Search

This paper investigates the safety of a novel ‘magnetic injection’ method of delivering therapy to the cochlea, in a rodent model. In this method of administration, a magnetic field is employed to actively transport drug-eluting superparamagnetic iron-oxide core nanoparticles into the cochlea, where they then release their drug payload (we delivered the steroid prednisolone). Our study design and selection of control groups was based on published regulatory guidance for safety studies that involve local drug delivery. We tested for both single and multiple delivery doses to the cochlea, and found that magnetic delivery did not harm hearing. There was no statistical difference in hearing between magnetically treated ears versus ears that received intra-tympanic steroid (a mimic of a standard-of-care for sudden sensorineural hearing loss), both 2 and 30 days after treatment. Since our treatment is local to the ear, the levels of steroid and iron circulating systemically after our treatment were low, below mass-spectrometry detection limits for the steroid and no different from normal for iron. No adverse findings were observed in ear tissue histopathology or in animal gross behavior. At 2 and 30 days after treatment, inflammatory changes examined in the ear were limited to the middle ear, were very mild in severity, and by day 90 there was ongoing and almost complete reversibility of these changes. There were no ear tissue scarring or hemorrhage trends associated with magnetic delivery. In summary, after conducting a pre-clinical safety study, no adverse safety issues were observed.

Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg(-1), 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.

Rare protein enrichment and sensitive detection hold great potential in biomedical studies and clinical practice. This work describes the use of aptamer-conjugated gold nanorods for the efficient enrichment of rare proteins from buffer solutions and human plasma. Gold nanorod (AuNR) surfaces were modified with a long PEG chain and a 15-mer thrombin aptamer for protein enrichment and detection. Studies of the effect of surface modification on enrichment efficiency of thrombin showed that a change of only one EG(6) linker unit, i.e, from 2EG(6) to 3EG(6), could increase thrombin protein capture efficiency by up to 47%. Furthermore, a 1 ppm sample of thrombin in buffer could be enriched with around 90% efficiency using a low concentration (0.19 nM) of gold nanorod probe modified with 3EG(6) spacer, and with the same probe, effective capture was achieved down to 10 ppb (1 ng) thrombin in plasma samples. In addition to alpha-thrombin enrichment, prothrombin was also efficiently captured from plasma samples via gold nanorods conjugated with 15-mer thrombin aptamer. Our work demonstrates efficient enrichment of rare proteins using aptamer-modified nanomaterials, which can be used in biomarker discovery studies.

Magnetic relaxation switch (MRSw) detection is based on aggregate formation or dissociation when magnetic nanoparticles (MNPs) bind to target molecules. In the aggregated state, the dephasing rate of nearby proton spins is higher than in the dispersed state, resulting in a decrease in the spin-spin relaxation time, T(2). In this work, an MRSw-based nanosensor for lysozyme (Lys) protein detection was achieved using iron oxide nanoparticles conjugated with either Lys aptamer or linker DNA, which can hybridize with the extended part of the aptamer to form clusters. Upon the addition of Lys, the aptamers bind with their targets, leading to disassembly of clusters and an increase in T(2). A detection limit in the nanomolar range was achieved for Lys detection in both buffer and human serum. The determination of Lys level in different types of cancer cell lysates was also performed to demonstrate detection in real clinical samples.

Although many different nanomaterials have been tested as substrates for laser desorption and ionization mass spectrometry (LDI-MS), this emerging field still requires more efficient multifuncional nanomaterials for targeting, enrichment and detection. Here, we report the use of gold-manganese oxide (Au@MnO) hybrid nanoflowers as an efficient matrix for LDI–MS. The nanoflowers were also functionalized with two different aptamers to target cancer cells and capture adenosine triphosphate (ATP), respectively. These nanoflowers were successfully used for metabolite extraction from cancer cell lysates. Thus, in one system, our multifunctional nanoflowers can 1) act as an ionization substrate for mass spectrometry, 2) target cancer cells, and 3) detect and analyze metabolites from cancer cells.

In this work, a DNA inter-strand replacement strategy for therapeutic activity has been successfully designed for multimodal therapy. In this multimodal therapy, chlorin e6 (Ce6) photosensitizer molecules are used for photodynamic therapy (PDT), while aptamer-AuNRs, are used for selective binding to target cancer cells and for photothermal therapy (PTT) with near infrared (NIR) laser irradiation. Aptamer Sgc8, which specifically targets leukemia T cells, is conjugated to an AuNR by a thiol-Au covalent bond. and then hybridized with a Ce6-labeled photosensitizer/reporter to form a DNA double helix. When target cancer cells are absent, Ce6 is quenched and shows no PDT effect. However, when target cancer cells are present, the aptamer changes structure to release Ce6 to produce singlet oxygen for PDT upon light irradiation. Importantly, by combining photosensitizer and photothermal agents, PTT/PDT dual therapy supplies a more effective therapeutic outcome than either therapeutic modality alone.

In this work, gold nanoparticles perform Boolean logic operations in response to two proangiogenic targets important in cancer diagnosis and treatment: PDGF and VEGF. In the absence of protein target, gold nanoparticles are initially dispersed as a red solution; the addition of target proteins causes nanoparticle aggregation, turning the solution blue, as well as the release of dye-labeled aptamer probes, which causes an increase in fluorescence. These outputs constitute an AND or OR gate for simultaneous protein detection. We believe this logic-gate-based detection system will become the basis for novel rapid, cheap, and reliable sensors for diagnostic applications.

Biocompatible magnetic nanosensors based on reversible self-assembly of dispersed magnetic nanoparticles into stable nanoassemblies have been used as effective magnetic relaxation switches (MRSw) for the detection of molecular interactions. We report, for the first time, the design of MRSw based on aptamer-conjugated magnetic nanoparticles (ACMNPs). The ACMNPs capitalize on the ability of aptamers to specifically bind target cancer cells, as well as the large surface area of MNPs to accommodate multiple aptamer binding events. The ACMNPs can detect as few as 10 cancer cells in 250 μL of sample. The ACMNPs’ specificity and sensitivity are also demonstrated by detection in cell mixtures and complex biological media, including fetal bovine serum (FBS), human plasma, and whole blood. Furthermore, by using an array of ACMNPs, various cell types can be differentiated through pattern recognition, thus creating a cellular molecular profile which will allow clinicians to accurately identify cancer cells at the molecular and single cell level.

Targeted chemotherapy and magnetic resonance imaging of cancer cells in vitro has been achieved using a smart multifunctional nanostructure (SMN) constructed from a porous hollow magnetite nanoparticle (PHMNP), a heterobifunctional PEG ligand, and an aptamer. The PHMNPs were prepared through a three-step reaction and loaded with the anticancer drug doxorubicin while being functionalized with PEG ligands. Targeting aptamers were then introduced by reaction with the PEG ligands. The pores of the PHMNPs are stable at physiological pH, but they are subject to acid etching. Specific binding and uptake of the SMN to the target cancer cells induced by aptamers was observed. In addition, multiple aptamers on the surface of one single SMN led to enhanced binding and uptake to target cancer cells due to the multivalent effect. Upon reaching the lysosomes of target cancer cells through receptor-mediated endocytosis, the relatively low lysosomal pH level resulted in corrosion of the PHMNP pores, facilitating the release of doxorubicin to kill the target cancer cells. In addition, the potential of using SMN for magnetic resonance imaging was also investigated.