Name: an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation
Uploaded: 2017-08-30T14:00:00
Description: Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

We thank the Nano Science and Technology Program of Academia Sinica and the Ministry of Science and Technology of Taiwan for the funding (101-2113-M-001-001-MY2; 103-2113-M-001-028-MY2).

NOTE: The protocol describes a detailed instrumentation modification for upconversion-assisted photoactivation, a synthetic procedure to generate caged PKA-UCNP, transmission electron microscopy (TEM) of the silica-coated UCNP and caged PKA-UCNP samples, UV and NIR photolysis setup, cell preparation, PKA-UCNP microinjection, a photoactivation study, and the stress fiber staining of REF52 cells.
1. Fluorimeter Setup for Upconversion Spectrum Measurement
<ol>
	<li>Install a 4X objective lens n...

<ol>
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Previously, Hofmann and coworkers found that dramatic morphological changes were observed in REF52 cells after the microinjection of the free PKA19. In another study, the Lawrence group demonstrated that caged PKA can be activated in vivo, leading to morphological changes and the disintegration of stress fibers when subjected to UV photolysis20. Earlier reports on exploiting upconverted UV light for photoactivation showed the activation of the several UCNP-assisted...

Chemically modified proteins that can be photoactivated (e.g., PKA caged proteins) have been developed as an emerging field in chemical biology to non-invasively manipulate intercellular biochemical processes1,2,3. Using light as a stimulus provides excellent spatiotemporal resolution when activating these caged proteins. However, UV light can cause undesired morphological changes, apoptosis, and DNA damage to cells4,5. Hence, recent developments in the design of photocaging groups focus on enabling photocleavage upon longer-wavelength or two-photon excitation to reduce phototoxicity, as well as to increase deep-tissue penetration6,7. Caging groups that respond to longer wavelength allowing us to choose suitable uncaging wavelengths (i.e., channels) to selectively activate bioeffectors when two or more caging groups are present7. Given these useful features, developing new red-light photocaging groups is very important upstream work in photochemical methodologies for biological studies ranging from probing the mechanisms of reactions to controlling cellular activities8. Nonetheless, a two-photon caging group is normally too hydrophobic due to the fused aromatic ring structure, and a visible-light caging group is normally organometallic, with aromatic ligands. This hydrophobic/aromatic property is not suitable when the bioeffector is a protein or enzyme, as it denatures the activation site of the enzyme/protein and causes loss of function, even if the conjugation and photolysis still work on the chemical level2,9.
UCNPs are effective transducers that convert the NIR excitation light to UV.This unique and fascinating property of UCNPs has offered realistic resolutions to address the challenges associated with photoactivation and triggered controlled release of small molecules, including folic acid10, cisplatin derivatives11, DNA/siRNA12, copolymer vesicles13, and hollow particles14. However, to the best of our knowledge, the UCNP-assisted photoactivation of enzymes or proteins has not been tested so far. Because there is no successful case of using red light or NIR to photoactive an enzyme, we were prompted to perform the NIR-triggered activation of a protein/enzyme construct composed of chemically modified caged enzyme complexes with a silica-coated, lanthanide-doped UCNP15. In this study, the UCNP was conjugated with a rapidly reacting signal transduction kinase in the form of caged PKA. PKA controls glycogen synthesis and cytoskeletal regulation that responds to external stimuli via cyclic adenosine phosphate (cAMP) regulation in the cytosol16. We studied the feasibility of enzyme activation in temporal and spatial manners in a cellular experiment after NIR irradiation. This UCNP-assisted photoactivation platform is a new methodology to photoactivate an enzyme using NIR and avoids the undesired signal transduction response from cells caused by conventional UV irradiation2,4.
It is very difficult to translocate large bioeffectors (e.g., proteins) across the cell membrane to control cellular activity. Although particle-immobilized protein may be easier to translocate via endocytosis into the cytosol, endocytosis may be damaged or degraded via endosomal entrapment and the consequent lysosomal degradation2,4. Even if the caged protein is still functional after membrane translocation, the translocated amounts may not be enough to trigger the cellular response2,17. In sharp contrast, microinjection is a direct and quantitative approach to deliver large bioeffectors to the cytoplasm of the cell. Moreover, UCNP-immobilized bioeffector requires upconverted light to be activated. Therefore, the optical instrumentation requires further modification to measure, visualize, and utilize the upconversion light. In this work, the delivery of a caged PKA-UCNP complex to a cell using microinjection and the following essential spectroscopy and microscopy modifications for NIR photoactivation will be described in detail.

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			<td height="20" style="height:20px;width:431px;">Reagent</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris(hydorxymethyl)aminomethane</td>
			<td>Sigma</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>31434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>12636</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yttrium acetate&#160;hydrate</td>
			<td>Sigma</td>
			<td>326046</td>
			<td>Y(C2H3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thulium(III) acetate hydrate</td>
			<td>Alfa Aesar</td>
			<td>14582</td>
			<td>Tm(CH3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ytterbium(III) acetate tetrahydrate</td>
			<td>Sigma</td>
			<td>326011</td>
			<td>Yb(C2H3O2)3 &#183; 4H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Octadecene</td>
			<td>Sigma</td>
			<td>O806</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid</td>
			<td>Sigma</td>
			<td>364525</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol&#160;</td>
			<td>macron</td>
			<td>304168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Sigma</td>
			<td>30620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium fluoride</td>
			<td>J.T.Baker</td>
			<td>69804</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGEPAL&#160;CO-520</td>
			<td>Sigma</td>
			<td>238643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyclohexane</td>
			<td>J.T.Baker</td>
			<td>920601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amomonium hydroxide (28%-30%)</td>
			<td>J.T.Baker</td>
			<td>972101</td>
			<td>Ammonia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetraethyl orthosilicate (TEOS)</td>
			<td>Sigma</td>
			<td>8658</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-hydroxymaleimide (NHM)</td>
			<td>Sigma</td>
			<td>226351</td>
			<td>PKA activity blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prionex protein stabilizer solution from hog collagen</td>
			<td>Sigma</td>
			<td>81662</td>
			<td>Protein stabilizer solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-nitrobenzyl bromide (NBB)</td>
			<td>Sigma</td>
			<td>107794</td>
			<td>PKA caging reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-(4-Chlorophenylthio)adenosine 3&#8242;,5&#8242;-cyclic monophosphate sodium salt</td>
			<td>Sigma</td>
			<td>C3912</td>
			<td>8-CPT-cAMP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td>
			<td>Sigma</td>
			<td>P0294</td>
			<td>PK/LDH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine 5&#39;-triphosphate disodium</td>
			<td>Sigma</td>
			<td>A2387</td>
			<td>ATP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-NADH reduced from dipotassium</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphoenolpyruvate</td>
			<td>Sigma</td>
			<td>P7127</td>
			<td>PEP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Protein Assay Reagent, 950 ml</td>
			<td>Thermo Scientific</td>
			<td>23200</td>
			<td>Bradford assay reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-dependent protein kinase</td>
			<td>Promega</td>
			<td>V5161</td>
			<td>PKA activity control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET15b-PKACAT plasmid</td>
			<td>Addgene</td>
			<td>#14921</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pKaede-MC1 plasmid</td>
			<td>CoralHue</td>
			<td>AM-V0012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leibovitz L-15 Medium</td>
			<td>Biological Industries</td>
			<td>01-115-1A</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>ACROS</td>
			<td>416785000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Nucleus staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 594-phalloidin</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>F-actin staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5(6)-Carboxyfluorescein</td>
			<td>Novabiochem</td>
			<td>8.51082.0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5(6)-Carboxytetramethylrhodamine&#160;</td>
			<td>Novabiochem</td>
			<td>8.51030.9999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Coomassie (Bradford) Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CelluSep T4 Tubings/Nominal filter rating MWCO 12000-14000 Da</td>
			<td>Membrane Filtration Products, Inc.</td>
			<td>1430-33</td>
			<td>Dialysis membrane</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HV Syringe Filter Unit, 0.45 &#181;m, PVDF, 13 mm, gamma sterilized</td>
			<td>EMD Milipore</td>
			<td>SLHVX13NL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynamic Light Scattering/Zetapotential Zetasizer nano-ZS</td>
			<td>Malvern</td>
			<td>M104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transmission Electron Microscope</td>
			<td>JEOL</td>
			<td>JEM-1400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Spectrophotometer</td>
			<td>Agilent Technologies</td>
			<td>10075200</td>
			<td>Cary Eclipse&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis Spectrophotometer</td>
			<td>Agilent Technologies</td>
			<td>10068900</td>
			<td>Cary 50&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Microscopy</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">950 nm longpass filter&#160;</td>
			<td>Thorlabs</td>
			<td>FEL0950</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">850 nm dichroic mirror shortpass</td>
			<td>Chroma</td>
			<td>NC265609</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RT3 color CCD system</td>
			<td>SPOT</td>
			<td>RT2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Illumination</td>
			<td>PRIOR</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">980nm Infra-red diode laser</td>
			<td>CNI</td>
			<td>MDL-N-980-8W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV LED Spot Light Source</td>
			<td>UVATA</td>
			<td>UVATA-UPS412</td>
			<td>With a UPH-056-365 nm LED at 200 mW/cm2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal pile sensor</td>
			<td>OPHIR</td>
			<td>12A-V1-ROHS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC-10 Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MANOMETER Digital pressure gauge</td>
			<td>Lutron</td>
			<td>PM-9100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">One-axis Oil Hydraulic Micromanipulator</td>
			<td>Narishige</td>
			<td>MMO-220A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heraeus Fresco 17 Centrifuge, Refrigerated</td>
			<td>Thermo Scientific</td>
			<td>75002421</td>
			<td></td>
		</tr>
	</tbody>
</table>

an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

The design of the caged enzyme-UCNP construct is illustrated in Figure 1. The PKA enzyme was first reacted with 2-nitrobenzyl bromide to generate an inactive caged PKA, and it was then electrostatically immobilized on the surface of UCNP. UCNPs emit the upconverted light and consequently photolytically cleave the o-nitrobenzyl groups on Cys 199 and Cys 343, generating the activated PKA. TEM images and Bradford assay confirmed that the PKA and caged PKA were i...

Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

The goal of this experiment is to demonstrate the feasibility to photoactivate enzyme using near infrared irradiation. Caged-protein kinase A is immobilized on this surface of upconversion nanoparticles micro-injected into cells and kinase induced response in living cells is observed after near infrared irradiation. Traditional photoactivation require phototoxic UV light and inevitably, will co-induce undesirable chemical reaction and similar response, however, in our friendly options, like near infrared irradiation cannot trigger reaction due to its low energy.The upconversion nanoparticle can effectively upconvert bio-friendly low energy near IR photon into high energy UV photon to trigger a chemical reaction near the particle surface without direct UV irradiation. Kinase plays a key role in oncogenesis, this technique can help us examine more signal transduction information with much lower interfering noise from UV phototoxicity and its side reaction. Generally, individuals new to this field will encounter difficulties in modifying instruments and optical setup for upconversion application.To begin, install a 4x objective lens next to the cuvette holder of the fluorospectrometer, so that the laser can be focused in the middle of the cuvette. Place a tunable 980 nanometer laser diode 90 degrees against the objective lens. Install a full reflectance mirror and the light path intersection of the objective lens and laser.Don't forget to place an anodized metal plate to block the excitation window of the spectrometer to protect the interior parts. Then place a cuvette of silica coated upconversion nanoparticle solution in the holder. A bright upconversion beam can be visualized when the laser turns on.Switch the spectroscopy to luminescence mode, with the lowest PMT voltage during the alignments. Adjust the mirror and laser stages so that the beam is through the middle of the cuvette for the PMT to pick up the strongest signal. After installing a dichroic mirror and an excitation filter, install a 980 nanometer laser diode in the lower tier light path.Add a collimator in between the laser and the mirror to help diffuse and enlarge the NIR irradiating area in the focal plane. Then use an upconversion nanoparticle solution to visualize the near infrared beam. Adjust the position of laser stage to center the near infrared light beam to finish the alignment.Perform caged-PKA synthesis and demobilize the caged-PKA/UCNP, to generate caged-PKA/UCNP as described in the text protocol. To measure the activity of both constructs before UV photolysis, measure the decrease in NADH absorbents in the assay mixture over time after the addition of two microliters of PKA solution to the assay mixture. Repeat the measurement for PKA/UCNP.Next, perform UV photolysis at 200 milliwatts per square centimeter of caged-PKA and caged-PKA/UCNP, in an ice water bath, to make sure PKA can be activated. PKA activity is determined by a pyruvate kinase lactate dehydrogenase enzyme coupled assay. With a decrease in NADH absorbents, is measured over time after the addition of PKA solution.Incubate the cells in 10%fetal bovine serum containing L15 medium during the microinjection period for stable pH control outside the incubator. Configure the needle puller for a cone shaped tip with a diameter of around 1.5 microns for later tip opening. For opening the tip, install the needle on the microinjector move the needle tip in contact with the edge of a slide and follow with a mild knob on the microscope stage.This vibration force is sufficient to slightly break the tip and enlarge the opening. Examine the needle opening to ensure that it has an external diameter from 1.3 to 1.5 microns which can be confirmed by microscope images using a 40x objective lens. Next, set the injection pressure from 50 to 75 hectopascals with a pressurized interval of 200 to 300 milliseconds and set the constant compensation pressure at 15 to 30 hectopascals to prevent medium backflow into the needle.Load the caged-PKA/UCNP complex with TAMRA dye at the back position of the needle. The loaded needle is then set up on one axis hydraulic micromanipulator, with an angle from 30 degrees to 45 degrees. Check the needle with microscope to make sure no bubble generated after sample loading.After immersing the needle into the medium, switch to fluorescence mode, to confirm a burst release of dye when injection pressure is applied and a slow release of dye when compensation pressure was constantly applied. Then switch to phase contrasted bright field mode, to start cell microinjection. The membrane shockwave can be observed in successful injections.Wash the cells twice and check the injected cells for co-injected TAMRA fluorescence to locate successfully microinjected cells. Cell with UCNP admission but without a co-injected dye signal, indicate the cell membrane was ruptured during injection and should be excluded from further experiments. Irradiate the cells with a 980 nanometer laser diode in the lower tier light path focused by a 10x objective lens for 15 minutes.Allow for a five minute dark interval after every five minutes of irradiation to avoid heating. If a shorter activation time is needed, such as seconds or even subseconds, simply switch to a 40x objective lens for higher focus photon density. Following photolysis and cell recovery, fix the cells in one milliliter or 4%paraformaldehyde in PBS for 20 minutes, before further alexa 594 followed within staining.Finally, visualize the NIR induced stress fiber disintegration. After NIR irradiation, cells microinjected with caged-PKA/UCNP as indicated by white arrows, show upconversion emission. Stress fiber disintegration caused by a successful PKA NIR photoactivation, exposes more f actin and consequently results in strong green staining near UCNP.A negative control where the cells are microinjected but remain in the dark, show no disintegration of stress fibers. The level of f actin exposure after NIR photoactivation can be stained with alexa 594 followed within and quantified by image J.Intensity curves of f actin staining along the red arrow were plotted, providing quantitative information of stress fiber disintegration which can be correlated to NIR induced PKA activity. Once familiar with microinjection, this technique can be done within one day, including preparation and the quality control of caged-PKA/UCNP.With our instructions for our instrument modifications and the experiments, the desired reaction can be triggered by near IR, in highly spatial temporal resolution like, traditional UV for titration methods, but without the phototoxicity, it's not contagious. After watching this video, hope you have a good understanding of how to deliver enzyme nanoparticle complex into a cell by microinjection and photoactivate the enzyme, using bell compatible near IR light. This technique paved the way for researchers in the field of chemical biology, who need to use light to trigger desired enzymatic reaction in living cells for spatial temporal resolution when the cell cannot tolerate UV phototoxicity.Following this procedure, other bio affectors can be associated to upconversion nanoparticle, to answer additional questions that need spatial temporal photoactivation. If the bio affector works extracellularly, microinjection is not even needed. The near IR photoactivation platform can also provide more penetration depth in tissue and can therefore be applied in small animals to trigger desired reactions at the given time and the location.

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Research

JoVE Journal

Bioengineering

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...