Tunable Resistive Pulse Sensing Used for Accurate Size, Charge and Concentration Analysis of Liposomes

Topics Covered

Introduction
Measuring Liposome Concentration
Measuring Liposome Size, Polydispersity, and Aggregation
Measuring Liposome Zeta-potential
Conclusion
About Izon

Introduction

Tunable resistive pulse sensing (TRPS) is an innovative technology that can be used to determine the properties of liposomal dispersions, which require precise and highly sensitive analysis. TRPS, which is built on particle-by-particle analysis, allows precise measurement of these properties from a single analysis run. In other words, it provides data on volume fraction and particle concentration of liposome delivered to the administered solution volume, which remains neutral to the polydispersity or size of the sample. Besides measuring concentration, the TRPS technique can also be used for studying liposome size distribution, which is important for liposome production, formulation as well as delivery applications. The TRPS feature of charge (zeta-potential) and particle-by-particle size analysis for differentiating liposome surface modification makes it a complete and precise analysis tool for liposomes and nanoparticles.

Liposomes and their derivatives are nano-scale particles that have gained significant interest as tunable tools for in vivo therapeutic delivery. Surface modification of liposomes and encapsulation of therapeutics can help in protecting and enhancing the circulation and solubility time of these therapies and also help in guiding their delivery in the body. Due to these aspects, liposomes have become important vesicular systems for drug delivery.

Measuring Liposome Concentration

Generally, the effectiveness of liposome therapeutic delivery relies on the properties of liposomes, such as their concentration, size, and charge. Owing to these reasons, there is a huge demand for instruments that can precisely determine the charge, concentration, and size of liposome solutions. In fact, a number of instrumentation methods were employed to differentiate liposome dispersions. These methods can be divided into three categories, namely, separation, ensemble and particle-by-particle counting.

Figure 1. Commercially available tunable resistive pulse sensors (TRPS). Izon qNano and qViro-X measure the size, charge and concentration of nano- and micro- size particles via a size-tunable pore. The pore, which is made in a four arm elastic membrane, is mounted on the adjustable jaws of the instruments. The pore size is tuned to the particle sample in real-time by adjusting the axial strain applied to the membrane. The properties of individual liposome particles (size, charge and concentration) are measured from the resistive pulse signal they generate as they pass through the pore.

TRPS, which is a particle-by-particle counting technique, can be used to calculate and collate the properties of individual liposome particles. These properties are calculated from the resistive pulse signal produced by liposome particles as they traverse through the pore. At present, the most common techniques used for measuring the concentration of liposome depend on indirect measurements. These techniques are usually built on the scattering measurements of liposome particle size or solution turbidity, or colorimetric light absorption techniques, like Stewart assayor.

Figure 2. Direct measurement of liposome concentration. The liposome concentration (particles / mL) is directly proportional to the particle count rate (particles / min) which is independent of the liposome size or polydispersity. Diluting the sample 1:10, 1:25, 1:50, 1:75, and 1:100 gives rise to a corresponding linear decrease in the particle count rate (red line). The measured count rates and corresponding concentration values are the average and standard deviation of three analysis runs.

Figure 3. Size-specific concentration. In addition to the total particle concentration, TRPS enables the overall liposome volume fraction and the size-specific concentration, that is number of liposomes of a specific measured size, to be calculated. For the undiluted liposome solution this was 5.80 ×1011 particles / mL and 0.24 mL liposome per mL of solution.

In this research, a total particle concentration of 5.80x1011 particles per mL was noted on the undiluted liposome sample. A subsequent deliverable volume fraction of 24% of liposome payload was delivered per mL of administered solution.

Measuring Liposome Size, Polydispersity, and Aggregation

Controlling the polydispersity and size of liposomes is important for their effective use in in vivo therapeutic delivery. A variety of techniques are now available to synthesize liposomes. One popular technique is lipid hydration and extrusion. In order to control the size and polydispersity of liposomes, different concentration, lipid type, filter pore size, temperature and flow rate can be used. These conditions are optimized by measuring the size of liposomes produced. Such optimization is critical for liposome production and quality control. TRPS technique is generally employed to quantitatively size biological and synthetic nanoparticles such as polymeric nanoparticles, liposomes and cells. The size of individual particles passing through the TRPS systems is determined from the linear correlation between the degree of the resistance pulse signal AR it generates and the particle volume. Interestingly, the Nucleopore filter measuring 100 nm produced liposomes that were smaller than the 200 nm filter; however, the size of an average liposome was not smaller than 100 nm. Liposomes are susceptible to aggregation and degradation, which can be caused by the presence of detergents or variations in osmotic pressure or temperature. Moreover, extreme physical force can also affect liposomes.

In this analysis, freeze-thawing produced a more polydisperese sample that had much bigger liposomes. This phenomenon is attributed to the splitting and reforming of liposomes that take place during freeze-thawing. Besides observing the simulated environmental effects and size on liposome stability, sensitive measurements of size are important for differentiating and understanding the interactions that liposomes experience in vivo.

Measuring Liposome Zeta-potential

Figure 4. Izon particle-by-particle size and charge analysis. The size and zeta-potential of individual liposome (blue) and PEGylated liposome (red) particles are shown in the 2D dot plot. The associated size (top) and zeta-potential (left) concentration histograms show the distribution of these properties over the whole liposome suspension. PEGylated liposomes are slightly larger and more negatively charged than the unmodified liposomes. The homogeneity of the PEGylation can be related back to the width of the size and zeta-potential distribution.

Generally, it is ideal to change the surface of liposome vehicles so as to enhance their performance in vivo drug delivery. These changes can comprise the inclusion of polyethylene glycol (PEG) chains to minimize aggregation and boost circulation time, cellular receptor recognition molecules such as the RGD peptide, and molecular targeting probes like antibodies. One simple way to detect the successful alteration of liposomes is to determine the modification in their electrophoretic mobility (zeta-potential) which tends to occur from the variation in the number of charged surface groups.

The phosphocoline lipids that constitute a huge majority of liposomes are zwitterionic, i.e. each molecule has an equivalent number of negative and positive charged groups, and hence they carry zero net surface charge.

When compared to unmodified liposomes, PEGylated liposomes are somewhat larger and more negatively charged. TRPS was employed to determine the size as well as the charge distribution of a ‘normal’ and PEGylated liposome solution. Both sets of particle exhibited identical size distributions with 90 nm and 95 nm modes for the normal and PEGylated liposomes, respectively. Besides the size increase, the existence of PEG substituted phospholipids in the PEGylated liposomes were shown by the additional negative shift in their zeta-potential values. Inevitably, normal liposomes exhibited a neutral zeta-potential as shown by their slight distribution and -5mV mode. This denotes that all liposomes integrate some amount of lipid that is modified by glycol; however, the magnitude of PEGylation is not homogenous across the system.

Conclusion

TRPS is a precise and complete analysis tool to determine and study the charge, size and concentration of liposomes in biologically related media. In addition to liposomes, the TRPS measurement technique can be easily applied to any particulate system, such as emulsions, nanobubbles, and polymeric or metallic particle materials.

About Izon

Izon is a nanotechnology company focused on measurement, analysis and single particle control. Since commencement in early 2005, Izon has developed the science, hardware and software to fabricate and control dynamically resizable nanopores. These are a practical and cost effective means for the detection, characterisation and control of nanoparticles down to the molecular scale.

Izon's nanopores can be opened, closed and changed in size in real time. The platform encompassing this technology is called SIOS (Scanning Ion Occlusion Spectroscopy).

Izon provides instrumentation and consumables for research uses. It is actively engaging worldwide in a number of research collaborations to develop new applications for the SIOS platform. Collaborations also target scientific discoveries enabled by this new technology. In due course out-licensing options will be available.

Izon's ultimate goal is for its resizable nanopore platform to form the basis of widely adopted molecular diagnostic solutions. These will be medically useful, practical in application and economical. We expect them to be developed in collaboration with key partners.

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

For more information on this source, please visit Izon.

Date Added: Dec 14, 2012 | Updated: Jun 11, 2013
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