Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications. Methods and Materials: Here, AuNPs of three sizes, 5 nm, 10 nm, and 20 nm, were coated with a lipid bilayer composed of sodium oleate, hydrogenated phosphatidylcholine, and hexanethiol. To understand how the physical features of AuNPs influence uptake through cellular membranes, sum frequency generation (SFG) was utilized to assess the interactions of the AuNPs with a biomimetic lipid monolayer composed of a deuterated phospholipid 1.2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC). Results and Discussion: SFG measurements showed that 5 nm and 10 nm AuNPs are able to phase into the lipid monolayer with very little energetic cost, whereas, the 20 nm AuNPs warped the membrane conforming it to the curvature of hybrid lipid-coated AuNPs. Toxicity of the AuNPs were assessed in vivo to determine how AuNP curvature and uptake influence cell health. In contrast, in vivo toxicity tested in embryonic zebrafish showed rapid toxicity of the 5 nm AuNPs, with significant 24 hpf mortality occurring at concentrations ≥20 mg/L, whereas the 10 nm and 20 nm AuNPs showed no significant mortality throughout the five-day experiment. Conclusion: By combining information from membrane models using SFG spectroscopy with in vivo toxicity studies, a better mechanistic understanding of how nanoparticles (NPs) interact with membranes is developed to understand how the physiochemical features of AuNPs drive nanoparticle-membrane interactions, cellular uptake, and toxicity.

Gold nanoparticles (AuNPs) are increasingly used in biomedical applications, hence understanding the processes that affect their biocompatibility and stability are of significant interest. In this study, we assessed the stability of peptide-capped AuNPs and used the embryonic zebrafish (Danio rerio) as a vertebrate system to investigate the impact of synthesis method and purity on their biocompatibility. Using glutathione (GSH) as a stabilizer, Au-GSH nanoparticles with identical core sizes were terminally modified with Tryptophan (Trp), Histidine (His) or Methionine (Met) amino acids and purified by either dialysis or ultracentrifugation. Au-GSH-(Trp)2 purified by dialysis elicited significant morbidity and mortality at 200 μg/mL, Au-GSH-(His)2 induced morbidity and mortality after purification by either method at 20 and 200 μg/mL, and Au-GSH-(Met)2 caused only sublethal responses at 200 μg/mL. Overall, toxicity was significantly reduced and ligand structure was improved by implementing ultracentrifugation purifications at several stages during the multi-step synthesis and surface modification of Au-GSH nanoparticles. When carefully synthesized at high purity, peptide-functionalized AuNPs showed high biocompatibility in biological systems.

Abstract:The preparation of gold nanoparticles (AuNPs) of high purity and stability remains a major challenge for biological applications. This paper reports a simple synthetic strategy to prepare water-soluble peptide-stabilized AuNPs. Reduced glutathione, a natural tripeptide, was used as a synthon for the growth of two peptide chains directly on the AuNP surface. Both nonpolar (tryptophan and methionine) and polar basic (histidine and dansylated arginine) amino acids were conjugated to the GSH-capped AuNPs. Ultracentrifugation concentrators with polyethersulfone (PES) membranes were used to purify precursor materials in each stage of the multi-step synthesis to minimize side reactions. Thin layer chromatography, transmission electron microscopy, UV-Visible, 1 H-NMR, and fluorescence spectroscopies demonstrated that ultracentrifugation produces high purity AuNPs, with narrow polydispersity, and minimal aggregation. More importantly, it allows for more control over the composition of the final ligand structure. Studies under conditions of varying pH and ionic strength revealed that peptide length, charge, and hydrophobicity influence the stability as well as solubility of the peptide-capped AuNPs. The synthetic and purification strategies used provide a facile route for developing a library of tailored biocompatible peptide-stabilized AuNPs for biomedical applications.

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The advancement of safe nanomaterials for use as optical coherence tomography (OCT) imaging and stem cell-labeling agents to longitudinally visually track therapeutic derived retinal stem cells to study their migration, survival rate, and efficacy is challenged by instability, intracellular aggregation, low uptake, and cytotoxicity. Here, we describe a series of hybrid lipid-coated gold nanorods (AuNRs) that could solve these issues. These nanomaterials were made via a layer-by-layer assembly approach, and their stability in biological media, mechanism, efficiency of uptake, and toxicity were compared with a commercially available set of AuNRs with a 5 nm mesoporous silica (mSiO 2 )-polymer coating. These nanomaterials can serve as stem cell labeling and OCT imaging agents because they absorb in the near-infrared (NIR) region away from biological tissues. Although both subtypes of AuNRs were taken up by retinal pigment epithelial, neural progenitor, and baby hamster kidney cells, slightly negatively charged hybrid lipid-coated AuNRs had minimal aggregation in biological media and within the cytoplasm of cells (∼3000 AuNRs/cell) as well as minimal impact on cell health. Hybrid lipid-coated AuNRs modified with cell-penetrating peptides had the least toxicological impact, with >92% cell viability. In contrast, the more "sticky" AuNRs with a 5 nm mSiO 2 -polymer coating showed significant aggregation in biological media and within the cytoplasm with lower-than-expected uptake of AuNRs (∼5400 of AuNRs/cell) given their highly positive surface charge (35+ mV). Collectively, we have demonstrated that hybrid lipid-coated AuNRs, which absorb in the NIR-II region away from biological tissues, with tuned surface chemistry can label therapeutic derived stem cells with minimal aggregation and impact on cell health as well as enhance uptake for OCT imaging applications.

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Gold nanorods (AuNRs) hold tremendous potential to improve the diagnosis and therapeutic options across the blood-retinal barrier to treat retinal diseases. For clinical ophthalmological translation, a fundamental understanding of how their physicochemical properties such as size, shape, charge, surface chemistry, and concentration, impact their stability biological environments, mechanism and efficiency of uptake, and toxicity is a necessity. Here we interrogated the uptake efficiency, biocompatibility, and stability of two subtypes of AuNRs with different types of surface coatings and varying charges, including a commercially available set of AuNRs with a 5 nm mSiO2-polymer coating and hybrid lipid-coated AuNRs developed in-house.Confocal and bright field microscopy images showed uptake of both subtypes of AuNRs in retinal pigment epithelium (RPE), neural progenitor (NP), and baby hamster kidney (BHK) cells.Transmission electron microscopy (TEM) confirms both types of AuNRs are taken up into the cytoplasm of the cells; however, larger aggregates of AuNRs are observed with the more positive and "sticky" AuNRs with a 5 nm mSiO2-polymer coating than the slightly negative hybrid lipidcoated AuNRs. Inductively Coupled Mass Spectroscopy (ICP-MS) confirm that ~3,000 of the slightly negative hybrid lipid-coated AuNRs cells and ~5,400 of the positively charged AuNRs with a 5 nm mSiO2-polymer coating (+35 mV) are taken up into RPE and BHK cell lines. Stability studies in a variety of cellular media showed that hybrid lipid-coated AuNRs are stable and disaggregated in water, 10 mM PBS buffer pH 7, and BHK media except for NP media. In contrast, the positively charged AuNRs with a 5 nm mSiO2-zeta polymer coating aggregated in all media, indicating more interactions with each other and components of the media. Bright-field and TEM confirm the presence of large aggregates of AuNRs on the surface and within the cytoplasm.Cytotoxicity studies both subtypes of AuNRs have an 80 ± 8 % cell viability, indicating mild toxicity. The hybrid lipid-coated AuNR with the cell-penetrating peptide had the least toxicological impact with a > 92 ± 7 % cell viability. Our study highlights the importance of evaluating the impact of the physicochemical features of each new nanoparticle design on their stability in biologically relevant environments and their impact on cellular uptake and toxicity in stem cell-derived therapeutic cells. Here we also provide a simple design strategy for tuning the surface chemistry of robust hybrid lipid-coated AuNRs to enhance cellular uptake to label stem cells with minimal aggregation and toxicity.

Gold nanorods (AuNRs) hold tremendous potential to improve the diagnosis and therapeutic options across the blood-retinal barrier to treat retinal diseases. For clinical ophthalmological translation, a fundamental understanding of how their physicochemical properties such as size, shape, charge, surface chemistry, and concentration, impact their stability biological environments, mechanism and efficiency of uptake, and toxicity is a necessity. Here we interrogated the uptake efficiency, biocompatibility, and stability of two subtypes of AuNRs with different types of surface coatings and varying charges, including a commercially available set of AuNRs with a 5 nm mSiO2-polymer coating and hybrid lipid-coated AuNRs developed in-house. Confocal and bright field microscopy images showed uptake of both subtypes of AuNRs in retinal pigment epithelium (RPE), neural progenitor (NP), and baby hamster kidney (BHK) cells. Transmission electron microscopy (TEM) confirms both types of AuNRs are taken up into the cytoplasm of the cells; however, larger aggregates of AuNRs are observed with the more positive and “sticky” AuNRs with a 5 nm mSiO2-polymer coating than the slightly negative hybrid lipid-coated AuNRs. Inductively Coupled Mass Spectroscopy (ICP-MS) confirm that ~3,000 of the slightly negative hybrid lipid-coated AuNRs cells and ~5,400 of the positively charged AuNRs with a 5 nm mSiO2-polymer coating (+35 mV) are taken up into RPE and BHK cell lines. Stability studies in a variety of cellular media showed that hybrid lipid-coated AuNRs are stable and disaggregated in water, 10 mM PBS buffer pH 7, and BHK media except for NP media. In contrast, the positively charged AuNRs with a 5 nm mSiO2-zeta polymer coating aggregated in all media, indicating more interactions with each other and components of the media. Bright-field and TEM confirm the presence of large aggregates of AuNRs on the surface and within the cytoplasm. Cytotoxicity studies both subtypes of AuNRs have an 80 ± 8 % cell viability, indicating mild toxicity. The hybrid lipid-coated AuNR with the cell-penetrating peptide had the least toxicological impact with a > 92 ± 7 % cell viability. Our study highlights the importance of evaluating the impact of the physicochemical features of each new nanoparticle design on their stability in biologically relevant environments and their impact on cellular uptake and toxicity in stem cell-derived therapeutic cells. Here we also provide a simple design strategy for tuning the surface chemistry of robust hybrid lipid-coated AuNRs to enhance cellular uptake to label stem cells with minimal aggregation and toxicity.