Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape.

Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles.

Ultrasound-sensitive nanoparticle aggregates for targeted drug delivery.

Here we describe injectable, ultrasound (US)-responsive, nanoparticle aggregates (NPAs) that disintegrate into slow-release, nanoscale, drug delivery systems, which can be targeted to selective sites by applying low-energy US locally. We show that, unlike microbubble based drug carriers which may suffer from stability problems, the properties of mechanical activated NPAs, composed of polymer nanoparticles, can be tuned by properly adjusting the polymer molecular weight, the size of the nanoparticle precursors as well as the percentage of excipient utilized to hold the NPA together. We then apply this concept to practice by fabricating NPAs composed of nanoparticles loaded with Doxorubicin (Dox) and tested their ability to treat tumors via ultrasound activation. Mouse studies demonstrated significantly increased efficiency of tumor targeting of the US-activated NPAs compared to PLGA nanoparticle controls (with or without US applied) or intact NPAs. Importantly, when the Dox-loaded NPAs were injected and exposed to US energy locally, this increased ability to concentrate nanoparticles at the tumor site resulted in a significantly greater reduction in tumor volume compared to tumors treated with a 20-fold higher dose of the free drug.

Detection and quantification of enzymatically active prostate-specific antigen in vivo.

Assays for blood levels of prostate-specific antigen (PSA), performed in prostate cancer detection, measure mostly inactive/complexed PSA and do not provide information regarding enzymatically active PSA, which is biologically more relevant. Thus, we designed and synthesized an enzymatically cleavable peptide sequence labeled with near-infrared (NIR) fluorophores (ex/em 740/770 nm) and coupled it to a pharmacokinetic modifier designed to improve its plasma kinetics. In its native state, the agent, PSA750 FAST™ (PSA750), is optically quenched (>95%) and only becomes fluorescent upon cleavage by active PSA, yielding a significant increase in signal. This activation is highly selective for PSA relative to a large panel of disease-relevant enzymes. Active PSA was detected in tumor frozen sections using PSA750 and this activity was abolished in the presence of the inhibitor, alpha-1 anti-chymotrypsin. In vivo imaging of tumor-bearing mice using fluorescence molecular tomography demonstrated a significantly higher fluorescent signal in PSA+ LNCaP tumors as compared to PSA- prostate cancer 3 tumors (13.0±3.7 versus 2.8±0.8 pmol, p=0.023). Ex vivo imaging of tumor sections confirms PSA750-derived NIR signal localization in nonvascular tissue. This is the first report that demonstrates the feasibility and effectiveness of noninvasive, real time, fluorescence molecular imaging of PSA enzymatic activity in prostate cancer.

In vivo imaging and quantification of carbonic anhydrase IX expression as an endogenous biomarker of tumor hypoxia.

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%-70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6-24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors.

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.

Noninvasive in vivo quantification of neutrophil elastase activity in acute experimental mouse lung injury.

We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions.

Dual in vivo quantification of integrin-targeted and protease-activated agents in cancer using fluorescence molecular tomography (FMT).

PURPOSE: Integrins, especially α(v)ß(3) and α(v)ß(5), are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of α(v)ß(3)/α(v)ß(5). PROCEDURES: A small peptidomimetic α(v)ß(3) antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo. RESULTS: The integrin NIRF agent showed strong selectivity towards α(v)ß(3/)α(v)ß(5) in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression. CONCLUSIONS: NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.