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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. 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'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 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 B as the hydrophobic bioactive agent.
Nascent discoidal high density lipoproteins (HDLs) are naturally occurring progenitors of the far more abundant spherical HDL present in the human circulatory system. These nascent particles, also referred to as pre-ß HDL, possess unique and distinctive structural properties1. Indeed, rather than existing as a spheroidal particle, nascent HDLs are disk-shaped. Extensive structural characterization studies on natural and reconstituted discoidal HDLs have revealed that they are comprised of a phospholipid bilayer whose perimeter is circumscribed by an amphipathic exchangeable apolipoprotein (apo), such as apoA-I. In human lipoprotein metabolism, circulating nascent HDLs accrue lipids from peripheral cells and mature into spherical HDLs in a process that is dependent upon key protein mediators, including the ATP binding cassette transporter A1 and lecithin:cholesterol acyltransferse2. This process represents a critical component of the reverse cholesterol transport pathway that is considered to be protective against heart disease. Armed with this knowledge and the ability to reconstitute discoidal HDLs, researchers have employed these particles as a therapeutic intervention to treat atherosclerosis3. In essence, the infusion of reconstituted HDL (rHDL) into patients promotes cholesterol efflux from plaque deposits and returns it to the liver for conversion to bile acids and excretion from the body. Several biotechnology/pharmaceutical companies are pursuing this treatment strategy4.
At the same time, the ability to generate these particles in the laboratory has sparked a flurry of research activities that has led to novel applications and new technologies. One prominent application involves the use of rHDL particles as a miniature membrane to house transmembrane proteins in a native-like environment5. To date, hundreds of proteins have been successfully incorporated into discoidal rHDL, and research has demonstrated that these proteins retain both native conformation and biological activity as receptors, enzymes, transporters, etc. These particles, referred to as "nanodiscs", have also been shown to be amenable to structural characterization, often at high resolution6. This approach to investigations of transmembrane proteins is recognized as superior to studies with detergent micelles or liposomes and, as a result, is rapidly advancing. It is important to recognize that two distinct methods have been reported that are capable of forming an rHDL. The "cholate dialysis" method13 is popular for applications related to the incorporation of transmembrane proteins in the rHDL bilayer5. Essentially, this method of formulation involves mixing a bilayer forming phospholipid, a scaffold protein, and the transmembrane protein of interest in a buffer containing the detergent sodium cholate (or sodium deoxycholate; micelle molecular weight [MW] of 4,200 Da). The detergent effectively solubilizes the different reaction components, permitting the sample to be dialyzed against buffer lacking detergent. During the dialysis step, as the detergent is removed from the sample, an rHDL spontaneously forms. When this approach is used to entrap a transmembrane protein of interest, the product particles have been termed nanodiscs5. Attempts to use this method to incorporate small molecule hydrophobic bioactive agents (MW <1,000 Da), however, have been largely unsuccessful. Unlike transmembrane proteins, small molecule bioactive agents are able to escape from the dialysis bag along with the detergent, greatly decreasing their incorporation efficiency into rHDLs. This problem was solved by omitting detergents from the formulation mixture14. Instead, the components are added to an aqueous buffer sequentially, beginning with the bilayer forming lipid, forming a stable bioactive agent containing rHDL, referred to as a nanodisk. Others have used rHDL for the incorporation and transport of in vivo imaging agents7. More recently, specialized rHDL comprised of an apolipoprotein scaffold and the anionic glycerophospholipid, cardiolipin, have been employed in ligand binding studies. These particles provide a platform for studies of the interaction of cardiolipin with various water soluble ligands, including calcium, cytochrome c, and the anticancer agent doxorubicin8.
The focus of the present study is on the formulation of rHDL that possess a stably incorporated hydrophobic bioactive agent (i.e. nanodisk). The ability of these agents to integrate into the lipid milieu of discoidal rHDL particles effectively confers them with aqueous solubility. As such, nanodisks have the potential for in vivo therapeutic applications. When formulating nanodisks, specific incubation/reaction conditions are required to successfully incorporate discrete hydrophobic bioactive agents into the product particle, and the goal of this report is to provide detailed practical information that can be used as a foundational template for creating novel nanodisk particles for specific applications. Thus, in the context of this manuscript the terms nanodisc and nanodisk are not interchangeable. Whereas nanodisc refers to an rHDL formulated to contain a transmembrane protein embedded in its lipid bilayer5, the term nanodisk refers to an rHDL formulated to incorporate low molecular weight (< 1,000 Da) hydrophobic bioactive agents, such as amphotericin B14.
A variety of methods are available for the acquisition of suitable scaffold proteins. It is possible to purchase scaffold proteins from manufacturers [e.g. apoA-I (SRP4693) or apoE4 (A3234)], however, the cost may be a limiting factor. A preferred approach is to express recombinant scaffold proteins in Escherichia coli. Protocols are published for human apoA-I9, apoE410, as well as the insect hemolymph protein apolipophorin-III11. For the purpose of the experiments described herein, recombinant human apoE4 N-terminal (NT) domain (amino acids 1-183) was used. The nucleotide sequence encoding human apoE4-NT was synthesized and inserted into a pET-22b (+) expression vector directly adjacent to the vector-encoded pelB leader sequence. This construct leads to the expression of a pelB leader sequence-apoE4-NT fusion protein. Following protein synthesis, the bacterial pelB leader sequence directs the newly synthesized protein to the periplasmic space where leader peptidase cleaves the pelB sequence. The resultant apoE4-NT protein, with no sequence tags or tails, subsequently escapes the bacteria and accumulates in the culture medium11,12, simplifying downstream processing.
1. Transformation, expression, and purification of scaffold protein component
2. Formulation of bioactive agent containing nanodisks
3. Spectral analysis of ampB-nanodisk samples
4. Yeast viability assay analysis
NOTE: Yeast viability assays were performed in order to evaluate the biological activity of ampB and determine whether the process of formulation or incorporation into nanodisks, affected its yeast growth inhibition activity.
Bioactive agent nanodisk formulation process
In the ampB-nanodisk formulation procedure described, the reaction is considered complete when the sample appearance transitions from turbid to clear (Figure 1). This change indicates that nanodisks have formed and that the bioactive agent has been solubilized. Oftentimes, bioactive agents absorb light in the visible wavelength region (e.g., ampB, curcumin, lutein, coenzyme Q10) and, in these cases, the sample ado...
Formulation of a bioactive agent containing nanodisks provide a convenient method to solubilize otherwise insoluble hydrophobic compounds. Because the product bioactive agent nanodisks are fully soluble in aqueous media, they provide a useful delivery method for a wide range of hydrophobic molecules (Table 1). These include small molecules, natural and synthetic drugs, phytonutrients, hormones, etc. The formulation strategy usually follows a standard protocol that must take into consideration the so...
The authors have nothing to disclose.
This work was supported by a grant from the National Institutes of Health (R37 HL-64159).
Name | Company | Catalog Number | Comments |
Amphotericin B | Cayman Chemical Company | 11636 | ND Formulation & Standard Preparation |
Ampicillin | Fisher Scientific | BP17925 | Transformation & Expansion |
ApoE4-NT Plasmid | GenScript | N/A | Transformation |
Baffled Flask | New Brunswick Scientific | N/A | Expansion & Expression |
BL21 competent E coli | New England Biolabs | C2527I | Transformation |
Centrifuge bottles | Nalgene | 3140-0250 | Expression |
Chloroform | Fisher Scientific | G607-4 | ND Formulation |
DMSO | Sigma Aldrich | 472301 | Standard Prepartation |
Dymyristoylphosphatidylcholine | Avanti Lipids | 850345P | ND Formulation |
Erlenmeyer flask | Bellco Biotechnology | N/A | Expansion & Expression |
Falcon Tubes | Sarstedt Ag & Co | D51588 | Yeast Viability Assay |
Glass borosilicate tubes | VWR | 47729-570 | ND Formulation |
GraphPad (Software) | Dotmatics | N/A | Yeast Viability Assay |
Heated Sonication Bath | VWR | N/A | ND Formulaton |
Heating and Nitrogen module | Thermo Scientific | TS-18822 | ND Formulation |
HiTrap Heparin HP (5 mL) | GE Healthcare | 17-0407-03 | Purification |
Isopropyl β-D-1-thiogalactopyranoside | Fisher Scientific | BP1755 | Expression |
J-25 Centrifuge | Beckman Coulter | J325-IM-2 | Expression |
JA-14 Rotor | Beckman Coulter | 339247 | Expression |
Lyophilizer | Labconco | 7755030 | ND Formulation |
Methanol | Fisher Scientific | A452-4 | ND Formulation |
Nitrogen gas | Praxair | UN1066 | ND Formulation |
NZCYM media | RPI Research Products | N7200-1000.0 | Expansion & Expression |
Pet-22B vector | GenScript | N/A | Transformation |
Petri dish | Fisher Scientific | FB0875718 | Transformation & Expansion |
Quartz Cuvettes | Fisher Brand | 14385 928A | Spectral Analysis |
Shaking Incubator | New Brunswick Scientific | M1344-0004 | Transformation, Expansion, & Expression |
Slide-A-Lyzer Buoys | Thermo Scientific | 66430 | Purification |
SnakeSkin Dialysis Tubing | Thermo Scientific | 68100 | Purification |
SnakeSkin Dialysis Tubing | Thermo Scientific | 88243 | Purification |
Sodium Chloride | Fisher Scientific | S271 | Purification |
Sodium Phosphate dibasic | Fisher Scientific | S374-500 | Purification |
Sodium Phosphate monobasic | Fisher Scientific | BP329-500 | Purification |
Spectra/POR Weighted Closures | Spectrum Medical Industries | 132736 | Purification |
Spectrophotometer | Shimadzu UV-1800 | 220-92961-01 | spectral analysis |
Tabletop Centrifuge | Beckman Coulter | 366816 | ND Formulation |
UVProbe 2.61 (Software) | Shimadzu | N/A | Spectral Analysis |
Vacuum filter | Millipore | 9004-70-0 | Expression & Purification |
Vacuum pump | GAST Manufacturing Inc | DOA-P704-AA | Expression & Purification |
Vortex | Fisher Scientific | 12-812 | ND Formulation |
Yeast | N/A | BY4741 | Yeast Viability Assay |
Yeast Extract-Peptone-Dextrose | BD | 242820 | Yeast Viability Assay |
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