Measuring High Concentration Liposome and Micelle Suspensions with Wave II Dynamic Light Scattering

This article describes micelles and lipoproteins, paying special attention to particle size measurement of liposomes in concentrated or dilute aqueous environments.

Water disperses and dissolves many compounds. Sodium chloride is one such ionic species which is dissolvable in water, and due to the dielectric properties of water, it can be easily separated into individually charged atoms. These charged atoms can be hydrated (solvated). The distance between the ions is inversely proportional to dielectric constant (D), while the ion charge is proportional to the attractive force. Thus, sodium chloride easily dissolves in water (D=80) while it is insoluble in benzene (D=2.3). Dispersal can also occur by hydrogen bonding of water molecules to polar functional groups in the chemical structure of sugars and alcohols. Salts or surfactants can change the structure of water which can be employed to "dissolve" proteins such as collagen, antibodies, hemoglobin, some hormones, and myosin. The protein composition generally dictates the dispersal of the proteins. Composition dictates which chemicals are needed to decrease the forces of attraction, preventing solubility. Biological chemicals can also be used to cause dispersal. Depending upon their structure, lipids will dissolve in polar or non-polar organic solvents, but generally, not in in water. Due to their water insolubility, lipids can enter into special chemical bonding to produce structures such as liposomes, blood lipoproteins, cell membranes, and micelles.

Cell Membranes

Biological tissues contain cells whose contents perform the biochemical reactions to provide living organisms with energy, respiration, tissue repair, immunity, etc. A cell consists of many different functional units called organelles. Each organelle offers a site for specific types of biological reactions. All organelles are located in the cell membrane which maintains protection of the organelles, allows a controlled environment for chemical reactions, and provides a "gate" through which all substances need to enter into or exit out of the cell. This action is enabled by a variety of complex mechanisms. The structure of the cell membrane has been investigated for many years. However, ranging from simple to complex models, a biological membrane’s structure has not been completely defined, but is still of great biotechnical interest, and certainly contains proteins and lipids. A cell membrane and other structural components of cells are formed by the interaction of these compounds. All compounds enter the cell by passing through the protein-lipid complex, via diffusion or other mechanisms, including mechanical means, where the cell preferentially "opens" and interacts with other membranes so as to allow the entry of components (reverse pinocytosis).

Cell Membrane

Cell Membrane

Liposomes and Micelles

An early study on the interaction of proteins and lipids to form structures was carried out by mixing water with specific polar lipids, (containing a water-loving portion in the molecule). These polar lipids may be insoluble in water or polar organic solvents, for example, alcohols. Phosphatidylcholine, classified as a polar lipid, is popular for preparing micelles or liposomes. However, due to their amphipathic (a molecule containing both highly polar portions and highly non-polar portions) nature, many polar lipids show dispersibility in both types of solvents. Such a mixture results in a two-phase system: oil on top and water on the bottom. If, by using an ultrasonic probe or homogenizer, the mixture is fiercely agitated, then the lipids will form a mixture with water. In this case, the molecule orients in such a manner that the polar portion interacts with water and the non-polar portion moves away from water. Similar orientation takes place when mixing water with surfactants (surface active agents). Surfactant molecules and phospholipids are alike in the respect that the molecule in surfactants also contains water-repelling and water-loving portions.

Phospholipid Molecule

Phospholipid Molecule

The molecules, when mixed with water at very high concentrations, can form discreet units called micelles. The general structural features of micelles are similar to that of liposomes and membranes in that the special orientation of the molecules is required for formation. When phospholipids are used to prepare micelles, the micelles do not contain water within the interior of the structure. These structures are used for prototype drug delivery systems, and preliminary modeling of complex biological structures. The micelle diameter may range from nanometers to micrometers, and the thickness of the structure can vary between 50 and 100 Å (1 Å = 0.1 nm).

Lipids such as phosphatidylcholine, (C-16 is shown), are used to prepare liposomes, which are often almost spherical. Similar to micelles, the non-polar tails of the molecules are thermodynamically driven to orient inside the completed liposome structure. The size of the completed structures usually varies from several microns (cosmetics) to 40 nm (transdermal drug delivery systems).

Drug Targeting and Delivery

The use of the above concepts is of particular interest to the pharmaceutical industry in drug targeting and drug delivery. A variety of polar lipids, including cholesterol and phosphatidylcholine, can be used to produce the lipid structures of the micelle. During drug targeting, it is necessary to encapsulate a drug species for the formation of the micelle. This structure is called a liposome, and was first described by Sessa and Weiss in the early 1970s. They used ultrasonic energy to treat an immiscible mixture, and the energy generates optically clear "solutions" containing closed (generally spherical) structures, (liposomes or vesicles). The chemical nature of the membrane makes it impermeable to almost all other water-soluble components. The characteristics of the liposomes can be made to vary as the polar lipids, especially phospholipids, can be negatively or positively charged. Liposomes consisting of several layers of lipids (multi-lamellar), separated by drug-water solution, have been produced to provide for time-release applications.

In a particular case, the polar phosphatidyl portion of the molecule orients toward the outside of the liposome. This structure is similar to that of a blood cell, and after injection, the polar phosphatidyl portion can pass through the blood, kidneys, and liver unassailed to a specific physiological site with no premature release of the drug and no potential side effects. Thus, a lower injection dosage achieves the same biological activity as a higher oral dosage. In addition, the formulation is protected from the effects of the gastrointestinal tract where inhibitors, enzymes, and pH effects are encountered.

The speed of delivery, or cell absorption by either cell membrane dissolution or diffusion through the skin, is also determined by the selection of the type of lipids. Thus, as discussed above, by reaction or selective interaction with the liposome shell, the targeted cell membrane of tissue organs acts as a gate to this drug. Additionally, the liposome can be chemically subjected to provide a timed drug release to maintain its efficacy. To explain this concept, Nema, et. al. describes the protection of lactate dehydrogenase (an enzyme/protein) against protease digestion by encapsulation in phosphatidyl choline. This approach is advantageous as vesicles are non-immunogenic, non-toxic, and biodegradable. These advantages are not necessarily provided by other protein protection approaches, such as polymer coating, complexation, and administration of protease inhibitors. Another method of specifically targeting liposomes to physiological sites is attaching specific receptors/antigens or monoclonal antibodies to the membrane, thus restricting recognition by other tissue membranes. Other benefits offered by liposomes are solubilizing recalcitrant drugs, carrying oil soluble and water soluble drugs in a single dose, controlled hydration, and protein stabilization.

Liposome Stability

Several factors need to be considered with regard to maintaining shelf life and liposome stability. Stabilizing or destabilizing (manifested as unacceptable shelf life) effects are caused by parameters such as pressure, temperature, drug stability, liposome composition, viscosity, and ionic strength. Particle size is also an important method used to measure instability. Typically, the size of liposomes should be smaller than 200 nm to pass through the blood system, without any obstruction. Additionally, as liposomes of this size are smaller than mold spores, bacteria, and fungi, a 200 nm filter can be employed to easily separate them from the microorganisms. In future, liposomes of maximum size 100 nm can be prepared for separation from most virus particles.

Size Measurement

It is vital to measure liposome size as a parameter in order to control, modify, and stabilize the liposomes. Thus, particle size measurement can enable the study of liposome agglomeration, growth of multi-lamellar structures, microbiological contamination, and membrane disruption, which are also of extreme importance. However, such measurements suffer from major drawbacks including: measurement duration, dilution requirement, and insufficient instrumentation capability. Dilution can be caused by destabilization of the preparation, or losses in particle-particle interactions, and can lead to misinterpreted data. Chromatographic approaches for measurements are time-consuming and can inhibit rapid assessment of characteristics, while the structure size may be misleadingly and adversely affected by chemicals used in the measurement. The liposomes are subject to Photon Correlation Spectroscopy (PCS) dilution requirements and the above-discussed issues, though PCS can be used to reduce measurement time (5 to 30 minutes) and to measure ultrafine particles (3 to 6000 nm).

Using the "Controlled Reference Method,” the Nanotrac overcomes these measurement issues. When compared to electrophoretic, chromatographic procedures or other DLS instruments, the measuring takes up only 30 to 100 seconds; a reduction of as much as 30 to 360 minutes. The measuring range of 0.8 to 6500 nm covers the size of very small micelles and proteins to the larger liposomes. The measurement does not depend on concentration, thus enabling full strength-measurements (typically 0.01 to 40%) can be carried out, avoiding the use of other chemicals or dilution, preventing the alteration of liposome-emulsion components which can cast doubts on test results.

Production personnel can remain close to the process while performing the measurement as the Wave II can withstand the production area environment. Lab time is freed-up to perform measurements that require special skills and training. The learning period is short while obtaining precision data, as no special training or technique is required.

Easy accessibility of data from the production area is also enabled. The data is also easily interpretable by personnel from various technical backgrounds, including quality control and engineering. Wave II software is exportable by and to all modern electronic venues, and also facilitates easy saving and recalling of data records. Wave II enables the saving of data to CDs, presentation as hard copy reports, pasting into PowerPoint and Word, exporting to EXCEL, transmitting by HTML and ASCII, or being tailored to meet any specific report or presentation needs.

A stability experiment conducted by employing the Wave II on micelles is shown below. The size of micelles increased with dextrose and saline dilution.

Wave II with Internal Cell

Wave II with Internal Cell

Wave II Capabilities

  • Patented “Controlled Reference Method” for true particle size distribution and high signal by frequency analysis
  • True background measurement prevents need for filtration operations or high purity diluent
  • Measures multimodal distributions without “fitting” or assumptions
  • Full database management capability exportable using ASCII, HTML, pdf formats and to all popular database managers and spreadsheets
  • Withstands plant environment
  • High concentration measurement up to 40% solids
  • Simplicity of operation
  • Compact, portable, and convenient
  • No selection of special distribution models where operator decides on cumulants, CONTIN, NNLS, or other complex choices
  • Extensive sample preparation not required
  • Compatible with many common aqueous solutions and organic solvents
  • Many models to choose from:
    • Cuvette cell (Wave Q) uses both reusable glass and disposable plastic cells
    • Internal cell (700 µl) available in stainless steel or Teflon
    • External probe a very good choice for:
      • Dip ‘N’ Run method (like making a pH measurement)
      • OEM applications

External Probe models measure directly into all types of containers or vessels

External Probe models measure directly into all types of containers or vessels

Wave II Q comes with a variety of polystyrene and glass cuvettes

Wave II Q comes with a variety of polystyrene and glass cuvettes

This information has been sourced, reviewed and adapted from materials provided by Microtrac.

For more information on this source, please visit Microtrac.

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