Francois Fay

Antibody-targeted nanoparticles for cancer therapy

In recent years, nanoparticulate-mediated drug delivery research has examined a full spectrum of nanoparticles that can be used in diagnostic and therapeutic cancer applications. A key aspect of this technology is in the potential to specifically target the nanoparticles to diseased cells using a range of molecules, in particular antibodies. Antibody-nanoparticle conjugates have the potential to elicit effective targeting and release of therapeutic targets at the disease site, while minimizing off-target side effects caused by dosing of normal tissues. This article provides an overview of various antibody-conjugated nanoparticle strategies, focusing on the rationale of cell-surface receptors targeted and their potential clinical application.

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery

Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality.

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Skin dendritic cell targeting via microneedle arrays laden with antigen-encapsulated poly-D,L-lactide-co-glycolide nanoparticles induces efficient antitumor and antiviral immune responses.

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8+ T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis

Significance This study shows that an endothelialized microfluidic chip with controllable permeability can serve as a model for nanoparticle translocation across the permeable endothelium. Integration of this in vitro model and an in vivo rabbit model revealed that the extravasation of nanoparticles across the endothelium in atherosclerotic plaques depends on microvascular permeability. This approach represents a unique method for the assessment of nanoparticle behavior across the atherosclerotic endothelium, and may also serve as a valuable tool to study nanomedicine accumulation in a variety of other diseases. Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid–polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.

Microneedle-mediated intradermal nanoparticle delivery: Potential for enhanced local administration of hydrophobic pre-formed photosensitisers

INTRODUCTION To date, 5-aminolevulinic acid (ALA) has been the most widely used agent in topical photodynamic therapy (PDT). However, owing to the poor penetration of ALA into skin, ALA-PDT is inappropriate for difficult-to-treat deep skin neoplasias, such as nodular basal cell carcinoma. An alternative strategy to ALA-PDT is to use pre-formed photosensitisers, which can be activated at longer wavelengths, facilitating enhanced light penetration into skin. Owing to their relatively high molecular weights and often high lipophilicities, these compounds cannot be effectively administered topically. This study aimed to deliver a model hydrophobic dye, Nile red, into the skin using novel microneedle (MN) technology. MATERIALS AND METHODS Nile red was incorporated into poly-lactide-co-glycolic acid (PLGA) nanoparticles using an emulsion and salting-out process. Polymeric MN arrays were prepared from aqueous blends of the mucoadhesive copolymer Gantrez(®) AN-139 and tailored to contain 1.0mg of Nile red-loaded PLGA nanoparticles. Intradermal delivery of Nile red was determined in vitro. RESULTS Uniform 150nm diameter PLGA nanoparticles were prepared containing 3.87μg Nile red / mg of PLGA. Tissue penetration studies using excised porcine skin revealed that high tissue concentrations of Nile red were observed at 1.125mm (382.63ng cm(-3)) following MN delivery. CONCLUSION For the first time, polymeric microneedles (MN) have been employed to deliver a model lipophilic dye, Nile red, into excised porcine skin. Importantly, this is a one-step delivery strategy for the local delivery of highly hydrophobic agents, which overcomes many of the disadvantages of current delivery strategies.

Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation.

Nanoparticle-based delivery of simvastatin inhibits plaque macrophage proliferation in apolipoprotein E–deficient mice. Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E–deficient mice (Apoe−/−) with advanced atherosclerotic plaques. This resulted in the rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an 8-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

Single Step Reconstitution of Multifunctional High-Density Lipoprotein-Derived Nanomaterials Using Microfluidics

High-density lipoprotein (HDL) is a natural nanoparticle that transports peripheral cholesterol to the liver. Reconstituted high-density lipoprotein (rHDL) exhibits antiatherothrombotic properties and is being considered as a natural treatment for cardiovascular diseases. Furthermore, HDL nanoparticle platforms have been created for targeted delivery of therapeutic and diagnostic agents. The current methods for HDL reconstitution involve lengthy procedures that are challenging to scale up. A central need in the synthesis of rHDL, and multifunctional nanomaterials in general, is to establish large-scale production of reproducible and homogeneous batches in a simple and efficient fashion. Here, we present a large-scale microfluidics-based manufacturing method for single-step synthesis of HDL-mimicking nanomaterials (μHDL). μHDL is shown to have the same properties (e.g., size, morphology, bioactivity) as conventionally reconstituted HDL and native HDL. In addition, we were able to incorporate simvastatin (a hydrophobic drug) into μHDL, as well as gold, iron oxide, quantum dot nanocrystals or fluorophores to enable its detection by computed tomography (CT), magnetic resonance imaging (MRI), or fluorescence microscopy, respectively. Our approach may contribute to effective development and optimization of lipoprotein-based nanomaterials for medical imaging and drug delivery.

Gold Nanocrystal Labeling Allows Low-Density Lipoprotein Imaging from the Subcellular to Macroscopic Level

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion-evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells.