A nanoparticle probe for the imaging of autophagic flux in live mice via magnetic resonance and near-infrared fluorescence.

Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model.

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

Positron annihilation localization by nanoscale magnetization.

In positron emission tomography (PET), the finite range over which positrons travel before annihilating with an electron places a fundamental physical limit on the spatial resolution of PET images. After annihilation, the photon pair detected by the PET instrumentation is emitted from a location that is different from the positron-emitting source, resulting in image blurring. Here, we report on the localization of positron range, and hence annihilation quanta, by strong nanoscale magnetization of superparamagnetic iron oxide nanoparticles (SPIONs) in PET-MRI. We found that positron annihilations localize within a region of interest by up to 60% more when SPIONs are present (with [Fe] = 3 mM) compared to when they are not. The resulting full width at half maximum of the PET scans showed the spatial resolution improved by up to [Formula: see text] 30%. We also found evidence suggesting that the radiolabeled SPIONs produced up to a six-fold increase in ortho-positronium. These results may also have implications for emerging cancer theranostic strategies, where charged particles are used as therapeutic as well as diagnostic agents and improved dose localization within a tumor is a determinant of better treatment outcomes.

A Radio-Nano-Platform for T1/T2 Dual-Mode PET-MR Imaging.

PURPOSE: This study aimed to develop a chelate-free radiolabeled nanoparticle platform for simultaneous positron emission tomography (PET) and magnetic resonance (MR) imaging that provides contrast-enhanced diagnostic imaging and significant image quality gain by integrating the high spatial resolution of MR with the high sensitivity of PET. METHODS: A commercially available super-paramagnetic iron oxide nanoparticle (SPION) (Feraheme®, FH) was labeled with the [89Zr]Zr using a novel chelate-free radiolabeling technique, heat-induced radiolabeling (HIR). Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and radio-thin layer chromatography (radio-TLC). Characterization of the non-radioactive isotope 90Zr-labeled FH was performed by transmission electron microscopy (TEM). Simultaneous PET-MR phantom imaging was performed with different 89Zr-FH concentrations. The MR quantitative image analysis determined the contrast-enhancing properties of FH. The signal-to-noise ratio (SNR) and full-width half-maximum (FWHM) of the line spread function (LSF) were calculated before and after co-registering the PET and MR image data. RESULTS: High RCY (92%) and RCP (98%) of the [89Zr]Zr-FH product was achieved. TEM analysis confirmed the 90Zr atoms adsorption onto the SPION surface (≈ 10% average radial increase). Simultaneous PET-MR scans confirmed the capability of the [89Zr]Zr-FH nano-platform for this multi-modal imaging technique. Relative contrast image analysis showed that [89Zr]Zr-FH can act as a dual-mode T1/T2 contrast agent. For co-registered PET-MR images, higher spatial resolution (FWHM enhancement ≈ 3) and SNR (enhancement ≈ 8) was achieved at a clinical dose of radio-isotope and Fe. CONCLUSION: Our results demonstrate FH is a highly suitable SPION-based platform for chelate-free labeling of PET tracers for hybrid PET-MR. The high RCY and RCP confirmed the robustness of the chelate-free HIR technique. An overall image quality gain was achieved compared to PET- or MR-alone imaging with a relatively low dosage of [89Zr]Zr-FH. Additionally, FH is suitable as a dual-mode T1/T2 MR image contrast agent.

A Chelate-Free Nano-Platform for Incorporation of Diagnostic and Therapeutic Isotopes.

PURPOSE: Using our chelate-free, heat-induced radiolabeling (HIR) method, we show that a wide range of metals, including those with radioactive isotopologues used for diagnostic imaging and radionuclide therapy, bind to the Feraheme (FH) nanoparticle (NP), a drug approved for the treatment of iron anemia. MATERIAL AND METHODS: FH NPs were heated (120°C) with nonradioactive metals, the resulting metal-FH NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), and r1 and r2 relaxivities obtained by nuclear magnetic relaxation spectrometry (NMRS). In addition, the HIR method was performed with [90Y]Y3+, [177Lu]Lu3+, and [64Cu]Cu2+, the latter with an HIR technique optimized for this isotope. Optimization included modifying reaction time, temperature, and vortex technique. Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and thin-layer chromatography (TLC). RESULTS: With ICP-MS, metals incorporated into FH at high efficiency were bismuth, indium, yttrium, lutetium, samarium, terbium and europium (>75% @ 120 oC). Incorporation occurred with a small (less than 20%) but statistically significant increases in size and the r2 relaxivity. An improved HIR technique (faster heating rate and improved vortexing) was developed specifically for copper and used with the HIR technique and [64Cu]Cu2+. Using SEC and TLC analyses with [90Y]Y3+, [177Lu]Lu3+ and [64Cu]Cu2+, RCYs were greater than 85% and RCPs were greater than 95% in all cases. CONCLUSION: The chelate-free HIR technique for binding metals to FH NPs has been extended to a range of metals with radioisotopes used in therapeutic and diagnostic applications. Cations with f-orbital electrons, more empty d-orbitals, larger radii, and higher positive charges achieved higher values of RCY and RCP in the HIR reaction. The ability to use a simple heating step to bind a wide range of metals to the FH NP, a widely available approved drug, may allow this NP to become a platform for obtaining radiolabeled nanoparticles in many settings.

Heat-induced radiolabeling and fluorescence labeling of Feraheme nanoparticles for PET/SPECT imaging and flow cytometry.

Feraheme (FH) nanoparticles (NPs) have been used extensively for treatment of iron anemia (due to their slow release of ionic iron in acidic environments). In addition, injected FH NPs are internalized by monocytes and function as MRI biomarkers for the pathological accumulation of monocytes in disease. We have recently expanded these applications by radiolabeling FH NPs for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging using a heat-induced radiolabeling (HIR) strategy. Imaging FH NPs using PET/SPECT has important advantages over MRI due to lower iron doses and improved quantitation of tissue NP concentrations. HIR of FH NPs leaves the physical and biological properties of the NPs unchanged and allows researchers to build on the extensive knowledge obtained about the pharmacokinetic and safety aspects of FH NPs. In this protocol, we present the step-by-step procedures for heat (120 °C)-induced bonding of three widely employed radiocations (89Zr4+ or 64Cu2+ for PET, and 111In3+ for SPECT) to FH NPs using a chelateless radiocation surface adsorption (RSA) approach. In addition, we describe the conversion of FH carboxyl groups into amines and their reaction with an N-hydroxysuccinimide (NHS) of a Cy5.5 fluorophore. This yields Cy5.5-FH, a fluorescent FH that enables the cells internalizing Cy5.5-FH to be examined using flow cytometry. Finally, we describe procedures for in vivo and ex vivo uptake of Cy5.5-FH by monocytes and for in vivo microPET/CT imaging of HIR-FH NPs. Synthesis of HIR-FH requires experience with working with radioactive cations and can be completed within <4 h. Synthesis of Cy5.5-FH NPs takes ∼17 h.

An Integrin-Targeted, Highly Diffusive Construct for Photodynamic Therapy.

Targeted antineoplastic agents show great promise in the treatment of cancer, having the ability to impart cytotoxicity only to specific tumor types. However, these therapies do not experience uniform uptake throughout tumors, leading to sub-lethal cell killing that can impart treatment resistance, and cause problematic off-target effects. Here we demonstrate a photodynamic therapy construct that integrates both a cyclic RGD moiety for integrin-targeting, as well as a 5 kDa PEG chain that passivates the construct and enables its rapid diffusion throughout tumors. PEGylation of the photosensitizer construct was found to prevent photosensitizer aggregation, boost the generation of cytotoxic reactive radical species, and enable the rapid uptake of the construct into cells throughout large (>500 µm diameter) 3D tumor spheroids. Replacing the cyclic RGD with the generic RAD peptide led to the loss of cellular uptake in 3D culture, demonstrating the specificity of the construct. Photodynamic therapy with the construct was successful in inducing cytotoxicity, which could be competitively blocked by a tenfold concentration of free cyclic RGD. This construct is a first-of-its kind theranostic that may serve as a new approach in our growing therapeutic toolbox.

Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions.

Chronic wounds affect 12-15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220+ /CD19+ /IgM+ /IgD+ B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2-3 days in wild-type mice and 5-6 days in obese diabetic mice. The treatment led to full closure in 43% of chronic diabetic wounds, as compared to only 5% in saline-treated controls. Applying equivalent numbers of T cells or disrupted B cells failed to reproduce these effects, indicating that live B cells mediated pro-healing responses. Topically applied B cell treatment was associated with significantly reduced scar size, increased collagen deposition and maturation, enhanced angiogenesis, and increased nerve growth into and under the healing wound. ß-III tubulin+ nerve endings in scars of wounds treated acutely with B cells showed increased relative expression of growth-associated protein 43. The improved healing associated with B cell treatment was supported by significantly increased fibroblast proliferation and decreased apoptosis in the wound bed and edges, altered kinetics of neutrophil infiltration, as well as an increase in TGF-ß and a significant reduction in MMP2 expression in wound granulation tissue. Our findings indicate that the timeline and efficacy of wound healing can be experimentally manipulated through the direct application of mature, naive B cells, which effectively modify the balance of mature immune cell populations within the wound microenvironment and accelerate the healing process.

Theranostic Nucleic Acid Binding Nanoprobe Exerts Anti-inflammatory and Cytoprotective Effects in Ischemic Injury.

Extracellular nucleic acids are proinflammatory molecules that have been implicated in a diverse range of diseases. We report here the development of a multivalent nucleic acid scavenging nanoprobe, where the fluorochrome thiazole orange (TO) is conjugated to a polymeric 40 kDa dextran carrier. Dextran-TO (Dex-TO) has nanomolar affinity for mammalian and bacterial nucleic acids and attenuates the production of inflammatory cytokines from activated macrophages exposed to DNA and RNA. Mice with myocardial ischemia reperfusion that were treated with Dex-TO showed a decrease in myocardial macrophage infiltration at 24 hours (p<0.05) and a decrease in infarct size (18% ± 9%, p<0.01) on day 7. Dex-TO allows sites of injury to be identified with fluorescence imaging, while simultaneously exerting an anti-inflammatory and cytoprotective effect. Dex-TO could be of significant diagnostic and therapeutic (theranostic) utility in a broad range of conditions including ischemia, trauma, burns, sepsis and autoimmune disease.

Heat-induced-radiolabeling and click chemistry: A powerful combination for generating multifunctional nanomaterials.

A key advantage of nanomaterials for biomedical applications is their ability to feature multiple small reporter groups (multimodality), or combinations of reporter groups and therapeutic agents (multifunctionality), while being targeted to cell surface receptors. Here a facile combination of techniques for the syntheses of multimodal, targeted nanoparticles (NPs) is presented, whereby heat-induced-radiolabeling (HIR) labels NPs with radiometals and so-called click chemistry is used to attach bioactive groups to the NP surface. Click-reactive alkyne or azide groups were first attached to the nonradioactive clinical Feraheme (FH) NPs. Resulting "Alkyne-FH" and "Azide-FH" intermediates, like the parent NP, tolerated 89Zr labeling by the HIR method previously described. Subsequently, biomolecules were quickly conjugated to the radioactive NPs by either copper-catalyzed or copper-free click reactions with high efficiency. Synthesis of the Alkyne-FH or Azide-FH intermediates, followed by HIR and then by click reactions for biomolecule attachment, provides a simple and potentially general path for the synthesis of multimodal, multifunctional, and targeted NPs for biomedical applications.

Heat-Induced Radiolabeling of Nanoparticles for Monocyte Tracking by PET.

Heat-induced radiolabeling (HIR) yielded (89) Zr-Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes in vitro. Using an IV injection of HIR (89) Zr-FH (rather than in vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.

Imaging PEG-like nanoprobes in tumor, transient ischemia, and inflammatory disease models.

The iron chelator deferoxamine (DFO), approved for the treatment of iron overload, has been examined as a therapeutic in a variety of conditions which iron may exacerbate. To evaluate the potential of DFO-bearing PEG-like nanoprobes (DFO-PNs) as therapeutics, we determined their pharmacokinetics (PK) in normal mice, and imaged their accumulation in a tumor model and in models of transient brain ischemia and inflammation. DFO-PNs consist of a DFO, a Cy5.5, and PEG (5 kDa or 30 kDa) attached to Lys-Cys scaffold. Tumor uptake of a [(89)Zr]:DFO-PN(10) (30 kDa PEG, diameter 10 nm) was imaged by PET, surface fluorescence, and fluorescence microscopy. DFO-PN(10) was internalized by tumor cells (fluorescence microscopy) and by cultured cells (by FACS). [(89)Zr]:DFO-PN(4.3) (5 kDa PEG, diameter 4.3 nm) concentrated at incision generated inflammations but not at sites of transient brain ischemia. DFO-PNs are fluorescent, PK tunable forms of DFO that might be investigated as antitumor or anti-inflammatory agents.

Cytoprotective nanoparticles by conjugation of a polyhis tagged annexin V to a nanoparticle drug.

We synthesized a cytoprotective magnetic nanoparticle by reacting a maleimide functionalized Feraheme (FH) with a disulfide linked dimer of a polyhis tagged annexin V. Following reductive cleavage of disulfide, the resulting annexin-nanoparticle (diameter = 28.0 ± 2.0 nm by laser light scattering, 7.6 annexin's/nanoparticle) was cytoprotective to cells subjected to plasma membrane disrupting chemotherapeutic or mechanical stresses, and significantly more protective than the starting annexin V. Annexin-nanoparticles provide an approach to the design of nanomaterials which antagonize the plasma membrane permeability characteristic of necrosis and which may have applications as cytoprotective agents.

PEG-like nanoprobes: multimodal, pharmacokinetically and optically tunable nanomaterials.

"PEG-like Nanoprobes" (PN's) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN's feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN's were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (111In-PN and SPECT). Clinical applications of PN's include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism.

Fluorescent and radiolabeled triphenylphosphonium probes for imaging mitochondria.

Triphenylphosphonium-fluorochromes (TPP-fluorochromes) are a new class of spectrally variable, mitochondrially targeted probes, with an [(18)F] labeling option which, when enabled, allows imaging of a cardiac perfusion deficit using PET/CT.

High efficiency diffusion molecular retention tumor targeting.

Here we introduce diffusion molecular retention (DMR) tumor targeting, a technique that employs PEG-fluorochrome shielded probes that, after a peritumoral (PT) injection, undergo slow vascular uptake and extensive interstitial diffusion, with tumor retention only through integrin molecular recognition. To demonstrate DMR, RGD (integrin binding) and RAD (control) probes were synthesized bearing DOTA (for (111) In(3+)), a NIR fluorochrome, and 5 kDa PEG that endows probes with a protein-like volume of 25 kDa and decreases non-specific interactions. With a GFP-BT-20 breast carcinoma model, tumor targeting by the DMR or i.v. methods was assessed by surface fluorescence, biodistribution of [(111)In] RGD and [(111)In] RAD probes, and whole animal SPECT. After a PT injection, both probes rapidly diffused through the normal and tumor interstitium, with retention of the RGD probe due to integrin interactions. With PT injection and the [(111)In] RGD probe, SPECT indicated a highly tumor specific uptake at 24 h post injection, with 352%ID/g tumor obtained by DMR (vs 4.14%ID/g by i.v.). The high efficiency molecular targeting of DMR employed low probe doses (e.g. 25 ng as RGD peptide), which minimizes toxicity risks and facilitates clinical translation. DMR applications include the delivery of fluorochromes for intraoperative tumor margin delineation, the delivery of radioisotopes (e.g. toxic, short range alpha emitters) for radiotherapy, or the delivery of photosensitizers to tumors accessible to light.

Fluorochrome-functionalized nanoparticles for imaging DNA in biological systems.

Attaching DNA binding fluorochromes to nanoparticles (NPs) provides a way of obtaining NPs that bind to DNA through fluorochrome mediated interactions. To obtain a nanoparticle (NP) that bound to the DNA in biological systems, we attached the DNA binding fluorochrome, TO-PRO 1 (TO), to the surface of the Feraheme (FH) NP, to obtain a fluorochrome-functionalized NP denoted TO-FH. When reacted with DNA in vitro, TO-FH formed microaggregates that were characterized by fluorescence, light scattering, and T2 changes. The formation of DNA/TO-FH microaggregates was also characterized by AFM, with microaggregates exhibiting a median size of 200 nm, and consisting of DNA and multiple TO-FH NPs whose individual diameters were only 25-35 nm. TO-FH failed to bind normal cells in culture, but treatment with chemotherapeutic agents or detergents yielded necrotic cells that bound TO-FH and vital fluorochromes similarly. The uptake of TO-FH by HT-29 xenografts (treated with 5-FU and oxaliplatin) was evident by surface fluorescence and MRI. Attaching multiple DNA binding fluorochromes to magnetic nanoparticles provides a way of generating DNA binding NPs that can be used to detect DNA detection by microaggregate formation in vitro, for imaging the DNA of necrotic cells in culture, and for imaging the DNA of a tumor treated with a chemotherapeutic agent. Fluorochrome functionalized NPs are a multimodal (magnetic and fluorescent), highly multivalent (n ≈ 10 fluorochromes/NP) nanomaterials useful for imaging the DNA of biological systems.

Use of a single hybrid imaging agent for integration of target validation with in vivo and ex vivo imaging of mouse tumor lesions resembling human DCIS.

UNLABELLED: Screening of biomarker expression levels in tumor biopsy samples not only provides an assessment of prognostic and predictive factors, but may also be used for selection of biomarker-specific imaging strategies. To assess the feasibility of using a biopsy specimen for a personalized selection of an imaging agent, the chemokine receptor 4 (CXCR4) was used as a reference biomarker. METHODS: A hybrid CXCR4 targeting peptide (MSAP-Ac-TZ14011) containing a fluorescent dye and a chelate for radioactive labeling was used to directly compare initial flow cytometry-based target validation in fresh tumor tissue to in vivo single photon emission computed tomography (SPECT) imaging and in vivo and ex vivo fluorescence imaging. RESULTS: Flow cytometric analysis of mouse tumor derived cell suspensions enabled discrimination between 4T1 control tumor lesions (with low levels of CXCR4 expression) and CXCR4 positive early, intermediate and late stage MIN-O lesions based on their CXCR4 expression levels; CXCR4(basal), CXCR4(+) and CXCR4(++) cell populations could be accurately discriminated. Mean fluorescent intensity ratios between expression in MIN-O and 4T1 tissue found with flow cytometry were comparable to ratios obtained with in vivo SPECT/CT and fluorescence imaging, ex vivo fluorescence evaluation and standard immunohistochemistry. CONCLUSION: The hybrid nature of a targeting imaging agent like MSAP-Ac-TZ14011 enables integration of target selection, in vivo imaging and ex vivo validation using a single agent. The use of biopsy tissue for biomarker screening can readily be expanded to other targeting hybrid imaging agents and can possibly help increase the clinical applicability of tumor-specific imaging approaches.

The PEG-fluorochrome shielding approach for targeted probe design.

We provide a new approach for fluorescent probe design termed "PEG-fluorochrome shielding", where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an (111)In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Fluorochrome-functionalized magnetic nanoparticles for high-sensitivity monitoring of the polymerase chain reaction by magnetic resonance.

Easy to find: magnetic nanoparticles bearing fluorochromes (red) that intercalate with DNA (green) form microaggregates with DNA generated by the polymerase chain reaction (PCR). These aggregates can be detected at low cycle numbers by magnetic resonance (MR).