Martin A. Philbert

Safe handling of nanotechnology

The pursuit of responsible nanotechnologies can be tackled through a series of grand challenges, argue Andrew D. Maynard and his co-authors.Take fiveThe spectre of possible harm — real or imagined — is threatening to slow the development of nanotechnology. In a Commentary this week a group of nanotechnologists outlines a series of five “grand challenges”. If they and their colleagues can rise to these challenges — which include development of new ways of measuring exposure to nanomaterials and assessing the health and environmental impact of that exposure — the true extent of any risks involved should become clear.

Vascular Targeted Nanoparticles for Imaging and Treatment of Brain Tumors

Purpose: Development of new therapeutic drug delivery systems is an area of significant research interest. The ability to directly target a therapeutic agent to a tumor site would minimize systemic drug exposure, thus providing the potential for increasing the therapeutic index. Experimental Design: Photodynamic therapy (PDT) involves the uptake of a sensitizer by the cancer cells followed by photoirradiation to activate the sensitizer. PDT using Photofrin has certain disadvantages that include prolonged cutaneous photosensitization. Delivery of nanoparticles encapsulated with photodynamic agent specifically to a tumor site could potentially overcome the drawbacks of systemic therapy. In this study, we have developed a multifunctional polymeric nanoparticle consisting of a surface-localized tumor vasculature targeting F3 peptide and encapsulated PDT and imaging agents. Results: The nanoparticles specifically bound to the surface of MDA-435 cells in vitro and were internalized conferring photosensitivity to the cells. Significant magnetic resonance imaging contrast enhancement was achieved in i.c. rat 9L gliomas following i.v. nanoparticle administration. Serial magnetic resonance imaging was used for determination of pharmacokinetics and distribution of nanoparticles within the tumor. Treatment of glioma-bearing rats with targeted nanoparticles followed by PDT showed a significant improvement in survival rate when compared with animals who received PDT after administration of nontargeted nanoparticles or systemic Photofrin. Conclusions: This study reveals the versatility and efficacy of the multifunctional nanoparticle for the targeted detection and treatment of cancer.

Photodynamic Characterization and In Vitro Application of Methylene Blue-containing Nanoparticle Platforms¶

Abstract This article presents the development and characterization of nanoparticles loaded with methylene blue (MB), which are designed to be administered to tumor cells externally and deliver singlet oxygen (1O2) for photodynamic therapy (PDT), i.e. cell kill via oxidative stress to the membrane. We demonstrated the encapsulation of MB, a photosensitizer (PS), in three types of sub-200 nm nanoparticles, composed of polyacrylamide, sol–gel silica and organically modified silicate (ORMOSIL), respectively. Induced by light irradiation, the entrapped MB generated 1O2, and the produced 1O2 was measured quantitatively with anthracene-9,10-dipropionic acid, disodium salt, to compare the effects of different matrices on 1O2 delivery. Among these three different kinds of nanoparticles, the polyacrylamide nanoparticles showed the most efficient delivery of 1O2, but its loading of MB was low. In contrast, the sol–gel nanoparticles had the best MB loading but the least efficient 1O2 delivery. In addition to investigating the matrix effects, a preliminary in vitro PDT study using the MB-loaded polyacrylamide nanoparticles was conducted on rat C6 glioma tumor cells with positive photodynamic results. The encapsulation of MB in nanoparticles should diminish the interaction of this PS with the biological milieu, thus facilitating its systemic administration. Furthermore, the concept of the drug-delivering nanoparticles has been extended to a new type of dynamic nanoplatform (DNP) that only delivers 1O2. This DNP could also be used as a targeted multifunctional platform for combined diagnostics and therapy of cancer.

A Novel Polyacrylamide Magnetic Nanoparticle Contrast Agent for Molecular Imaging using MRI

A novel polyacrylamide superparamagnetic iron oxide nanoparticle platform is described which has been synthetically prepared such that multiple crystals of iron oxide are encapsulated within a single polyacrylamide matrix (PolyAcrylamide Magnetic [PAM] nanoparticles). This formulation provides for an extremely large T2 and T2* relaxivity of between 620 and 1140 sec(-1) mM(-1). Administration of PAM nanoparticles into rats bearing orthotopic 9L gliomas allowed quantitative pharmacokinetic analysis of the uptake of nanoparticles in the vasculature, brain, and glioma. Addition of polyethylene glycol of varying sizes (0.6, 2, and 10 kDa) to the surface of the PAM nanoparticles resulted in an increase in plasma half-life and affected tumor uptake and retention of the nanoparticles as quantified by changes in tissue contrast using MRI. The flexible formulation of these nanoparticles suggests that future modifications could be accomplished allowing for their use as a targeted molecular imaging contrast agent and/or therapeutic platform for multiple indications.

Nanoscale probes encapsulated by biologically localized embedding (PEBBLEs) for ion sensing and imaging in live cells

This review discusses the development and recent advances of probes encapsulated by biologically localized embedding (PEBBLEs), and in particular the application of PEBBLEs as ion sensors. PEBBLEs allow for minimally intrusive sensing of ions in cellular environments due to their small size (20 to 600nm in diameter) and protect the sensing elements (i.e. fluorescent dyes) by encapsulating them within an inert matrix. The selectivity and sensitivity of these nanosensors are comparable to those of macroscopic ion selective optodes, and electrodes, while the response time and absolute detection limit are significantly better. This paper discusses the principles guiding PEBBLE design including synthesis, characterization, diversification, the advantages and limitations of the sensors, cellular applications and future directions of PEBBLE research.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Mechanisms of Injury in the Central Nervous System

Neurotoxicants with similar structural features or common mechanisms of chemical action frequently produce widely divergent neuropathologic outcomes. Methylmercury (MeHg) produces marked cerebellar dysmorphogenesis during critical periods of development. The pathologic picture is characterized by complete architectural disruption of neuronal elements within the cerebellum. MeHg binds strongly to protein and soluble sulphydryl groups. Binding to microtubular -SH groups results in catastrophic depolymerization of immature tyrosinated microtubules. However, more mature acetylated microtubules are resistant to MeHg-induced depolymerization. In contrast to MeHg, the structurally similar organotin trimethyltin (TMT) elicits specific apoptotic destruction of pyramidal neurons in the CA3 region of the hippocampus and in other limbic structures. Expression of the phylogenetically conserved protein stannin is required for development of TMT-induced lesions. Inhibition of expression using antisense oligonucleotides against stannin protects neurons from the effects of TMT, suggesting that this protein is required for expression of neurotoxicity. However, expression of stannin alone is insufficient for induction of apoptotic pathways in neuronal populations. The aromatic nitrocompound 1,3-dinitrobenzene (DNB) has 2 independent nitro groups that can redox cycle in the presence of molecular oxygen. Despite its ability to deplete neural glutathione stores, DNB produces edematous gliovascular lesions in the brain stem of rats. Glial cells are susceptible despite high concentrations of reduced glutathione compared with neuronal somata in the central nervous system (CNS). The severity of lesions produced by DNB is modulated by the activity of neurons in the affected pathways. The inherent discrepancy between susceptibility of neuronal and glial cell populations is likely mediated by differential control of the mitochondrial permeability transition in astrocytes and neurons. Lessons learned in the mechanistic investigation of neurotoxicants suggest caution in the evaluation and interpretation of structure-activity relationships, eg, TMT, MeHg, and DNB all induce oxidative stress, whereas TMT and triethyltin produce neuronal damage and myelin edema, respectively. The precise CNS molecular targets of cell-specific lipophilic neurotoxicants remain to be determined.

Subcellular optochemical nanobiosensors: probes encapsulated by biologically localised embedding (PEBBLEs)

Abstract Described here are arguably the worlds smallest stand-alone devices/sensors, consisting of multicomponent nano-spheres with radii as small as 10 nm, occupying ≈1 ppb of a typical mammalian cell’s volume. The probe is prepared from up to seven ingredients and is optimised for selective and reversible analyte detection, as well as sensor stability and reproducibility. Such a sensor probe encapsulated by biologically localised embedding (PEBBLE), is delivered into a cell by a variety of minimally-invasive techniques, including a pico-injector, a gene gun, liposomal incorporation and natural ingestion. These remote nano-optodes (PEBBLEs) have been prepared for pH, calcium, magnesium, potassium and oxygen. The sensor PEBBLEs can be inserted into a cell individually, in clusters (single analyte), in sets (multi-analyte) or in ensembles (single analyte, multiple locations).

Multicolored stain-free histopathology with coherent Raman imaging

Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.