Protein Stabilized Nanoparticle (PSN) Technology Background

Definition: An emulsion is a mixture of two immiscible substances (two substances that are not soluble in one another). One substance called the dispersed phase which is dispersed in the other phase called the continuous phase. There are many types of common examples of emulsions, including milk, butter and margarine, and mayonnaise. In butter and margarine, a continuous lipid phase surrounds droplets of water (water-in-oil emulsion) (1).

Emulsifiers are surface-active molecules that adsorb to the surface of freshly formed droplets of oil during homogenization, forming a protective membrane that prevents the droplets from coming close enough together to aggregate. Most emulsifiers are molecules having polar and nonpolar regions in the same molecule. The most common emulsifiers used in the food industry are amphophilic proteins (behaves as an acid or base), small-molecule surfactants, and monoglycerides, such as sucrose esters of fatty acids, citric acid esters of monodiglycerides, salts of fatty acids (2).

Emulsification is the process by which emulsions are prepared.

Typical physical properties of an emulsion include its general appearance as a cloudy or milky solution. This is a result of the many different phase interfaces of the oil (lipid) and water phases. The boundary between the phases is called the interface and light is scattered as it passes through the emulsion. In addition to its appearance, emulsions are inherently unstable and thus do not form spontaneously. Thus, an emulsion requires an energy input through shaking, stirring, homogenizers, or spray processes of oil (lipid) and water to form an emulsion. Azaya Therapeutics uses a high pressure fluidizer that homogenizes the oil and water phases. Over time, emulsions tend to revert to the stable state of oil separated from water. Surface active substances (surfactant) can increase the kinetic stability of emulsions greatly so that, once formed, the emulsion does not change significantly over a finite period of time. Protein stabilize nanoparticles uses the same principles, using human serum albumin (HSA) as a surfactant. A more basic analogy is homemade vinegar and oil salad dressing is an example of an unstable emulsion that will quickly separate unless shaken continuously. This phenomenon is called coalescence, and happens when small droplets recombine to form bigger ones. In scientific language this process is considered aggregation. Without a surfactant present our nanoparticles will aggregate to form larger particles (˜100 nm to <200 nm). In addition to aggregation, fluid emulsions can also suffer from creaming, the migration of one of the substances to the top of the emulsion under the influence of buoyancy or centripetal force when a centrifuge is used. Under ideal manufacturing methods Azaya Therapeutics can prepare a HSA stabilized nanoparticle product with a target vesicle size of 60-80 nm.

There are three types of emulsion instability: flocculation, where the particles form clumps (aggregation), creaming, where the particles concentrate towards the surface of the mixture while staying separated, or breaking, where the particles coalesce and form a layer of liquid. Flocculation or aggregation is a phenomenon that is slowed by the addition of HSA to the liposome, stabilizing the drug encapsulated nanoparticle. The key is to slow down this process of flocculation, creaming or breaking. For example, whole milk will begin to separate when it is stored in the refrigerator over a period of time. Much like ATI-1123, flocculation/aggregation will occur over a period of time producing larger particles.

In oil-in-water (o/w) emulsions, proteins are used mostly as surface active agents and emulsifiers. One of the food proteins used in o/w emulsions is whey proteins. The whey proteins include four proteins: β-lactoglobulin, α-lactalbumin, bovine serum albumin and immunoglobulin. Commercially, whey protein isolates (WPI) have an isoelectric point of pH ˜4.5-5.0 are used for o/w emulsion preparation. In the case of PSN technology, commercially available HSA has an isoelectric point of pH 5.16.

Isoelectric point (pI) is the pH at which a molecule carries no net electrical charge. In order to have a sharp isoelectric point, a molecule must be amphoteric, meaning it must have both acidic and basic functional groups. Proteins and amino acids are common molecules that meet this requirement; such is the case with HSA.

ATI-1123’s pH is adjusted to 6.5–6.8 before lyophilization, resulting in a net negatively charged liposomal product. Thus, the PSN stabilization occurs as a result of the HSA coated liposome with a net negative charge on the surface, repelling other particles with the same charge.

What does this all mean with regards to Azaya Therapeutics PSN technology? Historically, many individual researchers have had very little luck encapsulating hydrophobic molecules into a liposome. The primary reason is a result of drug retention in the liposome. Conventional methods require a resizing of the liposome after the product has been encapsulated. The shear force required to resize a liposome causes the API material to become disassociated with the liposome. Resulting in a net lose or lack of retention of the desired compound.

Azaya’s PSN technology uses a different principle for making its liposomes. In stead of resizing the liposome after loading, the liposomes are generated during the sizing process (homogenization in a fluidizer). This is done using all of the principles listed above. The oil and water layers are emulsified to an extremely small emulsion droplet <120 nm) that contains all of the parts required to form a liposome; lipids, cholesterol, and API. When the organics are removed the excipients and API self assemble to form a uniformly sized liposome (˜ 60-80 nm). This event only occurs when the organics are removed from the emulsion mixture. Therefore, the liposomes are sized based upon the initial sizing of the emulsion droplet.

If the Azaya’s PSN is subjected to resizing, the API material will escape the lipid layer of the liposome. The shear forces applied to a liposome will disrupt the lipid layer allowing the API material to escape.

Does the liposome particle size affect delivery of an API? It has been disclosed in literature that particle size is extremely important for tissue distribution. Any particle >200 nm will have a difficult time reaching its target for several reasons; 1. It is well known that tumors possess a leaky vasculature that allows the passage of colloidal particles in the range of 50–200 nm (3–5). Additionally, lympahtic drainage is generally impaired in the tumoral interstitium, favoring retention of the colloidal particles (6), 2. large colloidal particles > 200 nm in size have the potential of getting trapped in the key capillary beds resulting in a substantial impact on blood flow. In this regard, larger particles are more likely to get trapped in the key capillary beds during I.V. administration resulting in a substantial impact on blood flow. If this occurred in the lungs, animals could potentially have significant compromise of respiratory function resulting in sudden demise of the animal (7).

References:

  1. Wikipedia definition
  2. Webster’s dictionary
  3. F. Yuan, M. Leunig, S. K. Huang, D. A. Berk, D. Papahadjopoulos, and R. K. Jain. Microvascular permeability and interstitial penetration of sterically stabilized (Stealth) liposomes in a human tumor xenograft. Cancer Res. 54, 3352-3356, 1994.
  4. R. K. Jain. Delivery of molecular and cellular medicine to solid tumors. Adv. Drug Deliv. Rev. 26, 71-90, 1997.
  5. N. Z. Wu, D. Da, T. L. Rudoll, D. Needham, A. R. Whorton, and M. W. Dewhirst. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Res. 53, 3765-3770, 1993.
  6. M. N. Kahalid, P. Simard, D. Hoarau, A. Dragomir, and Jean-Christophe Leroux. Pharmaceutical Res. 23(4), 752-758, 2006.
  7. Correspondence with leading expert, Steve Weitman, 2006.