The overall goal of this protocol is to demonstrate the synthesis of a light responsive Liposomes and reveal the mechanism for the light controlled drug release from these Liposomes. This method helps answer the key questions in a figurative light response of Liposomes for drug delivery, where the key challenges are to synthesize the Liposomes and enhance the magnesium of controlled release. The main advantage of this technique is the cell synthesis of light responsive Liposomes with the commercially available materials.
The implication of this technology extends towards the superficial tissue therapy. The use of minimal evasive procedures as well as a part of the laser device, will make the tech platform more accessible to the patients. Besides providing insights into the top care and accurate drug delivery, our system can also be applied to other systems such as small ani-ma-mo-rose as well as mucus system through guided lasers.
First clean a 100 milliliter round bottom flask using Aqua regia. After washing with deionized water and autoclaving, dry the flask in a hot air oven at 100 degrees Celsius for 15 minutes. Then wrap and store the sterile flask until use.
Sterilize a hand-held mini-extruder set using 70%Ethanol. Next turn on the rotary evaporator and set the temperature of the hot water bath and the cooling tower to 37 and 4 degree Celsius, respectively. Prepare a 60 millimolar Calcium stock solution by dissolving 374 milligrams of Calcein and 10 milliliters of 0.1 millimolar Phosphate buffered saline or PBS.
Adjust the pH to 7.4 using one mollar Sodium Hydroxide solution. Remove the lipids from the freezer and thaw them to room temperature. Once the lipids have been thawed dissolve 15.9 milligrams of DPPC, 1.3 milligrams of MPPC and 2.8 milligrams of DSPE-PEG2000 and two milliliters of Chloroform.
After transferring the Chloroform solution to a sterile round bottom flask, evaporate the solvent using the rotary evaporator under reduced pressure to form a thin dry lipid layer. Following this, hydrate the lipid layer at 45 degree Celsius with two milliliters of Aqueous solution containing 1.95 milliliters of the previously prepared 60 millimolar Calcium solution and 50 microliters of Gold nanoparticles for 30 minutes. Pre-heat a heating block to 45 degree Celsius.
Place a 200 nanometer polycarbonate membrane filter between the filters supports and assemble the mini extruder set. Then check for potential leakage with deionized water. Fill one of the syringes with one milliliter of the previously prepared Liposome solution and extrude the sample by passing the solution through the syringe at the other end of the assembled mini extruder.
After repeating the previous step 11 times run the synthesized Liposomes through a PD-10 desalting Column to remove the free gold nano particles, lipids and Calcein, using PBS as the eluent according to the manufacturers protocol. When finished store the samples in sterile tubes at four degree Celsius. At this point, calculate the lipid molarity of the stock solution by adding up the molarities of DPPC, MPPC and DSPE-PEG2000.
Dilute the Liposome stock solution to a 5 millimolar lipid concentration using 0.1 millimolar PBS, then transfer the samples to 2 milliliters centrifuge tubes. Next, place the tubes in a hot water bath and raise the temperature gradually from 25 to 70 degrees Celsius. Collect 10 microliter aliquots at different temperature points.
At 10 microliters of two percent triton x-100 to a two milliliter aliquot of Liposomal solution to digest the Liposomes for 10 minutes at room temperature and to achieve complete release of Calcein. Following this, transfer 200 microliters of the Liposomal solution to each well in a 96 well microplate. Measure the fluorescence intensity of the collected samples using a fluorescence micro plate reader.
Taking the fluorescence intensity of the triton x-100 treated samples as 100 percent release, calculate the percentage of the released Calcein at each time point using the following formula:Transfer 100 microliters of the Liposome solution to a Quartz cuvette and place the cuvette in a cuvette holder. Guide the collimated laser beam through the cuvette such that the light passes through the Liposme solution. Then collect aliquots after various pulses.
Following this add 10 microliters of 2%triton x-100, to a 2 milliliter aliquot of Liposomal solution to digest the Liposomes for 10 minutes at room temperature and to achieve complete release of Calcein. To pre-measure the bleaching effect of the laser on the Calcein solution expose the solution to a post laser with a frequency of 1 Hertz for varying pulse numbers. Measure the fluorescence intensity of Calcein with excitation and emission wavelengths of 480 and 515 Nanometers respectively before and after laser exposure.
After calculating the amounts of bleaching use this factor to normalize the values obtained from Liposomes samples by multiplying the fluorescence intensity of the Liposomes with the factor. Next measure the fluorescence intensity of the collected sample using a fluorescence micro plate reader at excitation and emission wavelengths of 480 and 515 nanometers respectively. Taking the fluorescence intensity of the triton x-100 treated samples as 100 percent release, calculate the percentage of the released Calcein at each pulse number.
Place 100 microliters of the sample on a microscope slide then set the focus of the laser on to the sample. The laser is guided through the sample manually hence it is important to make sure that the focal point of the beam is well within the sample and not on the microscopic slide to avoid experiments later. Following this immerse a needle hydrophone into the solution.
The tip of the hydrophone is emerged into the sample at a sufficient distance away from the focal point of the laser. This is done to prevent any laser induced damages with the hydrophone. Irradiate the sample with the pulse laser with varying pulse numbers and pulse energy.
Finally record the pressure signals using a digital oscilloscope. No significant difference in the release percentage is observed for the Liposomes with and without gold nano-particles. The Liposomes were almost intact at physiological temperature with a leakage percentage of less than 10 percent with or without gold nano-particles.
When the temperature was increased to 42 degrees Celsius, 60 to 80 percent of the encapsulated Calcein was released from the Liposomes within two minutes. Liposomes with gold nano-particles released the encapsulated Calcein upon excitation in which the amount of released Calcein increased as the pulse numbers increased. The acoustic signals recorded for the gold nano-particles, the Liposomes with gold nano-particles and the Liposomes without gold nano-particles are shown here:The pressure impulse, contains both positive and negative phases and suggests that microbubbles form and disrupt in the solution.
As expected the acoustic signal amplitude gradually increased with the increasing laser energy and should be due to the increased intensity of the oscillation of the particles attributing to the subsequent thermoelastic expansion and contraction. Once mastered the fabrication of Liposomes can be done within 3 hours provided all glassware and equipment is prepared in advanced. While attempting the laser controlled release it is important to remember that the laser beam is focused completely unto the sample to avoid loss of laser energy.
Following this procedure other experiments like in vitro and in viol laser controlled release can be done to answer additional questions like how the physiological conditions will impact the laser controlled release. After this development, this technique will be resourceful in the field of reactivity to explore, localize and control the release to skin, eyes and mucus membrane.
