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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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 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. ...

targeted-plasma-membrane-delivery-hydrophobic-cargo-encapsulated

Research

JoVE Journal

Bioengineering

7.5K Views.  Naval Research Laboratory. 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.

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

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...