Name: preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation
Uploaded: 2017-05-22T15:00:00
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 aliquote...

<ol>
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Modulation of release rate.</a> J Control Release. 67 (2-3), 249-260 (2000).</li><li>Patel, A., Patel, M., Yang, X., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+protein+and+peptide+drug+delivery:+a+special+emphasis+on+polymeric+nanoparticles.">Recent advances in protein and peptide drug delivery: a special emphasis on polymeric nanoparticles.</a> Protein Pept lett. 21 (11), 1102-1120 (2014).</li><li>Kuai, R., Li, D., Chen, Y. E., Moon, J. 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G., F&#246;rtsch, V., Hodler, G., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+a+reconstituted+high+density+lipoprotein+for+therapeutic+applications.">Production and characterization of a reconstituted high density lipoprotein for therapeutic applications.</a> Vox Sang. 71 (3), 155-164 (1996).</li><li>Zhang, Z., Chen, J., Ding, L., Jin, H., Lovell, J. F., Corbin, I. R., Cao, W., Lo, P. C., Yang, M., Tsao, M. S., Luo, Q., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL-mimicking+peptide-lipid+nanoparticles+with+improved+tumor+targeting.">HDL-mimicking peptide-lipid nanoparticles with improved tumor targeting.</a> Small. 6 (3), 430-437 (2010).</li><li>Kim, Y., Fay, F., Cormode, D. P., Sanchez-Gaytan, B. L., Tang, J., Hennessy, E. J., Ma, M., Moore, K., Farokhzad, O. C., Fisher, E. A., Mulder, W. J., Langer, R., Fayad, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+step+reconstitution+of+multifunctional+high-density+lipoprotein-derived+nanomaterials+using+microfluidics.">Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics.</a> ACS Nano. 7 (11), 9975-9983 (2013).</li><li>Prathipati, P., Zhu, J., Dong, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+HDL-mimicking+&#945;-tocopherol-coated+nanoparticles+to+encapsulate+nerve+growth+factor+and+evaluation+of+biodistribution.">Development of novel HDL-mimicking &#945;-tocopherol-coated nanoparticles to encapsulate nerve growth factor and evaluation of biodistribution.</a> Eur J Pharm and Biopharm. 108, 126-135 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant Human Beta-NGF</td>
			<td>Creative Biomart</td>
			<td>NGF-05H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-a-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-&#945;-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 &#38; Technology</td>
			<td>16-16-120101</td>
			<td>1mg in 671 &#181;l 10 mM NH4HCO3, pH 7.4</td>
		</tr>
		<tr>
			<td>Sepharose 4B-CL</td>
			<td>Sigma</td>
			<td>CL4B200</td>
			<td>Cross-linked agarose,&#160; gel filtration chromatography column filling material</td>
		</tr>
		<tr>
			<td>Sandwich ELISA Kit for NGF</td>
			<td>R&#38;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>&#160;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 ...

Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

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 ...

The overall goal of this article is to introduce preparation and characterization methods for nerve growth factor-loaded, high-density lipoprotein-mimicking nanoparticles. Simple homogenization was used to prepare normal, high-density lipoprotein-mimicking nanoparticles. The procedure is simple and scalable.Moreover, the nanoparticle greatly improved entrapment efficiency, also NGF, and APOA1 in the nanoparticles. Various challenges to characterize a nanoparticle were overcome, including separate to the unloaded NGF from the NGF nanoparticles, conduct reliable in vitro radius studies, and the bioavailability of NGF nanoparticles. To begin, mix 10 microliters of NGF with 10 microliters of protamine USP in a 1.5 milliliter microcentrifuge tube and let it stand for 10 minutes at room temperature to form the complex.Next, add 59 microliters of phosphatidylcholine and 11 microliters of sphingomyelin to a glass vial. Also add four microliters of phosphatidylserine, 15 microliters of cholesteryl oleate, and 45 microliters of tocopheryl polyethylene glycol succinate. Mix and evaporate the ethanol under a gentle nitrogen stream for about five minutes.All excipients should form an oily thin film at the bottom of the glass vial. Add one milliliter of ultrapure water to the vial and homogenize at 9, 500 rpm for five minutes at room temperature to form the prototype nanoparticles, or NPs. Then, add the prepared complex to the prototype NPs.Incubate the suspension at 37 degrees Celsius for 30 minutes with stirring, by using a small stirring bar in the glass vial. Cool the NPs down by stirring at room temperature for another 30 minutes. After cooling, add 106 microliters or apolipoprotein A-1 and stir at room temperature overnight to form the final NGF HDL-mimicking NPs.Measure the particle size and zeta potential using a particle analyzer as per the manufacturer's instructions. A cross-linked gel filtration chromatographic column is used to separate the unloaded NGF from the NGF HDL-mimicking nanoparticles and determines entrapment efficiency of the NGF. For the column preparation, transfer 15 milliliters of sepharose 4B CL suspension to a 50 milliliter beaker.Stir the bead suspension and pour some into a column. Gently tap the column to get rid of bubbles. Allow the solvent to drain and the beads to settle for a few minutes.Continue to add the remaining suspension. Rinse the inside wall of the column to clean the beads. Drain the solvent until the solvent level is slightly above the top of the stationary phase.Then, wash and condition the column with 20 milliliters of 1X phosphate-buffered saline. To measure the entrapment efficiency of the NGF, load 200 microliters of NGF HDL-mimicking NPs onto the column and elute with 1X PBS. As before, collect a total of 12 one-milliliter fractions.Pretreat eight dialysis tubes by following the manufacturer's instructions. Add 30 milliliters of release medium to a 50 milliliter centrifuge tube. Then, warm it up to 37 degrees Celsius in a shaker with a 135 rpm shaking speed.Close the dialysis tube and place it into the release medium. Quickly withdraw 100 microliters of the release medium from outside the dialysis tube as the sample for time zero. Immediately put the sample into minus 20 degrees Celsius for later analysis.Add 100 microliters of fresh release medium into the centrifuge tube to replace the withdrawn sample. Then, start the timer. At 1, 2, 4, 6, 8, 24, 48, and 72 hours, withdraw 100 microliters of the release medium and replace it with 100 microliters of fresh medium.Immediately put the withdrawn samples into minus 20 degrees Celsius for later analysis. After a three-minute homogenization, consistent particle sizes were obtained for the prototype nanoparticles. The final NGF nanoparticles had particle sizes of 170 nanometers with a narrow and monodispersed size distribution.Unloaded NGF and nanoparticles were completely separated on the gel filtration column. Nanoparticles were eluded in fractions two through four. ELISA measurement indicated that free NGF was eluded in fraction 7 to fraction 10.The calculated entrapment efficiency of NGF was approximately 66%In vitro release of NGF nanoparticles is shown here. By adding PBS and BSA to the release medium, free NGF passed through the dialysis membrane with a recovery of over 85%Conversely, NGF nanoparticles showed a slow release profile with about 10%of NGF being released from the nanoparticles over 72 hours. NGF nanoparticles stimulated neurite outgrowth in PC12 cells to a similar extent as for free NGF.Neurite outgrowth was clearly observed under the microscope for both groups when dosed at 50 nanograms per milliliter. NGF nanoparticles considerably increased the plasma concentration and decreased the uptake of NGF in the mice liver, kidney, and spleen. HDL-mimicking nanoparticles assisted NGF, allowing for a long half-life in in vivo stability.Once mastered, monodispersed nanoparticles can be prepared in three minutes using a homogenization method if it is performed properly. While attempting this procedure, it is important to remember to use a gentle nitrogen stream to evaporate to the solvent and make sure no lipid residue sticking on the wall of the container after homogenization. After watching this video, you should have a good understanding of how to make HDL-mimicking nanoparticles using a homogenization method and how to characterize NGF loaded nanoparticles.

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