Jonathan Gunn

Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging.

Magnetic nanoparticles (MNPs) represent a class of non-invasive imaging agents that have been developed for magnetic resonance (MR) imaging. These MNPs have traditionally been used for disease imaging via passive targeting, but recent advances have opened the door to cellular-specific targeting, drug delivery, and multi-modal imaging by these nanoparticles. As more elaborate MNPs are envisioned, adherence to proper design criteria (e.g. size, coating, molecular functionalization) becomes even more essential. This review summarizes the design parameters that affect MNP performance in vivo, including the physicochemical properties and nanoparticle surface modifications, such as MNP coating and targeting ligand functionalizations that can enhance MNP management of biological barriers. A careful review of the chemistries used to modify the surfaces of MNPs is also given, with attention paid to optimizing the activity of bound ligands while maintaining favorable physicochemical properties.

Chitosan-based hydrogels for controlled, localized drug delivery.

Hydrogels are high-water content materials prepared from cross-linked polymers that are able to provide sustained, local delivery of a variety of therapeutic agents. Use of the natural polymer, chitosan, as the scaffold material in hydrogels has been highly pursued thanks to the polymers biocompatibility, low toxicity, and biodegradability. The advanced development of chitosan hydrogels has led to new drug delivery systems that release their payloads under varying environmental stimuli. In addition, thermosensitive hydrogel variants have been developed to form a chitosan hydrogel in situ, precluding the need for surgical implantation. The development of these intelligent drug delivery devices requires a foundation in the chemical and physical characteristics of chitosan-based hydrogels, as well as the therapeutics to be delivered. In this review, we investigate the newest developments in chitosan hydrogel preparation and define the design parameters in the development of physically and chemically cross-linked hydrogels.

Specific targeting of brain tumors with an optical/magnetic resonance imaging nanoprobe across the blood-brain barrier.

Nanoparticle-based platforms have drawn considerable attention for their potential effect on oncology and other biomedical fields. However, their in vivo application is challenged by insufficient accumulation and retention within tumors due to limited specificity to the target, and an inability to traverse biological barriers. Here, we present a nanoprobe that shows an ability to cross the blood-brain barrier and specifically target brain tumors in a genetically engineered mouse model, as established through in vivo magnetic resonance and biophotonic imaging, and histologic and biodistribution analyses. The nanoprobe is comprised of an iron oxide nanoparticle coated with biocompatible polyethylene glycol-grafted chitosan copolymer, to which a tumor-targeting agent, chlorotoxin, and a near-IR fluorophore are conjugated. The nanoprobe shows an innocuous toxicity profile and sustained retention in tumors. With the versatile affinity of the targeting ligand and the flexible conjugation chemistry for alternative diagnostic and therapeutic agents, this nanoparticle platform can be potentially used for the diagnosis and treatment of a variety of tumor types.

In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes.

Converging advances in the development of nanoparticle-based imaging probes and improved understanding of the molecular biology of brain tumors offer the potential to provide physicians with new tools for the diagnosis and treatment of these deadly diseases. However, the effectiveness of promising nanoparticle technologies is currently limited by insufficient accumulation of these contrast agents within tumors. Here a biocompatible nanoprobe composed of a poly(ethylene glycol) (PEG) coated iron oxide nanoparticle that is capable of specifically targeting glioma tumors via the surface-bound targeting peptide, chlorotoxin (CTX), is presented. The preferential accumulation of the nanoprobe within gliomas and subsequent magnetic resonance imaging (MRI) contrast enhancement are demonstrated in vitro in 9L cells and in vivo in tumors of a xenograft mouse model. TEM imaging reveals that the nanoprobes are internalized into the cytoplasm of 9L cells and histological analysis of selected tissues indicates that there are no acute toxic effects of these nanoprobes. High targeting specificity and benign biological response establish this nanoprobe as a potential platform to aid in the diagnosis and treatment of gliomas and other tumors of neuroectodermal origin.

Inhibition of tumor-cell invasion with chlorotoxin-bound superparamagnetic nanoparticles

Nanoparticles have been investigated as drug delivery vehicles, contrast agents, and multifunctional devices for patient care. Current nanoparticle-based therapeutic strategies for cancer treatment are mainly based on delivery of chemotherapeutic agents to induce apoptosis or DNA/siRNA to regulate oncogene expression. Here, a nanoparticle system that demonstrates an alternative approach to the treatment of cancers through the inhibition of cell invasion, while serving as a magnetic resonance and optical imaging contrast agent, is presented. The nanoparticle comprises an iron oxide nanoparticle core conjugated with an amine-functionalized poly(ethylene glycol) silane and a small peptide, chlorotoxin (CTX), which enables the tumor cell-specific binding of the nanoparticle. It is shown that the nanoparticle exhibits substantially enhanced cellular uptake and an invasion inhibition rate of approximately 98% compared to unbound CTX ( approximately 45%). Significantly, the investigation from flow cytometry analysis, transmission electron microscopy, and fluorescent imaging reveals that the CTX-enabled nanoparticles deactivated the membrane-bound matrix metalloproteinase 2 (MMP-2) and induced increased internalization of lipid rafts that contain surface-expressed MMP-2 and volume-regulating ion channels through receptor-mediated endocytosis, leading to enhanced prohibitory effects. Since upregulation and activity of MMP-2 have been observed in tumors of neuroectodermal origin, and in cancers of the breast, colon, skin, lung, prostate, ovaries, and a host of others, this nanoparticle system can be potentially used for non-invasive diagnosis and treatment of a variety of cancer types.

Polyblend nanofibers for biomedical applications: perspectives and challenges

Advances in disease treatment and tissue regeneration are buoyed by new, multifaceted materials that emulate and coercively interact with the local microenvironment. Polyblend nanofibers represent an emerging class of biomimetic nanostructures that can act as proxies of the native tissue, while providing topographical and biochemical cues that promote healing. These fibers are prepared with mixtures of synthetically and naturally derived polymers that can behave cooperatively to demonstrate unique combinations of mechanical, biochemical and structural properties. This flexibility has led to the application of polyblend nanofibers in a wide assortment of tissue engineering and drug delivery systems. In this review, we will examine design criteria and properties of polymer-blend nanofibers and their use in tissue engineering and local therapeutic delivery applications.

A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells

As conventional cancer therapies struggle with toxicity issues and irregular remedial efficacy, the preparation of novel gene therapy vectors could offer clinicians the tools for addressing the genetic errors of diseased tissue. The transfer of gene therapy to the clinic has proven difficult due to safety, target specificity, and transfection efficiency concerns. Polyethylenimine (PEI) nanoparticles have been identified as promising gene carriers that induce gene transfection with high efficiency. However, the inherent toxicity of the material and non-selective delivery are the major concerns in applying these particles clinically. Here, a non-viral nanovector has been developed by PEGylation of DNA-complexing PEI in nanoparticles functionalized with an Alexa Fluor 647 near infrared fluorophore, and the chlorotoxin (CTX) peptide which binds specifically to many forms of cancer. With this nanovector, the potential toxicity to healthy cells is minimized by both the reduction of the toxicity of PEI with the biocompatible copolymer and the targeted delivery of the nanovector to cancer cells, as evaluated by viability studies. The nanovector demonstrated high levels of targeting specificity and gene transfection efficiency with both C6 glioma and DAOY medulloblastoma tumor cells. Significantly, with the CTX as the targeting ligand, the nanovector may serve as a widely applicable gene delivery system for a broad array of cancer types.

A Multimodal Targeting Nanoparticle for Selectively Labeling T Cells

Cancer immunotherapy approaches, including vaccination,[1] adoptive cell transfer (ACT),[2,3] and combinational strategies,[4] have been developed to assist the bodys immune system to selectively recognize and kill malignant tumor cells. Currently, immunotherapies are evaluated by either function-based assays, such as enzyme-linked immunosorbent spot (ELISPOT) and limiting dilution studies,[5] or structure-based assays such as peptide-MHC tetramer labeling.[6] These assessment methods require invasive sample collection,[1-3] have not yielded strong correlations with clinical responses to treatment,[7] and provide limited in vivo T cell tracking information. Alternative visualization strategies have been developed, whereby T cells extracted from an animal and labeled ex vivo, are injected back into the animal to be monitored. This approach has been applied to positron emission tomography (PET), single-photon emission computed tomography (SPECT),[8] and multi-photon intravital microscopy.[9,10] More recently, magnetic nanoparticle labeling of T cells for in vivo tracking by MRI has received considerable attention, as MRI offers superior capabilities for deep-tissue, whole-body imaging at higher resolution than alternative imaging modalities.[11] These nanoparticles have been coupled with immunotherapy regimens as ex vivo T cell labels for ACT, inducing non-specific cellular uptake through conjugation with the transmembrane HIV-Tat peptide,[12,13] poly-L-lysine,[14] or using lipofection reagents.[15] While capable of labeling cells, these nanoparticles cannot specifically bind to cytotoxic T lymphocytes (CTLs) and thus, use of these nanoparticles in vitro requires either CTL isolation or prolonged CTL expansion before the labeling can be performed, and for in vivo tracking, is limited to externally tagged cells, neglecting endogenously recruited, vaccine-elicited or ad hoc labeling of adoptively transferred CTLs.

On-site alginate gelation for enhanced cell proliferation and uniform distribution in porous scaffolds.

High cell density and uniformity in a tissue-engineered construct is essential to expedite the formation of a uniform extracellular matrix. In this study, we demonstrated an on-site gelation approach to increase cellular population and uniformity through porous scaffolds using alginate as gelling material. The on-site gelation was triggered during cell seeding and was shown to effectively restrain the cells in the porous scaffold during subsequent cell cultivation. The initial demonstration of the effectiveness of this system was made with chondrocyte cells, targeted at functional restoration of damaged or dysfunctional cartilage. By limiting cellular mobility, cell population increased by 89% after 7 days of cell culture in scaffolds encapsulating alginate gel as opposed to a 36% increase in scaffolds without gel. The cell distribution throughout the gelled scaffold was found to be more uniform than in the nongelled scaffold. SEM analysis revealed that the cells exhibited typical chondrocytic morphology. Improved cellular functionality was verified by low levels of collagen type I gene expression and steady gene activity levels of collagen type II over 3 weeks of cell cultivation. Alternatively, cells seeded in scaffolds with the conventional cell-seeding method demonstrated increased levels of collagen type I gene expression, indicating the possibility of cell dedifferentiation over long-term cell culture. Success with the chitosan-alginate scaffold model suggested that this flexible on-site gelation method could be potentially applied to other cell and tissue types for enhanced tissue engineering development.

A pretargeted nanoparticle system for tumor cell labeling

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope.