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Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.
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Hidrozoários/genética , Hidrozoários/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Técnicas Biossensoriais , Cor , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrozoários/química , Proteínas Luminescentes/química , Modelos Moleculares , Imagem Óptica , Filogenia , Eletricidade EstáticaRESUMO
This Account summarizes recent advances in the chemistry of fluorocarbon nanoemulsion (FC NE) functionalization. We describe new families of fluorous molecules, such as chelators, fluorophores, and peptides, that are soluble in FC oils. These materials have helped transform the field of in vivo molecular imaging by enabling sensitive and cell-specific imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescence detection. FC emulsions, historically considered for artificial blood substitutes, are routinely used for ultrasound imaging in clinic and have a proven safety profile and a well-characterized biodistribution and pharmacokinetics. The inertness of fluorocarbons contributes to their low toxicity but makes functionalization difficult. The high electronegativity of fluorine imparts very low cohesive energy density and Lewis basicity to heavily fluorinated compounds, making dissolution of metal ions and organic molecules challenging. Functionalization is further complicated by colloidal instability toward heat and pH, as well as limited availability of biocompatible surfactants.We have devised new fluorous chelators that overcome solubility barriers and are able to bind a range of metal ions with high thermodynamic stability and biocompatibility. NE harboring chelators in the fluorous phase are a powerful platform for the development of multimodal imaging agents. These compositions rapidly capture metal ions added to the aqueous phase, thereby functionalizing NEs in useful ways. For example, Fe3+ encapsulation imparts a strong paramagnetic relaxation effect on 19F T1 that dramatically accelerates 19F MRI data acquisition times and hence sensitivity in cell tracking applications. Alternatively, 89Zr encapsulation creates a sensitive and versatile PET probe for inflammatory macrophage detection. Adding lanthanides, such as Eu3+, renders NE luminescent. Beyond chelators, this Account further covers our progress in formulating NEs with fluorophores, such as cyanine or BODIPY dyes, with their utility demonstrated in fluorescence imaging, biosensing, flow cytometry and histology. Fluorous dyes soluble in FC oils are also key enablers for nascent whole-body imaging technologies such as cryo-fluorescence tomography (CFT). Additionally, fluorous cell-penetrating peptides inserted on the NE surface increase the uptake of NE by â¼8-fold in weakly phagocytic stem cells and lymphocytes used in immunotherapy, resulting in significant leaps in detection sensitivity in vivo.
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Flúor/química , Imagem Molecular/métodos , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons/métodosRESUMO
The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides â¼4× increase in signal-to-noise and â¼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.
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Elementos da Série dos Lantanídeos , Microscopia Eletrônica de Transmissão por Filtração de Energia , Diagnóstico por Imagem , Elétrons , Coloração e RotulagemRESUMO
PURPOSE: A bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a non-invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations. METHODS: We performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors. RESULTS: By incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a >8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT. CONCLUSION: The peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes.
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Imagem por Ressonância Magnética de Flúor-19 , Fluorocarbonos/química , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Receptores de Antígenos Quiméricos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Rastreamento de Células/métodos , Peptídeos Penetradores de Células/química , Emulsões , Feminino , Glioblastoma/diagnóstico por imagem , Glioma/metabolismo , Glioma/patologia , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Linfócitos T/citologia , Distribuição TecidualRESUMO
Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.
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Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
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EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.
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Química Click , DNA/análise , Lipídeos/análise , Microscopia Eletrônica/métodos , Peptidoglicano/análise , RNA/análise , Aminobutiratos/química , DNA/química , DNA/metabolismo , Corantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Lipídeos/química , Listeria monocytogenes/metabolismo , Estrutura Molecular , Neurônios/química , Neurônios/metabolismo , Peptidoglicano/biossíntese , RNA/química , RNA/metabolismo , Oxigênio Singlete/químicaRESUMO
Fluorine-19 magnetic resonance imaging ((19)F MRI) probes enable quantitative in vivo detection of cell therapies and inflammatory cells. Here, we describe the formulation of perfluorocarbon-based nanoemulsions with improved sensitivity for cellular MRI. Reduction of the (19)F spin-lattice relaxation time (T1) enables rapid imaging and an improved signal-to-noise ratio, thereby improving cell detection sensitivity. We synthesized metal-binding ß-diketones conjugated to linear perfluoropolyether (PFPE), formulated these fluorinated ligands as aqueous nanoemulsions, and then metallated them with various transition and lanthanide ions in the fluorous phase. Iron(III) tris-ß-diketonate ('FETRIS') nanoemulsions with PFPE have low cytotoxicity (<20%) and superior MRI properties. Moreover, the (19)F T1 can readily be reduced by an order of magnitude and tuned by stoichiometric modulation of the iron concentration. The resulting (19)F MRI detection sensitivity is enhanced by three- to fivefold over previously used tracers at 11.7 T, and is predicted to increase by at least eightfold at the clinical field strength of 3 T.
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Compostos Férricos/química , Imagem por Ressonância Magnética de Flúor-19/métodos , Fluorocarbonos/química , Animais , Linhagem Celular Tumoral , Camundongos , Ratos , Sensibilidade e EspecificidadeRESUMO
PURPOSE: We explore the use of intravenously delivered fluorescent perfluorocarbon (PFC) nanoemulsion tracers and multi-spectral cryo-fluorescence tomography (CFT) for whole-body tracer imaging in murine inflammation models. CFT is an emerging technique that provides high-resolution, three-dimensional mapping of probe localization in intact animals and tissue samples, enabling unbiased validation of probe biodistribution and minimizes reliance on laborious histological methods employing discrete tissue panels, where disseminated populations of PFC-labeled cells may be overlooked. This methodology can be used to streamline the development of new generations of non-invasive, cellular-molecular imaging probes for in vivo imaging. PROCEDURES: Mixtures of nanoemulsions with different fluorescent emission wavelengths were administered intravenously to naïve mice and models of acute inflammation, colitis, and solid tumor. Mice were euthanized 24 h post-injection, frozen en bloc, and imaged at high resolution (~ 50 µm voxels) using CFT at multiple wavelengths. RESULTS: PFC nanoemulsions were visualized using CFT within tissues of the reticuloendothelial system and inflammatory lesions, consistent with immune cell (macrophage) labeling, as previously reported in in vivo magnetic resonance and nuclear imaging studies. The CFT signals show pronounced differences among fluorescence wavelengths and tissues, presumably due to autofluorescence, differential fluorescence quenching, and scattering of incident and emitted light. CONCLUSIONS: CFT is an effective and complementary methodology to in vivo imaging for validating PFC nanoemulsion biodistribution at high spatial localization, bridging the resolution gap between in vivo imaging and histology.
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In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.
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Peptídeos Penetradores de Células/metabolismo , Trombina/metabolismo , Animais , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos , Camundongos TransgênicosRESUMO
The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.
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The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.
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Ácidos Nucleicos , RNA/metabolismo , Microscopia Eletrônica , DNA , Nucléolo Celular/metabolismoRESUMO
We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.
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We describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling postemulsification functionalization via a click reaction with azide-containing ligands. The addition of DBCO-lipid to the surfactant in PFC nanoemulsions did not affect nanoemulsion size or nanoemulsion stability. As proof-of-concept, fluorescent dye-azides were conjugated to PFC nanoemulsions, demonstrating the feasibility of functionalization the by click reaction. Uptake of the fluorescent PFC by macrophages was demonstrated both in vitro in cultured macrophages and in situ in an acute inflammation mouse model, where fluorescence imaging and 1H/19F magnetic resonance imaging (MRI) were used for in vivo detection. Overall, these data demonstrate the potential of PFC nanoemulsions incorporating DBCO as a versatile platform for generating functionalized probes.
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Although WNT signaling is frequently dysregulated in solid tumors, drugging this pathway has been challenging due to off-tumor effects. Current clinical pan-WNT inhibitors are nonspecific and lead to adverse effects, highlighting the urgent need for more specific WNT pathway-targeting strategies. We identified elevated expression of the WNT receptor Frizzled class receptor 7 (FZD7) in multiple solid cancers in The Cancer Genome Atlas, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival. Moreover, we observed increased FZD7 protein expression in ovarian tumors compared with normal ovarian tissue, indicating that FZD7 may be a tumor-specific antigen. We therefore developed a novel antibody-drug conjugate, septuximab vedotin (F7-ADC), which is composed of a chimeric human-mouse antibody to human FZD7 conjugated to the microtubule-inhibiting drug monomethyl auristatin E (MMAE). F7-ADC selectively binds human FZD7, potently kills ovarian cancer cells in vitro, and induces regression of ovarian tumor xenografts in murine models. To evaluate F7-ADC toxicity in vivo, we generated mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the human-targeting F7-ADC. F7-ADC treatment of these mice did not induce acute toxicities, indicating a potentially favorable safety profile in patients. Overall, our data suggest that the antibody-drug conjugate approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic.
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Receptores Frizzled/genética , Imunoconjugados/metabolismo , Neoplasias Ovarianas/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologiaRESUMO
Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control.
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Imunoconjugados , Neoplasias , Aminobenzoatos , Anticorpos Antineoplásicos , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoterapia , Neoplasias/terapia , Oligopeptídeos , PeptídeosRESUMO
Recent advances in immunotherapy have revolutionized cancer therapy. Immunotherapies can engage the adaptive and innate arms of the immune system. Therapeutics targeting immune checkpoint inhibitors (i.e., CTLA-4; PD-1, and PD-L1) have shown efficacy for subsets of cancer patients by unleashing an adaptive antitumor immune response. Alternatively, small molecule immune modulators of the innate immune system such as toll-like receptor (TLR) agonists are being developed for cancer therapy. TLRs function as pattern recognition receptors to microbial products and are also involved in carcinogenesis. Reisquimod is a TLR 7/8 agonist that has antitumor efficacy. However, systemic delivery free resiquimod has proven to be challenging due to toxicity of nonspecific TLR 7/8 activation. Therefore, we developed a targeted peptide-drug conjugate strategy for systemic delivery of resiquimod. We designed an activatable cell penetrating peptide to deliver resiquimod specifically to the tumor tissue while avoiding normal tissues. The activatable cell penetrating peptide (ACPP) scaffold undergoes enzymatic cleavage by matrix metalloproteinases 2/9 in the extracellular matrix followed by intracellular lysosomal cathepsin B mediated release of the free resiquimod. Importantly, when conjugated to ACPP; the tumor tissue concentration of resiquimod was more than 1000-fold greater than that of surrounding non-cancerous tissue. Moreover, systemic ACPP-resiquimod delivery produced comparable therapeutic efficacy to localized free resiquimod in syngeneic murine tumors. These results highlight a precision peptide-drug conjugate delivery.
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Inflammation is associated with a range of serious human conditions, including autoimmune and cardiovascular diseases and cancer. The ability to image active inflammatory processes greatly enhances our ability to diagnose and treat these diseases at an early stage. We describe molecular compositions enabling sensitive and precise imaging of inflammatory hotspots in vivo. Methods: A functionalized nanoemulsion with a fluorocarbon-encapsulated radiometal chelate (FERM) was developed to serve as a platform for multimodal imaging probe development. The 19F-containing FERM nanoemulsion encapsulates 89Zr in the fluorous oil via a fluorinated hydroxamic acid chelate. Simple mixing of the radiometal with the preformed aqueous nanoemulsion before use yields FERM, a stable in vivo cell tracer, enabling whole-body 89Zr PET and 19F MRI after a single intravenous injection. Results: The FERM nanoemulsion was intrinsically taken up by phagocytic immune cells, particularly macrophages, with high specificity. FERM stability was demonstrated by a high correlation between the 19F and 89Zr content in the blood (correlation coefficient > 0.99). Image sensitivity at a low dose (37 kBq) was observed in a rodent model of acute infection. The versatility of FERM was further demonstrated in models of inflammatory bowel disease and 4T1 tumor. Conclusion: Multimodal detection using FERM yields robust whole-body lesion detection and leverages the strengths of combined PET and 19F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases.
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Macrófagos , Humanos , Inflamação , Imageamento por Ressonância MagnéticaRESUMO
PURPOSE: We aim to develop perfluorocarbon-based nanoemulsions with improved sensitivity for detection of inflammatory macrophages in situ using F-19 MRI. Towards this goal, we evaluate the feasibility of nanoemulsion formulation incorporating a metal chelate in the fluorous phase which shortens the F-19 longitudinal relaxation rate and image acquisition time. PROCEDURES: Perfluorinated linear polymers were conjugated to metal-binding tris-diketonate, blended with unconjugated polymers, and emulsified in water. Phospholipid-based surfactant was used to stabilize nanoemulsion and provide biocompatibility. Nanoemulsions were metalated with the addition of ferric salt to the buffer. Physical stability of surfactant and nanoemulsion was evaluated by mass spectrometry and dynamic light scattering measurements. Nanoemulsions were injected intravenously into a murine granuloma inflammation model, and in vivo19F/1H MRI at 11.7 T was performed. RESULTS: We demonstrated stability and biocompatibility of lipid-based paramagnetic nanoemulsions. We investigated potential oxidation of lipid in the presence of metal chelate. As a proof of concept, we performed non-invasive monitoring of macrophage burden in a murine inflammation model following intravenous injection of nanoemulsion using in vivo F-19 MRI. CONCLUSION: Lipid-based nanoemulsion probes of perfluorocarbon synthesized with iron-binding fluorinated ß-diketones can be formulated for intravenous delivery and inflammation detection in vivo.
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Imagem por Ressonância Magnética de Flúor-19/métodos , Fluorocarbonos/química , Inflamação/diagnóstico por imagem , Macrófagos/citologia , Imageamento por Ressonância Magnética/métodos , Nanoestruturas/química , Animais , Linhagem Celular , Modelos Animais de Doenças , Emulsões , Feminino , Compostos Férricos/química , Inflamação/imunologia , Inflamação/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Polímeros/químicaRESUMO
Despite the importance of understanding the regulation of microvascular blood flow in white matter, no data on subcortical capillary blood flow parameters are available, largely due to the lack of appropriate imaging methods. To address this knowledge gap, we employed two-photon microscopy using a far-red fluorophore Alexa680 and photon-counting detection to measure capillary red blood cell (RBC) flux in both cerebral gray and white matter, in isoflurane-anesthetized mice. We have found that in control animals, baseline capillary RBC flux in the white matter was significantly higher than in the adjacent cerebral gray matter. In response to mild hypercapnia, RBC flux in the white matter exhibited significantly smaller fractional increase than in the gray matter. Finally, during global cerebral hypoperfusion, RBC flux in the white matter was reduced significantly in comparison to the controls, while RBC flux in the gray matter was preserved. Our results suggest that blood flow in the white matter may be less efficiently regulated when challenged by physiological perturbations as compared to the gray matter. Importantly, the blood flow in the white matter may be more susceptible to hypoperfusion than in the gray matter, potentially exacerbating the white matter deterioration in brain conditions involving global cerebral hypoperfusion.