A Systematic comparison of in vitro cell uptake and in vivo biodistribution for three classes of gold nanoparticles with saturated PEG coatings.

A great deal of attention has been focused on nanoparticles for cancer therapy, with the promise of tumor-selective delivery. However, despite intense work in the field over many years, the biggest obstacle to this vision remains extremely low delivery efficiency of nanoparticles into tumors. Due to the cost, time, and impact on the animals for in vivo studies, the nanoparticle field predominantly uses cellular uptake assays as a proxy to predict in vivo outcomes. Extensive research has focused on decreasing macrophage uptake in vitro as a proxy to delay nanoparticle accumulation in the mononuclear phagocytic system (MPS), mainly the liver and spleen, and thereby increase tumor accumulation. We have recently reported novel synthetic methods employing small molecule crosslinkers for the controlled assembly of small nanoparticles into larger aggregates and found that these nanoaggregates had remarkably high surface coverage and low cell uptake, even in macrophages. We further found that this extremely low cellular uptake could be recapitulated on solid gold nanoparticles by densely coating their surface with small molecules. Here we report our studies on the biodistribution and clearance of these materials in comparison to more conventional PEGylated gold nanoparticles. It was expected that the remarkably low macrophage uptake in vitro would translate to extended blood circulation time in vivo, but instead we found no correlation between either surface coverage or in vitro macrophage cell uptake and in vivo blood circulation. Gold nanoaggregates accumulate more rapidly and to a higher level in the liver compared to control gold nanoparticles. The lack of correlation between in vitro macrophage uptake and in vivo blood circulation suggests that the field must find other in vitro assays to use as a primary proxy for in vivo outcomes or use direct in vivo experimentation as a primary assay.

Evaluation of size-based distribution of drug and excipient in amphotericin B liposomal formulation.

Conventional quantitation of drug content in the liposome formulation involves the breakdown of bulk liposomes, which ignores details on the distribution of the active pharmaceutical ingredient (API) and excipients in liposomes of different sizes. The objective of this study is to develop an analytical method which can separate the liposomes into different sizes and obtain information of the drug and excipient distribution in the different sized liposomes. We developed an asymmetric flow field-flow fractionation (AF4) method for size-based separation of AmBisome, an amphotericin B liposomal formulation, and a high-performance liquid chromatography ultraviolet-visible and charged aerosol detection (HPLC-UV-CAD) method for simultaneous quantitation of the API (Amphotericin B) and the lipid excipients [1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG), hydrogenated soy phosphatidylcholine (HSPC), and cholesterol]. The measured drug content in the bulk liposome formulation was consistent with the drug product labeling. Liposomes were separated using AF4 into eleven size fractions and the liposomes particles sizes of each fraction were measured with nanoparticle tracking analysis. The drug to total lipid ratios in fractionated liposomes increased from 0.1 to 0.45 when the liposome size increased from 75 nm to 124 nm, while the lipid composition remained constant throughout the fractioned size range (cholesterol:DSPG, 0.7 and HSPC:DSPG, 0.3). These study results suggest that, for liposomal formulations of Amphotericin B in liposomes, the drug to lipid ratio increases with the size of the liposomes. This new analytical method provided a more in-depth characterization of liposomes, i.e., determining drug and excipient distributions in different sizes of liposomes, in a more efficient manner with more specific size-based composition information.

Coating Metal Nanoparticle Surfaces with Small Organic Molecules Can Reduce Nonspecific Cell Uptake.

Elucidation of mechanisms of uptake of nanoparticles by cells and methods to prevent this uptake is essential for many applications of nanoparticles. Most recent studies have focused on the role of proteins that coat nanoparticles and have employed PEGylation, particularly dense coatings of PEG, to reduce protein opsonization and cell uptake. Here we show that small molecule coatings on metallic nanoparticles can markedly reduce cell uptake for very sparsely PEGylated nanoparticles. Similar results were obtained in media with and without proteins, suggesting that protein opsonization is not the primary driver of this phenomenon. The reduction in cell uptake is proportional to the degree of surface coverage by the small molecules. Probing cell uptake pathways using inhibitors suggested that the primary role of increased surface coverage is to reduce nanoparticles' interactions with the scavenger receptors. This work highlights an under-investigated mechanism of cell uptake that may have played a role in many other studies and also suggests that a wide variety of molecules can be used alongside PEGylation to stably passivate nanoparticle surfaces for low cell uptake.

Controlled Assembly of Biocompatible Metallic Nanoaggregates Using a Small Molecule Crosslinker.

By introducing a capping step and controlling the reaction parameters, assembly of metallic nanoparticle aggregates can be achieved using a small-molecule crosslinker. Aggregates can be assembled from particles of varied size and composition and the size of the aggregates can be systematically adjusted. Following cell uptake of 60 nm aggregates, the aggregates are stable and nontoxic to macrophage cells up to 55 × 10(-3) m Au.

Functionalized iron oxide nanoparticles for controlling the movement of immune cells.

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed "cell box" was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain.

Neural stem cell-mediated intratumoral delivery of gold nanorods improves photothermal therapy.

Plasmonic photothermal therapy utilizes biologically inert gold nanorods (AuNRs) as tumor-localized antennas that convert light into heat capable of eliminating cancerous tissue. This approach has lower morbidity than surgical resection and can potentially synergize with other treatment modalities including chemotherapy and immunotherapy. Despite these advantages, it is still challenging to obtain heating of the entire tumor mass while avoiding unnecessary collateral damage to surrounding healthy tissue. It is therefore critical to identify innovative methods to distribute an effective concentration of AuNRs throughout tumors without depositing them in surrounding healthy tissue. Here we demonstrate that AuNR-loaded, tumor-tropic neural stem cells (NSCs) can be used to improve the intratumoral distribution of AuNRs. A simple UV-vis technique for measuring AuNR loading within NSCs was established. It was then confirmed that NSC viability is unimpaired following AuNR loading and that NSCs retain AuNRs long enough to migrate throughout tumors. We then demonstrate that intratumoral injections of AuNR-loaded NSCs are more efficacious than free AuNR injections, as evidenced by reduced recurrence rates of triple-negative breast cancer (MDA-MB-231) xenografts following NIR exposure. Finally, we demonstrate that the distribution of AuNRs throughout the tumors is improved when transported by NSCs, likely resulting in the improved efficacy of AuNR-loaded NSCs as compared to free AuNRs. These findings highlight the advantage of combining cellular therapies and nanotechnology to generate more effective cancer treatments.

Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-κB.

Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression.

Matrix metalloproteinase-triggered denuding of engineered gold nanoparticles for selective cell uptake.

Targeted delivery of therapeutic agents to tumor sites increases efficacy and limits off-target toxicity. Nanoparticles are an emerging class of targeted drug delivery systems. Commonly, nanoparticles are coated with poly(ethylene glycol) (PEG) to reduce off-target uptake by cells of the mononuclear phagocyte system (MPS) and a targeting moiety to promote uptake at the desired location. This approach holds great promise, but such constructs still predominantly accumulate in the liver. Here we demonstrate a different approach to tumor targeting using nanoparticles functionalized with a PEG coating that is shed in the presence of matrix metalloproteinase-2 (MMP-2), which is overexpressed in many tumor microenvironments. There was very little uptake of intact particles by human breast adenocarcinoma cells, whereas, when the same cells were treated with particles in the presence of MMP-2, the resulting denuded particles were rapidly taken up by the cells. This system is remarkably simple as the core nanoparticles revealed by PEG cleavage are not modified; uptake is driven simply by revealing the nanoparticle surface. The cleavable linker is a modular component that, in the future, can be designed to respond to other stimuli. This approach could lead to improved imaging and targeted drug delivery for solid tumors.

Effect of orientation on plasmonic coupling between gold nanorods.

Radiative coupling of induced plasmonic fields in metal nanoparticles has drawn increasing attention in the recent literature due to a combination of improved experimental methods to study such phenomena and numerous potential applications, such as plasmonic nanoparticle rulers and plasmonic circuitry. Many groups, including ours, have used a near-exponential fit to express the size scaling of plasmonic coupling. First, we show experimental agreement between previously simulated nanorod coupling and plasmonic coupling in electron beam lithography (EBL) fabricated nanorods using the near-exponential expression. Next, we study the effect of nanoparticle orientation on plasmonic coupling using EBL and DDA simulations. We develop a mathematical relationship that is consistent with our findings and quantitatively describes plasmonic coupling between nanorods as a function of orientation, separation, induced dipole strength, and the dielectric constant of the medium. For applications utilizing plasmonic coupling to become viable with particle shapes that do not have spherical symmetry, such as nanoprisms and nanorods, comparison of the experimental and theoretical results of how particle orientation affects plasmonic coupling is essential.