The ultimate success of gene therapy depends largely on the development of suitably efficient vectors to carry foreign genetic material into target cells.
Non-viral vectors, although less efficient than viral vectors at introducing and maintaining foreign gene expression, have the advantage of being non-pathogenic and non-immunogenic.
Polycation-DNA complexes are a particularly attractive non-viral option. Their high positive charge density means they
self-assemble with DNA to generate condensed structures, 40–1000nm in diameter, that are capable of transfection. A number of novel cationic polymers are under development.
These polymers vary widely in their structure, ranging from linear to highly branched molecules such as dendrimers. Structural characteristics not only
influence their ability to complex DNA, but also their transfection efficiency and cytotoxicity.
A detailed structural characterisation thus forms an essential part of the development process for any cationic polymer with potential as a non-viral vector.
Here, we look at the use of dynamic light scattering (Zetasizer Nano, Malvern Instruments) to investigate the structural changes of dendritic poly(L-lysine) as a function of pH.
Non-invasive technique
Dynamic light scattering is a useful tool for the structural analysis of biological molecules in solution. A non-invasive technique, it is quick and easy, requires exceptionally small amounts of sample (as little as 12µl) and no reference material.
Dynamic light scattering, also known as photo-correlation spectroscopy (PCS) and quasi-electric light scattering (QELS), is a powerful technique for determining structural characteristics such as molecular weight, size, melting point and charge (zeta potential).
One of its great advantages is its ability to measure a large population of particles in a very short time, with no manipulation of the surrounding medium.
Colloidal dispersion
As its name implies, the technique is based on the scattering of a beam of light as it passes through a colloidal dispersion.
When the particles in the dispersion are very small compared with the wavelength of the light, the intensity of the scattered light is uniform in all directions (known as Rayleigh scattering). When the particles are larger (above approximately 250 nm diameter), the intensity of the scattered light is angle dependent (known as Mie scattering).
If the light source is coherent and monochromatic, as from a laser, it is possible to observe time-dependent fluctuations in the scattered light intensity.
These fluctuations arise from the fact that the dispersed particles are small enough to undergo random thermal motion, so the distance between them is constantly varying.
By analysing the time dependence of the intensity fluctuation it is possible to calculate the particles’ diffusion coefficient.
If the viscosity of the medium is known, it is then possible to calculate the particles’ hydrodynamic radius or diameter.
Lysine (K) is an amino acid with an isoelectric point of approximately 8.9. Dendritic poly(L-lysine) of the 6th generation (KG6) is a starburst polypeptide with an expected molar mass of (21+22+23+24+25+26)146~18400g/mol.
In common with other dendrimers, KG6 adopts conformations of different shape and density according to the conditions of the bulk solution (polarity, ionic strength and pH, for example).
Under acidic conditions, for example, KG6 is predicted to be positively charged and to exhibit an extended conformation as a result of electrostatic repulsion in its cationic charged branches.
When the solvent environment is changed such that the cationic charge neutralises and hence electrostatic repulsion of the arms is reduced, the molecule is predicted to ‘collapse’. Under such conditions there would be a higher influence of van der Waals attraction, stronger hydrogen bonds and an overall ‘shrinkage’ of the dendrimer.
Dynamic light scattering using a Zetasizer Nano (see The Zetasizer Nano) was used to determine how well these predictions of conformation change correlate in practice.
Experimental
KG6 was synthesised in the standard copolymer fashion and subsequently purified. Samples were dialysed in different pH buffers (pH5, 6, 7 and 8), filtered through a 200nm pore size Anodisk membrane (Whatman), and prepared at a concentration of 0.5mg/ml.
Dynamic light scattering measurements were performed using the Zetasizer Nano in automatic mode. The samples generated a strong light scattering signal from which it was possible to measure a correlation function.
By applying knowledge of the viscosity of the diffusing medium (in this case aqueous buffer) the diffusion coefficient was calculated and a size distribution generated for the hydrodynamic size of the KG6 particles (Fig.1). The computation of size is based on the size of an equivalent sphere that has the same diffusion coefficient as the polymer under study.
The results show molecules of KG6 to be smallest at high pH and largest at low pH. The size distributions for the intermediate pHs are in between.
A summary of the information generated is given in Table1 on this page.
Polydispersity index
With a polydispersity index (PDI) between 0.13 and 0.15, all peaks in the volume size distribution analysis appear relatively polydisperse.
The molecular estimates are based on the measured hydrodynamic size.
The Zetasizer Nano software has several molecular weight estimation models. In the case of poly(L-lysine) the starburst polymer and globular protein models were used.
At pH5.0, where KG6 is a stiff ‘star-polymer’, the starburst polymer model estimate of 22kDa was in reasonable agreement with the true 19kDa molecular weight.
At pH8.0, on the other hand, where KG6 forms a compact sphere, the globular protein model provided the best prediction. The intermediate samples were less well modelled since they neither behaved like a starburst polymer, nor a compact globular protein.
Summary
Dynamic light scattering provides a simple yet powerful tool for non-invasive structural studies of proteins in solution.
A highly sensitive technique, it is quick and easy to use and requires only a minimum of sample preparation.
Most importantly, however, it has the benefit of being able to provide structural information about the behaviour of the native molecule in solution. In keeping with the behaviour of the individual amino acid L-lysine, results with KG6 support the existence of a shape change as a function of pH. The protein is compact and globular at pH8, but ‘fluffy’ and starburst-like at pH5.
Ulf Nobbmann is Market Specialist for Biophysical Characterisation, Malvern Instruments Ltd, www.malvern.com.
Acknowledgements: Malvern Instruments would like to thank Assoc Professor Niidome, Faculty of Technology, Kyushu University, Japan, and Kohei Shiba, Sysmex, Japan who provided the data for this article.
