Austin M. Derfus

Probing the cytotoxicity of semiconductor quantum dots

With their bright, photostable fluorescence, semiconductor quantum dots show promise as alternatives to organic dyes for biological labeling. Questions about their potential cytotoxicity, however, remain unanswered. While cytotoxicity of bulk cadmium selenide (CdSe) is well documented, a number of groups have suggested that CdSe QDs are cytocompatible, at least with some immortalized cell lines. Using primary hepatocytes as a liver model, we found that CdSe-core QDs were indeed acutely toxic under certain conditions. Specifically, we found that the cytotoxicity of QDs was modulated by processing parameters during synthesis, exposure to ultraviolet light, and surface coatings. Our data further suggests that cytotoxicity correlates with the liberation of free Cd2+ ions due to deterioration of the CdSe lattice. When appropriately coated, CdSe-core QDs can be rendered non-toxic and used to track cell migration and reorganization in vitro. Our results inform design criteria for the use of QDs in vitro and especially in vivo where deterioration over time may occur.

Biomimetic amplification of nanoparticle homing to tumors

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

Systematic Surface Engineering of Magnetic Nanoworms for in vivo Tumor Targeting

In the design of nanoparticles that can target disease tissue in vivo, parameters such as targeting ligand density, type of target receptor, and nanoparticle shape can play an important role in determining the extent of accumulation. Herein, a systematic study of these parameters for the targeting of mouse xenograft tumors is performed using superparamagnetic iron oxide as a model nanoparticle system. The type of targeting peptide (recognizing cell surface versus extracellular matrix), the surface coverage of the peptide, its attachment chemistry, and the shape of the nanomaterial [elongated (nanoworm, NW) versus spherical (nanosphere, NS)] are varied. Nanoparticle circulation times and in vivo tumor-targeting efficiencies are quantified in two xenograft models of human tumors (MDA-MB-435 human carcinoma and HT1080 human fibrosarcoma). It is found that the in vivo tumor-targeting ability of the NW is superior to that of the NS, that the smaller, neutral CREKA targeting group is more effective than the larger, positively charged F3 molecule, that a maximum in tumor-targeting efficiency and blood half-life is observed with approximately 60 CREKA peptides per NW for either the HT1080 or the MDA-MB-435 tumor types, and that incorporation of a 5-kDa polyethylene glycol linker improves targeting to both tumor types relative to a short linker. It is concluded that the blood half-life of a targeting molecule-nanomaterial ensemble is a key consideration when selecting the appropriate ligand and nanoparticle chemistry for tumor targeting.

Quantum dots to monitor RNAi delivery and improve gene silencing

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell–cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.

The Compatibility of Hepatocytes with Chemically Modified Porous Silicon with Reference to In Vitro Biosensors

Porous Si is a nanostructured material that is of interest for molecular and cell-based biosensing, drug delivery, and tissue engineering applications. Surface chemistry is an important factor determining the stability of porous Si in aqueous media, its affinity for various biomolecular species, and its compatibility with tissues. In this study, the attachment and viability of a primary cell type to porous Si samples containing various surface chemistries is reported, and the ability of the porous Si films to retain their optical reflectivity properties relevant to molecular biosensing is assessed. Four chemical species grafted to the porous Si surface are studied: silicon oxide (via ozone oxidation), dodecyl (via hydrosilylation with dodecene), undecanoic acid (via hydrosilylation with undecylenic acid), and oligo(ethylene) glycol (via hydrosilylation with undecylenic acid followed by an oligo(ethylene) glycol coupling reaction). Fourier Transform Infrared (FTIR) spectroscopy and contact angle measurements are used to characterize the surface. Adhesion and short-term viability of primary rat hepatocytes on these surfaces, with and without pre-adsorption of collagen type I, are assessed using vital dyes (calcein-AM and ethidium homodimer I). Cell viability on undecanoic acid-terminated porous Si, oxide-terminated porous Si, and oxide-terminated flat (non-porous) Si are monitored by quantification of albumin production over the course of 8 days. The stability of porous Si thin films after 8 days in cell culture is probed by measuring the optical interferometric reflectance spectra. Results show that hepatocytes adhere better to surfaces coated with collagen, and that chemical modification does not exert a deleterious effect on primary rat hepatocytes. The hydrosilylation chemistry greatly improves the stability of porous Si in contact with cultured primary cells while allowing cell coverage levels comparable to standard culture preparations on tissue culture polystyrene.