Name: preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation
Uploaded: 2017-05-22T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

This work was supported by NIH R03 NS087322-01 to Dong, X.

NOTE: The animal studies included in all procedures have been approved by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center.
1. Preparation of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Dissolve the excipients, phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), cholesteryl oleate (CO), and D-&#945;-tocopheryl polyethylene glycol succinate (TPGS), in ethanol to prepare stock solutions at 1 mg/mL. 
	NOTE: The stock solutions were aliquoted and stored at -20 &#176;C. The cholesteryl oleate was stored in dark bottles. The PC, SM, PS, and CO stock solutions were stable for up to 6, 3, 12, and 12 months, respectively, at -20 &#176;C. The TPGS solution was stable for at least 12 months at -20 &#176;C.</li>
	<li>Mix 10 &#181;L of NGF (1 mg/mL in water) with 10 &#181;L of protamine USP (1 mg/mL in water) in a 1.5 mL microcentrifuge tube and let it stand for 10 min at room temperature to form the complex. 
	NOTE: The NGF and protamine stock solutions were aliquoted and stored at -20 &#176;C. Repeated freezing and thawing is not recommended for the NGF stock.</li>
	<li>Add 59 &#181;L of PC, 11 &#181;L of SM, 4 &#181;L of PS, 15 &#181;L of CO, and 45 &#181;L of TPGS to a glass vial. Mix and evaporate the ethanol under a gentle nitrogen stream for about 5 min; all excipients should form an oily, thin film at the bottom of the glass vial.</li>
	<li>Add 1 mL of ultrapure (type 1) water to the vial and homogenize at 9,500 rpm (8,600 x g) for 5 min at room temperature to form the prototype NPs.</li>
	<li>Add the complex prepared in step 1.2 to the prototype NPs and incubate at 37 &#176;C for 30 min with stirring by using a small stirring bar in the glass vial.
	<ol>
		<li>Cool the NPs down by stirring at room temperature for another 30 min. After this cools, add 106 &#181;L of Apo A-I (1.49 mg/mL) and stir at room temperature overnight to form the final NGF HDL-mimicking NPs.</li>
	</ol>
	</li>
</ol>
2. Characterization of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Measure the particle size and zeta potential using a particle analyzer (see the Materials Table) as per the manufacturer&#39;s instructions.</li>
	<li>Use a cross-linked agarose gel filtration chromatography column to separate the unloaded NGF from the NGF HDL-mimicking NPs and determine the entrapment efficiency of the NGF.
	<ol>
		<li>For the column preparation, transfer 15 mL of Sepharose 4B-CL suspension to a 50-mL beaker. Stir the bead suspension using a glass rod and pour some into a column (30 cm length &#215; 1 cm diameter, with a glass frit at the bottom). Gently tap the column to get rid of bubbles. Allow the solvent to drain and the beads to settle for a few min.</li>
		<li>Continue to add the remaining suspension. Rinse the inside wall of column to clean the beads. Drain the solvent until the solvent level is slightly above the top of the stationary phase. Wash and condition the column with 20 mL of 1x phosphate-buffered saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4).</li>
		<li>To determine the fractions containing unloaded NGF, load 200 &#181;L of NGF solution (10 &#181;g/mL) onto the gel filtration column (25 cm length x 1 cm diameter) and elute with 1x PBS.</li>
		<li>Collect a total of 12 fractions (1 mL for each fraction) and measure the concentration of NGF in each fraction using a Sandwich ELISA kit for NGF.</li>
		<li>Detect NGF from fractions 6 to 10 using the Sandwich ELISA kit. Obtain a chromatogram of unloaded NGF on the column. Wash the column with 20 mL of PBS.</li>
		<li>To determine the fractions containing NGF HDL-mimicking NPs, load 200 &#181;L of the NPs onto the column and elute with 1x PBS. Collect a total of 12 fractions (1 mL for each fraction) and measure the intensity of particles in each fraction using the particle size analyzer. Detect the NGF NPs from fractions 2 to 4. 
		NOTE: The intensity is a parameter measured by the particle analyzer, which tells how many nanoparticles may exist in the test solution. The more NPs exist in solution, the higher the intensity is. By this approach, we can confirm that the column can separate NGF NPs from unloaded NGF.</li>
		<li>To measure the entrapment efficiency of NGF, load 200 &#181;L of NGF HDL-mimicking NPs onto the column and elute with 1x PBS. Collect a total of 12 fractions (1 mL for each fraction).</li>
		<li>Measure the unloaded NGF concentration from fractions 6 to 10 using the Sandwich ELISA kit.</li>
		<li>Add the NGF amounts measured from fractions 6 to 10 together as the unloaded NGF and calculate the entrapment efficiency of NGF using the following equation. 
		%EE = (1 - unloaded NGF/total NGF added into NP) x 100% Equation (1)</li>
	</ol>
	</li>
</ol>
3. In Vitro Release of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Study free NGF (10 &#181;g/mL in water; n = 4) and NGF HDL-mimicking NPs (10 &#181;g/mL; n = 4) in parallel.</li>
	<li>Pre-treat 8 dialysis tubes (molecular weight cutoff: 300 kDa) by following the manufacturer&#39;s instructions.</li>
	<li>Prepare 5% bovine serum albumin (BSA) in PBS as the release medium. Add 30 mL of release medium to a 50-mL centrifuge tube and warm it up to 37 &#176;C in a shaker with a 135 rpm shaking speed. Place another 50 mL of release medium in the shaker for replacement.</li>
	<li>Add 400 &#181;L of release medium into the dialysis tube and quickly add 200 &#181;L of the tested sample to make enough volume for dialysis.</li>
	<li>Close the tube and quickly put the dialysis tube into the release medium (the centrifuge tube). Quickly withdraw 100 &#181;L of the release medium from the outside dialysis tube as the sample for time 0. Immediately put the sample into -20 &#176;C for later analysis. Add 100 &#181;L of fresh release medium into the centrifuge tube to replace the withdrawn sample. Start the timer.</li>
	<li>At 1, 2, 4, 6, 8, 24, 48, and 72 h, withdraw 100 &#181;L of the release medium and replace it with 100 &#181;L of fresh medium. Immediately put the withdrawn samples into -20 &#176;C for later analysis.</li>
	<li>After 72 h, take out all samples from -20 &#176;C and thaw them at room temperature. Measure the NGF concentrations of the release samples using the Sandwich ELISA kit.</li>
</ol>
4. Bioactivity of NGF HDL-mimicking Nanoparticles (Neurite Outgrowth Study)
<ol>
	<li>Culture PC12 cells in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 &#181;g/mL streptomycin, and 100 units/mL penicillin. Maintain the cells in a humidified incubator at 37 &#176;C and with 5% CO2.</li>
	<li>When the cells reach ~70-80% confluence, remove the culture medium and wash the cells with 1x PBS (approximately 2 mL per 10 cm2 culture surface area). Remove the wash solution. Trypsinize the cells by adding 0.25% Trypsin-EDTA solution (0.25% trypsin and 1 mM EDTA; 0.5 mL per 10 cm2 culture surface area) to the flask and incubate at 37 &#176;C until most cells are detached.</li>
	<li>Add two volumes of culture medium and spin down at 180 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 5 mL of culture medium. Filter the cells through a 22 G, &#189; inch needle to break cell clusters.</li>
	<li>Split the cells at a ratio of 1:3 into a new cell culture flask. After incubating overnight, remove unattached PC12 cells. Allow the attached cells to remain for further growth.</li>
	<li>Repeat this procedure for 3 passages to select the subculture of PC12 that has a strong adhesion property. Pre-coat a 6-well plate with 800 &#181;L/well of rat tail collagen type I (100 &#181;g/mL).</li>
	<li>Trypsinize the selected PC12 cells as described in step 4.2 and filter them through a 22 G, &#189; inch needle to break the cell clusters. Count the cells with a hemocytometer. Seed the cells overnight at a density of 10,000 cells/well in the pre-coated 6-well plate in step 4.5 to allow the cells to attach to the plate.</li>
	<li>Dilute free NGF (10 &#181;g/mL) and NGF HDL-mimicking NPs (10 &#181;g/mL) with the culture medium (described in step 4.1) to prepare 0.5, 1, 5, 10, 50, and 100 ng/mL concentrations. Remove 2 mL of medium from each well and replace with 2 mL of the diluted free NGF or NGF HDL-mimicking NPs. Culture for 4 days.</li>
	<li>On day 4, change the medium to fresh medium (described in step 4.1) containing the corresponding treatment and continue the treatment for another 3 days.</li>
	<li>On day 7, visualize the cells with an inverted light microscope and image each well at random spots under 10X magnification.</li>
</ol>
5. Biodistribution of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Use adult BALB/c mice (male, 25-30 g) to test the tissue distribution of NGF NPs. Randomly divide the mice into three groups of 3 mice per group. Use a cone-shaped plastic restraint (e.g., decapicone) to restrain a mouse and wipe the tail with ethanol to promote vasodilation and the visibility of the vein.</li>
	<li>Inject 100 &#181;L of either saline, free NGF, or NGF HDL-mimicking NPs to each group of mice (3 groups) through the tail veins at a dose of 40 &#181;g/kg of NGF. Use a 30&#189; G needle attached to a 1 mL syringe.</li>
	<li>At 30 min after the injection, anesthetize the mice using 3% inhaled isoflurane (in oxygen at flow rate of 2 L/min). Perform a tail and toe pinch to determine the depth of anesthesia. Collect blood by cardiac puncture.
	<ol>
		<li>Withdraw approximately 1 mL of blood from the heart. Euthanize each mouse by cervical dislocation.</li>
	</ol>
	</li>
	<li>Place the mouse carcass in dorsal recumbency. Open the abdomen using surgical scissors and move fat and intestine aside using a cotton swab to expose the liver, spleen, and kidney. Harvest these tissues and rinse them in 1x PBS to clean the blood.</li>
	<li>Immediately centrifuge the blood samples at 3,400 x g and 4 &#176;C for 5 min to obtain the plasma. Store the plasma and tissues at -80 &#176;C until the analyses.</li>
	<li>To analyze the tissue samples, move the samples from -80 &#176;C to 4 &#176;C and suspend 100 mg of tissue sample in a 10x volume of extraction buffer (0.05 M sodium acetate, 1.0 M sodium chloride, 1% triton X-100, 1% BSA, 0.2 mM phenylmethanesulfonyl fluoride, and 0.2 mM benzethonium chloride). Homogenize at 10,000 rpm and 4 &#176;C for 5 min.</li>
	<li>Sacrifice two untreated mice to collect blank plasma and tissues, as described in steps 5.3 and 5.4. Prepare NGF standard solutions using blank plasma or blank tissue homogenates.</li>
	<li>Determine the concentrations of NGF in the plasma and tissue homogenates using the Sandwich ELISA kit, as described above.</li>
</ol>

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The authors have nothing to disclose.

In this study, we demonstrate a simple method to prepare HDL-mimicking NPs for NGF encapsulation. Various NP delivery systems have been studied to deliver proteins. Currently, many NP preparations involve dialysis, solvent precipitation, and film hydration. These processes are generally complicated and challenging upon scale-up. During this NP development, it was determined that the lipids had strong adhesion to the glass wall of the container, which led to the difficulties in hydrating the thin film and efficiently mixi...

Macromolecules, such as proteins, peptides, and nucleic acids, have been emerging as promising medications and have gained considerable attention in past decades1,2. Due to their high efficacy and specific action modes, they exhibit great therapeutic potential for the treatments of cancer, immune disease, HIV, and related conditions3,4. However, physiochemical properties, such as their large molecular size, three-dimensional structure, surface charges, and hydrophilic nature, make the in vivo delivery of these macromolecules very challenging. This considerably impedes their clinical use4. Recent advancements in drug delivery systems, such as microparticles, polymer nanoparticles (NPs), liposomes, and lipid NPs, overcame these challenges and significantly improved the in vivo delivery of macromolecules. However, some drawbacks regarding these delivery cargoes have been revealed, including low drug loading capacity, low entrapment efficiency, short half-life, loss of bioactivity, and undesirable side effects5,6,7,8. Effective carrier systems remain an area of research interest. Moreover, the development of analytical methods to characterize drug-loaded NPs is more challenging for macromolecules than for small molecules.
High-density lipoprotein (HDL) is a natural NP composed of a lipid core that is coated by apolipoproteins and a phospholipid monolayer. Endogenous HDL plays a critical role in the transport of lipids, proteins, and nucleic acids through its interaction with target receptors, such as SR-BI, ABCAI, and ABCG1. It has been explored as a vehicle for the delivery of different therapeutic agents9,10,11,12. Various methods have been developed to prepare HDL-mimicking NPs. Dialysis is a popular approach. In this method, NPs are formed by hydrating a lipid film using sodium cholate solution. The salt is then removed through a 2-day dialysis with three buffers13. Sonication methods fabricate NPs by sonicating a lipid mixture for 60 min under a heating condition; the NPs are further purified through gel chromatography14. Microfluidics generates NPs via a microfluidic device, which mixes phospholipids and apolipoprotein A-I (Apo A-I) solutions by creating microvortices in a focusing pattern15. Clearly, these methods can be time consuming, harsh, and difficult for industrial scale-up.
In this article, we introduce the preparation and characterization of novel HDL-mimicking NPs for nerve growth factor (NGF) encapsulation. NGF is a disulfide-linked polypeptide homodimer containing two 13.6-kDa polypeptide monomers. A novel procedure to prepare the NPs by homogenization, followed by the encapsulation of NGF into the NPs, was developed. The NGF HDL-mimicking NPs were characterized for particle size, size distribution, zeta potential, and in vitro release. Their bioactivity was evaluated for neurite outgrowth in PC12 cells. The biodistribution of NGF HDL-mimicking NPs was compared with that of free NGF after intravenous injection in mice.

<table><tbody><tr><td>Recombinant Human Beta-NGF</td><td>Creative Biomart</td><td>NGF-05H</td><td></td></tr><tr><td>L-&amp;alpha;-Phosphatidylcholine (PC)</td><td>Avanti</td><td>131601P</td><td>95%, Egg, Chicken</td></tr><tr><td>Sphingomyelin (SM)</td><td>Avanti</td><td>860062P</td><td>Brain, Porcine</td></tr><tr><td>Phosphatidylserine (PS)</td><td>Avanti</td><td>840032P</td><td>Brain, Porcine</td></tr><tr><td>Cholesteryl oleate (CO)</td><td>Sigma</td><td>C9253</td><td></td></tr><tr><td>D-&amp;alpha;-Tocopheryl polyethylene glycol succinate (TPGS)</td><td>BASF</td><td>9002-96-4</td><td>Vitamin E Polyethylene Glycol Succinate</td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369</td><td>meets USP testing specifications</td></tr><tr><td>Apolipoprotein A1, Human plasma</td><td>Athens Research &amp;amp; Technology</td><td>16-16-120101</td><td>1 mg in 671 &amp;micro;L 10 mM NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;, pH 7.4</td></tr><tr><td>Sepharose 4B-CL</td><td>Sigma</td><td>CL4B200</td><td>Cross-linked agarose,&amp;nbsp; gel filtration chromatography column filling material</td></tr><tr><td>Sandwich ELISA Kit for NGF</td><td>R&amp;amp;D system</td><td>DY008</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>GE Healthcare Life Science</td><td>SH30096.02</td><td></td></tr><tr><td>Horse serum</td><td>GE Healthcare Life Science</td><td>SH30074.03</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>PC12 cells</td><td>ATCC</td><td>CRL-1721</td><td></td></tr><tr><td>Rat tail collagen type I</td><td>Sigma</td><td>C3867</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>S2889</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>31414</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Benzethonium chloride</td><td>Sigma</td><td>B8879</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Homogenizer</td><td>Tekmar</td><td>T 25-S1</td><td></td></tr><tr><td>Delsa Nano HC particle analyzer</td><td>Beckman-Coulter</td><td>Delsa Nano HC</td><td></td></tr><tr><td>Float-A-Lyzer G2 Dialysis Device</td><td>Spectrum Laboratories</td><td>G235036</td><td>Molecule Cutoff 300 kDa</td></tr><tr><td>Centrifuge</td><td>Eppendoff</td><td>5424R</td><td></td></tr><tr><td>Polytron homogenizer</td><td>Kinematica</td><td>PT 1200C</td><td></td></tr><tr><td>DecapiCone</td><td>&amp;nbsp;Braintree Scientific Inc.</td><td>DC-M200</td><td></td></tr></tbody></table>

preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

The engineering scheme of HDL-mimicking, &#945;-tocopherol-coated NGF NPs prepared by an ion-pair strategy is shown in Figure 1. To neutralize the surface charges of NGF, protamine USP was used as an ion-pair agent to form a complex with NGF. To protect the bioactivity, prototype HDL-mimicking NPs were engineered, first using homogenization; then, the NGF/protamine complex was encapsulated into the prototype NPs. Homogenization provided sufficient energy and successfully ...

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Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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8.3K Views.  University of North Texas Health Science Center. Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article. 

Video: Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemistry

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

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

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Cancer Research

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 ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

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 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 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Summary

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Chapters in this video

Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

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

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

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

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

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

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

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells