Name: Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier (Video) | JoVE Journal
Uploaded: 2017-02-08T15:00:00.000+00:00
Duration: 10 min 16 s
Description: Learn to use liquid crystal nanoparticles for targeted delivery of hydrophobic cargos to the plasma membrane of living mammalian cells. This method enhances cellular uptake efficiency while minimizing cytotoxicity compared to traditional approaches.

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5 minutes

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 droplets of the organic material surrounded by polymerizable surfactant in water.</li>
	<li>Heat the mixture to 64 &#176;C in an oil bath to initiate the polymerization of both the cross-linking agent and surfactant as the chloroform slowly evaporates, leaving a DiO-containing NP suspension that is stabilized by the surfactant.</li>
	<li>Filter the NP suspension (3 times) through a 0.2 &#181;m syringe filter to remove any aggregation. Store the filtered NP solution at 4 &#176;C until further use.</li>
	<li>Conjugation of PEG-Chol to DiO-LCNP via EDC coupling.
	<ol>
		<li>Dissolve Chol-PEG-NH2&#183;HCl (PEG-Chol, 0.9 mM) in 25 mM HEPES buffer (pH 7.0).</li>
		<li>Prepare a working solution containing N-hydroxy-sulfosuccinimide sodium salt (NHSS, 40 mM) and 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (EDC, 400 mM) in HEPES buffer from concentrated stock solutions.</li>
		<li>Immediately add 20 &#181;l of the freshly prepared working solution of NHSS/EDC to 1.0 ml of DiO-LCNP in HEPES buffer and stir for 5 min.</li>
		<li>Add 20 &#181;l of stock solution of Chol-PEG-NH2&#183;HCl to this mixture and stir for 2 hr.</li>
		<li>Briefly centrifuge the reaction mixture at maximum speed (~2,000 x g) for 30 sec using a tabletop mini centrifuge and pass the supernatant through a PD-10 size exclusion chromatography column13 equilibrated with Dulbecco&#39;s phosphate-buffered saline (DPBS, 0.1x).</li>
	</ol>
	</li>
</ol>
2.&#160; Characterization of DiO-LCNP and DiO-LCNP-PEG-Chol
<ol>
	<li>Confirm the successful covalent conjugation of PEG-Chol to the DiO-LCNP surface by gel electrophoresis.
	<ol>
		<li>Dissolve 0.5 g of agarose in 50 ml (1x) of tris-borate electrophoresis (TBE; 89 mM Tris (pH 7.6), 89 mM boric acid, and 2 mM EDTA) buffer. Heat the solution in a microwave oven to dissolve the agarose. Allow the solution to cool slightly and pour the contents into a gel plate in the electrophoresis box. Insert a gel comb to create sample wells.</li>
		<li>Once the gel has solidified, add the required amount (enough to submerge the gel in the chamber) of TBE running buffer to the chamber.</li>
		<li>Add 35 &#181;l of DiO-LCNP sample (amended with glycerol, 5% v/v) to the wells of the gel and run for 20 min at a voltage of 110 V.</li>
		<li>Image the gel using a gel imaging system with excitation and emission filters at 488 nm and 500-550 nm, respectively.</li>
	</ol>
	</li>
	<li>Assess particle size and distribution by dynamic light scattering (DLS) by diluting the DiO-LCNP solution (~200-fold dilution) in PBS (pH ~8, 0.1x) and measuring on a DLS instrument14. Measure the zeta-potential of the DiO-LCNPs using an appropriate zeta potential measurement instrument.</li>
</ol>
3. Preparation of Cell Culture Dishes for Delivery Experiments and Imaging
NOTE: DiO-LCNP labeling is performed on HEK 293T/17 human embryonic kidney cells (between passages 5 and 15) that are cultured as described previously4. Perform the delivery experiments and the subsequent cell imaging as described below.
<ol>
	<li>Prepare 35 mm diameter tissue culture dishes (fitted with 14 mm No. 1 coverglass inserts) by coating them with bovine fibronectin (~100 &#181;l at a concentration of 20 &#181;g/ml) in DPBS for 2 hr at 37 &#176;C.</li>
	<li>Remove the fibronectin coating solution from the culture dish. Harvest HEK 293 T1/7 cells from the T-25 flask by first washing the cell monolayer with 3 ml of DPBS and then by adding 2 ml of trypsin-EDTA (0.5% trypsin-0.25% EDTA).</li>
	<li>Incubate the flask at 37 &#176;C for ~3 min. Remove the trypsin-EDTA and return the flask to the incubator. Once cells are detached from the flask, neutralize the trypsin by adding 4 ml of complete medium (Dulbecco&#39;s Modified Eagle Medium; DMEM; amended to contain 10% fetal bovine serum, 5% sodium pyruvate, and 5% antibiotic/antimycotic) to the flask. Determine the cell concentration in the suspension by counting them in a cell counter.</li>
	<li>Adjust the cell concentration to ~8 x 104 cells/ml with growth medium. Add 3 ml of the cell suspension to the dish and culture in the incubator overnight; the next day, the cells should be at ~70% confluency and should be ready for use. Adequate cell density is critical for robust and efficient labeling of a high percentage of cells.</li>
</ol>
4. Cellular Delivery of DiO and DiO-LCNPs and Imaging of Fixed Cells
<ol>
	<li>Prepare 1 ml solutions of DiO, DiO-LCNP, and DiO-LCNP-PEG-Chol in delivery medium (HEPES-DMEM; DMEM containing 25 mM HEPES) by diluting stock solutions of DiOfree and DiO-LCNPs; appropriate DiO concentrations for incubation on cells will be ~1-10 &#181;M (expressed as either the concentration of DiOfree or DiO in the form of DiO-LCNP).</li>
	<li>Remove the growth medium from the culture dishes using a serological pipette and wash the cell monolayers (see step 3) two times with HEPES-DMEM (2 ml each wash). Perform the washes by gently adding and removing HEPES-DMEM using a pipette to/from the edge of the dishes.</li>
	<li>Add 0.2 ml of the prepared DiOfree or DiO-LCNP delivery solutions to the center of the culture dishes and return the dishes to the incubator for an appropriate period of time (typically 15 or 30 min, depending on the needs of the experiment). Longer incubation times add to more nonspecific cellular labeling of non-membranous areas (e.g., cytosol).</li>
	<li>After the incubation period, remove the delivery solutions using a serological pipette. Wash the cell monolayers two times with DPBS (2 ml each wash). Perform the washes by gently adding and removing DPBS to/from the edge of the dish.</li>
	<li>Fix the cell monolayers using 4% paraformaldehyde (prepared in DPBS) for 15 min at room temperature. 
	CAUTION: Paraformaldehyde is flammable, a respiratory irritant, and a suspected carcinogen.</li>
	<li>Remove the paraformaldehyde solution using a pipette and gently wash the cells 1 time with DPBS (2 ml) by adding and removing DPBS using pipette to/from the edge of the dishes.</li>
	<li>The fixed cells are ready to be imaged for the presence of a membranous fluorescence signal using confocal laser scanning microscopy (CLSM). Perform the imaging immediately or, alternatively, replace the medium with DPBS containing 0.05% NaN3and store the dishes at 4 &#176;C. 
	CAUTION: NaN3is toxic; exercise extreme caution when using NaN3. 
	NOTE: Fixed samples stored in this manner should be imaged within 48 hr for optimum results.</li>
</ol>
5. Cellular Delivery of DiO and DiO-LCNPs and FRET Imaging in Live Cells
NOTE: In this method, cells are colabeled with 6 &#181;M each DiO-LCNP-PEG-Chol (where DiO is a FRET donor) and 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiIfree, where DiI is a FRET acceptor). The release of DiO from DiO-LCNP-PEG-Chol and its incorporation into the plasma membrane is confirmed by an observed increase in energy transfer from the DiO donor to the DiI acceptor.
<ol>
	<li>Label cells sequentially with DiO-LCNP-PEG-Chol and DiIfree using the method described in step 4.</li>
	<li>After staining, wash the cells 1 time with DPBS (2 ml) using a serological pipette and replace the wash buffer with 2 ml of live cell imaging solution (LCIS).</li>
	<li>On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope (60X objective) with a FRET imaging configuration at 30 min intervals over a 4 hr period by exciting the DiO donor at 488 nm and collecting full emission spectra of both the DiO donor and the DiI acceptor from 490-700 nm with a 32-channel spectral detector.</li>
	<li>NOTE: For more information on FRET imaging, please see reference15.</li>
	<li>Determine the time-resolved emission intensity of both the DiO donor and the DiI acceptor from cells stained with DiO-LCNP-PEG-Chol and DiI. Calculate the time-resolved acceptor/donor FRET ratio (DiIemi/DiOemi) for the images, which will steadily increase and eventually plateau once the amount of DiO donor partitioned into the membrane has reached a maximum16.</li>
</ol>
6. Cytotoxicity Assay of DiO and DiO-LCNPs to the HEK 293T/17 Cells
NOTE: The cytotoxicity of the DiO-LCNP materials is assessed using a tetrazolium dye-based proliferation assay17. Cells are cultured in a multiwell plate in the presence of varying concentrations of the materials under conditions that emulate delivery/labeling. The cells are then cultured for 72 hr to allow for proliferation. A dye (MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is then added to the wells, and metabolically active cells convert the dye into a blue formazan product. The amount of color formation is directly proportional to the number of viable cells.
<ol>
	<li>Harvest HEK 293 T1/7 cells from the T-25 flask by following the procedure described in step 3.2.Seed HEK 293T/17 cells (5,000 cells/100 &#181;l/well) to the wells of a 96-well tissue culture-treated plate and culture for 24 hr.</li>
	<li>Completely remove the culture media from the wells using a micropipette and add 50 &#181;l of HEPES-DMEM containing DiOfree, DiO-LCNPs, or DiO-LCNP-PEG-Chol at increasing concentrations to replicate wells. Incubate on the cells at 37 &#176;C for 30 min. 
	NOTE: Typically, replicates done in triplicate or quadruplicate are sufficient to yield statistically reliable data.
	<ol>
		<li>After incubation, remove the delivery medium containing the materials using a micropipette and replace it with 100 &#181;l of growth medium. Culture the cells for 72 hr.</li>
	</ol>
	</li>
	<li>Add 20 &#181;l of the tetrazolium substrate to each well, return the plate to the incubator, and allow color formation to proceed at 37 &#176;C for 4 hr. Read the absorbance (abs) of the formazan product at 570 nm (absorption minima for the DiO-LCNPs used in this study) and 650 nm (for subtraction of nonspecific background absorbance) using a microtiter plate reader.</li>
	<li>Plot the differential absorbance value (abs570 - abs650) versus material concentration and report the results as percent of control cell proliferation (degree of proliferation of cells in cell culture medium only).</li>
</ol>
7. Data Analysis
<ol>
	<li>Statistically analyze the data with a univariate analysis of variance (ANOVA). For multiple comparisons, apply the Bonferroni&#39;s post hoc test. Provide all average values as &#177; standard error of mean (SEM) unless otherwise mentioned. 
	NOTE: The acceptable probability for significance was p &#60; 0.05.</li>
</ol>

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<li>Timmers, M., Vermijlen, D., Vekemans, K., De Zanger, R., Wisse, E., Braet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracing+DiO-labelled+tumour+cells+in+liver+sections+by+confocal+laser+scanning+microscopy%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">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/pubmed?term=((Tracing+neuronal+connections+in+postmortem+human+hippocampal+complex+with+the+carbocyanine+dye+DiI%5BTitle%5D)+AND+%22Neurobiol+Aging%22%5BJournal%5D)">Tracing neuronal connections in postmortem human hippocampal complex with the carbocyanine dye DiI.</a> Neurobiol Aging. 11 (6), 649-653 (1990).</li>
<li>Köbbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+concepts+in+neuroanatomical+tracing%5BTitle%5D)+AND+%22Prog.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Dil+and+DiO%3A+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((A+study+in+developing+visual+systems+with+a+new+method+of+staining+neurones+and+their+processes+in+fixed+tissue%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Labeling+with+fluorescent+carbocyanine+dyes+of+cultured+endothelial+and+smooth+muscle+cells+by+growth+in+dye-containing+medium%5BTitle%5D)+AND+%22Histochemistry%22%5BJournal%5D)">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/pubmed?term=((Elevation+of+intracellular+glucosylceramide+levels+results+in+an+increase+in+endoplasmic+reticulum+density+and+in+functional+calcium+stores+in+cultured+neurons%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGarrett%2C+RH+%22Biochemistry%22">Biochemistry</a>. , 5th, Brooks/Cole Cengage Learning. Belmont, CA. (2013).</li>
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<li>Chen, H., Kim, S., Li, L., Wang, S., Park, K., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Release+of+hydrophobic+molecules+from+polymer+micelles+into+cell+membranes+revealed+by+Fo%CC%88rster+resonance+energy+transfer+imaging%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6596-6601 (2008).</li>
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The authors declare that they have no competing financial interests.

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><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&amp;prime;-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&amp;prime;&amp;#8288;,3&amp;prime;-tetramethylindocarbocyanine perchlorate (DiI)</td><td>Sigma Aldrich</td><td>468495-100MG</td><td>Hazardous; make stock solution in DMSO</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ThermoFisher</td><td>21063045</td><td>Warm in 37 &amp;deg;C before use</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>ThermoFisher</td><td>14040182</td><td>Warm in 37 &amp;deg;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 1 mg/ml using DPBS. Use 20-30 &amp;micro;g/ml for coating MetTek dish, 2 hr at 37 &amp;deg;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&amp;reg; CRL-11268&amp;trade;</td><td></td></tr><tr><td>Live cell imaging solution (LCIS)</td><td>ThermoFisher</td><td>A14291DJ</td><td>Warm in 37 &amp;deg;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 &amp;deg;C before use</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-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%)&amp;nbsp;</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&amp;trade; 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

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...

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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Learn to use liquid crystal nanoparticles for targeted delivery of hydrophobic cargos to the plasma membrane of living mammalian cells. This method enhances cellular uptake efficiency while minimizing cytotoxicity compared to traditional approaches.

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

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