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.

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.

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.

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.

Multimodal interventional molecular imaging of tumor margins and distant metastases by targeting αvß3 integrin.

α(v)ß(3) integrin is involved in (tumor-induced) angiogenesis and is a promising candidate for the specific visualization of both primary tumors and of their distant metastases. Combination of radioactive and fluorescent imaging labels in a single multimodal, or rather hybrid, RGD-based imaging agent enables integration of pre-, intra-, and postoperative angiogenesis imaging. A hybrid imaging agent targeting the α(v)ß(3) integrin--(111)In-MSAP-RGD (MSAP = multifunctional single-attachment-point reagent), which contains a targeting moiety, a pentetic acid (DTPA) chelate, and a cyanine dye--was evaluated for its potential value in combined lesion detection and interventional molecular imaging in a 4T1 mouse breast cancer model. SPECT/CT and fluorescence imaging were used to visualize the tumor in vivo. Tracer distribution was evaluated ex vivo down to the microscopic level. The properties of (111)In-MSAP-RGD were compared with those of (111)In-DTPA-RGD. Biodistribution studies revealed a prolonged retention and increased tumor accumulation of (111)In-MSAP-RGD relative to (111)In-DTPA-RGD. With (111)In-MSAP-RGD, identical features could be visualized preoperatively (SPECT/CT) and intraoperatively (fluorescence imaging). As well as the primary tumor, (111)In-MSAP-RGD also enabled detection and accurate excision of distant metastases in the head and neck region of the mice. Therefore, the hybrid RGD derivative (111)In-MSAP-RGD shows potential in preoperative planning and fluorescence-based surgical intervention.

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).

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.

Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Synthesis and evaluation of a bimodal CXCR4 antagonistic peptide.

The antagonistic Ac-TZ14011 peptide, which binds to the chemokine receptor 4, has been labeled with a multifunctional single attachment point reagent that contains a DTPA chelate and a fluorescent dye with Cy5.5 spectral properties. Flow cytometry and confocal microscopy showed that the bimodal labeled peptide gave a specific receptor binding that is similar to monofunctionalized peptide derivatives. Therefore, the newly developed bimodal peptide derivative can be used in multimodal imaging applications.

Hybrid peptide dendrimers for imaging of chemokine receptor 4 (CXCR4) expression.

The chemokine receptor 4 (CXCR4), which is overexpressed in many types of cancer, is an emerging target in the field of molecular imaging and therapeutics. The CXCR4 binding of several peptides, including the cyclic Ac-TZ14011, has already been validated. In this study mono-, di- and tetrameric Ac-TZ14011-containing dendrimers were prepared and functionalized with a multimodal (hybrid) label, consisting of a Cy5.5-like fluorophore and a DTPA chelate. Confocal microscopy revealed that all three dendrimers were membrane bound at 4 °C, consistent with CXCR4 binding in vitro. The unlabeled dimer and tetramer had a somewhat lower affinity for CXCR4 than the unlabeled monomer. However, when labeled with the multimodal label the CXCR4 affinity of the dimer and tetramer was considerably higher compared to that of the labeled monomer. On top of that, biodistribution studies revealed that the additional peptides in the dimer and tetramer reduced nonspecific muscle uptake. Thus, multimerization of the cyclic Ac-TZ14011 peptide reduces the negative influence of the multimodal label on the receptor affinity and the biodistribution.

Facile Synthesis of Monofunctional Pentamethine Carbocyanine Fluorophores.

A high yield route to symmetric, conjugatable pentamethine carbocyanine dyes with far-red/near infrared (NIR) emission between 650 and 700 nm is reported. The dyes are prepared via condensation of indolium or benz[e]indolium inner salts with an alkyl carboxylic acid derivatized malonaldehyde dianil or alternatively in a one-pot reaction without isolation of the malonaldehyde intermediate. The fluorophores are water-soluble, have bright fluorescence emission, are easily prepared in good yield, and are promising candidates for use in a variety of biochemical and in vivo imaging applications.

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.

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.

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 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.

A wortmannin-cetuximab as a double drug.

Double drugs are obtained when two pharmacologically active entities are covalently joined to improve potency. We conjugated the viridin Wm with a self-activating linkage to cetuximab and demonstrated the retention of immunoreactivity by the conjugate. Though cetuximab lacked a growth inhibitory activity against A549 cells, the Wm-cetuximab conjugate had an antiproliferative IC(50) of 155 nM in vitro. The chemistry of attaching a self-releasing Wm to clinically approved antibodies is general and, in selected instances, may yield antibody-based double drugs with improved efficacy.

A stabilized demethoxyviridin derivative inhibits PI3 kinase.

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26min versus Wm's half-life of 3470min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1min. To overcome Dmv's instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218min in PBS and 64min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC(50) of 44nM, compared to Wm's IC(50) of 12nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems.

Covalent reactions of wortmannin under physiological conditions.

Wortmannin (Wm), a steroid-like molecule of 428.4 Da, appears to be unstable in biological fluids (apparent chemical instability), yet it exhibits an antiproliferative activity in assays employing a 48 hr incubation period (prolonged bioactivity), a situation we refer to as the "wortmannin paradox." Under physiological conditions, Wm covalently reacts with nucleophiles such as the side chains of cysteine, N-methyl hexanoic acid, lysine, or proline at the C20 position on the furan ring. Like Wm, WmC20 amino acid derivatives had significant antiproliferative activities. Three Wm derivatives, WmC20-proline, WmC20-cysteine, and a WmC20-N-methyl hexanoic acid, generated Wm that then reacted with lysine in an exchange-type reaction. This unusual, reversible, covalent reaction of Wm with nucleophiles under physiological conditions provides an explanation for the wortmannin paradox.

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.

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.