Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Summary

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Abstract

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 μm, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

Introduction

Microbubbles composed of protein, polymer, and lipid shells have been developed as vectors for drug delivery, gene therapy, and ultrasound contrast agents^1-5. Because these therapeutic uses require intravascular microbubble persistence, such microbubbles are commonly filled with inert, high molecular-weight gases such as perfluorocarbons⁶, which have low solubility in blood and stabilize the bubble^3,4.

Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver therapeutic doses of oxygen, which may preserve end-organ oxygen delivery and prevent hemodynamic instability during periods of ai....

Protocol

1. System Set-up

The system consists of a holding and concentrating tank (HCT) fitted with a single stage mixer, an in-line high shear homogenizer, a roller pump to move fluid between the HCT and the homogenizer, and a heat-exchanger (Figure 1).

1. Place a sterilized, wide-mouthed 4 L glass collection vessel fitted with 2 base ports and 3 side ports beneath the single stage mixer. Lower the mixer head to the mouth of the vessel and ensure a gas-tight fitting using rubber seals or tape (in order to prevent ambient air from contaminating the head space).
2. Fit one of the base ports (Figure 1,....

Results

High shear homogenization enables the efficient (i.e. within one afternoon) production of sufficient LOMs for an animal study and does not require technical expertise. Once proficient, up to 2 L of concentrated LOMs can be manufactured in 90 min.

Microbubble size and morphology was assessed by light microscopy and by light obscuration. When a 10 μl sample of LOMs was visualized, spherical LOMs were noted, as well as a relative paucity of lipid debris (Figure 3A). This.......

Discussion

The most important steps to creating concentrated, highly oxygenated LOMs include: 1) ensuring that the headspace within the HCT remains fully oxygenated; 2) ensuring that the purity of the lipid excipients is optimal (including storage conditions and use of GMP products); 3) ensuring that the powdered lipids mix completely with the aqueous phase prior to priming the system; and 4) paying close attention to the increase in gas fraction within the HCT to ensure that the volume fraction of gas does not exceed 70%.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Avanti Polar Lipids	770365	Alternate product: non-GMP from NOF America (Coatsome MC-8080)
Cholesterol	Sigma Aldrich	C75209
Plasma-Lyte A	VWR	80089-818	Alternatively can use NaCl
Glass collection vessel	Specialty Glass, Inc.	Custom	Contact: Pam Zurbrick - 281-595-2210
Gas composition (oxygen) monitor	Precision Medical	PM5900L
Sarns 8000 roller pump	Calicut Medical	16407	Part of a modular perfusion system
BIOtherm Heat Exchanger	Medtronic	ECMOtherm-II
Verso laboratory in-line mixer	Silverson Machines, Inc	TH-IL-102-VERSO	Use multistage workheads and front-end extension with T piece
T-piece for Silverson Verso inlet port	Process Innovations	Custom	Contact: Brian Leavitt - 508-423-2266
L5M-A laboratory mixer	Silverson Machines, Inc	NC0136483	Use mesh emulsor screen (fine)
Rochester-Ochsner toothed forceps	Fisher Scientific	13-812-18
140 ml syringe	Kendall Healthcare Monoject	8881114030	Ensure there is a luer lock.
IX71 Inverted light microscope	Olympus	IX71
Retiga-2000R microscope camera	QImaging	RET-2000R-F-M-12
Accusizer 780A Autodilution	PSS-NICOMP Particle Sizing Systems	Out of production