Clinical translation of photoacoustic imaging (PAI) has been limited by the lack of near-infrared (NIR) contrast agents with low toxicity required for regulatory approval. Herein, J aggregates of indocyanine green (ICG) with strong NIR absorbance were encapsulated at high loadings within small 77 nm polymersomes (nanocapsules) composed of poly­(lactide-co-glycolide-b-poly­(ethylene glycol)) (PLGA-b-PEG) bilayers, thus enabling PAI of of breast and ovarian cancer cells with high specificity and a sensitivity at the level of ∼100 total cells. All of the major components of the polymersomes are FDA approved and used in the clinic. During formation of polymersomes with a water-in-oil-in-water double emulsion process, loss of ICG from the ICG J aggregates was minimized by coating them with a layer of branched polyethylenimine and by providing excess “sacrificial” ICG to adsorb at the oil–water interfaces. The encapsulated J aggregates were protected against dissociation by the polymersome shell for 24 h in 100% fetal bovine serum, after which the polymersomes biodegraded and the J aggregates dissociated to ICG monomers.

Nanoparticle (NP) clusters with diameters ranging from 20 to 100 nm are reversibly assembled from 5 nm gold (Au) primary particles coated with glutathione (GSH) in aqueous solution as a function of pH in the range of 5.4 to 3.8. As the pH is lowered, the GSH surface ligands become partially zwitterionic and form interparticle hydrogen bonds that drive the self-limited assembly of metastable clusters in <1 min. Whereas clusters up to 20 nm in size are stable against cluster-cluster aggregation for up to 1 day, clusters up to 80 nm in size can be stabilized over this period via the addition of citrate to the solution in equal molarity with GSH molecules. The cluster diameter may be cycled reversibly by tuning pH to manipulate the colloidal interactions; however, modest background cluster-cluster aggregation occurs during cycling. Cluster sizes can be stabilized for at least 1 month via the addition of PEG-thiol as a grafted steric stabilizer, where PEG-grafted clusters dissociate back to starting primary NPs at pH 7 in fewer than 3 days. Whereas the presence of excess citrate has little effect on the initial size of the metastable clusters, it is necessary for both the cycling and dissociation to mediate the GSH-GSH hydrogen bonds. In summary, these metastable clusters exhibit significant characteristics of equilibrium self-limited assembly between primary particles and clusters on time scales where cluster-cluster aggregation is not present.

The self-assembly of citrate-capped Au nanoparticles (5 nm) resulted in branched nanochains by adding CaCl2 versus spherical nanoclusters for NaCl. These assemblies were formed between 1 s to 30 min by tuning the electrostatic repulsion and the interparticle bridging attraction between the cations and citrate ligands as a function of electrolyte concentration. For dilute Ca(2+), strong interparticle bridging favored particle attachment at chain ends. This resulted in the formation of small, branched chains with lengths as short as 20 nm, due to the large Debye length for the diffuse counterions. Furthermore, the bridging produced very small interparticle spacings and sintering, as evident in high-resolution TEM despite the low temperature. This morphology produced a large red shift in the surface plasmon resonance, as characterized by a broad extinction peak with NIR absorption out to 1000 nm, which is unusual for such small particles. Whereas these properties were seen for primary particles with partial citrate monolayers, the degrees of sintering and NIR extinction were small in the case of citrate multilayers. The ability to design the size and shape of nanoparticle clusters as well as the interparticle spacing by tuning bridging and electrostatic interactions may be expected to be quite general and of broad applicability in materials synthesis.

Reversible NIR-active nanoparticle clusters with controlled size from 20 to 100 nm were assembled from 5 nm gold nanoparticles (Au NP), with either citrate (CIT) or various binary ligands on the surface, by tuning the electrostatic repulsion and the hydrogen bonding via pH. The nanoclusters were bound together by vdW forces between the cores and the hydrogen bonds between the surface ligands and dissociated to primary nanoparticles over a period of 20 days at pH 5 and at pH 7. When high levels of citrate ligands were used on the primary particle surfaces, the large particle spacings in the nanoclusters led to only modest NIR extinction. However, a NIR extinction (E) ratio of up to ∼0.4 was obtained for nanoclusters with binary ligand mixtures composed of citrate and either cysteine (CYS), glutathione (GSH), or thioctic acid zwitterion (TAZ) while maintaining full reversibility to primary particles. The optimum ligand ratio for both an E of ∼0.4 and full reversibility decreased with increasing length of the secondary ligand (1.5/1 for CYS/CIT, 0.75/1 for GSH/CIT, and 0.5/1 for TAZ/CIT) because a longer secondary ligand maintains a sufficient interparticle spacing required for dissociation more effectively. Interestingly, the zeta potential and the first-order rate constant for nanocluster dissociation were similar for all three systems at the optimum ligand ratios. After incubation in 10 mM GSH solution (intracellular concentration), only the TAZ/CIT primary nanoparticles were resistant to protein opsonization in 100% fetal bovine serum, as the bidentate binding and zwitterion tips of TAZ resisted GSH exchange and protein opsonization, respectively.

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