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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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+HDL+nanoparticles+delivering+docetaxel+and+CpG+for+chemoimmunotherapy+of+colon+adenocarcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statin-loaded+reconstituted+high-density+lipoprotein+nanoparticle+inhibits+atherosclerotic+plaque+inflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+studies+of+amphotericin+B+solubilized+in+nanoscale+bilayer+membranes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+stabilized+amphotericin+B+nanodisks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodisks+derived+from+amphotericin+B+lipid+complex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphotericin+B+induces+interdigitation+of+apolipoprotein+stabilized+nanodisk+bilayers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobiotechnology+applications+of+reconstituted+high+density+lipoprotein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a+nanodisks+promote+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-CD20+single+chain+variable+antibody+fragment-apolipoprotein+A-I+chimera+containing+nanodisks+promote+targeted+bioactive+agent+delivery+to+CD20-positive+lymphomas.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid+nanodisks+as+an+siRNA+delivery+vehicle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+transformation+of+cardiolipin+nanodisks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+release+of+cytochrome+c+from+cardiolipin+nanodisks:+Implications+for+apoptosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+binding+assay+reveals+doxorubicin+preference+for+DNA+versus+cardiolipin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+nanodisks+confer+protection+against+doxorubicin-induced+mitochondrial+dysfunction.">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

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 ado...

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Formulation and Characterization of Bioactive Agent Containing Nanodisks

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. ...

formulation-characterization-bioactive-agent-containing

Research

JoVE Journal

Biochemistry

991 Views.  University of Nevada. 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. 

Video: Formulation and Characterization of Bioactive Agent Containing Nanodisks

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

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

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, 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-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemistry

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug in aqueous solution. To overcome this, a nanostructured lipid carrier (NLC) consisting of glyceryl palmitostearate and isopropyl myristate was developed to encapsulate Dac. NLCs with controlled size were achieved using high shear dispersion (HSD) following solidification of oil-in-water emulsion. The synthesis parameters, including surfactant concentration, the speed and time of HSD were optimized to achieve the smallest NLC with size, polydispersion index and zeta potential of 155 &#177; 10 nm, 0.2 &#177; 0.01, and -43.4 &#177; 2 mV, respectively. The optimal parameters were also employed for Dac-loaded NLC preparation. The resultant NLC loaded with Dac possessed size, polydispersion index and zeta potential of 190 &#177; 10 nm, 0.2 &#177; 0.01, and -43.5 &#177; 1.2 mV, respectively. The drug encapsulation efficiency and drug loading reached 98% and 14%, respectively. This is the first report on encapsulation of Dac using NLC, implying that NLC could be a new potential candidate as drug carrier to improve the therapeutic profile of Dac.

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.

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

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

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;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.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

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

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

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.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...