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><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

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.  Vloeibaar kristal nanodeeltje (LCNP) Nanodrager wordt benut als middel voor de gecontroleerde afgifte van een hydrofoob lading naar de plasmamembraan van levende cellen. 

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

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

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

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

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

Chemistry

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

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

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

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

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

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

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

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

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

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

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

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

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

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

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

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

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