomol Engn, Raleigh, NC 27695 USA
Mem Sloan Kettering Canc Ctr, Sloan Kettering Inst Canc Res, New York, NY 10021 USAMore Effective Nanomedicines through Particle DesignSmallSmall
SmallSmallSmall1919-1931714nanoparticle-cell interactionstargeted drug-deliveryprostate-cancer cellsgpi-anchored proteinsin-vivogold nanoparticlestransferrin receptorendocytic pathwaycross-linkinggene delivery2011Jul 181613-6810WOS:000293636600005<Go to ISI>://WOS:000293636600005English18 In the design phase, knowledge of the type of ligand that binds to the surface of AuNPs, ligand surface-packing density, and its molecular conformation at the surface is the key to modify and optimize AuNP-NEP performance. Surface packing density is determined by a combination of the binding affinity of ligands with the AuNP surface, the equilibrium ligand/AuNP concentration used in the formulation, and the size/structure of the ligand (e.g., steric effects can reduce packing density). Molecular conformation is determined by the interactions between the surface-bound ligands and the biological medium, including the number of ligands existing on the Au surface. In general, AuNPs grafted with a high-packing-density, neutral polymer corona (e.g., thiolated polyethylene glycol, SH-PEG) on the surface are more transportable and less likely to be recognized by the RES, resulting in an increased efficacy in the delivery of drug ligands. ADDIN EN.CITE ADDIN EN.CITE.DATA 1,10,11,19,20 In addition to the transport properties, a variety of desirable functionalities can be transferred to AuNPs by modifying the Au surface through ligand conjugation. For example, the drug cisplatin has been studied for its application in chemo-radiation therapy. ADDIN EN.CITE Lee201341414117Lee, S. M.Tsai, D. H.Hackley, V. A.Brechbiel, M. W.Cook, R. F.Cook, RF
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
NCI, NIH, Bethesda, MD 20892 USASurface-engineered nanomaterials as X-ray absorbing adjuvant agents for Auger-mediated chemo-radiationNanoscaleNanoscaleNanoscaleNanoscaleNanoscaleNanoscale5252-5256512coated gold nanoparticlescomputed-tomographylow-energycancerdeliveryphenhancementadsorptioncomplexeselectrons20132040-3364WOS:000319756200009<Go to ISI>://WOS:000319756200009Doi 10.1039/C3nr00333gEnglish6 Through design and engineering of the AuNP surface with ligands that have both thiol and carboxylic end groups, cisplatin can be complexed to the AuNP-based platform, delivered to the tumor cells, and released on target or enhanced by the synergetic stimulated emission of photoelectrons and Auger electrons from both the Au and Pt components. With the concept of surface modification, the effective dosing and targeting can be improved relative to the therapeutic application of traditional small molecule drugs.
Colloidal stability of AuNPs is an important factor for the production and application AuNP-NEPs. Agglomeration/aggregation is the principal result of poor or uncontrolled colloidal instability, occurring due to the strong van der Waals attractive force between AuNPs. The physical dimensions (mainly secondary size) generally change over time once agglomeration/aggregation begins. ADDIN EN.CITE ADDIN EN.CITE.DATA 6,13,14,21 This non-equilibrium process directly leads to decline of both the transport properties and imaging/diagnostic capacity (e.g., via quenching of the SPR effect), which cause difficulties in the construction of correlations between the designed material properties and the therapeutic performance. Because of the issues described above, colloidal stability has to be considered at the outset in the design and formulation of AuNP-NEPs, not just in the native suspension but under use conditions and in biological fluids.
Another important factor is the concentration of particles needed to deliver a required dose in therapeutics, or to exhibit the required sensitivity for imaging and diagnostic applications. ADDIN EN.CITE ADDIN EN.CITE.DATA 6,21 As discussed previously, the surface area of AuNPs is a key factor for dosing, as it sets limits for the maximum number of ligands that can be carried by a single particle. An increase in the concentration of primary particles is expected to increase the total surface area in NEPs and concomitantly the therapeutic load. To monitor both dose and biodistribution, it is necessary to have particle concentration above the detection limit for the measurement of choice. ADDIN EN.CITE ADDIN EN.CITE.DATA 13,14 In general, these properties are correlated or interrelated, making it a challenge to differentiate dependencies and develop clear structure-property-performance criteria. For example, particle size and number concentration of AuNPs can be strongly affected by colloidal stability over time; on the other hand, surface properties significantly impact the colloidal stability and the resulting physical state. ADDIN EN.CITE ADDIN EN.CITE.DATA 6,11,21
In this work, we propose an orthogonal multi-technique characterization approach as a viable strategy to interrogate correlations between key material properties and the efficacy and safety of AuNP-NEPs. We focus on investigations of particle size and size distribution, number-based and mass-based concentration, surface properties (e.g., packing density, binding affinity, and conformation), and colloidal stability. As a test bed, we employ SH-PEG-conjugated AuNPs with different terminal groups, due to the relevance of PEG in biomedical applications. ADD I N E N . C I T E A D D I N E N . C I T E . D A T A 1 , 1 0 , 1 1 , 1 9 , 2 0 W e a l s o e x a m i n e t h e c h a n g e s t h a t o c c u r d u r i n g e x t e n d e d c o l d s t o r a g e ( H"4 o C ) o v e r a p e r i o d o f H" 5 0 m o n t h s , s h e d d i n g l i g h t o n t h e c h e m i c a l s t a b i l i t y o f t h e l i g a n d - A u c o m p l e x a n d i t s r e l a t i o n s h i p t o c o l l o i d a l stability of the functionalized particles. For nanomedicine platforms, long term storage, particularly at reduced temperatures, could potentially alter the physical and chemical state, thus impacting their therapeutic efficacy. Studies of long term cold storage of functionalized nanomaterials in aqueous-based suspensions are virtually non-existent in the peer reviewed literature. To our knowledge, this is also the longest cold storage study of functionalized AuNPs reported in the literature. Our objectives are (1) to develop a methodology to perform pre-screening prior to AuNP-NEP use in biomedical applications and clinical evaluations, and (2) to identify and quantify key properties of these materials that may provide scientific insight into the correlations between material properties and therapeutic performance (e.g., the size effect).
2. Experimental Methods
2.1. Materials
Four types of surface-modified AuNP colloids were obtained from Nanopartz ADDIN EN.CITE 90909017The identification of any commercial product or trade name does not imply endorsement or recommendation by the National Institute of Standards and Technology. The identification of any commercial product or trade name does not imply endorsement or recommendation by the National Institute of Standards and Technology.22 (Loveland, CO, USA): citrate-stabilized (unconjugated), methyl-terminated-SH-PEG (m-SH-PEG-AuNPs), carboxyl-terminated-SH-PEG (c-SH-PEG-AuNP), and amine-terminated-SH-PEG (a-SH-PEG-AuNP). The diameter of the AuNP core is H"2 0 n m f o r a l l s a m p l e s . T h e s t a r t i n g c o n c e n t r a t i o n ( a t t = 0 ) i s u n k n o w n f o r a l l s a m p l e s . T h e m o l a r m a s s o f t h e S H - P E G , M m , i s 5 k D a ( i . e . , S H - P E G 5 K , b a s e d o n t h e v e n d o r s i n f o r m a t i o n ) . A q u e o u s a m m o n i u m a c e t a t e ( S i g m a - A l d r i c h , > 9 8 % , U S A ) s o l u t i o n w a s u s e d t o a d j u s t t h e i o n i c s t r e n g t h o f s a m p l e s a n d t o p e r f o r m e l e c t r o s p r a y i o n i z a t i o n . A l l c h e m i c a l s w e r e u s e d a s r e c e i v e d w i t h o u t f u r t h e r p u r i f i c a t i o n . B i o l o g i c a l g r a d e 1 8 . 2 M c m d e i o n i z e d w a t e r ( M i l l i p o r e , B i l l e r i c a , M A , U S A ; A q u a S o l u t i o n s , J a s p e r , G A , U S A ) was used to prepare solutions and colloidal suspensions. The pH of the unconjugated AuNP, m-SH-PEG-AuNP, c-SH-PEG-AuNP, and a-SH-PEG-AuNP suspensions, was measured as 7.8, 7.0. 7.3 and 3.9, respectively. These materials were analyzed and compared before and af t e r l o n g t e r m c o l d s t o r a g e ( H" 5 0 m o n t h s ) a t 4 o C ; t = 0 m o n t h s r e f e r s t o a s - r e c e i v e d m a t e r i a l p r i o r t o c o l d s t o r a g e .
2 . 2 . E l e c t r o s p r a y - d i f f e r e n t i a l m o b i l i t y a n a l y s i s
T h e e l e c t r o s p r a y - d i f f e r e n t i a l m o b i l i t y a n a l y s i s ( E S - D M A ) w a s u s e d t o o b t a i n a n u m b e r - b a s e d particle size distribution. ADDIN EN.CITE ADDIN EN.CITE.DATA 11,23,24 Briefly, the electrospray (ES) aerosol generator (model 3480, TSI Inc., Shoreview, MN, USA) was used to aerosolize AuNPs. Then the aerosolized AuNPs were delivered to an electrostatic classifier (model 3081, TSI Inc.), where the particles were classified based on their electric mobility under an applied DC electric field. Particles of a specific mobility size, dp,m, that exited the electrostatic classifier were counted by a condensation particle counter (CPC, model 3775 or model 3025, TSI Inc.). The step size used in the particle size measurements was 0.2 nm and the time interval between each step size was 10 s. Sample flow rate (Qsamp) in the DMA was set to 1.2 L min-1 and sheath flow rate (Qsh) in the DMA was 10.0 L min-1 or 30 L min-1. The uncertainty in the measurement of dp,m was estimated to be 0.3 nm in this study, using one standard deviation of triplicate measurements.
2.3. Asymmetric-flow field flow fractionation
The asymmetric-flow field flow fractionation (A4F) system consists of a high-performance isocratic pump (1100 series, Agilent Technologies, Santa Clara, CA, USA), manual injection valve (Rheodyne 7725i, IDEX Corporation, Oak Harbor, WA, USA) with a 100 L stainless steel sample loop, field/flow control module and A4F separation channel (Eclipse 2, Wyatt Technology, Santa Clara, CA, USA), multi-angle light scattering (MALS) detector (Dawn Heleos, Wyatt Technology) and ultraviolet-visible (UV-Vis) diode array detect o r ( 1 2 0 0 D A D , A g i l e n t T e c h n o l o g i e s ) . R e g e n e r a t e d c e l l u l o s e m e m b r a n e ( m o l a r m a s s c u t - o f f = 1 0 k D a , W y a t t T e c h n o l o g y ) w a s u s e d f o r a l l A 4 F e x p e r i m e n t s . A 3 5 0 m s p a c e r w a s u s e d t o d e f i n e t h e d e p t h o f t h e A 4 F c h a n n e l f o r a l l s e p a r a t i o n e x p e r i m e n t s r e p o r t e d h ere. The mobile phase for A4F separation was 0.02 % aqueous sodium azide solution. The cross flow rate was 2 mL min-1 and the channel flow was 0.5 mL min-1. [Details of the A4F method, including the estimation of particle size based on retention time, have been described in our previous publications and are summarized in the Electronic Supporting Information (ESI)]. ADDIN EN.CITE Tsai201118181817Tsai, D. H.Cho, T. J.DelRio, F. W.Taurozzi, J.Zachariah, M. R.Hackley, V. A.Hackley, VA
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
NIST, Mat Measurement Lab, Gaithersburg, MD 20899 USA
Univ Maryland, Dept Mech Engn, College Pk, MD 20740 USA
Univ Maryland, Dept Chem, College Pk, MD 20740 USAHydrodynamic Fractionation of Finite Size Gold Nanoparticle ClustersJournal of the American Chemical SocietyJ Am Chem SocJournal of the American Chemical SocietyJ Am Chem SocJournal of the American Chemical SocietyJ Am Chem Soc8884-888713323trimersdimers2011Jun 150002-7863ISI:000291667600029<Go to ISI>://000291667600029Doi 10.1021/Ja203328jEnglish23 At least two replicate fractograms were measured by A4F for each condition.
2.4. Electron and atomic force microscopy
The primary structures of AuNP samples were imaged using a transmission electron microscope (TEM, JEM-2100HT, JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV. The drop-cast method was used to prepare samples for TEM analysis. To image and understand the extent of particle aggregation, we use a scanning electron microscope (SEM, Hitachi SU8010, Hitachi, Japan) operated at 10 kV and an atomic force microscope (AFM, Dimension 3100, Veeco, Santa Barbara, CA, USA) operated in intermittent-contact (tapping) mode. Positively-charged aerosolized AuNPs were delivered to an electrostatic precipitator and deposited onto a silicon chip operated at a sample flow rate of H" 1 . 5 L m i n - 1 a n d a n e l e c t r i c f i e l d o f ( 2 t o 5 ) k V c m - 1 . A D D I N E N . C I T E <