Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The overall goal of this procedure is to prepare an activat liposome that enables in vivo imaging of inflammation with a high sensitivity. This is accomplished by first preparing a lipid film composed of phospholipids in the second step. Spontaneous vesicles are formed by hydrating the phospholipid film with a quenched concentration solution of a near infrared fluorescent dye.

The spontaneously formed vesicles or SFV are extruded to ensure homogeneity of the liposomal vesicles and then the liposomes are purified from the residual non encapsulated dye molecules. Ultimately, spectrometric measurements of absorbance and fluorescence emission are used to show the quench state of the encapsulated dye while in vivo. Near infrared fluorescence imaging is used to demonstrate the potential of the liposomes for imaging inflammatory processes.

The main advantage of this technology over other existing ones is that we can determine the signal directly after internalization of the contrast agent into the cells. That means that they're activated in the cell so that the signal what is detected comes exclusively from from the cells. The implication of this technique extend toward diagnosis and therapy of cancer.

Since phagocytes, such as tumor associated macrophages can take up and degrade the liposomes releasing therapeutic drugs or imaging probes within the tumors. Generally individuals new to this method will struggle because the encapsulation efficiency is influenced by the properties of the DAI use and the effectiveness of the hydration process To initiate spontaneously formed vesical dispersion begin by adding approximately three milliliters of chloroform to a round bottom flask, followed by the appropriate volume of freshly prepared phospholipid stalk solutions at a molar ratio of 6.5 to three to 0.5 for double fluorescent labeling of the liposomes. Next, add 0.3 molar percent N-B-D-D-O-P-E to the lipid solution.

Then use a rotary evaporator to evaporate the chloroform from the organic phospholipid solution under reduced pressure at 300 millibar and 55 degrees Celsius. After a homogenous phospholipid film is formed, reduce the pressure to 10 millibar for one to two hours to remove any residual chloroform deposit while the chloroform is evaporating. Dissolve fluorescent dye carboxylic acid interest buffer and fill a doer vessel with liquid nitrogen.

Then switch on an ultrasonic bath set at 50 degrees Celsius. Now transfer an appropriate volume of the dye carboxylic acid to the round bottom flask to hydrate the dry phospholipid film and vortex to flask vigorously until a spontaneously formed vesicle dispersion forms, making sure that all of the phospholipids are dispersed to avoid lipid loss. Then carefully freeze the round bottom flask containing the SFV dispersion in liquid nitrogen.

And after three to five minutes, place the flask into the ultrasonic bath to thaw the dispersion and vortex the flask vigorously for another one to two minutes. Next, to facilitate the formation of homogenous vesicles, aspirate the SFV dispersion into a one milliliter syringe, and then use a lipo sofas basic extruder to pass it through a 100 nanometer polycarbonate membrane into a second one milliliter syringe. Extrude the dispersion back into the first syringe, repeating the transfer 10 times.

The solution should turn from a hazy appearance to a clear dispersion. After the 10th extrusion cycle, remove the second syringe and extrude the dispersion for the last time directly into a sterile 1.5 milliliter reaction tube. Now transfer 0.5 milliliters of the extruded vesicle dispersion onto the gel bed of a 28 centimeter gel chromatography column loaded with G 25 beads soaked in tris buffer and let the sample drain into the gel matrix.

Elute the liposomes with tris buffer and collect the liposome fraction until the liposome drains completely out. Then concentrate the eluded liposomes by ultracentrifugation and disperse them in an adequate volume of sterile tris buffer electron micrographs of the liposomes should reveal a mostly unal morphology of the liposomal vesicles to analyze the fluorescence quenching and activation abilities of the liposomes. Next, transfer 100 nanomolar total of the appropriate lipids into two 1.5 milliliter micro centrifuge tubes.

Then transfer a free di carboxylic acid equivalent to the dye content of lip Q in another 1.5 milliliter tube and incubate one tube of each probe at four degrees Celsius and the second at negative 80 degrees Celsius. The next morning, set to heating block to 30 degrees Celsius. Fill a cooling box with crushed ice and equilibrate an aliquot of tris buffer to room temperature.

Next, quickly thaw the probes from the negative 80 degrees Celsius storage at 30 degrees Celsius for five minutes while equilibrate the probes from four degrees Celsius to room temperature for five minutes. Then chill all the probes on ice for one minute before transferring them back to room temperature. Now add tris buffer to each of the probes to a final volume of 100 microliters.

And then after another 10 minute incubation at room temperature, pipette 80 microliters of each probe into a low volume glass vet and measure the absorption from 400 to 900 nanometers on a spectrometer. Return the probes to their corresponding tubes and then transfer 80 microliters of each into suitable glass cuvettes. To measure the fluorescence emissions on a spectral fluorimeter by exciting the probes at 674 nanometers and measuring the fluorescence from 694 to 800 nanometers.

To evaluate the inflammation detection properties of the liposomes, fill one syringe with 50 microliters of xin a solution, one with 50 microliters of isotonic saline solution and one with the experimental probe solution, all diluted an HBSS to a final 150 microliter volume. After confirming sedation by toe pinch, apply eye cream to the experimental animal and then place the mouse on a warm mat. Inject the xon a solution subcutaneously on the animal's right hind leg and the saline solution on the left hind leg.

Then immediately inject the probe intravenously and image the animal thereafter. Finally, save the resulting images as image cubes. Imaging the animals every two hours for 10 hours post-injection, and then again at 24 hours.

Liposomal probes reveal a predominant phagocytic uptake that is inhibited by energy depletion as can be seen via the uptake of lip Q by highly phagocytic mirroring microphage cell lines and mo phagocytic human glioblastoma cell lines. Consistent with the phagocytic uptake observed in cultured cell lines, the intravenous injection of LIP Q leads to a time-dependent increase in the fluorescence intensity of edema in mice models with very low background fluorescence and with a maximum fluorescence intensity detected at eight to 10 hours post-injection. In contrast, a relatively strong near infrared fluorescence of the whole animal is seen after application of the free carboxylic acid or the always on liposome lip DQ compared to the lip Q.Rapid perfusion and clearance of the free carboxylic acid as seen from zero to four hours post-injection, interferes with the imaging making reliable detection of the edema not possible.

Furthermore, the non quenched liposome lip DQ reveals a maximum fluorescence of edema within two to four hours post-injection that remains almost constant till eight hours and then gradually decreases in similar manner to the quenched lip. Q based edema fluorescence imaging of the organs of mice euthanized 24 hours after probe administration reveals a mild ex vivo fluorescence of the liver, gallbladder, and kidneys, and a very low to no fluorescence of the spleen, lungs, and heart serving as evidence for the elimination of the probes through the patho biliary route. While attending this procedure, it is important to remember to use a high concentration of the flu sense quench dye and to disperse the phospholipids properly.

After watching this video, you should have a good understanding of how to prepare activat liposomes for in vivo imaging of inflammation with a high sensitivity.Okay? Don't forget that for utilization of animals in research, you have to follow the international guidelines, and this means that you have to apply for the utilization of animals in in experiments. You need the approval from the local ethical committee.