This work was supported by the NRL Base Funding Program (Work Unit MA041-06-41-4943). ON is supported by a National Research Council Postdoctoral Research Associateship.

1. Preparation of DiO-LCNP and DiO-LCNP-PEG-Chol
<ol>
	<li>Dissolve liquid crystalline diacrylate cross-linking agent (DACTP11, 45 mg), 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO, 2 mg), and a free radical initiator (azobisisobutyronitrile, 1 mg) for polymerization in 2 ml of chloroform. Add this to an aqueous solution of acrylate-functionalized surfactant (AC10COONa, 13 mg in 7 ml).</li>
	<li>Stir the mixture for 1 hr and sonicate at 80% amplitude for 5 min to produce a miniemulsion consisting of small dro...

<ol>
	<li>Nag, O. K., Field, L. D., Chen, Y., Sangtani, A., Breger, J. C., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+actuation+of+therapeutic+nanoparticles:+an+update+on+recent+progress.">Controlled actuation of therapeutic nanoparticles: an update on recent progress.</a> Ther. Deliv. 7 (5), 335-352 (2016).</li><li>Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+low-dose+toxicity+of+the+universal+solvent+DMSO.">Unexpected low-dose toxicity of the universal solvent DMSO.</a> FASEB J. 28 (3), 1317-1330 (2014).</li><li>Spillmann, C. M., Naciri, J., Anderson, G. P., Chen, M. S., Ratna, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+tuning+of+organic+nanocolloids+by+controlled+molecular+interactions.">Spectral tuning of organic nanocolloids by controlled molecular interactions.</a> ACS Nano. 3 (10), 3214-3220 (2009).</li><li>Spillmann, C. M., Naciri, J., Algar, W. R., Medintz, I. L., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+Liquid+Crystal+Nanoparticles+for+Intracellular+Fluorescent+Imaging+and+Drug+Delivery.">Multifunctional Liquid Crystal Nanoparticles for Intracellular Fluorescent Imaging and Drug Delivery.</a> ACS Nano. 8 (7), 6986-6997 (2014).</li><li>Timmers, M., Vermijlen, D., Vekemans, K., De Zanger, R., Wisse, E., Braet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+DiO-labelled+tumour+cells+in+liver+sections+by+confocal+laser+scanning+microscopy.">Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy.</a> J. Microsc. 208 (Pt 1), 65-74 (2002).</li><li>Mufson, E. J., Brady, D. R., Kordower, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+neuronal+connections+in+postmortem+human+hippocampal+complex+with+the+carbocyanine+dye+DiI.">Tracing neuronal connections in postmortem human hippocampal complex with the carbocyanine dye DiI.</a> Neurobiol Aging. 11 (6), 649-653 (1990).</li><li>K&#246;bbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog. Neurobiol. 62 (4), 327-351 (2000).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+DiO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 12 (9), 333-341 (1989).</li><li>Gan, W. B., Bishop, D. L., Turney, S. G., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye.">Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.</a> J. Neurosci. Methods. 93 (1), 13-20 (1999).</li><li>Godement, P., Vanselow, J., Thanos, S., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+in+developing+visual+systems+with+a+new+method+of+staining+neurones+and+their+processes+in+fixed+tissue.">A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.</a> Development. 101 (4), 697-713 (1987).</li><li>Ragnarson, B., Bengtsson, L., Haegerstrand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+with+fluorescent+carbocyanine+dyes+of+cultured+endothelial+and+smooth+muscle+cells+by+growth+in+dye-containing+medium.">Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium.</a> Histochemistry. 97 (4), 329-333 (1992).</li><li>Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M., Futerman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+glucosylceramide+levels+results+in+an+increase+in+endoplasmic+reticulum+density+and+in+functional+calcium+stores+in+cultured+neurons.">Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.</a> J. Biol. Chem. 274 (31), 21673-21678 (1999).</li><li>Garrett, R. H., Grisham, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. , (2013).</li><li>Berne, B. J., Pecora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Light Scattering. , 41155-41159 (2000).</li><li>Kremers, G. J., Piston, D. W., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basics of FRET Microscopy. , (2016).</li><li>Chen, H., Kim, S., Li, L., Wang, S., Park, K., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+hydrophobic+molecules+from+polymer+micelles+into+cell+membranes+revealed+by+Fo&#776;rster+resonance+energy+transfer+imaging.">Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Fo&#776;rster resonance energy transfer imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6596-6601 (2008).</li><li>Campling, B. G., Pym, J., Galbraith, P. R., Cole, S. P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+MTT+assay+for+rapid+determination+of+chemosensitivity+of+human+leukemic+blast+cells.">Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.</a> Leukemia Res. 12, 823-831 (1988).</li><li>Nag, O. K., Naciri, J., Oh, E., Spillmann, C. M., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+raft-mediated+membrane+tethering+and+delivery+of+hydrophobic+cargos+from+liquid+crystal-based+nanocarriers.">Lipid raft-mediated membrane tethering and delivery of hydrophobic cargos from liquid crystal-based nanocarriers.</a> Bioconjug. Chem. 27 (4), 982-993 (2016).</li><li>Karve, 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=Revival+of+the+abandoned+therapeutic+wortmannin+by+nanoparticle+drug+delivery.">Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery.</a> Proc. Natl. Acad. Sci. U. S. A. 109 (21), 8230-8235 (2012).</li></ol>

A continuing goal of NMDD is the controlled targeting and delivery of drug formulations to cells and tissues, combined with simultaneous improved drug efficacy. One specific class of drug molecules for which this has posed a significant challenge is hydrophobic drugs/imaging agents that have sparingly to no solubility in aqueous media. This problem has plagued the transition of potent drugs from in vitro cell culture systems to the clinical setting and has resulted in a number of promising drug molecules being &...

Since the advent of interfacing nanomaterials (materials &#8804;100 nm in at least one dimension) with living cells, a continuing goal has been to take advantage of the unique size-dependent properties of nanoparticles (NPs) for various applications. These applications include cell and tissue labeling/imaging (both in vitro and in vivo), real-time sensing, and the controlled delivery of drugs and other cargos1. Examples of such relevant NP properties include the size-dependent emission of semiconductor nanocrystals (quantum dots, QDs); the photothermal properties of gold nanoparticles; the large loading capacity of the aqueous core of liposomes; and the ballistic conductivity of carbon allotropes, such as single-wall carbon nanotubes and graphene.
More recently, significant interest has arisen in the use of NPs for the controlled modulation of drugs and other cargos, such as contrast/imaging agents. Here, the rationale is to significantly enhance/optimize the overall solubility, delivered dose, circulation time, and eventual clearance of the drug cargo by delivering it as an NP formulation. This has come to be known as NP-mediated drug delivery (NMDD), and there are currently seven FDA-approved NP drug formulations for use in the clinic to treat various cancers and hundreds more in various stages of clinical trials. In essence, the goal is to &#34;achieve more with less;&#34; that is, to use the NP as a scaffold to deliver more drug with fewer dosing administrations by taking advantage of the large surface area:volume (e.g., hard particles, such as QDs and metal oxides) of NPs or their large interior volume for loading large cargo payloads (e.g., liposomes or micelles). The purpose here is to reduce the necessity for multiple systemically delivered dosing regimens while at the same time promoting aqueous stability and enhanced circulation, particularly for challenging hydrophobic drug cargos that, while highly effective, are sparingly soluble in aqueous media.
Thus, the goal of the work described herein was to determine the viability of using a novel NP scaffold for the specific and controlled delivery of hydrophobic cargos to the lipophilic plasma membrane bilayer. The motivation for the work was the inherent limited solubility and difficulty in the delivery of hydrophobic molecules to cells from aqueous media. Typically, the delivery of such hydrophobic molecules requires the use of organic solvents (e.g., DMSO) or amphiphilic surfactants (e.g., Poloxamers), which can be toxic and compromise cell and tissue viability2, or micelle carriers, which can have limited internal loading capacities. The NP carrier chosen here was a novel liquid crystal NP (LCNP) formulation developed previously3 and that had been shown previously to achieve a ~40-fold improvement in the efficacy of the anticancer drug doxorubicin in cultured cells4.
In the work described herein, the representative cargo selected was the potentiometric membrane dye, 3,3&#39;-dioctadecyloxacarbocyanine perchlorate (DiO). DiO is a water-insoluble dye that has been used for anterograde and retrograde tracing in living and fixed neurons, membrane potential measurements, and for general membrane labeling5,6,7,8,9. Due to its hydrophobic nature, DiO is typically added directly to cell monolayers or tissues in a crystalline form10, or it is incubated at very high concentrations (~1-20 &#181;M) after dilution from a concentration stock solution11,12.
Here, the approach was use to the LCNP platform, a multifunctional NP whose inner core is completely hydrophobic and whose surface is simultaneously hydrophilic and amenable to bioconjugation, as a delivery vehicle for DiO. DiO is incorporated into the LCNP core during synthesis, and the NP surface is then functionalized with a PEGylated cholesterol moiety to promote the membrane binding of the DiO-LCNP ensemble to the plasma membrane. This approach resulted in a delivery system that partitioned the DiO into the plasma membrane with greater fidelity and membrane residence time than the free form of DiO delivered from bulk solution (DiOfree). Further, this method showed that the LCNP-mediated delivery of DiO substantially modulates and drives the rate of specific partitioning of the dye into the lipophilic plasma membrane bilayer. This is achieved while concomitantly reducing the cytotoxicity of the free drug by ~40% by delivering it as an LCNP formulation.
It is anticipated that the methodology described herein will be a powerful enabling technique for researchers whose work involves or requires the cellular delivery of highly hydrophobic cargos that are sparingly soluble or completely insoluble in aqueous solution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide hydrochloride (EDCA)</td>
			<td>ThermoFisher</td>
			<td>E2247</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO)</td>
			<td>Sigma Aldrich</td>
			<td>D4292-20MG</td>
			<td>Hazardous/ make stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Cholesterol poly(ethylene glycol) amine hydrochloride</td>
			<td>Nanocs, Inc.</td>
			<td>PG2-AMCS-2k</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>ThermoFisher</td>
			<td>C10227</td>
			<td></td>
		</tr>
		<tr>
			<td>Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate (DiI)</td>
			<td>Sigma Aldrich</td>
			<td>468495-100MG</td>
			<td>Hazardous/ make stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>ThermoFisher</td>
			<td>21063045</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>ThermoFisher</td>
			<td>14040182</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Dynamic light scattering instrument</td>
			<td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Bovine Protein, Plasma</td>
			<td>ThermoFisher</td>
			<td>33010018</td>
			<td>Make stock solution 1mg/mL using DPBS. Use 20-30 &#181;g/mL for coating MetTek dish, 2 h@ 37&#176;C</td>
		</tr>
		<tr>
			<td>Formaldehyde (16%, W/V)</td>
			<td>ThermoFisher</td>
			<td>28906</td>
			<td>Hazardous, dilute to 4% using DPBS</td>
		</tr>
		<tr>
			<td>Human embryonic kidney cells (HEK 293T/17)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC&#174; CRL-11268&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Live cell imaging solution (LCIS)</td>
			<td>ThermoFisher</td>
			<td>A14291DJ</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>MatTek 14 mm # 1.0 coverglass insert cell culture dish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Modified Eagle Medium (DMEM) containing 25 mM HEPES</td>
			<td>ThermoFisher</td>
			<td>21063045</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>N-hydroxysulfosuccinimide sodium salt (NHSS)</td>
			<td>ThermoFisher</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1si spectral confocal microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>ThermoFisher</td>
			<td>T10282</td>
			<td>mix as a 50% to the cell suspension before counting the cells</td>
		</tr>
		<tr>
			<td>Zeta potential instrument</td>
			<td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Processor</td>
			<td>Sonics and Materials Inc</td>
			<td>GEX 600-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cetntrifuge</td>
			<td>Benchmark</td>
			<td>Mini-fuge-04477</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-10 Sephadex&#8482; G-25 Medium</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad ChemiDoc XRS Imaging System</td>
			<td>Bio-RAD</td>
			<td>76S/07434</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA(0.25%), phenol red</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted plasma membrane delivery of a hydrophobic cargo encapsulated in a liquid crystal nanoparticle carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

LCNPs were prepared in which the hydrophobic core of the NP was loaded with a representative membrane-labeling probe to demonstrate the utility of the LCNP as an efficient delivery vehicle for hydrophobic cargos. For this purpose, the cargo chosen was the highly water-insoluble potentiometric membrane-labeling dye, DiO. DiO-loaded LCNPs (DiO-LCNPs) were synthesized using a two-phase mini-emulsion technique with the chemical components DACTP11, AC10COONa, and DiO, as shown in Figure 1<sup...

Watch this Scientific Journal Video about Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier at JoVE.com

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

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.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

The overall goal of this experimental procedure is to use a liquid crystal based nanoparticle as a delivery platform for the controlled delivery of a hydrophobic dicargo to the lipophilic portion of the plasma membrane of living mammalian cells. This method can help answer key questions in the drug delivery field, such as how to efficiently enable the delivery and cellular uptake of poorly water soluble hydrophobic cargos. The main advantage of this technique is that it allows more specific delivery of membrane targeted hydrophobic cargos, to the plasma membrane, coupled with their reduced cytotoxicity.Demonstrating the procedure will be Dr.Okhil Nag, a postdoc from my laboratory. Begin this procedure with preparation of DiO liquid crystal nanoparticle as described in the text protocol. Then, add 20 microliters of freshly prepared working solution of NHSS/EDC to 1.0 millileters of the DiO liquid crystal nanoparticle in HEPES buffer.And stir for five minutes. Next, add 20 microleters of a stock solution of cholesterol PEG amine to this mixture and stir for two hours. After two hours, briefly centrifuge the reaction mixture at maximum speed for 30 seconds using a tabletop mini centrifuge.Then pass the supernatant through a PD 10 size exclusion chromatography column equilibrated with Dulbecco's phosphate buffered saline. To confirm the successful covalent conjugation of PEG cholesterol to the DiO liquid crystal nanoparticle surface by gel electrophoresis, prepare an agarose gel as described in the text protocol. Once the gel has solidified, add enough TBE running buffer to submerge the gel in the chamber.Then, add 35 microleters of DiO liquid crystal nanoparticle sample to the wells of the gel and run for 20 minutes at a voltage of 110 volts. Image the gel using a gel imaging system with an excitation filter at 488 nanometers and emission filters of 500 to 550 nanometers. Prepare 35 millimeter diameter tissue culture dishes fitted with 14 millimeter number one cover glass inserts by coating them with bovine fibronectin in DPBS for two hours at 37 degrees Celsius.After two hours, remove the fibronectin coating solution from the culture dish. Harvest HEK 293 T17 cells from the T25 flask by first washing the cell monolayer with three milliliters of DPBS, and then adding two milliliters of trypsin EDTA. Incubate the flask at 37 degrees Celsius for approximately three minutes.Once cells are detached from the flask, neutralize the trypsin by adding four milliliters of complete medium to the flask. Determine the cell concentration in the suspension by counting them in a cell counter. Then adjust the cell concentration to approximately 80, 000 cells per milliliter with growth medium.Add three milliliters of the cell suspension to the dish, and culture in the incubator overnight. The next day, the cells should be at approximately 70 percent confluency and ready for use. Adequate cell density is critical for robust and efficient labeling of a high percentage of cells.Prepare one milliliter solutions of DiO, DiO liquid crystal nanoparticle, and DiO liquid crystal nanoparticle PEG cholesterol, in delivery medium by diluting the corresponding stock solutions. Remove the growth medium from the culture dishes using a serological pipette, and wash the cell monolayers two times with HEPES DMEM. Perform the washes by using a pipette to gently add HEPES DMEM to the edge of the dishes, and then removing it.Add 0.2 milliliters of the prepared DiO free or DiO liquid crystal nanoparticle delivery solutions to the center of the culture dishes. Then return the dishes to the incubator for an appropriate period of time. Longer incubation times lead to nonspecific cellular labeling of non membraneous areas.After the incubation period, remove the delivery solutions using a serological pipette. Wash the cell monolayers two times with DPBS using two milliliters for each wash. Fix the cell monolayers using four percent paraformaldehyde for 15 minutes at room temperature.Remove the paraformaldehyde solution using a pipette and gently wash the cells one time with two milliliters of DPBS. Now the fixed cells are ready to be imaged for the presence of a membraneous fluorescent signal, using confocal laser scanning microscopy. On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope to assess membrane labeling.If the imaging is not performed immediately, replace the medium with DPBS containing 0.05 percent sodium azide and store the dishes at four degrees Celsius. In this method, cells are colabeled with DiO as a FRET donor, and DiI as a FRET acceptor. Label the sells sequentially with DiO liquid crystal nanoparticle PEG cholesterol, and DiI free as before.After staining, wash the cells one time with two millileters of DPBS using a serological pipette and replace with two milliliters of the live cell imaging solution. On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope with a FRET imaging. Configure the experiment for 30 minute intervals over a four hour period by exciting the DiO donor at 488 nanometers, and collecting full emission spectra of both the DiO donor and the DiI acceptor from 490 to 700 nanometers, with a 32 channel spectral detector.Then, determine the time resolved emission intensity of both the DiO donor and the DiI acceptor from cells stained with DiO liquid crystal nanoparticle PEG cholesterol, and DiI. Finally, calculate the time resolved acceptor donor FRET ratio for the images as described in the text protocol. Free DiO accumulates nonspecifically in the cell interior, rather than the plasma membranes of the cells which were counterstained with a dilabeled membrane phospholipid.This can be visualized with the merged images. Conversely, the DiO liquid crystal nanoparticle PEG cholesterol specifically labels just the plasma membrane of the cell, as visualized by colocalization with the dilabeled membrane phospholipid. FRET confirms diopartitioning from the liquid crystal nanoparticle into the membrane lipid bilayer through an observed increase in energy transfer from the DiO donor to the DiI acceptor.FRET between DiO and DiI, as well as DiO and DiI emission changes, confirm increased FRET from DiO to DiI as a function of time. The DiO liquid crystal nanoparticle PEG cholesterol nanoparticles exhibit minimal cytotoxicity at 90 percent viability, as compared to significant toxicity for free DiO at approximately 50 percent viability. After watching this video, you should have a good understanding of how to synthesize cargo loaded liquid crystal nanoparticles, biofunctionalize them for cellular targeting, and image the cargo loaded cells for successful cellular delivery.Generally, individuals new to this method will struggle because the specific delivery of DiO from aqueous solution to the plasma membrane is a significant challenge. We first had the idea for this method after we used the liquid crystal nanoparticle platform for the cellular delivery of the anticancer drug doxorubicin, and saw its ability to modulate the drug's cellular uptake kinetics. Following this procedure, this approach can be implemented for the cellular delivery of a wide array of hydrophobic cargos that pose a particular challenge with respect of their delivery to cells from aqueous media.This approach can be utilized to achieve more efficient delivery of membrane targeted drugs and bioactive molecules, such as voltage sensitive dyes for brain imaging, or photodynamic drugs for photonic assisted drug therapy.

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JoVE Journal

Bioengineering

7.4K visualizações.  Naval Research Laboratory. The overall goal of this experimental procedure is to use a liquid crystal based nanoparticle as a delivery platform for the controlled delivery of a hydrophobic dicargo to the lipophilic portion of the plasma membrane of living mammalian cells. This method can help answer key questions in the drug delivery field, such as how to efficiently enable the delivery and cellular uptake of poorly water soluble hydrophobic cargos. The main advantage of this technique is that it allows more specific d...