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

Summary

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.

Abstract

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.

Introduction

Macromolecules, such as proteins, peptides, and nucleic acids, have been emerging as promising medications and have gained considerable attention in past decades^1,2. Due to their high efficacy and specific action modes, they exhibit great therapeutic potential for the treatments of cancer, immune disease, HIV, and related conditions^3,4. 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. ....

Protocol

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

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,....

Results

The engineering scheme of HDL-mimicking, α-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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Recombinant Human Beta-NGF	Creative Biomart	NGF-05H
L-α-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	1 mg in 671 µL 10 mM NH4HCO3, pH 7.4
Sepharose 4B-CL	Sigma	CL4B200	Cross-linked agarose,  gel filtration chromatography column filling material
Sandwich ELISA Kit for NGF	R&D system	DY008
Bovine Serum Albumin	Sigma	A2153
RPMI-1640 medium	GE Healthcare Life Science	SH30096.02
Horse serum	GE Healthcare Life Science	SH30074.03
Fetal bovine serum	Gibco	10082147
PC12 cells	ATCC	CRL-1721
Rat tail collagen type I	Sigma	C3867
Sodium acetate	Sigma	S2889
Sodium chloride	Sigma	31414
Triton X-100	Sigma	T8787
Phenylmethanesulfonyl fluoride (PMSF)	Sigma	P7626
Benzethonium chloride	Sigma	B8879
Name	Company	Catalog Number	Comments
Equipment
Homogenizer	Tekmar	T 25-S1
Delsa Nano HC particle analyzer	Beckman-Coulter	Delsa Nano HC
Float-A-Lyzer G2 Dialysis Device	Spectrum Laboratories	G235036	Molecule Cutoff 300 kDa
Centrifuge	Eppendoff	5424R
Polytron homogenizer	Kinematica	PT 1200C
DecapiCone	Braintree Scientific Inc.	DC-M200