Interfacing Nanomaterials with Biology through Ligand Engineering.

Nanoparticles (NPs) have incredible potential in biology and biomedicine. Gold nanoparticles (AuNPs) have become a cornerstone of the nanomedicine revolution due to their ease of synthesis, inertness, and versatility. The widespread use of AuNPs can be traced to the development of accessible, bottom-up wet synthesis methods that emphasized the role of ligands in controlling the size, dispersity, and stability of colloids in solution. Decoration of AuNPs with organic ligands can be used to dictate the interactions of these nanomaterials with biosystems on multiple scales. The tunability of the AuNP ligand monolayer via covalent and noncovalent approaches allows the use of AuNPs in a broad range of biomedical fields.In this Account, we describe our use of AuNPs to answer a central question in the ligand engineering of colloidal nanoparticles: can we fabricate NPs that are nontoxic, modular, and functional in biological environments? We explored spherical AuNPs of different sizes and ligand structures, empirically exploring the AuNP-biomolecule interaction. We show here how the atom-by-atom control provided by organic synthesis can be used to create engineered ligands. Presenting these ligands on the surface of AuNPs creates multivalent constructs with unique and useful properties. Ligand design is a key feature of these AuNPs. We have developed ligands that have three distinct structural segments: 1) a hydrophobic alkanethiol interior that imparts stability; 2) a tetra(ethylene glycol) segment that creates a noninteracting tabula rasa surface; and 3) ligand headgroups that dictate how the AuNP interacts with the outside world. Our research into the design principles of ligands on AuNPs and their interactions with biological systems can be translated to other nanoparticle systems.This Account also summarizes the trajectory of ligand engineering in our laboratory and further afield. At the outset, experimental and theoretical fundamental studies were focused on the interactions between AuNPs and cellular components, such as proteins and lipid membranes. Understanding these behaviors provided the direction for investigating how ligands mediate the interface of AuNPs with mammalian and bacterial cells. In these experiments, it was particularly noteworthy that the ligand hydrophobicity and charge play a significant role in the uptake and toxicity of AuNPs. These revelations formed a basis for translating AuNPs to physiological environments. We present how we have integrated our synthetic abilities to construct AuNPs for biomedical applications, including delivery, bioorthogonal catalysis, antimicrobial and antitumor therapeutics, and biosensing.Overall, we hope that this Account will give the reader insight into how our research has evolved, changing AuNPs from synthetic curiosities into functional nanoplatforms for nanomedicine, all through the power of ligand design and synthesis.

Turning on the Antimicrobial Activity of Gold Nanoclusters Against Multidrug-Resistant Bacteria.

In this work, we show that the addition of thiourea (TU) initiated broad-spectrum antimicrobial activity of otherwise inactive D-maltose-capped gold nanoclusters (AuNC-Mal). For example, AuNC-Mal/TU was effective against multidrug-resistant Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) of 1 µg mL-1 (2.5 µM [Au]) while having 30-60 times lower in vitro cytotoxicity against mammalian cells. The reaction of AuNC-Mal and TU generated the antimicrobial species of [Au(TU)2 ]+ and smaller AuNCs. TU increased the accumulation of Au in bacteria and helped maintain the oxidation state as AuI (vs. AuIII ). The modes of action included the inhibition of thioredoxin reductase, interference with the CuI regulation and depletion of ATP. Moreover, the antimicrobial activity did not change in the presence of colistin or carbonyl cyanide 3-chlorophenylhydrazone, suggesting that AuNC-Mal/TU was indifferent to the outer membrane barrier and to bacterial efflux pumps.

Synthesis of Carbohydrate-Grafted Glycopolymers Using a Catalyst-Free, Perfluoroarylazide-Mediated Fast Staudinger Reaction.

Glycopolymers have gained increasing importance in investigating glycan-lectin interactions, as drug delivery vehicles and in modulating interactions with proteins. The synthesis of these glycopolymers is still a challenging and rigorous exercise. In this regard, the highly efficient click reaction, copper (I)-catalyzed alkyne-azide cycloaddition, has been widely applied not only for its efficiency but also for its tolerance of the appended carbohydrate groups. However, a significant drawback of this method is the use of the heavy metal catalyst which is difficult to remove completely, and ultimately toxic to biological systems. In this work, we present the synthesis of carbohydrate-grafted glycopolymers utilizing a mild and catalyst-free perfluorophenyl azide (PFPA)-mediated Staudinger reaction. Using this strategy, mannose (Man) and maltoheptaose (MH) were grafted onto the biodegradable poly(lactic acid) (PLA) by stirring a PFAA-functionalized PLA with a phosphine-derivatized Man or MH in DMSO at room temperature within an hour. The glycopolymers were characterized by ¹H-NMR, 19F-NMR, 31P-NMR and FTIR.

Controlled Bio-Orthogonal Catalysis Using Nanozyme-Protein Complexes via Modulation of Electrostatic Interactions.

Bio-orthogonal chemistry provides a powerful tool for drug delivery systems due to its ability to generate therapeutic agents in situ, minimizing off-target effects. Bio-orthogonal transition metal catalysts (TMCs) with stimuli-responsive properties offer possibilities for controllable catalysis due to their spatial-, temporal-, and dosage-controllable properties. In this paper, we fabricated a stimuli-responsive bio-orthogonal catalysis system based on an enhanced green fluorescent protein (EGFP)-nanozyme (NZ) complex (EGFP-NZ). Regulation of the catalytic properties of the EGFP-NZ complex was directly achieved by modulating the ionic strength of the solution. The dielectric screening introduced by salt ions allows the dissociation of the EGFP-NZ complex, increasing the access of substrate to the active site of the NZs and concomitantly increasing nanozyme activity. The change in catalytic rate of the NZ/EGFP = 1:1 complex was positively correlated with salt concentration from 0 mM to 150 mM.

Engineering of bioorthogonal polyzymes through polymer sidechain design.

Synthetic polymer scaffolds can encapsulate transition metal catalysts (TMCs) to provide bioorthogonal nanocatalysts. These 'polyzymes' catalyze the in situ generation of therapeutic agents without disrupting native biological processes. The design and modification of polymer scaffolds in these polyzymes can enhance the catalytic performance of TMCs in biological environments. In this study, we explore the hydrophobic design space of an oxanorborneneimide-based polymer by varying the length of its carbon side chain to engineer bioorthogonal polyzymes. Activity studies indicate that modulating the hydrophobicity of the polymer scaffold can be used to enhance the catalyst loading efficacy, catalytic activity, and serum stability of polyzymes. These findings provide insight into the structural elements contributing to improving polymeric nanocatalysts for a variety of applications.

Gold Nanoclusters as Nanoantibiotic Auranofin Analogues.

Auranofin, a gold(I)-complex with tetraacetylated thioglucose (Ac4 GlcSH) and triethylphosphine ligands, is an FDA-approved drug used as an anti-inflammatory aid in the treatment of rheumatoid arthritis. In repurposing auranofin for other diseases, it was found that the drug showed significant activity against Gram-positive but was inactive against Gram-negative bacteria. Herein, the design and synthesis of gold nanoclusters (AuNCs) based on the structural motif of auranofin are reported. Phosphine-capped AuNCs are synthesized and glycosylated, yielding auranofin analogues with mixed triphenylphosphine monosulfonate (TPPMS)/Ac4 GlcSH ligand shells. These AuNCs are active against both Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Notably, an auranofin analogue, a mixed-ligand 1.6 nm AuNC 4b, is more active than auranofin against Pseudomonas aeruginosa, while exhibiting lower toxicity against human A549 cells. The enhanced antibacterial activity of these AuNCs is characterized by a greater uptake of Au by the bacteria compared to AuI complexes. Additional factors include increased oxidative stress, moderate inhibition of thioredoxin reductase (TrxR), and DNA damage. Most intriguingly, the uptake of AuNCs are not affected by the bacterial outer membrane (OM) barrier or by binding with the extracellular proteins. This contrasts with AuI complexes like auranofin that are susceptible to protein binding and hindered by the OM barrier.

Synthesis and solution isomerization of water-soluble Au9 nanoclusters prepared by nuclearity conversion of [Au11(PPh3)8Cl2]Cl.

Water-soluble gold nanoclusters (AuNCs) are popular in biomedical applications such as bioimaging, labelling, drug delivery, and biosensing. Despite their widespread applications, the synthesis of water-soluble phosphine-capped AuNCs is not as straightforward as their organic-soluble equivalents. Organic soluble phosphine-passivated [Au9(L)8]3+ are 6-electron closed-shell AuNCs that are generally prepared via the reduction of a phosphine-Au(I) complex by NaBH4. A similar approach attempted for the water-soluble ligand triphenylphosphine monosulfonate (TPPMS) using [AuTPPMS]Cl resulted in a mixture of cluster sizes that required gel electrophoresis or fractional precipitation to isolate the Au9 product. In this work, we report the synthesis of water-soluble [Au9(L)8]3+ nanoclusters in high yield through the biphasic ligand exchange of [Au11(PPh3)8Cl2]Cl with water-soluble phosphines such as TPPMS and 4-(diphenylphosphino)benzoic acid (DPPBA). The small molecule byproducts can be completely removed by size-based separation methods, like size exclusion chromatography or dialysis, as confirmed by 31P and 1H nuclear magnetic resonance (NMR) as well as diffusion ordered spectroscopy (DOSY). Furthermore, [Au9(DPPBA)8]Cl3 underwent a visible pH- and temperature-induced isomerization in ethanol between the 'crown' and 'butterfly' isomers of [Au9(L)8]3+ which has not been previously reported. Cytotoxicity evaluation of these water-soluble nanoclusters gave CC50 values of 36 µg mL-1 and 70 µg mL-1 against A549 human alveolar epithelial cells, and 30 µg mL-1 and 40 µg mL-1 against NIH/3T3 mouse fibroblast cells for [Au9(TPPMS)8]Cl3 and [Au9(DPPBA)8]Cl3, respectively. For comparison, auranofin, an FDA-approved gold drug, is more than an order of magnitude more toxic with a CC50 value of 7.7 µg mL-1 against A549 cells.

Maltoheptaose-Presenting Nanoscale Glycoliposomes for the Delivery of Rifampicin to E. coli.

Liposomes, a nanoscale drug delivery system, are well known for their ability to improve pharmacokinetics and reduce drug toxicity. In this work, maltoheptaose (G7)-presenting glycoliposomes were synthesized and evaluated in the delivery of the antibiotic rifampicin. Two types of liposomes were prepared: nonfluid liposomes from l-α-phosphatidylcholine (PC) and cholesterol, and fluid liposomes from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol). G7-derivatized glycolipid, G7-DPPE (DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), was incorporated into the liposomes at 21 and 14 µmol/mg to form nanoparticles of 75 ± 12 and 146 ± 14 nm for the nonfluid and fluid G7-glycoliposomes, respectively. The multivalent G7-glycoliposomes were characterized by lectin binding with concanavalin A (Con A). The dissociation constant K d between Con A and the nonfluid or fluid G7-glycoliposomes was 0.93 or 0.51 µM, which represented ~900- or 1600-fold stronger affinity than the binding between Con A and G7. The G7-glycoliposomes were loaded with rifampicin at 6.6 and 16 wt % encapsulation for the nonfluid and fluid G7-glycoliposomes, respectively. Introducing a carbohydrate in the liposomes slowed down the release of rifampicin, with the G7-glycoliposomes having the slowest release rate and the lowest permeability coefficient among the liposome formulations. The fluid G7-glycoliposomes lowered the minimal inhibitory concentration (MIC) of rifampicin against E. coli ORN208 by about 3 times, whereas liposomes without G7 or Man (d-mannose)-glycoliposomes showed no improvement in MIC. The rifampicin-loaded fluid G7-glycoliposomes demonstrated the best sustained antibacterial activity against E. coli, with up to 2 log reduction in the colony forming units at 4 × MIC after 24 h. Fluorescence resonance energy transfer and confocal fluorescence microscopy revealed stronger interactions of the bacterium with the fluid G7-glycoliposomes than other liposome formulations.