Useful discussions with Isja de Feijter and Matt Baker are gratefully acknowledged.

1. Synthesis of the Sensors
<ol>
	<li>In the following section we provide a procedure for the conjugation of pH indicators to PAMAM dendrimers. The same protocol can be applied with minimal modification to alternative amine-bearing dendrimers.5,17,13,14 Commercially available dendrimers and dyes can be used without further purifications.</li>
	<li>Dissolve the dendrimer in anhydrous DMSO (50 &#956;M final concentration). Prepare 10mM stock solutions of fluorescein-NHS and tetramethyl-rhodamine-NHS (TMR) in a...

<ol>
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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+new+generation+of+ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of biological chemistry. 260 (6), 3440-3450 (1985).</li><li>Han, J., Burgess, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+intracellular+pH.">Fluorescent indicators for intracellular pH.</a> Chemical reviews. 110 (5), 2709-2728 (2010).</li><li>Silver, R. A., Whitaker, M., Bolsover, S. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+dendrimers+for+biological+applications.">Designing dendrimers for biological applications.</a> Nature Biotechnology. 23 (12), 1517-1526 (2005).</li><li>Lee, C. C., Gillies, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dose+of+doxorubicin-functionalized+bow-tie+dendrimer+cures+mice+bearing+C-26+colon+carcinomas.">A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas.</a> Proceedings of the National Academy of Sciences. 103 (45), 16649-16654 (2006).</li><li>Caminade, A. -. M., Turrin, C. -. O., Majoral, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimers+and+DNA:+combinations+of+two+special+topologies+for+nanomaterials+and+biology.">Dendrimers and DNA: combinations of two special topologies for nanomaterials and biology.</a> Chemistry (Weinheim an der Bergstrasse, Germany). 14 (25), 7422-7432 (2008).</li><li>Rakow, N. A., Suslick, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+sensor+array+for+odour+visualization.">A colorimetric sensor array for odour visualization.</a> Nature. 406 (6797), 710-713 (2000).</li><li>Armada, M. P. G., Losada, J., Zamora, M., Alonso, B., Cuadrado, I., Casado, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocatalytical+properties+of+polymethylferrocenyl+dendrimers+and+their+applications+in+biosensing.">Electrocatalytical properties of polymethylferrocenyl dendrimers and their applications in biosensing.</a> Bioelectrochemistry (Amsterdam, Netherlands). 69 (1), 65-73 (2006).</li><li>Finikova, O., Galkin, A., Rozhkov, V., Cordero, M., H&#228;gerh&#228;ll, C., Vinogradov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrin+and+tetrabenzoporphyrin+dendrimers:+tunable+membrane-impermeable+fluorescent+pH+nanosensors.">Porphyrin and tetrabenzoporphyrin dendrimers: tunable membrane-impermeable fluorescent pH nanosensors.</a> Journal of the American Chemical Society. 125 (16), 4882-4893 (2003).</li><li>Albertazzi, L., Serresi, M., Albanese, A., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer+internalization+and+intracellular+trafficking+in+living+cells.">Dendrimer internalization and intracellular trafficking in living cells.</a> Molecular pharmaceutics. 7 (3), 680-688 (2010).</li><li>Albertazzi, L., Mickler, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bioactivity+of+internally+functionalized+cationic+dendrimers+with+PEG+cores.">Enhanced bioactivity of internally functionalized cationic dendrimers with PEG cores.</a> Biomacromolecules. , (2012).</li><li>Albertazzi, L., Fernandez-Villamarin, M., Riguera, R., Fernandez-Megia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+Functionalization+of+Dendrimers+Regulates+Internalization+and+Intracellular+Trafficking+in+Living+Cells.">Peripheral Functionalization of Dendrimers Regulates Internalization and Intracellular Trafficking in Living Cells.</a> Bioconjugate chemistry. , (2012).</li><li>Sakadzi&#263;, S., Roussakis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nature. 7 (9), 755-759 (2010).</li><li>Bizzarri, R., Arcangeli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+GFP-based+ratiometric+excitation+and+emission+pH+indicator+for+intracellular+studies.">Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.</a> Biophysical journal. 90 (9), 3300-3314 (2006).</li><li>Albertazzi, L., Brondi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+fluorescent+indicators:+in+vitro+and+in+vivo+applications.">Dendrimer-based fluorescent indicators: in vitro and in vivo applications.</a> PloS one. 6 (12), e28450 (2011).</li><li>Amir, R. J., Albertazzi, L., Willis, J., Khan, A., Kang, T., Hawker, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+Trackable+Dendritic+Scaffolds+and+Delivery+Agents.">Multifunctional Trackable Dendritic Scaffolds and Delivery Agents.</a> Angewandte Chemie International Edition. 50 (15), 3425-3429 (2011).</li><li>Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intracellular+chloride+and+pH+measurements+using+a+GFP-based+sensor.">Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.</a> Nature methods. 7 (7), 516-518 (2010).</li><li>Brondi, M., Sato, S. S., Rossi, L. F., Ferrara, S., Ratto, G. 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The critical steps for successful pH imaging with dendrimer-based sensors are: i) the selection of the correct dendritic scaffold and the number of indicators conjugated to it and ii) the optimization of sensor delivery protocol in cells or in vivo.
The synthetic procedure is fairly easy and can be applied virtually to every amine-bearing hyperbranched polymer. The sensors can be obtained from commercially available dendrimers and NHS-activated dyes in one single step. We believe that...

The use of fluorescent molecules to label specific biologically-relevant molecules has completely changed the way we study biological systems. Widefield and confocal microscopy allowed for a real-time high-resolution visualization of biological processes and nowadays are among the most popular techniques to study biological events in vitro, in cells and in vivo.1 A relevant improvement was represented by the development of fluorescence indicators, i.e. dyes whose fluorescence is dependent on the concentration of a specific molecular entity. pH and calcium indicators in particular had a dramatic impact on the study of cell physiology due to the enormous relevance of H+ and Ca2+ ions in biology.2,3
However, most of the sensing dyes present several intrinsic limitations related to their small molecule behavior such as: i) difficulties in subcellular targeting; ii) poor solubility in water and consequently poor biocompatibility; and iii) cell leakage and thus lack of long time-lapse imaging ability.4 Moreover, the signal of many probes cannot be corrected for the dependence on the dye concentration (non-ratiometric imaging) and therefore, an absolute measurement in cells or in vivo is not possible.
We recently described a simple and effective methodology to overcome these limitations, based on the conjugation of sensing dyes on a dendrimer scaffold.5 Dendrimers are monodisperse hyperbranched polymers with very appealing properties for biological applications.6 In particular several dendritic architectures have been developed and used for drug7 and gene delivery.8 Only very recently several groups started to explore the potential of these molecules as scaffold for sensing devices.9,10,11
We previously described an easy synthetic route towards the functionalization of different polyamidoamine (PAMAM) scaffolds based on NHS-activated esters.12 Conjugates can be obtained in a single step by means of dialysis as only purification. Interestingly this approach can easily be applied to a variety of dendritic or polymeric scaffolds.13,14
To achieve ratiometric imaging dendrimers were double-labeled with two sets of dyes: i) a pH indicator (i.e. fluorescein) and ii) a pH-independent fluorescent moiety (i.e. rhodamine). This allowed us to perform accurate pH imaging as the ratio between fluorescein and rhodamine is only dependent on the pH and no more on the concentration of the probe. Another interesting approach to this issue is represented by the use of lifetime-based probes.15 As the lifetime does not depend on probe concentration these measurements do not need a ratiometric correction. However, lifetime measurements require a more complicated instrumental setup and their temporal resolution is sub-optimal for fast physiological processes, thus limiting their potential applications.
In order to perform intracellular imaging, the probe needs to be delivered across the plasma membrane into the cytosol. As the dendrimers are not membrane permeable due to their size and hydrophilicity, intracellular delivery could be achieved through electroporation. By means of this technique, widely used in biology for transfection, labeled macromolecules can be effectively delivered into cells to perform high quality imaging. Moreover, with electroporation the complications related to dendrimer endocytosis can be avoided as the macromolecules are directly delivered to the cytoplasm. Interestingly after electroporation different dendrimers shows distinct localizations inside the cells even in absence of any specific targeting sequence.5 This passive targeting, only due to the physicochemical properties of the dendrimer, can be exploited to achieve organelle-specific pH imaging.
Ratiometric imaging can be performed using confocal microscopy. Fluorescein and rhodamine, covalently conjugated to the dendritic scaffold, were separately imaged and a pixel-by-pixel ratio map was created. Several procedures to control intracellular pH in living cells by means of ionophores were reported. Ionophores are small hydrophobic molecules able to transport ions across the plasma membrane; ionophores for H+ ion, such as nigericin, are available and can be used to calibrate dendrimer-based sensors.16 These measurements revealed a linear response to pH similarly to what observed in vitro. On the basis of the calibration intracellular pH could be accurately measured. These measurements demonstrated that dendrimer-based sensor can be a valuable tool in study H+ homeostasis in living cells and pathological processes that involve pH regulation malfunctions.
We recently demonstrated that dendrimer-based pH sensors can also be applied in vivo, performing pH imaging in the brain of anesthetized mice.17 Due to the complex environment of living tissues a high quality in vivo sensing is technically challenging. Here we show a detailed description of the experimental procedure for in vivo pH imaging with emphasis of the crucial issues to be addressed to perform an accurate pH imaging in the brain. Two-photon microscopy has been employed for two main reasons: i) the use of infrared light allows to overcome the lack of tissue penetration of standard confocal microscopy; ii) the broad two-photon absorption of fluorescein and rhodamine allow their simultaneous excitation avoiding the complications related to the use of two wavelengths for excitation. pH measurements in mouse brain were successfully carried out; sensors readily respond to hypoxia induce change of pH in the brain extracellular space. These measurements demonstrate that dendrimer-based indicators can be successfully used to highlight physiological and pathological change of pH in vivo in an animal model.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
		 <td>Company</td>
		 <td>Catalogue number</td>
		 <td>Comments (optional)</td>
 </tr>
	 <tr>
	 <td>PAMAM G4</td>
		 <td>Sigma-Aldrich</td>
		 <td>412449</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Carboxyfluorescein NHS ester</td>
		 <td>Life technologies</td>
		 <td>C-1311</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>TMR NHS ester</td>
		 <td>Life technologies</td>
		 <td>C-1171</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>DMSO</td>
		 <td>Sigma-Aldrich</td>
		 <td>D8418</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Dyalsis bags</td>
		 <td>Spectrum Labs</td>
		 <td>132117</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>WillCo Dishes</td>
		 <td>WillCo Wells</td>
		 <td>GWSt-3512</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Urethane</td>
		 <td>Sigma-Aldrich</td>
		 <td>U2500</td>
		 <td></td>
	 </tr>
 </tbody>
</table>

synthesis, cellular delivery and in vivo application of dendrimer-based ph sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Figure 1 shows a schematic representation of the conjugation of sensing dyes to different dendritic scaffolds. The resulting indicators can be obtained in one easy synthetic step from commercially available products. Amine-bearing dendrimers are reacted with NHS-activated dyes in DMSO and purified by dialysis. This general procedure has already been successfully used for labeling of several dendrimers: i) PAMAM dendrimer generation 2, 4 and 6;12 pegylated PAMAM dendrimers17 an...

Watch this Scientific Journal Video about Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors at JoVE.com

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

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11.6K vistas.  Eindhoven University of Technology & NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR.  Sensores de fluorescencia son herramientas poderosas en ciencias de la vida. Aquí se describe una metodología para sintetizar y utilizar sensores fluorescentes basadas en dendrímeros para medir el pH en las células vivas e in vivo. El andamio dendríticas mejora las propiedades de los tintes fluorescentes conjugados que conducen a propiedades mejoradas de detección. 