Associate Professor University of Oklahoma, United States
Introduction: We generated predictive models for gold nanoparticle (AuNP) synthesis and designed a novel method of imparting biocompatibility to CTAC-coated AuNPs. AuNPs are used as model systems to characterize interactions between nanoparticles and cells. Nanoparticle diameter and colloidal monodispersity are important characteristics that influence nanoparticle interactions with cells.1 We have previously demonstrated how CTAC-coated AuNPs possess improved monodispersity compared to biocompatible citrate-coated AuNPs.2 However, no quantitative relationship between CTAC-coated AuNP diameter and synthesis reaction inputs exists to allow for accessible synthesis of a wide range of CTAC-coated AuNP sizes. Further, the innate cytotoxicity of CTAC-coated AuNPs needs to be overcome prior to their use in nanomedicine applications. Our study addresses these challenges through developing predictive mathematical models connecting AuNP reaction input with final AuNP diameter as measured by single-particle analytical methods. Further, we create a novel approach for replacing CTAC with biocompatible citrate that imparts biocompatibility and allows for surface modification of originally CTAC-coated AuNPs. Our results demonstrate the applicability of predictive models in nanoparticle synthesis and the potential for using monodisperse originally CTAC-coated AuNPs in biomedical research.
Materials and
Methods: Citrate-coated and CTAC-coated AuNPs were synthesized using established seed-mediated growth reactions.2 In order to produce AuNPs of different diameters, AuNP synthesis was carried out using different molar ratios of AuNP seeds to ionic gold. Following initial characterization and purification by centrifugation, single-particle analysis using transmission electron microscopy (TEM) and single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) was used to quantify mean AuNP diameter. Mean AuNP diameter values from single-particle analysis were compared with synthesis molar ratios to identify the relationship between reaction inputs and final AuNP diameter. Predictive models generated were used to synthesize AuNPs with target mean diameters of 30 nm, 60 nm, and 90 nm. 60 nm AuNPs were synthesized under normal and scaled-up reaction conditions to assess predictive model accuracy with scale-up. To improve biocompatibility, synthesized CTAC-coated AuNPs underwent a new citrate physical replacement (citrate-PR) process involving extensive vortexing and centrifugation in a 1% Tween20, 0.17mM sodium citrate solution. Post-citrate-PR AuNPs were characterized by TEM and SP-ICP-MS to identify changes in colloidal stability. Aliquots of post-citrate-PR AuNPs were surface conjugated with polyethylene glycol (PEG) or heparosan (HEP) and used in a 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) assay to assess biocompatibility. RAW 264.7 murine macrophages were treated with 0.02 nM of AuNPs and incubated for 24 hours before quantifying cell viability.
Results, Conclusions, and Discussions: Results and
Discussion: Our predictive synthesis models (Figure 1A) demonstrate high coefficient of determination (R2) values for both citrate-coated AuNPs (R2=0.9993) and for CTAC-coated AuNPs (R2=0.9986). Additionally, for both AuNP types and for multiple target diameters (30 nm, 60 nm, and 90 nm), AuNP synthesis using our predictive models produced particles with ≤5% deviation from the target diameter for both normal and scaled-up reactions (Figure 1B). Our citrate-PR method was shown to successfully remove CTAC according to zeta potential measurements and Fourier transform infrared (FTIR) spectroscopy while maintaining monodispersity as measured by SP-ICP-MS. Additionally, we found that both with and without PEG or HEP conjugated, post-citrate-PR AuNPs exhibited biocompatibility as quantified by an XTT assay (Figure 1C). ANOVA statistical testing showed no statistical significance between citrate-coated AuNP groups, citrate-PR AuNP groups, and the cell-only control (p=0.2755). By comparison, treating cells with unmodified CTAC-coated AuNPs resulted in significant cell death compared to all other AuNP groups and cell-only controls (p < 0.0001).
Conclusions: We show that our predictive synthesis models demonstrate significant accuracy when used to synthesize citrate- or CTAC-coated AuNPs. Additionally, our CTAC-coated AuNP modification using citrate-PR imparts biocompatibility to originally cytotoxic CTAC-coated AuNPs, allowing for use of more monodisperse AuNPs in nanomedicine research. Future directions include applying our predictive model generation workflow to additional nanoparticle types to improve translation of and collaboration within clinical nanomedicine research efforts.
Acknowledgements (Optional): 1. Dheyab, M. et al. Int. J. Mol. Sci. (2022) 23 (13) 7400 2. Frickenstein, A. et al. Anal. Bioanal. (2023) 415, 4353–4366
