There are variety of ways to make microbubbles, each yields products of various size and consistency.  The most suitable fabrication and loading method must be carefully chosen for the application at hand.  Some important characteristics to consider are the shell material, size, dispersion, biological properties, drug properties, and drug loading capacity.  A few common fabrication methods are detailed below.

Mechanical Agitation

Mechanical agitation is a two-step process to create phospholipid-shelled microbubbles.  First, the phospholipids are created from conventional methods such as thin phospholipid film hydration, phase inversion, or ethanol injection.  Ideally, the drugs of interested are incorporated into the phospholipids before MB formation. The “lipsomal dispersion” is placed into vials with the remaining headspace filled with the gas to be captured within the microbubbles.  The vials are then agitated at several thousand oscillations per minute.  Drug loading after formation requires incubation of the drug and microbubbles.  This method is versatile and gentle on fragile drug substances and targeting ligands.  Important considerations for microbubble formation via mechanical agitation include “agitation time, proportion of vial head-space to liquid volume, viscosity of the liposomal dispersion or microemulsion, temperature during agitation, and concentration of drug and phospholipids.” [1]

Emulsification Method

This method creates oil-in-water (O/W) emulsions as a means of encapsulating lipophillic drugs.  The inner layer is typically an organic solvent.  These can be created by freeze-drying an emulsion of a lyophilizable water-immiscible organic solvent and subsequently removing the aqueous and organic phases. This leaves the emulsion matrix in the frozen vial which is then filled with the microbubble core gas.  The lipophillic drugs are incorporated into the inner layer during the organic emulsion phase.  The microbubble will immediately form in the injected gas. To improve biocompatibility, hydrophillic biomaterials are sometimes included in the aqeuous emulsion phase to coat the microspheres. This method can also be used to create double phase W/O/W emulsions for including lipophillic drugs. [1]

Important considerations for the emulsification method include: control of microbubble size (regulated by high-pressure extrusion), molecular weight of the polymeric shell material, and the amount of shell material to regulated shell thickness. [1]

Probe-type Sonication Method

This method is extremely common and can create microbubbles with denatured protein or phospholipid shells.  Probe-type sonication uses low frequency ultrasound (US) at high intensity in an aqueous solution of microbubble core gas and shell material to disperse the gas.  During this procedure the cavitation creates chemically reactive free-radicals and causes high temperatures (up to 808°C) that denature the protein shell material and create stable covalent cross-bridging of protein thiol groups.  This process creates stable shells with a high affinity for DNA and a wide range of drug molecules.  Because of the high chemical and thermal stresses in making the microbubbles, therapeutic drugs are usually only loaded on the surface of preformed bubbles by drug incubation. [1]

Spray-Drying Method

Spray-drying is used to produce polymer, protein, or phosopholipid shelled microbubbles.  In this method a liquid or slurry is rapidly dried with a hot gas into a dry powder with a consistent particle size.  To form pores or cavities in the particles, one can use a volatile ammonium salt or enclose volatile organic liquids into the spray-dry medium.  As the solvent evaporates, the droplets shrink isotropically and the shell-material accumulates at the liquid-water interface. The outer shell material solidifies and the remaining solvent evaporates leaving a bubble.  It is unclear whether the cavities are of uniform size or if there are multiple small voids in each bubble.  Drugs can be volume-loaded into the shell by mixing with the shell material before microbubble formation.  Compared to probe-type sonication, this method is quite gentle and provides a dry, stable product. [1]

Flow-Focusing

There are additional methods being develop to create microbubbles specifically for UTMD, such as flow focusing.  To create these bubbles a core gas and the shell material are sent through a fine nozzel into a water bath.  A more precise and consistent method is also being developed by the engineerings in the BioMiNT laboratory at UCI.  This group uses microfluidic dual hydrodynamic focusing to create monodispersed droplets.  The droplets have a gas core, inner oil shell layer, and outer phospholipid layer.  The oil allows for uniform loading in high concentrations of hyrdrophobic and toxic drugs.  Ligands are also uniformly attached to the outer shell in the fabrication process. [5]

NOTE: Quality Control

It is important to characterize the quality and consistency of the microbubbles for any fabrication method.  Some important characteristics are the particle size distribution, chemical integrity of drugs and excipients, stability, the zeta potential, phase separation of shell components, and drug localization.  The zeta potential is a characteristic of microbubbles that provides information on colloidal dispersion, stability, biological characteristics, and drug loading capacity.  Phase separation of the shell components and drug localization are important because unequal distributions will hinder microbubble stability and loading capacity. [1]