Encapsulating Nerve Growth Factor in High-Density Lipoprotein-Mimicking Nanoparticles

Abstract

Source: Zhu, J., et. al., Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation. J. Vis. Exp. (2017)

This video demonstrates the stepwise assembly of NGF-loaded HDL-mimicking nanoparticles using protamine-assisted encapsulation and lipid-based self-assembly for potential neurotrophic delivery applications.

Protocol

1. Preparation of NGF HDL-mimicking Nanoparticles

1. Dissolve the excipients, phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), cholesteryl oleate (CO), and D-α-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 °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 °C. The TPGS solution was stable for at least 12 months at -20 °C.
2. Mix 10 µL of NGF (1 mg/mL in water) with 10 µL 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 °C. Repeated freezing and thawing are not recommended for the NGF stock.
3. Add 59 µL of PC, 11 µL of SM, 4 µL of PS, 15 µL of CO, and 45 µL 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.
4. 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 nanoparticles (NPs).
5. Add the complex prepared in step 1.2 to the prototype NPs and incubate at 37 °C for 30 min with stirring by using a small stirring bar in the glass vial.
6. Cool the NPs down by stirring at room temperature for another 30 min. After this cools, add 106 µL of Apo A-I (1.49 mg/mL) and stir at room temperature overnight to form the final NGF HDL-mimicking NPs.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Recombinant Human Beta-NGF	Creative Biomart	NGF-05H
L-a-Phosphatidylcholine (PC)	Avanti	131601P	95%, Egg, Chicken
Sphingomyelin (SM)	Avanti	860062P	Brain, Porcine
Phosphatidylserine (PS)	Avanti	840032P	Brain, Porcine
Cholesteryl oleate (CO)	Sigma	C9253
D-α-Tocopheryl polyethylene glycol succinate (TPGS)	BASF	9002-96-4	Vitamin E Polyethylene Glycol Succinate
Protamine sulfate	Sigma	P3369	meets USP testing specifications
Apolipoprotein A1, Human plasma	Athens Research & Technology	16-16-120101	1mg in 671 µl 10 mM ammonium bicarbonate, pH 7.4
Homogenizer	Tekmar	T 25-S1
Polytron homogenizer	Kinematica	PT 1200C