The preparation of liposomal nanocapsules by an extrusion technique will be performed step-by-step in the entrapment of tarin 48 kilodaltons globular in hydrophilic protein purified from colocasia esculenta will be demonstrated. All the procedures for characterization of the nanocapsules include the scanning electron microscopy and the dynamic light scattering will be also followed. Capsulation procedures can face some challenges from the lipids choice to the achievement of high efficiency encapsulation, keeping the bio activity of the entrapped molecules.
Protocol that will be described can overcome some of these difficulties. Weigh the liposome components using an analytical balance. Dissolve the lipid components in chloroform using a volumetric flask that fits in a rotary evaporator to avoid loss of material.
Stir the mixture at 150 RPM for 15 minutes. Remove the chloroform using a rotary evaporator. Adjust the volumetric flask mouth to the standard position for best efficiency, while in contact with the water from the heating bath.
Set the parameters according to the described protocol. After about 25 minutes, remove the flask, and discard the evaporated solvent appropriately. A thin and opaque film is formed and can be easily visualized.
Hydrate the lipid film in an ammonium sulfate solution containing tarin at one milligram per milliliter. Stir the mixture for 40 minutes and incubate overnight at four degrees Celsius. Then, sonicate the suspension for one minute at 25 degrees Celsius, room temperature.
To reduce vesicle size and avoid aggregation, place the polycarbonate membrane between two pre-wet filter supports and place it into the holder. Insert one empty syringe into the device, fill the other with one milliliter of the liposomal suspension, and insert it on the opposite side. Perform a 12 cycle extrusion through a 2 micrometer polycarbonate pore membrane.
Push the sample from one syringe to another slowly. At the end, the liposomal suspension should become clear during the excursion process, as a result of size reduction. About 2 milliliters of the sample can be lost during this step.
Separate the liposomes by ultracentrifugation. First, weigh the sample into the titanium tube and balance the tubes. Check the minimum volume to be filled, and if necessary, adjust the volume with ammonium sulfate.
Fit the titanium tubes into the swing bucket. Open the centrifuge door and place the rotor inside. Close the centrifuge door, press vacuum, and wait until the vacuum reaches from 200 to less than 20 microns.
Adjust the parameters in the ultracentrifuge display. Press recall, check the conditions, and start the run. After 90 minutes, release the vacuum by clicking the vacuum button and open the centrifuge door.
Remove the rotor from inside. Maintain the ultracentrifuge sample on ice and then separate the supernatant from the pellet, by turning the tube upside down, into a disposable 59 mil centrifuge to separate the supernatant from the pellet. Store the supernatant containing the unencapsulated protein at four degrees Celsius.
This will be used to determine the encapsulation efficiency. The pellet is presented as a translucent jelly. Suspend the pellet and heat HEPES buffered saline solution.
Please check the protocol for more information. Determine the encapsulation efficiency by Peterson's protocol, in order to avoid lipid interference in protein quantification. Samples should be analyzed in triplicate.
In a microfuge tube, dilute the lipsomal supernatant or BSA at one milligram per milliliter with water to a final volume of one milliliter. Add DOC and incubate for 10 minutes. Add 1 milliliter of 72%TCA and mix well.
Centrifuge at 3, 000 g for 15 minutes at room temperature. Carefully discard the supernatant by averting the tube downwards, laying it on an absorbent paper and letting it dry. Suspend the pellet in in one milliliter of water.
Mix thoroughly to make sure the pellet is dissolved. Then, transfer the sample to test tubes. Add one milliliter of reagent A, mix and incubate for 10 minutes at room temperature.
Add 5 milliliters of reagent B, mix and incubate for 30 minutes at room temperature, protected from light. Determine the absorbance at 750 nanometers and calculate the encapsulation efficiency. The size average and size distribution is determined by dynamic light scattering.
Transfer the liposomal nanovesicles to a disposable sizing cuvette. Fit the cuvette inside the equipment. Set the equipment parameters.
For stability determination, store the liposomes at four degrees Celsius and check size distribution and size average regularly. Liposome characterization by scanning electron microscope is performed according to Murta and Ramasamy in triplicate. Fix the glass cover slips in the bottom of a Petri dish with a tape.
Coat the cover slips with Poly-L-lysine. Place a wet filter paper inside the Petri dish to maintain moisture and incubate for one hour. Rinse with distilled water.
Add a drop of the sample and allow it to dry for one hour, at room temperature. To fix the samples, cover them with glutaraldehyde, prepared in phosphate buffer. Place wet filter papers inside the Petri dish.
It is important to seal the Petri dish. Incubate at four degrees Celsius for 48 hours. Rinse the cover slips three times for five minutes each, with the same phosphate buffer.
Dehydrate the samples as follows by washing in an increasing ethanol concentration solution from 35%to absolute ethanol. Chemically dry the samples with hexamethyldisilazane for 10 minutes. The hexamethyldisilazane should be carefully manipulated inside a fume hood.
Samples should be allowed to dry overnight inside a desiccator or inside the fume hood at room temperature. Mount the dried samples on a stub, with a carbon conductive adhesive tape. Sputter the stubs with gold.
Record SEM images with a scanning electron microscope, SEM, at low vacuum mode and low voltage, 20 kilovolts. Figure one describes the nano liposome preparation. Phospholipids, dope, peg, and chems, the main liposome constituents, were first dissolved in chloroform to obtain the lipid film.
The lipid film was then rehydrated in a saline buffer, containing the hydrophilic protein, tarin, to be entrapt, and the incubation should be preformed overnight. Then, sonication and extrusion are applied to generate small unilamellar vesicles. The ultracentrifugation step separates liposomal preparation from free lipids, and unencapsulated protein.
While the supernatant is used for the determination of entrapment efficiency. Nanoliposomes produced using the aforementioned methodology exhibit a size distribution ranging from 51 to 396 nanometers, and an average size of 155 nanometers. The preparation is homogeneous, since the polidispersity index is of 168.
A high entrapment efficiency of 83%is reached if the liposomes are extruded through a 2 micrometer pore size membrane. Morphological nanoliposome characteristics were evaluated by scanning electron microscopy. Liposomes should be finally treated similarly to living cells, to obtain better quality images.
Fixation and drying procedures are important to ensure the visualization of smaller intact vesicles that support over 20 kilovolts under vacuum conditions. Figures 2A and 2B display round shaped liposomal vesicles in the range of 121 nanometers, analyzed at 20 kilovolts. Whereas figures 2C and 2D, display inadequately prepared samples.
Mistreated samples allowed only for the observation of larger and or damaged vesicles, which cannot resist vacuum and or voltage conditions. Protocol described, here in, allowed for the production of reproducible vesicles displaying a round shape, small surface, and average size of approximately 150 nanometers. The method can be used to entrap complex and large molecules, as a tetrameric protein, exhibiting a hydrophilic corrector.
Deficient self entrapment is higher than 80%and the leakage rate is very low, inferior to 1.5%When the nanocapsules are stored at four Celsius degrees. We hope that this video would help you in the encapsulation of big molecules, such as teri.
