Formulation and Characterization of Bioactive Agent Containing Nanodisks

This protocol provides practical knowledge, useful for formulating nanoscale particles, i.e. nanodisks, that stably incorporate a wide range of hydrophobic bioactive molecules. This method has many practical applications.

The ability to confer aqueous solubility to otherwise insoluble bioactive compounds permits the use of nanodisks in drug delivery applications. The method described is straightforward. It is preferred to conduct studies at the scale described, five milligrams phospholipid, two milligrams scaffold protein, so that the formation of nanodisks is readily monitored by visual inspection.

Demonstrating the procedure will be Michael Karo and Matthew Swackhammer, undergraduate student researchers in the Ryan lab. To prepare a phospholipid aliquot, weigh five milligrams of DMPC and transfer it to a glass test tube. Dissolve the phospholipid by adding 300 microliters of chloroform and 100 microliters of methanol for a ratio of three to one.

Evaporate the organic solvent by placing the glass test tube under a gentle stream of nitrogen gas for 10 to 15 minutes, such that a thin film of dried phospholipid forms along the walls of the bottom portion of the tube. To formulate amphotericin B or AmpB-Nanodisks, pipette 450 microliters of PBS to the lyophilized phospholipid aliquot, and vortex for 30 seconds to disperse the lipid. Pipette 50 microliters of 20 milligrams per milliliter stock AmpB solution into the dispersed phospholipid sample and vortex.

Add 500 microliters of four milligrams per milliliter ApoE4 NT scaffold protein to the glass test tube containing the dispersed phospholipid and AmpB. Bath sonicate the sample at 24 degrees Celsius until the solution clarifies. For AmpB-Nanodisk dialysis, prepare a section of dialysis tubing by thoroughly soaking it in distilled deionized water for 10 minutes.

Using a dialysis tubing clamp, clamp one end of the soaked dialysis tubing, ensuring no liquid can escape. Insert a narrow neck funnel into the open end of the dialysis tubing and transfer the AmpB-Nanodisk sample into the dialysis tubing. Remove the funnel, and clamp the end of the dialysis tubing with another dialysis tube clamp.

Place a foam dialysis float onto one of the sealed ends of the dialysis tubing, and place the assembled dialysis tubing into the beaker containing freshly prepared one liter PBS buffer. Place a magnetic stir bar into the bottom of the beaker and adjust the stirring control to low, ensuring not to produce a vortex. Allow dialysis to continue overnight at four degrees Celsius.

Turn on the spectrophotometer by flipping the power switch and connect to a corresponding support computer by pressing PC control. On the support computer, open the software labeled UVProbe 2.61, and connect to the spectrophotometer by clicking connect"in the bottom left corner. Click on the spectrum, then click method"in the top toolbar.

Click on the measurement tab and input 500 into the start"text box located under wavelength range, and 300 into the end"text box. Click the dropdown menu next to the tab labeled scan speed"and set it to medium. Prepare a sample blank by transferring one milliliter of DMSO into two quartz cuvettes.

Load both cuvettes into the respective sample ports of the spectrophotometer. Click auto blank"and record a spectrum from 300 to 500 nanometers by clicking start"in the bottom left corner. For AmpB standard spectral analysis, remove the cuvette from the front sample port and add 20 microliters of one milligram per milliliter AmpB stock solution.

Load the cuvette back into the sample board and click start"to record the sample absorbance. After recording sample absorbance, remove the cuvette and decant the liquid contents into a waste container. Thoroughly rinse the cuvette with three washes of deionized water, followed by three washes with 70%ethanol.

For spectral analysis of disrupted AmpB-Nanodisk, prepare the sample by pipetting 20 microliters of one milligram per milliliter AmpB-Nanodisk stock into one milliliter of DMSO, and incubate for one minute before recording the spectrum. Load the cuvette into the front sample port and click start"to record the sample absorbance. For spectral analysis of a PBS buffer blank, prepare a blank by transferring one milliliter of PBS into two quartz cuvettes.

Load the cuvettes into the sample port. Click auto blank"and record the spectrum from 300 to 500 nanometers. For spectral analysis of a non-disrupted AmpB-Nanodisk sample, remove the cuvette from the front sample port and add 20 microliters of a one milligram per milliliter AmpB-Nanodisk sample into the PBS buffer.

Load the cuvette back into the sample board. Click start"and record the sample spectrum. The AmpB-Nanodisk formulation reaction is considered complete when the sample appearance transitions from turbid to clear.

UV visible absorption spectroscopy of AmpB samples in DMSO and PBS is shown. In DMSO, three distinctive absorbance maxima at 372, 392, and 415 nanometers, peaks indicative of AmpB, were observed. The absorbance spectra of AmpB-Nanodisks in PBS showed a single major absorbance peak at a shorter wavelength.

In comparison, the absorbance spectra of AmpB-Nanodisks in DMSO appeared similar to the spectra of stock AmpB. Biological activity of AmpB-Nanodisks was performed by assessing yeast growth inhibition assays. Control samples like PBS, DMSO, and reconstituted high density lipoproteins demonstrated that nanodisks components other than AmpB have no discernible effect on yeast growth.

The positive control confirmed that AmpB is an efficient inhibitor of yeast growth. In the case of AmpB-Nanodisks, concentration-dependent growth inhibition activity was observed. Not all nanodisk preparations are the same.

Some may require more time, different lipids, or different scaffold proteins. They only indistinct}is the clarification of the nanodisk sample. This technique has led to novel nanoparticles used to study Doxorubicin-induced cardiotoxicity, apoptosis, ligand interactions, and delivered numerous aqueous insoluble bioactive molecules, including lutein and curcumin.