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 anhydrous DMSO.</li>
	<li>Add to the dendrimer solution the desired amount of fluorescein and TMR. The molar ratio in the mixture will reflect the amount of dyes loaded on the dendrimer. Typically 1 ml of G4 PAMAM dendrimer solution in a microcentrifuge tube is reacted with 8 eq (40 &#956;l) of fluorescein and 8 eq (40 &#956;l) of TMR. Stir the solution at room temperature for 12 hr.</li>
	<li>Dilute 1:10 with deionized water and load the reaction mixture in a dialysis bag (MWCO=10 kDa). Dialyze for 24 hr against deionized water replacing frequently the water in the reservoir.</li>
	<li>Transfer the solution to a vial and freeze-dry for 24 hr. A purple powder should be obtained. Weight the obtained solid and dissolve it in milliQ water at a final concentration of 10 &#956;M. Aliquot the solution and store at -20 &#176;C.</li>
</ol>
2. In Vitro pH Measurements
<ol>
	<li>For in vitro calibration prepare a solution 500nM of dendrimer in PBS (2 mM phosphate) in a quartz cuvette. The use of a very dilute PBS buffer (2 mM) to avoid abrupt changes of pH during the titration is recommended.</li>
	<li>Measure the emission spectra of fluorescein (exc 488 nm) and TMR (exc 550 nm) and optimize the optical settings of the fluorimeter to achieve a good signal-to-noise ratio.</li>
	<li>Perform a pH titration by adding small volumes of NaOH 0.1 N and HCl 0.1 N. After every addition shake the cuvette for mixing, wait 1 min for equilibration and measure the pH by means of a pH microelectrode. Emission spectra of fluorescein and TMR should be recorded for every step without any change in the optical settings.</li>
	<li>Plot the fluorescence intensity vs pH for the titration. The rhodamine signal should be unaffected by pH (&#60;10%). The fluorescein signal should be a sigmoidal curve and should be fitted with a single-binding model with pK = 6.4.</li>
</ol>
3. Cell Culture and Electroporation
<ol>
	<li>Cultivate HeLa cells in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Keep cell culture at 37 &#176;C in a humidified 5% CO2 atmosphere.</li>
	<li>For dendrimer electroporation, when cells are confluent, remove the media and wash cells using DPBS (Dulbecco&#39;s Phosphate-Buffered Saline). Remove the DPBS and add trypsin-EDTA. Neutralize the trypsin by adding medium containing serum but not antibiotics. Centrifuge at 900-1,200 rpm for 2 min at room temperature. Remove the media and rinse the pellets using DPBS.</li>
	<li>Count the cells and take 4*106 cells. Centrifuge at 1,200-1,500 rpm for 2 min at room temperature.</li>
	<li>Resuspend the cell pellet in 200 &#956;l of microporation buffer (provided by microporator manufacturer) and transfer cells to a 1.5 ml microcentrifuge tube.</li>
	<li>Add dendrimer aqueous solution into resuspended cells. The amount of dendrimer required per sample is dependent on the PAMAM type (typically 250 nM for cationic and 2 &#956;M for neutral dendrimers).</li>
	<li>Add electroporation buffer (provided by microporator manufacturer) in a microporation tube. Pipette the cells and dendrimers mixtures with electroporation tip of 100 &#956;l volume-sized. Insert the microporator pipette into the pipette station. Set the pulse condition for microporation: pulse voltage = 1,005 V; pulse width = 35 msec; pulse number = 2.</li>
	<li>After the pulse transfer cells to a 1.5 ml microcentrifuge tube and centrifuge cells for 5 min at 1,200 rpm to remove the excess of dendrimer in the medium. Plate 10 &#956;l of electroporated cells onto 35 mm glass-bottom dishes (WillCo-dish GWSt-3522) with fresh medium w/o antibiotics.</li>
</ol>
4. pH Sensing in Living HeLa Cells
<ol>
	<li>Image cells with a confocal microscope 12 hr after electroporation.</li>
	<li>Standard filter set for fluorescein and rhodamine can be used. If tunable filters are available set a green channel from 500 - 550 nm and a red channel from 580 nm to 650 nm. Excitation at 488nm is optimal for fluorescein while rhodamine could be imaged either with the 543nm or the 561 laser line.</li>
	<li>Focus on the specimen and adjust lasers power and detectors gain to maximize the signal-to-noise ratio. If the electroporation was successful cells should be brightly fluorescent in both channels. The localization depends on the size and charge of the dendrimer used. Often some lysosomal localization (small perinuclear vesicles) is present due to endocytosis or compartmentalization. If the lysosomal localization is predominant, i.e. most of the fluorescence is localized inside vesicles and poor signal is observed in the cytosol, this signifies toxicity and measurement should be discarded. Acquire sequentially the two channels, if needed acquire several images and average the images to improve image quality.</li>
	<li>For calibration clamp cell pH using buffers with ionophores at different pH and acquire at least 20 cells per pH as described above. For a detailed description of the procedure and the composition of the buffers please refer to Bizzarri and Coworkers.16 We suggest to measure at least 5 points from pH = 5.5 to pH = 7.5. pH below 6 are toxic to cells but tolerated for short amount of time, we suggest to acquire the images as quick as possible. If cells demonstrate signs of apoptosis, discard the cells and restart.</li>
	<li>Use ImageJ or analogous software for data analysis. Import the images of the green and red channel, subtract background and create a pixel by pixel ratio imaging with the tool &#34;Image calculator&#34;.</li>
	<li>Draw a region of interest (ROI) selecting the desired cell and measure the intracellular green-to-red ratio. Analyze all the images acquired and then plot the ratio versus the pH. In the range from 5.5 to 7.5 the trend should be linear. The linear fit of the points obtained will give the calibration curve that will be used to convert green-to-red ratio to pH.</li>
	<li>As further control acquire several untreated cells (no ionophores) and try to calculate pH with the obtained calibration curve. A value between 7.2 and 7.4 should be obtained.</li>
</ol>
5. In Vivo Sample Preparation
<ol>
	<li>Experiments were performed on C57Bl/6J (males and females) between postnatal day 28 and 70. Anesthetized the mouse with an intraperitoneal injection of Urethane (i.e. ethyl carbamate) (20% w/v in physiological saline, 20 mg/Kg urethane). Animals were sacrificed after experiment with an overdose of urethane followed by an intracardiac injection of the same anesthetic.</li>
	<li>Perform an intramuscular injection of dexamethasone sodium phosphate (2 mg/kg body weight) to reduce the cortical stress response and cerebral edema during the surgery.</li>
	<li>Shave the animal&#39;s head and apply 2.5% lidocaine gel to the scalp.</li>
	<li>Use scissors to cut the flap of skin covering the skull of both hemispheres</li>
	<li>Wash the exposed bone with saline and gently remove the periosteum using forceps. This will provide a better base for glue and dental cement to adhere with.</li>
	<li>Apply a custom-made steel head post with a central imaging chamber and glue it with cyanoacrylate in a plane approximately parallel with the skull over the cortical region of interest and fix it in place with white dental cement (Paladur).</li>
	<li>Fix the head of the mouse in order to perform a craniotomy of 2-3 mm in diameter drilled over the region of interest.</li>
	<li>Try to minimize heating of the cortex during surgery, dural tears, or bleeding.</li>
	<li>Keep the cortex superfused with sterile ACSF (126 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2.4 mM CaCl2, 15 mM glucose, 1.2 mM HEPES in distilled H2O, pH = 7.4).</li>
</ol>
6. pH imaging in Mouse Brain
<ol>
	<li>During the experiment aid animal respiration providing O2-enriched air. Oxygen is enriched up to 80%. O2 partial pressure and flow is frequently adjusted to obtain a proper respiration aid. Keep body temperature constant at 37 &#176;C with a feedback controlled heating blanket.</li>
	<li>Fix the animal through the steel post under the objective of a two-photon imaging setup.</li>
	<li>In order to inject the sensor in the brain cortex load a glass pipette containing an AgCl electrode (tip diameter 4 mm) with the dendrimer solution (1 &#956;M). The electrode will allow to record extracellular field potentials.</li>
	<li>With a microinjection setup insert the pipette in the cortex at approximately 150 &#956;m depth. Inject for 1-2 min at a pressure of 0.5 psi.</li>
	<li>Optimize the optical setup for imaging. Laser power should be adjusted to minimize photobleaching and photodamage. Typically laser power employed is around 20 mW and PMT gain was kept constant at 667 V since previous calibrations showed that this voltage gives the best S/N ratio.</li>
	<li>For imaging excite the sensor at 820 nm and detect simultaneously fluorescein and rhodamine fluorescence through standard FITC and TRITC filters.</li>
	<li>For time resolved measurements acquire time lapse series at 2 Hz.</li>
	<li>For background correction acquire a dark frame with the laser shutter closed to measure the mean thermal noise arising in the PMTs and the pedestal usually added by the electronics.</li>
	<li>For data analysis follow the same procedure reported in section 4.</li>
</ol>

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Default: Authors have nothing to disclose.

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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Acid-base and Solubility Equilibria

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11.7K vues.  Eindhoven University of Technology & NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR.  capteurs de fluorescence sont des outils puissants en sciences de la vie. Nous décrivons ici une méthode pour synthétiser et utiliser des capteurs fluorescents à base de dendrimères pour mesurer le pH dans les cellules vivantes et in vivo. L'échafaudage dendritique améliore les propriétés de colorants fluorescents conjugués conduisant à des propriétés de détection améliorées. 

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

Synthesis of Hypervalent Iodonium Alkynyl Triflates for the Application of Generating Cyanocarbenes

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent reactions with azides to form cyanocarbene intermediates. The synthesis of hypervalent iodonium alkynyl triflates can be facile, but difficulties stem from their isolation and reactivity. In particular, the necessity to use filtration under inert atmosphere at -45 &deg;C for some HIATs requires special care and equipment. Once isolated, the compounds can be stored and used in reactions with azides to form cyanocarbene intermediates. 
The evidence for cyanocarbene generation is shown by visible extrusion of dinitrogen as well as the characterization of products that occur from O-H insertion, sulfoxide complexation, and cyclopropanation. A side reaction of the cyanocarbene formation is the generation of a vinylidene-carbene and the conditions to control this process are discussed. There is also potential to form a hypervalent iodonium alkenyl triflate and the means of isolation and control of its generation are provided. The O-H insertion reaction involves using a HIAT, sodium azide or tetrabutylammonium azide, and methanol as solvent/substrate. The sulfoxide complexation reaction uses a HIAT, sodium azide or tetrabutylammonium azide, and dimethyl sulfoxide as solvent. The cyclopropanations can be performed with or without the use of solvent. The azide source must be tetrabutylammonium azide and the substrate shown is styrene.

Herein is described the synthesis, isolation, and reactions of hypervalent iodonium alkynyl triflates (HIATs) with azides to generate cyanocarbenes. The procedures will involve air-sensitive and cryogen techniques, including cold filtration under an inert atmosphere. Handling and safety of the synthesized compounds will also be discussed.

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...