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Colloidal Stability of Citrate and Mercaptoacetic Acid Capped Gold Nanoparticles Upon Lyophilization: Effect of Capping Ligand Attachment and Type of Cryoprotectants Alaaldin M Alkilany, Samer R Abulateefeh, Kayla Mills, Alaa Bani Yaseen, Majd Hamaly, Hatim Alkhatib, Khaled Aiedeh, and John W. Stone Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504000v • Publication Date (Web): 30 Oct 2014 Downloaded from http://pubs.acs.org on November 2, 2014
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Colloidal Stability of Citrate and Mercaptoacetic Acid Capped Gold Nanoparticles Upon Lyophilization: Effect of Capping Ligand Attachment and Type of Cryoprotectants Alaaldin M. Alkilany*‡, Samer R. Abulateefeh‡, Kayla K. Mills†, Alaa I. Bani Yaseen‡, Majd A. Hamaly‡, Hatim S. Alkhatib‡, Khaled M. Aiedeh‡, John W. Stone† ‡
Department of Pharmaceutics & Pharmaceutical Technology, Faculty of
Pharmacy, The University of Jordan, Amman 11942, Jordan; †Department of Chemistry, Georgia Southern University, USA KEYWORDS: gold nanoparticles, Freeze-drying, lyophilization, cryoprotectants, colloidal stability
ABSTRACT
For various applications of gold nanotechnology, long-term nanoparticle stability in solution is a major challenge. Lyophilization (freeze-drying) is a widely used
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process to convert labile protein and various colloidal systems into powder for improved long-term stability. However, the lyophilization process itself may induce various stresses resulting in nanoparticle aggregation. Despite a plethora of studies evaluating lyophilization of proteins, liposomes, and polymeric nanoparticles, little is known about the stability of gold nanoparticles (GNPs) upon lyophilization. Herein, the effects of lyophilization and freeze-thaw cycles on the stability of two types of GNPs: Citrate-capped GNPs (stabilized via weakly physisorbed citrate ions, Cit-GNPs) and mercaptoacetic acid-capped GNPs (stabilized via strongly chemisorbed mercaptoacetic acid, MAA-GNPs) are investigated. Both types of GNPs have similar core size and effective surface charge as evident from transmission electron microscopy and zeta potential measurements, respectively. Plasmon absorption of GNPs and its dependence on nanoparticle aggregation was employed to follow stability of GNPs in combination with dynamic light scattering analysis. Plasmon peak broadening index (PPBI) is proposed herein for the first time to quantify GNPs aggregation using non-linear Gaussian fitting of GNPs UV-vis spectra. Our results indicate that Cit-GNPs aggregate irreversibly upon freeze-thaw cycles and lyophilization. In contrast, MAA-GNPs exhibits remarkable stability under the same conditions. Cit-GNPs exhibit no significant aggregation in the presence of cryoprotectants (molecules that are typically used to protect labile ingredients during lyophilization) upon freeze-thaw cycles and lyophilization. The effectiveness of the cyroprotectants evaluated was in the order of trehalose or sucrose > sorbitol > mannitol. The ability of cryoprotectants to prevent GNPs aggregation was
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dependent on their chemical structure and their ability to interact with the GNPs as assessed with zeta potential analysis.
INTRODUCTION
Gold nanoparticles (GNPs) have attracted a vast research interest with various promising applications.1, 2 Unique optical properties (extensive plasmon absorption/scattering), ease of synthesis, biocompatibility, and the ability to functionalize their surfaces, make these nanostructures an excellent platform for sensing, imaging, controlled drug delivery, and photothermal-based cancer ablation.1, 2, 3, 4 However, colloidal instability of GNP in solution is a major challenge considering their high surface area and their tendency to aggregate upon exposure to various stresses.1 Aggregation of GNPs has a dramatic effect on their physical, chemical and biomedical properties.1, 5 Efforts to improve the colloidal stability of GNPs in solutions via surface modification with physisorbed (physically-bound) or chemisorbed (covalently-bound) ligands are abundant in the literature.6 An alternative approach to preserve the colloidal stability and to extend their shelf life is to convert GNPs solutions into dried powders, and thus minimizing their physical aggregation and/or chemical degradation. Upon request, GNPs powder can be reconstituted with water or buffer solution to prepare GNPs solutions with similar physiochemical and optical properties to the initial solutions. This approach may allow for the development of “reference nanomaterials” that can be used by different laboratories at various times with constant physical and chemical properties of GNPs, as was demonstrated
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recently for silver nanoparticles by researchers at the National Institute of Standards and Technology (NIST).7 Another importance of this approach is applied when nanoparticles are conjugated to biological moieties such as enzymes, proteins and antibodies, which have superior stability as dried powder compared to solution. Lyophilization or freeze-drying, a process to remove water from frozen solution by sublimation, is a routinely employed in pharmaceutical, food and agricultural industries to preserve/stabilize labile ingredients and increase their storage shelf life.8 Lyophilization has been also applied to various colloidal systems such as proteins, liposomes, and polymeric nanoparticles.9 In a similar direction, manufacturers of GNPs are moving toward supplying their product in the form of lyophilized powder.7 For example, GYC-LyoFTM (Ocean NanoTech LLC.) and Nanogold® (Nanoprobes Inc.) are commercially available lyophilized GNPs powders for long-term storage and enhanced colloidal stability. However, little literature is available on lyophilization of inorganic nanoparticles and specifically GNPs.7, 9, 10, 11, 12 Despite the advantage of lyophilization in improving colloidal stability upon storage of various nanoformulations, the process itself may induce various physical stresses resulting in nanoparticle aggregation.9 Lyophilization involves freezing of nanoparticle solutions followed by drying the frozen mass under vacuum (sublimation) to remove frozen water at low temperature. Both freezing and drying processes can induce stress to the suspended nanoparticles and result in undesired nanoparticle aggregation.9 Stresses during lyophilization include: increased local concentration of nanoparticles as a result of ice 4 ACS Paragon Plus Environment
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formation and solute exclusion, mechanical stress by the formed ice crystals, pH/ionic strength changes, and surface dehydration.9 Cryoprotectants are routinely used excipients to reduce stresses during lyophilization. Mono- and disaccharides, surfactants, amino acids, and polymers are examples of cryoprotectants that have been utilized to protect drug molecules, proteins, and polymeric nanoparticles during freeze-drying. The current study aimed to evaluate the colloidal stability of GNPs, upon freeze-thaw cycles and lyophilization, with two different surface chemistries: 1) weakly capped GNPs (citrate-capped GNPs, Cit-GNPs) and; 2) strongly capped GNPs (mercaptoacetic acid-capped GNPs, MAA-GNPs). Moreover, the colloidal stability of Cit-GNPs and MAA-GNPS in the presence of commonly used cryoprotectants (mannitol, sorbitol, trehalose and sucrose) was evaluated and discussed. In this report, we chose “simple and classical” types of GNPs (capped with citrate ions or thiolated small molecules) at constant nanoparticle’s size and cryoprotectant level to make the focus of this work on the effect of the nature of capping agent attachment to nanoparticle’s surface and the type of evaluated cryoprotectant. Moreover, MAA was selected to ensure that both types of GNPs (Cit-GNPs and MAA-GNPs) are terminated with carboxylate moieties with different attachment nature to nanoparticles (physisorbed versus chemisorbed).
EXPERIMENTAL SECTION
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Materials. Chloroauric acid (HAuCl4 .3H2O, 99.9%), mercaptoacetic acid (MAA) (≥98%), Poly(ethylene glycol) methylether thiol, PEG-SH, Mn=2000 Da, mannitol, sorbitol, sucrose, trehalose, trisodium citrate were obtained from Sigma-Aldrich and used as received. FITC-PEG-SH (MW of 2000 Da) was purchased from Nanocs, Inc. (Boston, USA). All solutions were prepared with purified 18 MΩ.cm water. Glasswares were cleaned with aqua regia and rinsed thoroughly with 18 MΩ.cm water. Dialysis tubes of MWCO=10,000 Da were purchased from Spectra Laboratories (Milpitas, CA). Amicon Ultra-15 centrifugal filter units with NMWL of 100 kDa were purchased from Millipore Inc. (Billerica, MA) (the units retain only GNPs and allow the free polymer/molecules to pass with the filtrate). Instrumentation. Lyophilization was carried out using a bench top manifold freeze-drier (HumanLab Instrument Co, Korea). Solutions were placed in glass vials and submersed in a liquid nitrogen bath for complete freezing. Frozen solutions were then placed in glass flasks attached to the drying chamber without shelf-temperature control. The temperature of the vapor collector chamber was kept at -80 oC throughout lyophilization. Vacuum pressure within the drying chamber was below 1x10-3 bar. Absorption spectra were recorded using a UV/VIS spectrophotometer (Spectrascan 80D, Biotech Eng., UK). Emission fluorescence spectra were recorder using a SLM Aminco Spectrofluorometer (SLM Instruments, Urbana, IL) with excitation wavelength of 494 nm using Quartz cuvettes at 25 oC. Emission spectra were collected in the range of (500-650 nm) and area under the curve (AUC) values in the range of (510-625 nm) were used in all calculations. 6 ACS Paragon Plus Environment
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Transmission electron microscopy (TEM) images were obtained with a Morgagni (Philips, Netherlands) 268 FEI electron microscope operating at 40 kV attached to MegaViewG2 Olympus Soft Imaging Solutions. TEM grids were prepared by dropcasting 10 µL of the purified GNPs solutions on Formvar coated copper TEM grids (300 mesh, Ted Pella Inc., Redding, CA), and allowing them to dry in air. Zeta potential and dynamic light scattering measurements were performed on a Zetasizer Nano ZS (Malvern instruments, UK) with 1:10 dilution with deionized water prior to analysis. For zeta potential measurements, universal dip cell with applied voltage less than 5 V was used and the Smoluchowski approximation was employed by the instrument’s software to calculate zeta potential values for aqueous solutions of GNPs (F(Ka) value=1.5). Dynamic light scattering measurements were carried out using disposable polystyrene cuvettes and a 173o scattering angle. A micro centrifuge (Eppendorf 5418, Hamburg, Germany) and a LabQuake® shaker (Barnstead-Thermolyne, Dubuque, IA) were used in GNPs synthesis and purification as detailed below. Preparation of Citrate-Capped Gold Nanoparticles (Cit-GNPs). Cit-GNPs were synthesized using Frens method.13 An aqueous solution of 0.25 mM HAuCl4, 100 mL, was heated in a conical flask and brought to boil. To the boiling solution, 3.0 mL of an aqueous solution of 1% (w/w) sodium citrate was added. The heating was maintained until a deep ruby red color appeared (10 minutes), indicating the formation of GNPs. The resulting nanoparticles were purified once by centrifugation to get rid of excess citrate ions and other impurities. Solution’s pH was adjusted to 7.4 with 0.1 M sodium hydroxide solution. Transmission 7 ACS Paragon Plus Environment
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electron microscopy, Zeta potential analyzer, and UV-vis spectrophotometry measurements were used to evaluate the prepared nanoparticles. Preparation of Mercaptoacetic acid-Capped Gold Nanoparticles (MAAGNPs). The prepared Cit-GNPs were functionalized with mercaptoacetic acid based on the well-documented gold-thiol chemistry with major modification to published protocols.14 Direct addition of MAA to Cit-GNPs resulted in severe aggregation of GNPs. Observed aggregation was not avoided by optimizing the molar ratio of Au:MAA or the pH of GNP solutions. In this regard, we employed two-round functionalization approach using PEG-SH as an intermediate. First, 50 mL as prepared Cit-GNPs were centrifuged and the resulting pellets (i.e. concentrated suspension of GNPs at the bottom of centrifugation vials after removing supernatant) were collected and diluted with DIW to final volume of 5.0 mL. The resulting GNPs solution (5.0 mL) was added dropwise to 2.0 mL solution of Poly(ethylene glycol) methylether thiol (10.0 mg/mL, PEG-SH, Mn=2000 Da, Sigma-Aldrich) and stirred for about 12 hours and then dialyzed against 4.0 L of deionized water for 48 hours to remove excess PEG-SH molecules using dialysis tube with 10,000 Da MWCO. Aqueous solutions of mercaptoacetic acid (MAA, 50mM) were prepared and 1.0 mL of this solution was added to 9.0 mL PEGGNPs to ensure excess MAA and force thiol displacement on the surface of GNPs (concentration of PEG-GNPs is 0.5 nM in particles or 58.4 µg/L). The mixture was stirred overnight allowing MAA to replace PEG molecules resulting in the formation of covalently linked MAA shell on the nanoparticle surface. MAA8 ACS Paragon Plus Environment
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GNPs were purified by three rounds of centrifugation (10,000 rpm, 20 minutes) and the resulting pellets were resuspended with deionized water followed by dialysis against 4.0 L of deionized water for 48 hours to remove excess PEG-SH molecules using dialysis tube with 10,000 Da MWCO. The displacement reaction was repeated three times to ensure a complete displacement of PEG-SH by MAA molecules. Solution pH was adjusted to 7.4 with 0.1 M sodium hydroxide solution. Quantification of displaced PEG-SH by MAA molecules on GNPs A fluorescent PEG-SH derivative was used (FITC-PEG-SH, MW of 2000 Da) to follow the displacement of PEG-SH by MAA molecules on GNPs. Cit-GNPs (200 mL) were centrifuged and the resulting pellets were collected in 5.0 mL deionized water. Collected GNP solutions or a control vial containing deionized water (5.0 mL) were added dropwise to 2.0 mL solution of FITC-PEG-SH (10.0 mg/mL). GNPs solutions (sample and control) were stirred for 12 hours in the dark at room temperature. To quantify the amount of FITC-PEG-SH bound to GNPs, both sample and control solutions were centrifuged using Amicon Ultra-15 centrifugal filter units with NMWL of 100 kDa (2000 rcf for 20 minutes) and the resulting filtrates were collected for fluorescence analysis. The control vial was used to compensate for any flourophore loss by adsorption to vial surface and/or filtration membranes. Amount of bound FITC-PEG-SH to GNPs was calculated by the difference of FITC-PEG-SH amount in both sample and control filtrates. To follow up displacement of PEG-SH by MAA, Cit-GNPs (200 mL) were centrifuged and the resulting pellets were collected in 5.0 mL deionized water. 9 ACS Paragon Plus Environment
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Collected GNPs pellets were added dropwise to 2.0 mL solution of FITC-PEGSH (10.0 mg/mL) followed by dialyzed against 4.0 L of deionized water for 48 hours to remove excess FITC-PEG-SH using dialysis tubing with 10,000 Da MWCO. Sample from the purified FITC-PEG-GNPs solution was filtrated using Amicon Ultra-15 centrifugal filter units with NMWL of 100 kDa (2000 rcf for 20 minutes) and resulting filtrate was collected for fluorescence analysis to ensure the absence of free FITC-PEG-SH in the GNP solutions (the units retain only the nanoparticles and allow the free polymer to pass with the filtrate). Aqueous solutions of mercaptoacetic acid (MAA, 50mM) were prepared and 1.0 mL of this solution was added to 9.0 mL of dialyzed FITC-PEG-GNPs. The mixture was stirred for 12 hours in the dark allowing MAA to replace FITC-PEG molecules and then sample from this mixture was filtrated using Amicon Ultra-15 centrifugal filter unit with NMWL of 100 kDa (2000 rcf for 20 minutes) to quantify displaced FITC-PEG-SH with fluorescence analysis of the filtrate. MAA-GNPs were purified by three rounds of centrifugation (10,000 rpm, 20 minutes) and the resulting pellets were resuspended with deionized water followed by dialysis against 4.0 L of deionized water for 48 hours to remove excess PEG-SH molecules using dialysis tubing with 10,000 Da MWCO. A sample of purified MAA-GNP solution was filtrated using Amicon Ultra-15 centrifugal filter unit with NMWL of 100 kDa (2000 rcf for 20 minutes) and resulting filtrate was collected for fluorescence analysis to ensure absence of displaced FITC-PEG-SH in the final solution. All filtrates were lyophilized and reconstituted with PBS buffer (pH
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7.4) prior to fluorescence analysis to minimize artifacts that originate from pHdependent fluorescence of FITC molecules. Freeze-Thaw Cycles. In glass vials containing GNP solutions (2.0 mL, 0.5 nM in particles or 58.4 µg/L), 0.2 g cryoprotectants (mannitol, sucrose, sorbitol, or trehalose) was dissolved. The resulting solutions were frozen by fast immersion in liquid nitrogen for 10 minutes and then thawed (30 minutes) at room temperature. No sonication was applied before further evaluation. For each preparation, freeze-thaw cycles were repeated six times. The thawed solutions were analyzed using UV-vis spectrophotometry, DLS, and effective surface charge analysis (zeta potential). Lyophilization (Freeze-Drying) of GNP solutions. In glass vials containing 2.0 mL of GNP solutions (0.5 nM in particles or 58.4 µg/L), 0.2 g cryoprotectant (mannitol, sucrose, sorbitol, or trehalose) was dissolved. The resulting solutions were frozen by fast immersion in liquid nitrogen for 5 minutes and then connected immediately to freeze-drier (HumanLab Instrument Co, Korea) with applied vacuum (
