Name: Formulazione e caratterizzazione di agenti bioattivi contenenti nanodischi (Video) | JoVE Journal
Uploaded: 2023-03-17T14:15:00.000+00:00
Duration: 7 min 58 s
Description: Scopri come formulare nanodischi per incorporare molecole bioattive idrofobiche e migliorare la solubilità acquosa di composti insolubili. Questo metodo semplice consente applicazioni avanzate nel drug delivery.

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This work was supported by a grant from the National Institutes of Health (R37 HL-64159).

1. Transformation, expression, and purification of scaffold protein component
<ol>
	<li>BL21 bacterial transformation with apoE4-NT containing plasmid
	<ol>
		<li>Thaw a tube of BL21 (DE3) competent cells on ice for 10 min.</li>
		<li>Once all the ice has melted, mix gently and carefully pipette 50 &#181;L of the cells into a transformation tube on ice.</li>
		<li>Add 5 &#181;L containing 50 ng of plasmid DNA (for sequence, see Supplemental Table 1) to the cell mixture. Carefully flick the tube four or five times to mix. Do not vortex.</li>
		<li>Place the mixture on ice for 30 min.</li>
		<li>Heat shock the mixture at exactly 42 &#176;C for 10 s.</li>
		<li>Place on ice for 5 min.</li>
		<li>Pipette 950 &#181;L of S.O.C. medium into the tube at room temperature.</li>
		<li>Place the tube in a 37 &#176;C shaking incubator for 60 min with oscillation set to 250 rpm.</li>
		<li>Mix the cells thoroughly by flicking and inverting, then spread 100 &#181;L of cell solution onto a 20 mL Luria Broth + Agar selection plate treated with ampicillin at a concentration of 0.1 mg/mL.</li>
		<li>Incubate overnight at 37 &#176;C, or until visible colonies have grown.</li>
	</ol>
	</li>
	<li>Using an electronic pipettor, transfer 25 mL of sterile, NZCYM medium into a sterile 250 mL conical flask at room temperature and add ampicillin to a final working concentration of 0.1 mg/mL.</li>
	<li>Using a sterile inoculation loop, transfer one individual colony from the selection plate containing the appropriately transformed bacterial strain and add directly to the flask containing 25 mL of NZCYM media + ampicillin.</li>
	<li>Place the 25 mL of NZCYM seed culture flask into a 37 &#176;C shaking incubator with orbital oscillation set to 250 rpm. 
	NOTE: It is recommended that this seed culture be grown overnight to reach a suitable bacterial concentration (~1.5-2.0 optical density units), as measured using a spectrophotometer set to wavelength = 600 nm.</li>
	<li>Transfer 250 mL of sterile NZCYM medium into four 1 L baffled flasks and add ampicillin antibiotic to a working concentration of 0.1 mg/mL.</li>
	<li>Under sterile conditions, transfer 5 mL of saturated seed culture into each of the four 250 mL culture containing flasks and place in a 37 &#730;C shaking incubator with orbital oscillation set to 250 rpm. 
	NOTE: At this point, the culture will be referred to as an expansion culture and exponential growth will be monitored using a UV1800 spectrophotometer. Growth is allowed to proceed until the culture optical density at 600 nm (OD600) reaches a value between 0.6-0.8.</li>
	<li>Once the expansion culture has reached the desired OD600 value of 0.6-0.8, add 59.6 mg of isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to each 250 ml culture containing flask for a final concentration of 1 mM to induce protein expression. At this point, the expansion culture is referred to as the expression culture. Commence expression for a period of 5 h.</li>
	<li>At the end of the 5 h expression period, remove the four flasks from the shaking incubator and pipette 125 mL into six 250 ml centrifuge bottles (750 mL total).</li>
	<li>Balance each centrifuge bottle to ensure the total mass is within &#177;0.5 mg of one another. 
	NOTE: This step is essential to ensure safe operation of the centrifuge device.</li>
	<li>Prepare the centrifuge device by first flipping the power switch to the on position. Click the button labeled &#34;Set/Actual&#34; and adjust the parameters to &#34;JA-14&#34;, &#34;9,400 x g&#34;, 20.00 min, and 4 &#176;C using the corresponding dials.</li>
	<li>Load the six balanced centrifuge bottles into an appropriately sized centrifuge rotor and place into the centrifuge ensuring&#160;any specific safety parameters are followed. 
	NOTE: Continue this step until all bacterial cell culture has been centrifuged and supernatants collected.</li>
	<li>Filter the isolated bacterial supernatant through a vacuum filtration or equivalent apparatus fitted with a 0.45 micron filter to remove any residual debris.</li>
	<li>Heparin purification of the recombinant apoE4-NT scaffold protein 
	NOTE: The column purification procedure is conducted at room temperature.
	<ol>
		<li>Equilibrate the Hi-trap Heparin column by applying 10 column volumes (50 mL) of 10 mM sodium phosphate buffer, pH 7.2, and eluting at a rate of 5 mL/min.</li>
		<li>Apply apoE4-NT enriched medium to the column at a rate of 2.5 mL/min until the entire 1 L volume of medium has been applied and discard the flowthrough.</li>
		<li>Wash the column with 10 column volumes (50 mL) of 10 mM sodium phosphate buffer, pH 7.2, and discard the flowthrough.</li>
		<li>Elute the desired apoE4-NT protein by applying three column volumes (15 mL) of elution buffer (10 mM sodium phosphate buffer + 1.5 M NaCl, pH 7.2) to the column and collect the eluate.</li>
	</ol>
	</li>
	<li>Dialysis of apoE4-NT eluate
	<ol>
		<li>Prepare a section of dialysis tubing (dimensions of dialysis tubing used were 22 cm in length, 12 mm internal diameter, and 10,000 MWCO) by thoroughly soaking in distilled deionized (ddi) water for 10 min.</li>
		<li>Prepare 1 L of phosphate buffered saline (PBS) (10 mM sodium phosphate + 150 mM NaCl, pH 7.2) in a 1 L plastic beaker or equivalent.</li>
		<li>Using a dialysis tubing clamp, clamp one end of the soaked dialysis tubing, ensuring the clamp is secured and no liquid can escape.</li>
		<li>Insert a narrow neck funnel into the open end of the dialysis tubing and pour the 15 mL of apoE4-NT eluate into the dialysis tubing. 
		NOTE: It is imperative to check for any leaks at this step.</li>
		<li>Remove the funnel and clamp the end of the dialysis tubing with another dialysis tube clamp.</li>
		<li>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 1 L of PBS buffer.</li>
		<li>Place a magnetic stir bar into the bottom of the beaker and adjust the stirring control to &#34;low&#34;, ensuring a vortex is not formed.</li>
		<li>After dialysis has concluded, decant the retentate into a 50 mL conical tube and store at -20 &#176;C. 
		NOTE: It is recommended that this dialysis be conducted at 4 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
2. Formulation of bioactive agent containing nanodisks
<ol>
	<li>Preparation of phospholipid aliquot
	<ol>
		<li>Weigh 5 mg of an appropriate phospholipid (e.g., dimyristoylphosphatidylcholine [DMPC]) and transfer to a glass test tube.</li>
		<li>Dissolve the 5 mg of phospholipid by adding 300 &#181;L of CHCl3 and 100 &#181;L of CH3OH for a total ratio of 3:1 v/v.</li>
		<li>Evaporate the organic solvent by placing the glass test tube under a gentle stream of N2 gas for 10-15 min, such that a thin film of dried phospholipid forms along the walls of the bottom portion of the tube.</li>
	</ol>
	</li>
	<li>Lyophilization of phospholipid aliquots
	<ol>
		<li>Prepare phospholipid aliquots for lyophilization by covering the glass test tube opening with parafilm.</li>
		<li>Perforate the parafilm using a 24 G needle approximately 10-15 times.</li>
		<li>Place the perforated aliquots into an appropriate lyophilization container and ensure the rubber lid is sealed correctly.</li>
		<li>Attach the lyophilization container to the vacuum manifold located at the top of the lyophilization machine and ensure all the other valves are closed tightly.</li>
		<li>Turn on the lyophilization machine by flipping the power switch and pressing the vacuum initialization button.</li>
		<li>After the system has reached total vacuum, click the refrigeration initialization button to begin the freeze drying process. 
		NOTE: It is recommended that samples be allowed to lyophilize overnight.</li>
	</ol>
	</li>
	<li>Formulation of amphotericin-B (amp-B) nanodisks
	<ol>
		<li>Pipette 0.45 mL of PBS to the lyophilized phospholipid aliquot and vortex for ~30 s to disperse the lipid. 
		NOTE: The resulting sample will appear opaque and turbid.</li>
		<li>Pipette 50 &#181;L of a 20 mg/mL stock ampB solution (20 mg of ampB dissolved in 1 mL of dimethyl sulfoxide [DMSO], stored at -20 &#176;C in a closed amber vessel) into the dispersed phospholipid sample and vortex. 
		NOTE: When selecting a solvent to use in preparing a stock solution of the hydrophobic bioactive agent, the two overarching considerations are 1) the solubility of the bioactive agent and 2) the miscibility of the solvent with the aqueous buffer used in the formulation. While DMSO is often used15,16,17,18,19, dimethylformamide20,21 or tetrahydrofuran22 have also been successfully employed23.</li>
		<li>Pipette 0.5 mL of apoE4-NT scaffold protein (concentration of ~4 mg/mL) to the glass test tube containing the dispersed phospholipid and ampB. The final volume of the sample should be approximately 1 mL.</li>
		<li>Bath sonicate the sample at 24 &#176;C until the solution clarifies (generally 10-15 min).</li>
	</ol>
	</li>
	<li>Nanodisk centrifugation
	<ol>
		<li>Transfer the clarified ampB-nanodisk solution to a sterile 1.7 mL micro-centrifuge tube.</li>
		<li>Place the tube into a tabletop micro-centrifuge rotor with the addition of a balance tube placed directly opposite.</li>
		<li>Tighten the rotor cap and close the centrifuge lid.</li>
		<li>Program the centrifuge to spin for 10 min at ~11,000 x g using the corresponding dials located on the front of the micro-centrifuge unit. 
		NOTE: At this point, a pellet may be visible. This pellet consists of unincorporated phospholipid and/or ampB.</li>
		<li>Remove the supernatant and transfer it to another clean 1.7 mL micro-centrifuge tube.</li>
	</ol>
	</li>
	<li>Dialysis of ampB-nanodisk sample
	<ol>
		<li>Prepare a section of dialysis tubing (dimensions of dialysis tubing used were 3 cm in length, 16 mm internal diameter, and 10,000 MWCO) by thoroughly soaking in ddi water for 10 min.</li>
		<li>Prepare a 1 L PBS buffer solution (10 mM sodium phosphate + 150 mM NaCl, pH 7.2) in a 1 L plastic beaker or equivalent.</li>
		<li>Using a dialysis tubing clamp, clamp one end of the soaked dialysis tubing, ensuring the clamp is secured and no liquid can escape.</li>
		<li>Insert a narrow neck funnel into the open end of the dialysis tubing and transfer the ampB-nanodisk sample into the dialysis tubing.</li>
		<li>Remove the funnel and clamp the end of the dialysis tubing with another dialysis tube clamp.</li>
		<li>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 the 1 L of PBS buffer.</li>
		<li>Place a magnetic stir bar into the bottom of the beaker and adjust the stirring control to &#34;low&#34;, ensuring a vortex is not formed. Allow dialysis to continue overnight at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Spectral analysis of ampB-nanodisk samples
<ol>
	<li>Spectrophotometer initialization followed by auto blank
	<ol>
		<li>Turn on the spectrophotometer by flipping the power switch and connect to a corresponding support computer by pressing the button labeled &#34;PC Control&#34;.</li>
		<li>On the support computer, open the software labeled &#34;UVProbe 2.61&#34; and connect to the spectrophotometer by clicking the button labeled &#34;Connect&#34; in the bottom left corner.</li>
		<li>Click the button labeled Spectrum on the top toolbar within the UVProbe software.</li>
		<li>Click the button labeled Method on the top toolbar.</li>
		<li>Click on the Measurement tab and input &#34;500&#34; into the Start textbox located under &#34;Wavelength Range (nm)&#34; and &#34;300&#34; into the &#34;End&#34; textbox.</li>
		<li>Click the drop-down menu next to the tab labeled Scan Speed and set it to Medium.</li>
		<li>Prepare a sample blank by transferring 1 ml of DMSO into two quartz cuvettes (QS 1.000).</li>
		<li>Load both cuvettes into the respective sample ports of the spectrophotometer, click Autoblank, and record a spectrum from 300-500 nm by clicking Start in the bottom left corner.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of ampB standard
	<ol>
		<li>Prepare 20 &#181;g/mL ampB standard by removing the cuvette from the front sample port and adding 20 &#181;L of a 1 mg/mL ampB stock solution (20 &#181;g of ampB total).</li>
		<li>Load the cuvette back into the sample port of the spectrophotometer and click Start to record the sample absorbance.</li>
		<li>Remove the cuvette from the sample port and decant the liquid contents into an appropriately labeled waste container.</li>
		<li>Thoroughly rinse the cuvette with three washes of deionized water followed by three washes with 70% ethanol.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of disrupted ampB-nanodisk sample
	<ol>
		<li>Prepare a disrupted ampB-nanodisk sample (containing 20 &#181;g/mL as ampB) by pipetting 20 &#181;L of a 1 mg/mL ampB-nanodisk stock into 1 mL of DMSO. Incubate for at least 1 min prior to recording the spectrum.</li>
		<li>Load the cuvette into the front sample port of the spectrophotometer and click Start to record the sample absorbance.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of a PBS buffer blank
	<ol>
		<li>Prepare a PBS buffer blank by transferring 1 mL of PBS buffer into two quartz cuvettes (QS 1.000).</li>
		<li>Load the cuvettes into the respective sample ports of the spectrophotometer, click Autoblank, and record the spectrum from 300-500 nm.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of a non-disrupted ampB-nanodisk sample
	<ol>
		<li>Prepare a non-disrupted ampB-nanodisk sample (containing 20 &#181;g/mL as ampB) by removing the cuvette from the front sample port and introducing 20 &#181;L of a 1 mg/mL ampB-nanodisk sample into the PBS buffer.</li>
		<li>Load the cuvette back into the sample port of the spectrophotometer, click Start, and record the sample spectrum. 
		&#8203;NOTE: All chemical waste should be disposed of in an appropriately labeled waste container following accepted guidelines.</li>
	</ol>
	</li>
</ol>
4. Yeast viability assay analysis
NOTE: Yeast viability assays were performed in order to evaluate the biological activity of ampB and determine whether the process of formulation or incorporation into nanodisks, affected its yeast growth inhibition activity.
<ol>
	<li>Formulate two ampB-nanodisk samples (final volume = 1 mL) to contain 1 mg of ampB per 5 mg of DMPC and 0.1 mg of ampB per 5 mg of DMPC following the previously described method (2.3-2.4.1). 
	NOTE: To make a 1 mg/mL and 0.1 mg/mL ampB per 5 mg of DMPC, pipette 50 &#181;L and 5 &#181;L, respectively, of a 20 mg/mL ampB in DMSO stock into each sample vial.</li>
	<li>Preparation of a saturated yeast culture
	<ol>
		<li>Transfer 25 mL of sterile Yeast Extract-Peptone-Dextrose (YPD) medium into a sterile 50 mL conical tube.</li>
		<li>Use a sterile inoculation loop to transfer a single Saccharomyces cerevisiae (BY4741) colony into the 25 mL of YPD medium.</li>
		<li>Culture the yeast in a shaking incubator set at 30 &#176;C, with oscillation set to 200 rpm for 18 h.</li>
	</ol>
	</li>
	<li>Preparation of individual growth inhibition assay samples
	<ol>
		<li>Transfer 5 mL of sterile YPD medium into 30 sterile 13 mL culture tubes.</li>
		<li>Treat three sets of culture tubes by adding 20, 10, and 5 &#181;L of the 1 mg/mL ampB-nanodisk sample, representing 20, 10, and 5 &#181;g of ampB, respectively.</li>
		<li>Treat three sets of culture tubes by adding 20, 10, and 5 &#181;l of the 0.1 mg/mL ampB-nanodisk sample, representing 2, 1, and 0.5 &#181;g of ampB, respectively.</li>
		<li>Treat four sets of culture tubes by adding 20 &#181;L of PBS, DMSO, control rHDL (no ampB), or 20 &#181;g of ampB in DMSO. 
		NOTE: Each set of culture tubes represents an independent replicate. Therefore, following this methods results in an overall n value of n = 3.</li>
	</ol>
	</li>
	<li>Yeast viability assay initialization
	<ol>
		<li>Initiate the experiment by inoculating each sample with 100 &#181;L (2% vol/vol) of the saturated yeast culture</li>
		<li>Culture the yeast using a shaking incubator set to 30 &#176;C, with oscillation set to 200 rpm for a maximum of 18 h.</li>
	</ol>
	</li>
	<li>Yeast viability measurements
	<ol>
		<li>Turn on the spectrophotometer by flipping the power switch, then click the button labeled Go To WL and set the value to 600 nm.</li>
		<li>Transfer 1 mL of sterile YPD medium into two plastic cuvettes, load into the respective sample ports on the spectrophotometer, and click Autoblank.</li>
		<li>Transfer 1 mL of each sample into a plastic cuvette, making sure to change cuvettes each time and load the cuvette into the front sample port of the spectrophotometer.</li>
		<li>Measure and record each sample&#39;s optical density at 600 nm and dispose of each expended cuvette into a properly labeled biohazard waste container.</li>
	</ol>
	</li>
</ol>

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<li>Zhao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sphingadienes+show+therapeutic+efficacy+in+neuroblastoma+in+vitro+and+in+vivo+by+targeting+the+AKT+signaling+pathway%5BTitle%5D)+AND+%22Investigational+New+Drugs%22%5BJournal%5D)">Sphingadienes show therapeutic efficacy in neuroblastoma in vitro and in vivo by targeting the AKT signaling pathway.</a> Investigational New Drugs. 36 (5), 743-754 (2018).</li>
<li>Moschetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+and+characterization+of+biocompatible+coenzyme+Q10+enriched+lipid+nanoparticles%5BTitle%5D)+AND+%22Lipids%22%5BJournal%5D)">Assembly and characterization of biocompatible coenzyme Q10 enriched lipid nanoparticles.</a> Lipids. 55 (2), 141-149 (2020).</li>
<li>Krishnamoorthy, A., Witkowski, A., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutlin-3a+nanodisks+induce+p53+stabilization+and+apoptosis+in+a+subset+of+cultured+glioblastoma+cells%5BTitle%5D)+AND+%22Journal+of+Nanomedicine+and+Nanotechnology%22%5BJournal%5D)">Nutlin-3a nanodisks induce p53 stabilization and apoptosis in a subset of cultured glioblastoma cells.</a> Journal of Nanomedicine and Nanotechnology. 8 (4), 454(2017).</li>
<li>Moschetti, A., Fox, C. A., McGowen, S., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lutein+nanodisks+protect+retinal+pigment+epithelial+cells+from+UV+light+induced+damage%5BTitle%5D)+AND+%22Frontiers+in+Nanotechnology%22%5BJournal%5D)">Lutein nanodisks protect retinal pigment epithelial cells from UV light induced damage.</a> Frontiers in Nanotechnology. 4, 955022(2022).</li>
<li>Scheetz, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+HDL+nanoparticles+delivering+docetaxel+and+CpG+for+chemoimmunotherapy+of+colon+adenocarcinoma%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Synthetic HDL nanoparticles delivering docetaxel and CpG for chemoimmunotherapy of colon adenocarcinoma.</a> International Journal of Molecular Sciences. 21 (5), 1777(2020).</li>
<li>Duivenvoorden, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+statin-loaded+reconstituted+high-density+lipoprotein+nanoparticle+inhibits+atherosclerotic+plaque+inflammation%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation.</a> Nature Communications. 5, 3065(2014).</li>
<li>Hargreaves, P. L., Nguyen, T. S., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectroscopic+studies+of+amphotericin+B+solubilized+in+nanoscale+bilayer+membranes%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes.</a> Biochimica et Biophysica Acta. 1758 (1), 38-44 (2006).</li>
<li>Tufteland, M., Pesavento, J. B., Bermingham, R. L., Hoeprich Jr, P. D., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide+stabilized+amphotericin+B+nanodisks%5BTitle%5D)+AND+%22Peptides%22%5BJournal%5D)">Peptide stabilized amphotericin B nanodisks.</a> Peptides. 28 (4), 741-746 (2007).</li>
<li>Tufteland, M., Ren, G., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanodisks+derived+from+amphotericin+B+lipid+complex%5BTitle%5D)+AND+%22Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Nanodisks derived from amphotericin B lipid complex.</a> Journal of Pharmaceutical Sciences. 97 (10), 4425-4432 (2008).</li>
<li>Nguyen, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphotericin+B+induces+interdigitation+of+apolipoprotein+stabilized+nanodisk+bilayers%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers.</a> Biochimica et Biophysica Acta. 1778 (1), 303-312 (2008).</li>
<li>Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanobiotechnology+applications+of+reconstituted+high+density+lipoprotein%5BTitle%5D)+AND+%22Journal+of+Nanobiotechnology%22%5BJournal%5D)">Nanobiotechnology applications of reconstituted high density lipoprotein.</a> Journal of Nanobiotechnology. 8, 28(2010).</li>
<li>Lalefar, N. R., Witkowski, A., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt3a+nanodisks+promote+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells%5BTitle%5D)+AND+%22Journal+of+Nanobiotechnology%22%5BJournal%5D)">Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells.</a> Journal of Nanobiotechnology. 14 (1), 66(2016).</li>
<li>Crosby, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-CD20+single+chain+variable+antibody+fragment-apolipoprotein+A-I+chimera+containing+nanodisks+promote+targeted+bioactive+agent+delivery+to+CD20-positive+lymphomas%5BTitle%5D)+AND+%22Biochemistry+and+Cell+Biology%22%5BJournal%5D)">Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.</a> Biochemistry and Cell Biology. 93 (4), 343-350 (2015).</li>
<li>Ghosh, M., Ren, G., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cationic+lipid+nanodisks+as+an+siRNA+delivery+vehicle%5BTitle%5D)+AND+%22Biochemistry+and+Cell+Biology%22%5BJournal%5D)">Cationic lipid nanodisks as an siRNA delivery vehicle.</a> Biochemistry and Cell Biology. 92 (3), 200-205 (2014).</li>
<li>Fox, C. A., Ellison, P., Ikon, N., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-induced+transformation+of+cardiolipin+nanodisks%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta.+Biomembranes%22%5BJournal%5D)">Calcium-induced transformation of cardiolipin nanodisks.</a> Biochimica et Biophysica Acta. Biomembranes. 1861 (5), 1030-1036 (2019).</li>
<li>Fox, C. A., Lethcoe, K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-induced+release+of+cytochrome+c+from+cardiolipin+nanodisks%3A+Implications+for+apoptosis%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+Biomembranes%22%5BJournal%5D)">Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.</a> Biochimica et Biophysica Acta Biomembranes. 1861 (12), 183722(2021).</li>
<li>Fox, C. A., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dye+binding+assay+reveals+doxorubicin+preference+for+DNA+versus+cardiolipin%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.</a> Analytical Biochemistry. 594, 113617(2020).</li>
<li>Fox, C. A., Romenskaia, I., Dagda, R. K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiolipin+nanodisks+confer+protection+against+doxorubicin-induced+mitochondrial+dysfunction%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+Biomembranes%22%5BJournal%5D)">Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.</a> Biochimica et Biophysica Acta Biomembranes. 1864 (10), 183984(2022).</li>
</ol>

The authors have nothing to disclose.

Formulation of a bioactive agent containing nanodisks provide&#160;a convenient method to solubilize otherwise insoluble hydrophobic compounds. Because the product bioactive agent nanodisks are fully soluble in aqueous media, they provide a useful delivery method for a wide range of hydrophobic molecules (Table 1). These include small molecules, natural and synthetic drugs, phytonutrients, hormones, etc. The formulation strategy usually follows a standard protocol that must take into consideration the so...

Nascent discoidal high density lipoproteins (HDLs) are naturally occurring progenitors of the far more abundant spherical HDL present in the human circulatory system. These nascent particles, also referred to as pre-&#223; HDL, possess unique and distinctive structural properties1. Indeed, rather than existing as a spheroidal particle, nascent HDLs are disk-shaped. Extensive structural characterization studies on natural and reconstituted discoidal HDLs have revealed that they are comprised of a phospholipid bilayer whose perimeter is circumscribed by an amphipathic exchangeable apolipoprotein (apo), such as apoA-I. In human lipoprotein metabolism, circulating nascent HDLs accrue lipids from peripheral cells and mature into spherical HDLs in a process that is dependent upon key protein mediators, including the ATP binding cassette transporter A1 and lecithin:cholesterol acyltransferse2. This process represents a critical component of the reverse cholesterol transport pathway that is considered to be protective against heart disease. Armed with this knowledge and the ability to reconstitute discoidal HDLs, researchers have employed these particles as a therapeutic intervention to treat atherosclerosis3. In essence, the infusion of reconstituted HDL (rHDL) into patients promotes cholesterol efflux from plaque deposits and returns it to the liver for conversion to bile acids and excretion from the body. Several biotechnology/pharmaceutical companies are pursuing this treatment strategy4.
At the same time, the ability to generate these particles in the laboratory has sparked a flurry of research activities that has led to novel applications and new technologies. One prominent application involves the use of rHDL particles as a miniature membrane to house transmembrane proteins in a native-like environment5. To date, hundreds of proteins have been successfully incorporated into discoidal rHDL, and research has demonstrated that these proteins retain both native conformation and biological activity as receptors, enzymes, transporters, etc. These particles, referred to as &#34;nanodiscs&#34;, have also been shown to be amenable to structural characterization, often at high resolution6. This approach to investigations of transmembrane proteins is recognized as superior to studies with detergent micelles or liposomes and, as a result, is rapidly advancing. It is important to recognize that two distinct methods have been reported that are capable of forming an rHDL. The &#34;cholate dialysis&#34; method13 is popular for applications related to the incorporation of transmembrane proteins in the rHDL bilayer5. Essentially, this method of formulation involves mixing a bilayer forming phospholipid, a scaffold protein, and the transmembrane protein of interest in a buffer containing the detergent sodium cholate (or sodium deoxycholate; micelle molecular weight [MW] of 4,200 Da). The detergent effectively solubilizes the different reaction components, permitting the sample to be dialyzed against buffer lacking detergent. During the dialysis step, as the detergent is removed from the sample, an rHDL spontaneously forms. When this approach is used to entrap a transmembrane protein of interest, the product particles have been termed nanodiscs5. Attempts to use this method to incorporate small molecule hydrophobic bioactive agents (MW &#60;1,000 Da), however, have been largely unsuccessful. Unlike transmembrane proteins, small molecule bioactive agents are able to escape from the dialysis bag along with the detergent, greatly decreasing their incorporation efficiency into rHDLs. This problem was solved by omitting detergents from the formulation mixture14. Instead, the components are added to an aqueous buffer sequentially, beginning with the bilayer forming lipid, forming a stable bioactive agent containing rHDL, referred to as a nanodisk. Others have used rHDL for the incorporation and transport of in vivo imaging agents7. More recently, specialized rHDL comprised of an apolipoprotein scaffold and the anionic glycerophospholipid, cardiolipin, have been employed in ligand binding studies. These particles provide a platform for studies of the interaction of cardiolipin with various water soluble ligands, including calcium, cytochrome c, and the anticancer agent doxorubicin8.
The focus of the present study is on the formulation of rHDL that possess a stably incorporated hydrophobic bioactive agent (i.e. nanodisk). The ability of these agents to integrate into the lipid milieu of discoidal rHDL particles effectively confers them with aqueous solubility. As such, nanodisks have the potential for in vivo therapeutic applications. When formulating nanodisks, specific incubation/reaction conditions are required to successfully incorporate discrete hydrophobic bioactive agents into the product particle, and the goal of this report is to provide detailed practical information that can be used as a foundational template for creating novel nanodisk particles for specific applications. Thus, in the context of this manuscript the terms nanodisc and nanodisk are not interchangeable. Whereas nanodisc refers to an rHDL formulated to contain a transmembrane protein embedded in its lipid bilayer5, the term nanodisk refers to an rHDL formulated to incorporate low molecular weight (&#60; 1,000 Da) hydrophobic bioactive agents, such as amphotericin B14.
A variety of methods are available for the acquisition of suitable scaffold proteins. It is possible to purchase scaffold proteins from manufacturers [e.g. apoA-I (SRP4693) or apoE4 (A3234)], however, the cost may be a limiting factor. A preferred approach is to express recombinant scaffold proteins in Escherichia coli. Protocols are published for human apoA-I9, apoE410, as well as the insect hemolymph protein apolipophorin-III11. For the purpose of the experiments described herein, recombinant human apoE4 N-terminal (NT) domain (amino acids 1-183) was used. The nucleotide sequence encoding human apoE4-NT was synthesized and inserted into a pET-22b (+) expression vector directly adjacent to the vector-encoded pelB leader sequence. This construct leads to the expression of a pelB leader sequence-apoE4-NT fusion protein. Following protein synthesis, the bacterial pelB leader sequence directs the newly synthesized protein to the periplasmic space where leader peptidase cleaves the pelB sequence. The resultant apoE4-NT protein, with no sequence tags or tails, subsequently escapes the bacteria and accumulates in the culture medium11,12, simplifying downstream processing.

<table><tbody><tr><td>Amphotericin B</td><td>Cayman Chemical Company</td><td>11636</td><td>ND Formulation &amp;amp; Standard Preparation</td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP17925</td><td>Transformation &amp;amp; Expansion</td></tr><tr><td>ApoE4-NT Plasmid</td><td>GenScript</td><td>N/A</td><td>Transformation</td></tr><tr><td>Baffled Flask</td><td>New Brunswick Scientific</td><td>N/A</td><td>Expansion &amp;amp; Expression</td></tr><tr><td>BL21 competent E coli</td><td>New England Biolabs</td><td>C2527I</td><td>Transformation</td></tr><tr><td>Centrifuge bottles</td><td>Nalgene</td><td>3140-0250</td><td>Expression</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>G607-4</td><td>ND Formulation</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>472301</td><td>Standard Prepartation</td></tr><tr><td>Dymyristoylphosphatidylcholine</td><td>Avanti Lipids</td><td>850345P</td><td>ND Formulation</td></tr><tr><td>Erlenmeyer flask</td><td>Bellco Biotechnology</td><td>N/A</td><td>Expansion &amp;amp; Expression</td></tr><tr><td>Falcon Tubes</td><td>Sarstedt Ag &amp;amp; Co</td><td>D51588</td><td>Yeast Viability Assay</td></tr><tr><td>Glass borosilicate tubes</td><td>VWR</td><td>47729-570</td><td>ND Formulation</td></tr><tr><td>GraphPad (Software)</td><td>Dotmatics</td><td>N/A</td><td>Yeast Viability Assay</td></tr><tr><td>Heated Sonication Bath</td><td>VWR</td><td>N/A</td><td>ND Formulaton</td></tr><tr><td>Heating and Nitrogen module</td><td>Thermo Scientific</td><td>TS-18822</td><td>ND Formulation</td></tr><tr><td>HiTrap Heparin HP (5 mL)</td><td>GE Healthcare</td><td>17-0407-03</td><td>Purification</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1755</td><td>Expression</td></tr><tr><td>J-25 Centrifuge</td><td>Beckman Coulter</td><td>J325-IM-2</td><td>Expression</td></tr><tr><td>JA-14 Rotor</td><td>Beckman Coulter</td><td>339247</td><td>Expression</td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>7755030</td><td>ND Formulation</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>ND Formulation</td></tr><tr><td>Nitrogen gas</td><td>Praxair</td><td>UN1066</td><td>ND Formulation</td></tr><tr><td>NZCYM media</td><td>RPI Research Products</td><td>N7200-1000.0</td><td>Expansion &amp;amp; Expression</td></tr><tr><td>Pet-22B vector</td><td>GenScript</td><td>N/A</td><td>Transformation</td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>FB0875718</td><td>Transformation &amp;amp; Expansion</td></tr><tr><td>Quartz Cuvettes</td><td>Fisher Brand</td><td>14385 928A</td><td>Spectral Analysis</td></tr><tr><td>Shaking Incubator</td><td>New Brunswick Scientific</td><td>M1344-0004</td><td>Transformation, Expansion, &amp;amp; Expression</td></tr><tr><td>Slide-A-Lyzer Buoys</td><td>Thermo Scientific</td><td>66430</td><td>Purification</td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo Scientific</td><td>68100</td><td>Purification</td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo Scientific</td><td>88243</td><td>Purification</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271</td><td>Purification</td></tr><tr><td>Sodium Phosphate dibasic</td><td>Fisher Scientific</td><td>S374-500</td><td>Purification</td></tr><tr><td>Sodium Phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-500</td><td>Purification</td></tr><tr><td>Spectra/POR Weighted Closures</td><td>Spectrum Medical Industries</td><td>132736</td><td>Purification</td></tr><tr><td>Spectrophotometer</td><td>Shimadzu UV-1800</td><td>220-92961-01</td><td>spectral analysis</td></tr><tr><td>Tabletop Centrifuge</td><td>Beckman Coulter</td><td>366816</td><td>ND Formulation</td></tr><tr><td>UVProbe 2.61 (Software)</td><td>Shimadzu</td><td>N/A</td><td>Spectral Analysis</td></tr><tr><td>Vacuum filter</td><td>Millipore</td><td>9004-70-0</td><td>Expression &amp;amp; Purification</td></tr><tr><td>Vacuum pump</td><td>GAST Manufacturing Inc</td><td>DOA-P704-AA</td><td>Expression &amp;amp; Purification</td></tr><tr><td>Vortex</td><td>Fisher Scientific</td><td>12-812</td><td>ND Formulation</td></tr><tr><td>Yeast</td><td>N/A</td><td>BY4741</td><td>Yeast Viability Assay</td></tr><tr><td>Yeast Extract-Peptone-Dextrose</td><td>BD</td><td>242820</td><td>Yeast Viability Assay</td></tr></tbody></table>

formulation and characterization of bioactive agent containing nanodisks

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Bioactive agent nanodisk formulation process 
In the ampB-nanodisk formulation procedure described, the reaction is considered complete when the sample appearance transitions from turbid to clear (Figure 1). This change indicates that nanodisks have formed and that the bioactive agent has been solubilized. Oftentimes, bioactive agents absorb light in the visible wavelength region (e.g., ampB, curcumin, lutein, coenzyme Q10) and, in these cases, the sample adopt...

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

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 amphot

formulation-characterization-bioactive-agent-containing

Research

JoVE Journal

Biochemistry

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer.  We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Procedure for Fabricating Biofunctional Nanofibers

The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles.  The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

The most commonly used method for nanostructured lipid carrier (NLC) synthesis involves oil-in-water emulsion, homogenization and solidification. This was modified here by applying high shear dispersion after solidification to achieve a NLC with desirable size, improved drug encapsulation and drug loading efficiency as a potential carrier for dacarbazine delivery.

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

The present work aims to serve as a practical guide enabling researchers to prepare their own DMAP devices using the solvent-casting method, also known as micromolding, and to perform basic characterization.

Dissolving Microneedle Array Patches Manufactured By Solvent Casting Technique and Essential Characterization of Microneedle-Based Biomedical Devices

This study outlines the procedure for reconstituting the membrane protein TWIK-related acid-sensitive K+ channel 2 (Task2) into nanodiscs. The successful assembly was confirmed by single-particle cryo-electron microscopy, which yielded well-defined two-dimensional class averages.

A Technique for Stabilizing Membrane Proteins in Nanodiscs

Scopri come formulare nanodischi per incorporare molecole bioattive idrofobiche e migliorare la solubilità acquosa di composti insolubili. Questo metodo semplice consente applicazioni avanzate nel drug delivery.

Formulation and Characterization of Bioactive Agent Containing Nanodisks

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