Jennifer Sherwood

Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles

Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow easy conjugation of other bioactive molecules.

Short- and Long-Term Effects of Prenatal Exposure to Iron Oxide Nanoparticles: Influence of Surface Charge and Dose on Developmental and Reproductive Toxicity

Iron oxide nanoparticles (NPs) are commonly utilized for biomedical, industrial, and commercial applications due to their unique properties and potential biocompatibility. However, little is known about how exposure to iron oxide NPs may affect susceptible populations such as pregnant women and developing fetuses. To examine the influence of NP surface-charge and dose on the developmental toxicity of iron oxide NPs, Crl:CD1(ICR) (CD-1) mice were exposed to a single, low (10 mg/kg) or high (100 mg/kg) dose of positively-charged polyethyleneimine-Fe2O3-NPs (PEI-NPs), or negatively-charged poly(acrylic acid)-Fe2O3-NPs (PAA-NPs) during critical windows of organogenesis (gestation day (GD) 8, 9, or 10). A low dose of NPs, regardless of charge, did not induce toxicity. However, a high exposure led to charge-dependent fetal loss as well as morphological alterations of the uteri (both charges) and testes (positive only) of surviving offspring. Positively-charged PEI-NPs given later in organogenesis resulted in a combination of short-term fetal loss (42%) and long-term alterations in reproduction, including increased fetal loss for second generation matings (mice exposed in utero). Alternatively, negatively-charged PAA-NPs induced fetal loss (22%) earlier in organogenesis to a lesser degree than PEI-NPs with only mild alterations in offspring uterine histology observed in the long-term.

Developmental and Reproductive Effects of Iron Oxide Nanoparticles in Arabidopsis thaliana.

Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%–6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed.

The responses of immune cells to iron oxide nanoparticles.

Immune cells play an important role in recognizing and removing foreign objects, such as nanoparticles. Among various parameters, surface coatings of nanoparticles are the first contact with biological system, which critically affect nanoparticle interactions. Here, surface coating effects on nanoparticle cellular uptake, toxicity and ability to trigger immune response were evaluated on a human monocyte cell line using iron oxide nanoparticles. The cells were treated with nanoparticles of three types of coatings (negatively charged polyacrylic acid, positively charged polyethylenimine and neutral polyethylene glycol). The cells were treated at various nanoparticle concentrations (5, 10, 20, 30, 50 μg ml−1 or 2, 4, 8, 12, 20 μg cm−2) with 6 h incubation or treated at a nanoparticle concentration of 50 μg ml−1 (20 μg cm−2) at different incubation times (6, 12, 24, 48 or 72 h). Cell viability over 80% was observed for all nanoparticle treatment experiments, regardless of surface coatings, nanoparticle concentrations and incubation times. The much lower cell viability for cells treated with free ligands (e.g. ~10% for polyethylenimine) suggested that the surface coatings were tightly attached to the nanoparticle surfaces. The immune responses of cells to nanoparticles were evaluated by quantifying the expression of toll‐like receptor 2 and tumor necrosis factor‐α. The expression of tumor necrosis factor‐α and toll‐like receptor 2 were not significant in any case of the surface coatings, nanoparticle concentrations and incubation times. These results provide useful information to select nanoparticle surface coatings for biological and biomedical applications. Copyright

Shape-dependent cellular behaviors and relaxivity of iron oxide-based T1 MRI contrast agents

Recent research efforts about iron oxide nanoparticles has focused on the development of iron oxide-based T1 contrast agents for magnetic resonance imaging (MRI), such as ultrasmall iron oxide nanospheres (USNPs <4 nm) and ultrathin nanowires (NW, diameter <4 nm). In this paper, we report the cellular uptake behaviors of these two types of ultrasmall scale nanostructures on HepG2 cells. Both these two nanostructures were functionalized with tannic acid and their physical and chemical properties were carefully analyzed before cellular tests. Both USNPs and NWs exhibited strong paramagnetic signals, a property suitable for T1 MRI contrast agents. The distinct shapes also caused much difference in their cellular uptake behaviors. Specifically, the uptake of USNPs was five times higher than that of NWs after 72 hours incubation. The shape-dependent cellular uptake can potentially lead to different blood circulation times, and subsequently different applications of these two types of ultrasmall nanostructures.

Magnetic iron oxide nanoparticles as T1 contrast agents for magnetic resonance imaging

Ultrasmall iron oxide nanoparticles have recently attracted much attention as T1 (positive) contrast agents for magnetic resonance imaging to serve as safer alternatives to gadolinium-based T1 contrast agents. This review will discuss several key aspects regarding this newly developed contrast agent, including synthetic strategies, parameters affecting T1 signal (e.g., size, surface, and environment), and emerging applications. The integration with other imaging modalities will be discussed as well, such as dual T1/T2 imaging, positron emission tomography, and computed tomography. Finally, perspectives and outlook about the future development and concerns will be included.

Citric Acid Capped Iron Oxide Nanoparticles as an Effective MALDI Matrix for Polymers

AbstractA new matrix-assisted laser desorption ionization (MALDI) mass spectrometry matrix is proposed for molecular mass determination of polymers. This matrix contains an iron oxide nanoparticle (NP) core with citric acid (CA) molecules covalently bound to the surface. With the assistance of additives, the particulate nature of NPs allows the matrix to mix uniformly with polar or nonpolar polymer layers and promotes ionization, which may simplify matrix selection and sample preparation procedures. Several distinctively different polymer classes (polyethyleneglycol (PEG), polywax/polyethylene, perfluoropolyether, and polydimethylsiloxane) are effectively detected by the water or methanol dispersed NPCA matrix with NaCl, NaOH, LiOH, or AgNO3 as additives. Furtheremore, successful quantitative measurements of PEG1000 using polypropylene glycol 1000 as an internal standard are demonstrated. Graphical Abstractᅟ

Development of an iron quantification method using nuclear magnetic resonance relaxometry

Biocompatibility has prompted a great amount of research in iron oxide nanoparticles (IONPs) as alternative magnetic resonance imaging (MRI) contrast agents. Iron concentration analysis is a key parameter to determine the relaxivities of IONPs as MRI contrast agents. Currently available methods for iron quantification are mainly inductively coupled plasma mass spectrometry (ICP-MS) and ferrozine-based iron assays. ICP spectrometry may not be easily accessible for routine analysis while iron assays are highly sensitive to sample preparation. In this paper, we present an alternative method for quantifying iron concentration using nuclear magnetic resonance (NMR) relaxometry, a technique commonly used for developing MRI contrast agents. To quantify iron concentration with NMR, a standard curve of relaxation rate versus iron concentrations was created to obtain the relaxivity of Fe3+ iron in solution. After dissolving IONPs in an acid, the iron concentration of the solution can be obtained using the relaxatio...

Ultrasound-Triggered Delivery of Anticancer Therapeutics from MRI-Visible Multilayer Microcapsules

Although imaging‐guided drug delivery represents a noninvasive alternative to both surgical resection and systemic methods, it has seen limited clinical use due to the potential toxicity and fast clearance of currently available imaging agents. Herein, we introduce theranostic biocompatible microcapsules as efficient contrast‐enhanced imaging agents that combine magnetic resonance imaging (MRI) with ultrasound‐triggered drug release for real‐time tracking and targeted delivery in vivo. The 3‐μm diameter capsules are assembled via layer‐by‐layer deposition of the natural polyphenol tannic acid and poly(N‐vinylpyrrolidone) with 4 nm iron oxide nanoparticles incorporated in the capsule wall. The nanoparticle‐modified capsules exhibit excellent T1 and T2 MRI contrast in a clinical 3T MRI scanner. Loaded with the anticancer drug doxorubicin, these capsules circulate in the blood stream for at least 48 h, which is a remarkable improvement compared to nonencapsulated nanoparticles. The application of focused ultrasound results in targeted drug release with a 16‐fold increase in doxorubicin localization in tumors compared to off‐target organs in a mouse model of breast cancer. Owing to the active contrast, long circulation, customizable size, shape, composition, and precise delivery of high payload concentrations, these materials present a powerful and safe platform for imaging‐guided precision drug delivery.

A coiled-coil strategy for the directional display of multiple proteins on the surface of iron oxide nanoparticles

Current methods for attachment of proteins to inorganic nanoparticle surfaces utilize strategies that can destroy the tertiary structure of large proteins and/or do not allow for directional attachment, thereby diminishing functionality. We report a novel approach for the conjugation of proteins to iron oxide nanoparticles (IONPs) through coiled-coil interactions. With this strategy, coiled-coiled interactions drive attachment of the desired protein, fused to a K-coil peptide, and E-coil peptides linked to the IONP surface via a nanoparticle attachment peptide (NAP). Importantly, this method allows for directional attachment of proteins without the use of either chemical conjugation or extreme pH solutions and, thus, allows maintenance of correct protein structure with functional orientation relative to the IONP surface. In our studies, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (EGFP) were used as model proteins to demonstrate the coiled-coil approach can be utilized to attach large proteins to IONP surfaces while maintaining complex tertiary structures. These proof-of-concept studies showed EGFP- and mRFP-K-coil fusion proteins interacted specifically with E-coil-coated nanoparticles and multiple different proteins could be displayed on individual IONPs. We did not observe fluorescence after conjugation of fluorescent proteins to either E-coil-coated IONPs or dopamine-coated IONPs, which was likely due to quenching or FRET activity induced by the iron oxide core. However, substituting an E-coil-bound chromatography resin for E-coil-coated IONPs allowed the preservation of protein fluorescence following coiled-coil attachment of K-coil-fused mRFP, demonstrating the coiled-coil attachment strategy provided maintenance of the mRFP tertiary structure required for fluorescence. The coiled-coil nanoparticle attachment strategy developed herein should have broad applications in nanomedicine.