Name: solid lipid nanoparticles (slns) for intracellular targeting applications
Uploaded: 2015-11-17T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1. Processing of SLNs
<ol>
	<li>Synthesis of SLNs 
	Note: Use the PIT method 1,17,20 to prepare the sub-20 nm SLNs 1.
	<ol>
		<li>Use aseptic technique, bioreagents or cell culture grade reagents, and sterile materials (i.e., pipettes, spatulas, vials, etc.).</li>
		<li>Use a biosafety cabinet for the synthesis of SLNs (Figure 2).</li>
		<li>Combine 0.6 mg of fluorescent dye (Nile Red (NiR), NiR concentration is approximately 0.3 mg/ml) and 0.10 g of Heneicosane (Lipid, 5 wt/wt% lipid concentration) into a vial (15 ml), co-melt at 90 &#176;C, and stir.</li>
		<li>Add 0.11 g polyoxyethylene (10) oleyl ether (Surfactant, 5.5 wt/wt% surfactant concentration). Co-melt the resulting mixture at 90 &#176;C and stir.</li>
		<li>Add 1.79 g of sterile water to the mixture, heat to 90 &#176;C, and stir until a transparent nanoemulsion is formed. 
		Note: Under continued stirring and controlled cooling, SLNs are created. These conditions cause an inversion of the water-in-oil emulsion to an oil-in-water emulsion. Lipophilic molecules such as NiR partition to the lipid droplets.</li>
		<li>Prepare a nanoemulsion in parallel without adding the fluorescent dye, in order to serve as a control sample. Combine 0.10 g of Heneicosane and 0.11 g polyoxyethylene (10) oleyl ether into a vial (15 ml) co-melt at 90 &#176;C, and stir.
		<ol>
			<li>Add 1.79 g of sterile water to the mixture, heat to 90 &#176;C, and stir until a transparent nanoemulsion is formed.</li>
		</ol>
		</li>
		<li>Further sterilize each nanoemulsion using a sterile 0.2 &#181;m filter.</li>
	</ol>
	<img alt="Figure 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> 
	Figure 2. Schematic of synthesis of SLNs. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
	<li>Particle Size and Polydispersity
	<ol>
		<li>Use dynamic light scattering (DLS) to measure the particle size and polydispersity of the SLNs 1 using a glass cuvette and an instrument designed to measure particle size.</li>
		<li>Prior to the measurements of the samples, measure the particle sizes of two known standards following the manufacturer&#8217;s protocol for the standards preparation and measurement.</li>
		<li>Fill the glass cuvette with 1.5 ml of standard solution and measure the standard solution using the same measurement conditions as the samples. 
		Note: Previous step was repeated for each standard.</li>
		<li>Measure the samples as prepared. For each, use the following experimental parameters: a run time of 100 sec, refractive index of water (1.33), viscosity of water at 20 &#176;C (1.002 mPa&#183;sec). 
		Note: This technique uses frequency shifted light to measure the size of the nanoparticles.</li>
		<li>Report the average particle diameter and polydispersity of particle size distribution.</li>
	</ol>
	</li>
	<li>Melting Point and Latent Heat of Melting
	<ol>
		<li>Use a differential scanning calorimeter (DSC) equipped with an auto-sampler and liquid nitrogen cooling source to investigate the thermal behavior of SLNs 1.</li>
		<li>Pipette the as-prepared SLNs into a 40 &#181;l aluminum pan with a mass of approximately 25 mg and hermetically seal the pan using a universal crimper press to minimize moisture loss during the DSC scan.</li>
		<li>Measure each nanoemulsion from 5 to 80 &#176;C.</li>
		<li>Report the melting point and the latent heat of melting from the DSC plot, where the valley represents the melting point and the integral of the area under the curve in the DSC plot divided by the amount of material represents the latent heat of melting.</li>
	</ol>
	</li>
</ol>
2. SLNs Interaction with Fibroblasts and Dendritic Cells
<ol>
	<li>Culture of Primary Human Fibroblasts
	<ol>
		<li>Culture primary human fibroblasts according to the manufacturer&#8217;s instructions in liquid medium for the culture of human dermal fibroblasts (medium 106), supplemented with the low serum growth supplement (LSGS) kit. Maintain cells in a humidified atmosphere at 37 &#176;C, 5% CO2 and subculture upon reaching 80% confluence.</li>
		<li>For both MTT and imaging analysis, seed cells in a 96-well plate, at a density of 2 x 104 cells per well, and allow to equilibrate at 37 &#176;C O/N prior to SLN exposure.</li>
		<li>At time zero, dilute SLNs in complete medium as indicated (Figure 4), with respect to lipid concentration, and add 10 &#956;l per well to each replicate well in the 96-well plate.</li>
		<li>Maintain cells at 37 &#176;C, 5% CO2, during the incubation period.</li>
	</ol>
	</li>
	<li>MTT Toxicity Assay
	<ol>
		<li>After 24 hr of incubation with SLNs, determine cellular viability using the MTT assay, according to the manufacturer&#8217;s protocol.</li>
		<li>Wash cells once and add fresh medium to each well (100 &#956;l/well). Kill control wells by incubating in a 70% methanol solution for 10 min before replacing with fresh medium.</li>
		<li>Add MTT reagent (10 &#956;l per well) to each well of the microplate and incubate O/N at 37 &#176;C.</li>
		<li>After O/N incubation, solubilize intracellular formazin crystals with the provided detergent solution (100 &#956;l per well) according to manufacturer&#8217;s protocol.</li>
		<li>After 3 hr of incubation at RT, obtain absorbance values at a measurement wavelength of 570 nm using a microplate reader. 
		Note: MTT reagent used should be less than 1% (w/v) 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyl-tetrazolium bromide.</li>
	</ol>
	</li>
	<li>Fluorescence Microscopy
	<ol>
		<li>At specific time points following dosing of fibroblasts with SLN, wash the fibroblasts twice with sterile phosphate buffered saline (PBS) and fix for 10 min with cold 70% methanol.</li>
		<li>After 10 min, replace the methanol with PBS and capture images using an inverted microscope, using a band pass (BP) 690/50 nm filter set.</li>
	</ol>
	</li>
	<li>Culture of Mouse Bone Marrow Dendritic Cells
	<ol>
		<li>Induce mouse bone marrow-derived dendritic cells (BMDC) as previously described 23,24. Briefly, plate single cell suspensions of C57BL/10 bone marrow in 100 mm Petri dishes at 2 x 106/plate with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 300 U/ml) and recombinant murine IL-4 (B cell stimulating factor, 200 U/ml). Change media every 3 days.</li>
		<li>On day 7, add NiR-loaded SLNs to the dishes at a final concentration of 0.5 and 5.0 &#181;g/ml lipid and maintain the cells in a 37 &#176;C, 5% CO2 incubator for the desired length of exposure.</li>
		<li>Collect cells from the dishes by aspirating all the media in the plate, collecting it in a 50 ml tube, followed by thorough washing of the plate (to dislodge loosely adherent cells) with 5 ml of phosphate buffered saline (PBS) containing 4 mM ethylenediaminetetraacetic acid (EDTA). Collect the obtained cell suspension in the same 50 ml tube.</li>
	</ol>
	</li>
	<li>Assessment of SLNs Incorporation via Flow Cytometry
	<ol>
		<li>Determine the concentration of the cell suspension obtained in point 2.4.3 and distribute 3x105 cells into a 12 mm x&#160;75&#160;mm round bottom FACS tube.</li>
		<li>Wash the cells by adding 1 ml of PBS and then centrifuging the tubes for 5 min at 400 x g, at a temperature of 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet with 100 &#181;l of PBS/2 mM EDTA/0.1% bovine serum albumin (BSA) (staining buffer) containing anti-CD11c-FITC monoclonal antibody (0.25 &#956;g/ml final). Incubate for 30 min on ice in the dark.</li>
		<li>Wash the cells with 1 ml of PBS and then centrifuge the tubes for 5 min at 400 x g, at a temperature of 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each pellet with 400 &#181;l of staining buffer. The cells are now ready for acquisition of the samples with a flow cytometer (possibly equipped with blue and red lasers).</li>
		<li>Using flow cytometry analysis software, assess the level of incorporation of SLNs by DC by gating on the population of CD11c+ cells (FITC positive) and comparing the intensity of SLNs-associated fluorescence (measured in the PerCP-Cy5.5 or APC channels).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this study, the synthesis of SLNs and their applicability for intracellular targeting applications were explored. These biocompatible nanoparticles have shown promise as delivery vehicles for multiple applications including drug delivery, gene silencing, and vaccine technologies 25-30. Ultra-small SLNs were synthesized using a facile process, and their interactions with primary skin cells and primary immune cells were explored. SLNs were designed to include encapsulation of a fluorescent dye (NiR), which se...

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. These vehicles can be loaded with drugs, proteins, and nucleic acids designed to impact cellular responses and achieve a desired effect in target tissues. Many types of nanocarriers have been explored for therapeutic and diagnostic benefit including lipids, polymers, silicon, and magnetic materials. These systems are attractive due to their potential for localized drug delivery, increased therapeutic concentration in target tissues, and reduction of systemic toxicity.
Solid lipid nanoparticles (SLNs) are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle in recent years. SLNs can be readily formulated for multiple applications including bio-sensing 1, cosmetics 2, and therapeutic delivery 3-7. Their utility stems from the fact that they are comprised entirely of resorbable, nontoxic lipids, resulting in enhanced biocompatibility. During synthesis, lipophilic drugs can be incorporated into SLN vehicles, thereby increasing drug solubility and suitability for parenteral administration. SLN vehicles also help to stabilize encapsulated therapeutics, reducing their degradation and clearance, and maximizing therapeutic action. These vehicles are particularly well suited for long acting, controlled-release preparations due to their stability at body temperature 3,4,8,9. Importantly, encapsulation of drugs in lipid nanoparticles alters the intrinsic pharmacokinetic profiles of the drug molecules. This provides a potential advantage by allowing the controlled release of drugs with a narrow therapeutic index. The release rate of SLN-incorporated therapeutics can be tuned based on the lipid degradation rate or the drug diffusion rate in the lipid matrix.
SLNs are often engineered to accumulate in specific target tissues. For example, their size (typically greater than 10 nm) potentiates retention in the circulation, where the leaky vasculature of tumor tissue facilitates deposition. In addition, the route of particle administration has been shown to alter biodistribution with the potential to target specific physiological structures such as lymph nodes 10,11. Upon deposition in target tissues, achieving appropriate cellular interactions and eventual internalization of nanoparticles is challenging due to the ability of cell membranes to selectively control the flow of ions and molecules into and out of the cell 12. To facilitate cellular uptake, it is possible to modify nanocarriers with specific ligands including peptides, small molecules, and monoclonal antibodies13,14. Several mechanisms including both passive penetration and active transport of nanoparticles across the cell membrane have been previously described 3,12,15. In general, it has been demonstrated that cell-nanoparticle interactions are influenced by the physicochemical properties of the nanoparticles including size, shape, surface charge and surface chemistry, in addition to cell-specific parameters such as cell type or cell cycle phase 12.
A previous investigation demonstrated the synthesis of sub-10 nm SLNs for topical 16 and biomarker detection applications 1 using the phase inversion temperature (PIT) method 17. This is a gentle synthesis method where 2 the composition remains constant while the temperature is gradually changed. Continuous stirring of the heated solution, as it cools to RT results in a nanoemulsion. This process results in the synthesis of SLNs with smaller particle size1 than that previously reported using various methods for the synthesis of lipid nanoparticles17-22. The resulting size scale, less than 20 nm, provides an advantage for intracellular targeting applications due to increased surface area and the potential for enhanced cellular interactions.
A schematic of SLNs, designed to deliver a fluorescent dye or therapeutic, is shown in Figure 1. The SLNs consist of a lipid interior (e.g., linear alkane) allowing the incorporation of lipophilic compounds (e.g., dyes or therapeutics) and a surfactant exterior (e.g., linear nonionic surfactant) surrounded by water. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. Primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time in order to characterize interactions with respect to toxicity and particle uptake. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels. Fluorescence microscopy and flow cytometry were two methods employed to examine particle uptake in vitro.
<img alt="Figure 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> 
Figure 1. Schematic of SLN showing the major constituents. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Nile Red (NiR)</td><td>Sigma</td><td>19123</td><td>BioReagent, suitable for fluorescence, &amp;ge;98.0%</td></tr><tr><td>Heneicosane</td><td>Aldrich</td><td>286052</td><td>98%</td></tr><tr><td>Brij O10</td><td>Sigma</td><td>P6136</td><td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td>Sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>Syringe Filter 0.2 &amp;micro;m Supor Membrane Low Protein Binding</td><td>Life Sciences</td><td>PN4612</td><td>Non-Pyrogenic</td></tr><tr><td>Nanotrac Ultra&amp;nbsp;</td><td>Microtrac</td><td>serial number U1985IS</td><td>Instrument</td></tr><tr><td>Differential Scanning Calorimeter</td><td>Mettlet-Toledo</td><td></td><td>Instument</td></tr><tr><td>Primary human fibroblasts&amp;nbsp;</td><td>Life Technologies</td><td>C-004-5C</td><td>Neonatal (HDFn)</td></tr><tr><td>Medium 106</td><td>Life Technologies</td><td>M-106-500</td><td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td></tr><tr><td>Low Serum Growth Supplement Kit (LSGS Kit)</td><td>Life Technologies</td><td>S-003-K</td><td>All the components of complete LSGS</td></tr><tr><td>MTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4890-025-K</td><td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td></tr><tr><td>Safire2 microplate reader</td><td>Tecan</td><td>----</td><td>Instrument</td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Sigma</td><td>P5493</td><td>For molecular biology</td></tr><tr><td>Recombinant murine GM-CSF&amp;nbsp;</td><td>Peprotech</td><td>315-03</td><td>&gt;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td></tr><tr><td>Recombinant murine IL-4&amp;nbsp;</td><td>Peprotech</td><td>214-14</td><td>&gt;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td></tr></tbody></table>

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.

The PIT method was used to synthesize the SLNs and the phase inversion temperature was determined utilizing a water bath. The samples were slowly heated and gently agitated until the solution appeared clear. The phase inversion temperature for the SLNs made using heneicosane lipid is 45 &#176;C. Table 1 summarizes the particle size, polydispersity, melting point and latent heat of melting for the SLNs. The SLNs synthesized using the processing conditions described above resulted in control SLNs and NiR-l...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

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.

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 ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

16.9K Views.  The Johns Hopkins Applied Physics Laboratory. 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. 

Video: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

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

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 ...

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.

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. ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

Biochemistry

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 ...

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

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

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

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

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Summary

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Chapters in this video

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

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

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

Formulation and Characterization of Bioactive Agent Containing Nanodisks

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

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

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

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

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy