Il lavoro è stato sostenuto dalle concessioni 1K25MH089851, 1P01DA028555-01A1, NS034239 2R01, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570 e P01 NS43985 dal National Institutes of Health. Gli autori ringraziano la signora Robin Taylor per la lettura critica del manoscritto ed eccezionale supporto grafico e letterario. Gli autori desiderano inoltre ringraziare Erin McIntyre, Melissa Mellon, e Lindsay Rice per il loro supporto tecnico.

<ol>
	<li>Nowacek, A., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoART+neuroAIDS+and+CNS+drug+delivery.">NanoART neuroAIDS and CNS drug delivery.</a> Nanomedicine (Lond). 4, 557-574 (2009).</li><li>Nowacek, A., Kosloski, L. M., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+disorders+and+nanoformulated+drug+development.">Neurodegenerative disorders and nanoformulated drug development.</a> Nanomedicine (Lond). 4, 541-555 (2009).</li><li>Nowacek, A. S., Miller, R. L., McMillan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoART+synthesis,+characterization,+uptake,+release+and+toxicology+for+human+monocyte-macrophage+drug+delivery.">NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.</a> Nanomedicine (Lond). 4, 903-917 (2009).</li><li>Herbein, G., Varin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+in+HIV-1+infection:+from+activation+to+deactivation.">The macrophage in HIV-1 infection: from activation to deactivation.</a> Retrovirology. 7, 33-33 (2010).</li><li>Qian, B. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+diversity+enhances+tumor+progression+and+metastasis.">Macrophage diversity enhances tumor progression and metastasis.</a> Cell. 141, 39-51 (2010).</li><li>Nathan, C., Ding, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonresolving+inflammation.">Nonresolving inflammation.</a> Cell. 140, 871-882 (2010).</li><li>McNeill, E., Channon, K. M., Greaves, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cell+recruitment+in+cardiovascular+disease:+murine+models+and+potential+clinical+applications.">Inflammatory cell recruitment in cardiovascular disease: murine models and potential clinical applications.</a> Clin Sci (Lond). 118, 641-655 (2010).</li><li>Bondeson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+synovial+macrophages+as+targets+for+osteoarthritis+drug+therapy.">Activated synovial macrophages as targets for osteoarthritis drug therapy.</a> Curr Drug Targets. 11, 576-585 (2010).</li><li>Benoit, L. A., Holtzman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+immune+pathways+from+chronic+post-viral+lung+disease.">New immune pathways from chronic post-viral lung disease.</a> Ann N Y Acad Sci. 1183, 195-210 (2010).</li><li>Beduneau, A., Ma, Z., Grotepas, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitated+monocyte-macrophage+uptake+and+tissue+distribution+of+superparmagnetic+iron-oxide+nanoparticles.">Facilitated monocyte-macrophage uptake and tissue distribution of superparmagnetic iron-oxide nanoparticles.</a> PLoS One. 4, e4343-e4343 (2009).</li><li>Dou, H., Grotepas, C. B., McMillan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+delivery+of+nanoformulated+antiretroviral+drug+to+the+brain+in+a+murine+model+of+neuroAIDS.">Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS.</a> J Immunol. 183, 661-669 (2009).</li><li>Liu, Y., Uberti, M. G., Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingress+of+blood-borne+macrophages+across+the+blood-brain+barrier+in+murine+HIV-1+encephalitis.">Ingress of blood-borne macrophages across the blood-brain barrier in murine HIV-1 encephalitis.</a> J Neuroimmunol. 200, 41-52 (2008).</li><li>Hetherington, H. P., Chu, W. J., Gonen, O., Pan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+fully+automated+shimming+of+the+human+brain+for+high-field+1H+spectroscopic+imaging.">Robust fully automated shimming of the human brain for high-field 1H spectroscopic imaging.</a> Magn Reson Med. 56, 26-33 (2006).</li><li>Hasan, K. M., Parker, D. L., Alexander, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gradient+encoding+schemes+for+diffusion-tensor+MRI.">Comparison of gradient encoding schemes for diffusion-tensor MRI.</a> J Magn Reson Imaging. 13, 769-780 (2001).</li><li>Hasan, K. M., Basser, P. J., Parker, D. L., Alexander, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+computation+of+the+eigenvalues+and+eigenvectors+in+DT-MRI.">Analytical computation of the eigenvalues and eigenvectors in DT-MRI.</a> J Magn Reson. 152, 41-47 (2001).</li><li>Neeman, M., Freyer, J. P., Sillerud, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+obtaining+cross-term-free+images+for+diffusion+anisotropy+studies+in+NMR+microimaging.">A simple method for obtaining cross-term-free images for diffusion anisotropy studies in NMR microimaging.</a> Magn Reson Med. 21, 138-143 (1991).</li><li>Uberti, M. G., Boska, M. D., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automatic+image+segmentation+method+for+extraction+of+brain+volume+from+in+vivo+mouse+head+magnetic+resonance+imaging+using+Constraint+Level+Sets.">A semi-automatic image segmentation method for extraction of brain volume from in vivo mouse head magnetic resonance imaging using Constraint Level Sets.</a> J of Neurosci Methods. 179, 338-344 (2009).</li><li>Liu, Y., Uberti, M. G., Dou, H., Mosley, R. L., Gendelman, H. E., Boska, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Image+Warping+Technique+for+Rodent+Brain+MRI-Histology+Registration+Based+On+Thin-Plate+Splines+with+Landmark+Optimization.">An Image Warping Technique for Rodent Brain MRI-Histology Registration Based On Thin-Plate Splines with Landmark Optimization.</a> Proc Soc Photo Opt Instrum Eng. 7259, 72592-K-72597-K (2009).</li><li>Uberti, M. G., Liu, Y., Dou, H., Mosley, R. L., Gendelman, H. E., Boska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+in+vivo+MR+to+histology+of+rodent+brains+using+blockface+imaging.">Registration of in vivo MR to histology of rodent brains using blockface imaging.</a> Proc Soc Photo Opt Instrum Eng. 7262, 726213-726213 (2009).</li><li>Basser, P. J., Mattiello, J., LeBihan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+effective+self-diffusion+tensor+from+the+NMR+spin+echo.">Estimation of the effective self-diffusion tensor from the NMR spin echo.</a> J Magn Reson B. 103, 247-254 (1994).</li><li>Hasan, K. M., Narayana, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+measurement+of+the+diffusion+tensor+eigenvalues+from+diffusion+anisotropy+and+mean+diffusivity+in+DTI.">Retrospective measurement of the diffusion tensor eigenvalues from diffusion anisotropy and mean diffusivity in DTI.</a> Magn Reson Med. 56, 130-137 (2006).</li><li>Ratiney, H., Sdika, M., Coenradie, Y., Cavassila, S., Ormondt, D. v. a. n., Graveron-Demilly, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-domain+semi-parametric+estimation+based+on+a+metabolite+basis+set.">Time-domain semi-parametric estimation based on a metabolite basis set.</a> NMR Biomed. 18, 1-13 (2005).</li><li>Naressi, A., Couturier, C., Castang, I., Beer, R. d. e., Graveron-Demilly, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Java-based+graphical+user+interface+for+MRUI,+a+software+package+for+quantitation+of+in+vivo/medical+magnetic+resonance+spectroscopy+signals.">Java-based graphical user interface for MRUI, a software package for quantitation of in vivo/medical magnetic resonance spectroscopy signals.</a> Comput Biol Med. 31, 269-286 (2001).</li><li>Boska, L. e. w. i. s., Destache, T. B., J, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+1H+magnetic+resonance+spectroscopic+imaging+determines+therapeutic+immunization+efficacy+in+an+animal+model+of+Parkinson's+disease.">Quantitative 1H magnetic resonance spectroscopic imaging determines therapeutic immunization efficacy in an animal model of Parkinson's disease.</a> J Neurosci. 25, 1691-1700 (2005).</li><li>Mosley, R. L., Benner, E. J., Kadiu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation,+Oxidative+Stress+and+the+Pathogenesis+of+Parkinson's+Disease.">Neuroinflammation, Oxidative Stress and the Pathogenesis of Parkinson's Disease.</a> Clin Neurosci Res. 6, 261-281 (2006).</li><li>Nelson, J. A., Dou, H., Ellison, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coregistration+of+quantitative+proton+magnetic+resonance+spectroscopic+imaging+with+neuropathological+and+neurophysiological+analyses+defines+the+extent+of+neuronal+impairments+in+murine+human+immunodeficiency+virus+type-1+encephalitis.">Coregistration of quantitative proton magnetic resonance spectroscopic imaging with neuropathological and neurophysiological analyses defines the extent of neuronal impairments in murine human immunodeficiency virus type-1 encephalitis.</a> J Neurosci Res. 80, 562-575 (2005).</li><li>Boska, H. a. s. a. n., Kibuule, K. M., D, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+diffusion+tensor+imaging+detects+dopaminergic+neuronal+degeneration+in+a+murine+model+of+Parkinson's+disease.">Quantitative diffusion tensor imaging detects dopaminergic neuronal degeneration in a murine model of Parkinson's disease.</a> Neurobiol Dis. 26, 590-596 (2007).</li><li>Liu, W., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+magnetically+labeled+cells+by+cellular+MRI.">Detection and quantification of magnetically labeled cells by cellular MRI.</a> Eur J Radiol. 70, 258-264 (2009).</li><li>Balchandani, P., Yamada, M., Pauly, J., Yang, P., Spielman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-refocused+spatial-spectral+pulse+for+positive+contrast+imaging+of+cells+labeled+with+SPIO+nanoparticles.">Self-refocused spatial-spectral pulse for positive contrast imaging of cells labeled with SPIO nanoparticles.</a> Magn Reson Med. 62, 183-192 (2009).</li><li>Stuber, M., Gilson, W. D., Schar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+contrast+visualization+of+iron+oxide-labeled+stem+cells+using+inversion-recovery+with+ON-resonant+water+suppression+(IRON).">Positive contrast visualization of iron oxide-labeled stem cells using inversion-recovery with ON-resonant water suppression (IRON).</a> Magn Reson Med. 58, 1072-1077 (2007).</li><li>Suzuki, Y., Cunningham, C. H., Noguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+serial+evaluation+of+superparamagnetic+iron-oxide+labeled+stem+cells+by+off-resonance+positive+contrast.">In vivo serial evaluation of superparamagnetic iron-oxide labeled stem cells by off-resonance positive contrast.</a> Magn Reson Med. 60, 1269-1275 (2008).</li><li>Kim, Y. B., Bae, K. H., Yoo, S. S., Park, T. G., H, P. a. r. k. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+contrast+visualization+for+cellular+magnetic+resonance+imaging+using+susceptibility-weighted+echo-time+encoding.">Positive contrast visualization for cellular magnetic resonance imaging using susceptibility-weighted echo-time encoding.</a> Magn Reson Imaging. 27, 601-610 (2009).</li><li>Zhou, R., Idiyatullin, D., Moeller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWIFT+detection+of+SPIO-labeled+stem+cells+grafted+in+the+myocardium.">SWIFT detection of SPIO-labeled stem cells grafted in the myocardium.</a> Magn Reson Med. 63, 1154-1161 .</li></ol>

Nessun conflitto di interessi dichiarati.

La registrazione accurata di istologia in vivo con risultati di imaging è un passo fondamentale nello sviluppo di biomarcatori di imaging per il rilevamento e la stadiazione della malattia neuronale. Alcuni parametri di imaging possono essere correlati con i cambiamenti morfologici lordi compresi i cambiamenti nelle proprietà magnetiche rilassamento dei tessuti utilizzati per rilevare la presenza di malattie della sostanza bianca e tumori. Altri metodi più sottili, come DTI, sono suscettibili di individuare precocemente cambiamenti cellulari che possono non essere rilevabili come i cambiamenti istologici causati dalla malattia non compaiono più tardi le fasi della malattia. Marcatori ancora altri, come marker spettroscopici, possono essere indicatori di cambiamenti precoci e reversibili, che precedono anche le più sottili alterazioni cellulari. Biodistribuzione può essere determinata in modo non invasivo utilizzando una varietà di metodi. Il primario metodi non invasivi sono la tomografia ad emissione di positroni (PET), emissione di singolo fotone SPECT (tomografia computerizzata), imaging ottico, e la risonanza magnetica. Medicina nucleare a base di imaging (PET e SPECT) sono stati utilizzati nel corso degli anni per la biodistribuzione molti, ma questi metodi sono limitati dalla vita della metà dei radiotraccianti utilizzati per l&#39;etichettatura dei composti o nanomateriali, soprattutto per i traccianti PET. L&#39;imaging ottico può essere usato per piccoli roditori, ma non può essere tradotto per l&#39;uso nell&#39;uomo, tranne per le regioni facilmente accessibili, come i tumori superficiali a causa di assorbimento della luce e la dispersione della luce. Inoltre, è difficile quantificare i segnali ottici per queste stesse ragioni. La RM utilizza tag persistenti come SPIO che possono essere rintracciati nel corpo per un periodo di settimane. Anche questo deve essere usato con cautela, in quanto l&#39;etichetta può essere trasferito alle cellule diverse o essere riassorbito dal corpo. Specificità di rilevamento per SPIO a risonanza magnetica può essere fornito da una varietà di metodi. Metodi di rilevamento, che forniscono segnali positivi e negativi, sono utilizzati per migliorare la specificità della risonanza magnetica per rilevare la presenza di SPIO nei tessuti. Il metodo di sottrazione utilizzato in questo lavoro è stato utilizzato da altri, oltre 28. Altri approcci sono fuori rilevamento risonanza 29-31, fase di imaging sensibile che produce un particolare modello vicino vuoti SPIO 32, immagine e zero tempo di eco ponderazione T1 che utilizza per produrre un positivo segnale di intensità nella regione di SPIO 33. L&#39;avanzamento di questi metodi per migliorare la quantificazione di etichetta, la sensibilità e la specificità è un&#39;area di ricerca attiva oggi.

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<td>Isoflurane</td>
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<td>Isoflurane vaporizer</td>
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<td>Rodent gas anesthesia mask</td>
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<td>MRI compatible Stereotactic head holder</td>
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<td>Syringe</td>
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<td>Polyethylene catheter tubing</td>
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<td>Perfusion media</td>
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<td>OCT compound for embedding tissue</td>
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<td>MRI system</td>
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<td>Digital Camera</td>
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<td>Tissue Sectioning Cryostat</td>
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registered bioimaging of nanomaterials for diagnostic and therapeutic monitoring

Nanomedications può essere trasportato da ematica monociti-macrofagi nel sistema reticolo-endoteliale (RES, milza, fegato, linfonodi) e per finire organi. Questi ultimi includono i polmoni, RES, e il cervello e sono operative durante il virus dell&#39;immunodeficienza umana di tipo uno (HIV-1) infezioni. Ingresso dei macrofagi nei tessuti è notevole in aree di attivi replicazione dell&#39;HIV-1 e siti di infiammazione. Al fine di valutare il potenziale dei macrofagi come nanovettori, superparamagnetiche di ossido di ferro e / o droga particelle cariche rivestite con tensioattivi parenterale sono state iniettate in topi da HIV-1 encefalitiche. Ciò è stato fatto per valutare quantitativamente particelle e biodistribuzione di droga. Risonanza magnetica (MRI) i risultati dei test sono stati convalidati da coregistrazione istologici e di elaborazione delle immagini migliorato. Organo terminale della malattia come caratterizzata da istologia cervello alterato sono stati valutati mediante risonanza magnetica. La dimostrazione della migrazione robusta nanoformulations in aree di encefalite focale fornisce &#39;&quot;proof of concept&quot; per l&#39;utilizzo di avanzate tecniche di bioimmagini di monitorare la migrazione dei macrofagi. È importante sottolineare che le aberrazioni istopatologici nel cervello di correlazione con i parametri di bioimmagini rendendo l&#39;utilità generale della RM negli studi di distribuzione delle cellule nella malattia fattibile. Noi ipotizzare che l&#39;utilizzo di tali metodi in grado di fornire un indice in tempo reale di tutte le malattie e l&#39;efficacia terapeutica con un potenziale traslazionali per l&#39;uomo.

Watch this Scientific Journal Video about Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring at JoVE.com

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimmagini metodi utilizzati per valutare biodistribuzione delle cellule di nanoparticelle sono applicabili per il monitoraggio terapeutico e diagnostico di composti nanoformulated. I metodi descritti nel presente documento sono sensibili e specifici, quando valutati da coregistrazione istologici. Le metodologie di fornire un percorso traslazionale da roditore a applicazioni sull&#39;uomo.

Bioimmagini registrato di Nanomateriali per il monitoraggio diagnostico e terapeutico

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

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10.3K Views.  University of Nebraska Medical Center. Bioimmagini metodi utilizzati per valutare biodistribuzione delle cellule di nanoparticelle sono applicabili per il monitoraggio terapeutico e diagnostico di composti nanoformulated. I metodi descritti nel presente documento sono sensibili e specifici, quando valutati da coregistrazione istologici. Le metodologie di fornire un percorso traslazionale da roditore a applicazioni sull'uomo.

Video: Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Basso Peso Molecolare proteina di arricchimento sul film sottili di silice mesoporosi per la scoperta di biomarcatori

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

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Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Hollow microago-based sensore per Multiplexed transdermico elettrochimica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Massa Citometria: Protocollo per l&#39;ottimizzazione giornaliera e campioni di cellule in esecuzione su un citometro Messa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinazione del tasso di trasporto di xenobiotici e nanomateriali attraverso la placenta con il<em&gt; Ex vivo</em&gt; Umano Modello perfusione placentare

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Rapida e basso costo Prototipazione di dispositivi medici che usano 3D Stampo Stampato per stampaggio ad iniezione Liquid

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolore fluorescenza Detection per Droplet microfluidica tramite fibre ottiche

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

La sintesi di RGD-funzionalizzati idrogeli come strumento per applicazioni terapeutiche

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Sintesi di cationizzata Magnetoferritin per la magnetizzazione ultra-veloce di celle

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

Sintesi dei batteriofagi infettive in un e. coli -basato sistema di espressione senza cellula

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Bioimmagini registrato di Nanomateriali per il monitoraggio diagnostico e terapeutico

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