Joseph Hardie

Promises and Pitfalls of Intracellular Delivery of Proteins

The direct delivery of functional proteins into the cell cytosol is a key issue for protein therapy, with many current strategies resulting in endosomal entrapment. Protein delivery to the cytosol is challenging due to the high molecular weight and the polarity of therapeutic proteins. Here we review strategies for the delivery of proteins into cells, including cell-penetrating peptides, virus-like particles, supercharged proteins, nanocarriers, polymers, and nanoparticle-stabilized nanocapsules. The advantages and disadvantages of these approaches including cytosolar delivery are compared and contrasted, with promising pathways forward identified.

Chemically Engineered Nanoparticle-Protein Interface for Real-Time Cellular Oxidative Stress Monitoring.

A co-engineered nanoparticle/protein peroxide detector is created. This system features a gold nanoparticle functionalized with a galactose headgroup (AuNP-Gal) that reacts covalently with a boronate-modified green fluorescent protein (PB-GFP). Boronate acid-saccharide complexation between PB-GFP and AuNP-Gal affords a highly stable assembly. This complex is disrupted by peroxide, allowing quantitative and selective monitoring of hydrogen peroxide production in real time.

Progress and perspective of inorganic nanoparticle-based siRNA delivery systems

ABSTRACT Introduction: Small interfering RNA (siRNA) is an effective method for regulating the expression of proteins, even “undruggable” ones that are nearly impossible to target through traditional small molecule therapeutics. Delivery to the cell and then to the cytosol is the primary requirement for realization of therapeutic potential of siRNA. Areas covered: We summarize recent advances in the design of inorganic nanoparticle with surface functionality and physicochemical properties engineered for siRNA delivery. Specifically, we discuss the main approaches developed so far to load siRNA into/onto NPs, and NP surface chemistry engineered for enhanced intracellular siRNA delivery, endosomal escape, and targeted delivery of siRNA to disease cells and tissues. Expert Opinion: Several challenges remain in developing inorganic NPs for efficient and effective siRNA delivery. Getting the material to the chosen site is important, however the greatest hurdle may well be delivery into the cytosol, either through efficient endosomal escape or by direct cytosolic siRNA delivery. Effective delivery at the organismic and cellular level coupled with biocompatible vehicles with low immunogenic response will facilitate the clinical translation of RNAi for the treatment of genetic diseases.

Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules

We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death.

Effects of engineered nanoparticles on the innate immune system

Engineered nanoparticles (NPs) have broad applications in industry and nanomedicine. When NPs enter the body, interactions with the immune system are unavoidable. The innate immune system, a non-specific first line of defense against potential threats to the host, immediately interacts with introduced NPs and generates complicated immune responses. Depending on their physicochemical properties, NPs can interact with cells and proteins to stimulate or suppress the innate immune response, and similarly activate or avoid the complement system. NPs size, shape, hydrophobicity and surface modification are the main factors that influence the interactions between NPs and the innate immune system. In this review, we will focus on recent reports about the relationship between the physicochemical properties of NPs and their innate immune response, and their applications in immunotherapy.

RNAi Nanomaterials and Nanovehicles: Inorganic Nanoparticles for RNAi

Abstract The discovery of RNA interference (RNAi) has been enormously beneficial as a biochemical tool, and holds the potential to become an effective therapy for a variety of diseases, including cancer. To harness this capability, a safe and efficient short interfering RNA (siRNA) delivery system is required. The development of such an entity has posed a critical challenge to the drug delivery field. In recent years, advancements in the fabrication and functionalization of inorganic materials have provided an array of alternative tools for siRNA delivery in vitro and in vivo. The combination of the appropriate selection of siRNA targets, along with the use of increasingly sophisticated inorganic nanocarriers, will pave the way to expanding the therapeutic potential of RNAi, facilitating its translation to clinical use.

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment

&NA; The use of nanoparticle‐stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor‐&agr; efficiently silenced TNF‐&agr; expression in LPS‐challenged mice, demonstrating efficacy in modulating immune response in an organ‐selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Graphical abstract Nanoparticle‐stabilized nanocapsules (NPSCs) are used to deliver TNF‐&agr; targeted siRNA for inflammation therapy. NPSC siRNA delivery effectively prevents cytokine production in LPS‐stimulated mice. Figure. No Caption available. HighlightsImmunomodulation is an effective strategy to treat a wide range of immune diseases.RNA interference (RNAi) is a safe, specific method for reducing the cytokine expression.Nanocapsules deliver siRNA direct to the cytosol for effective RNAi.Nanocapsules loaded with anti‐inflammatory siRNA diminished immune response in mice.Anti‐inflammatory NPSCs have potential to ameliorate inflammatory diseases.

Surface-Modified Macrophages Facilitate Tracking of Breast Cancer-Immune Interactions

The immune system has been found to play key roles in cancer development and progression. Macrophages are typically considered to be pro-inflammatory cells but can also facilitate pro-oncogenic activities via associations with tumors and metastases. The study of macrophages and their interactions within the context of cancer microenvironments is stymied by the lack of a system to track them. We present a cell-based strategy for studying cancer-immune cell interactions by chemically modifying the surfaces of macrophages with fluorophores. Two widely used methods are employed, affecting cell surface proteins and glycans via NHS-ester and Staudinger ligation reactions, respectively. We show that these modifications do not interfere with macrophage responses to chemoattractants and that interactions with cancer cells can be readily monitored. This work describes the development of macrophage-based imaging agents for tumor detection and assessment of interactions between immune cells and cancers.