Nanoparticles are being employed in an increasingly wide range of biological applications, including as fluorescent markers and as vehicles for drug delivery. Their size allows them to cross physiological barriers and access different tissues, and they are easily taken up by cells by the process of endocytosis. Nanoparticles can also be optimized for a particular purpose through surface modification via the addition of ligands, to produce ‘functionalized nanoparticles’.1-3
The suspension stability of these functional nanoparticles is important in many bioapplications. The work described here illustrates the effect on both particle size and charge of adding different surface functionality to silica nanoparticles, and looks at how the particles might then behave in the human body. The Zetasizer Nano from Malvern Instruments was used to measure particle size and zeta potential.
Zeta potential and stability
The efficiency of cellular uptake decreases with increasing particle size.4,5 Particles with diameters from 100 to 200 nm or below display the highest uptake.5,6 Since cell membranes are normally negatively charged, there are also advantages in using positively charged particles.
Silica nanoparticles are of considerable interest, being both biocompatible and chemically inert under a wide range of conditions. They can also be produced with a tunable particle size and pore structure.7,8 Silica is negatively charged over the pH range of biological interest, with an isoelectric point (IEP), the point of zero zeta potential, in the range 2 to 3.
Zeta potential is a physical property exhibited by all particles in suspension that can be quantified using an electrophoretic mobility (electrophoresis) measurement. Zeta potential has long been recognized as a good index of the magnitude of the charge interaction between colloidal particles, and its measurement is commonly used to assess the stability of colloidal systems.
The zeta potential of silica can be controlled easily by attaching various functional groups. This is important for both biological activity and particle stability, and is especially critical in the human body where electrolyte concentration is high, resulting in decreased electrostatic repulsion between particles. To maintain the stability of a colloidal system the repulsive forces must dominate. If there is no repulsive mechanism then eventually flocculation or coagulation will occur.
The surface modification of stable particles can influence their zeta potential and could therefore lead to agglomeration. Problems with dispersion stability would be expected for zeta potential values between +30 and -30mV. The magnitude of the zeta potential will depend on the acidic or basic strength of the surface groups and on the pH of the solution.
Adding surface functionality
The initial building blocks for the studies described here were monodisperse silica particles (SiO2) with a diameter of 240 nm, determined by scanning electron microscopy (SEM). Sample subsets were then modified by adding a negatively charged polymer polyethyleneimine (PEI) ion followed by either succinic acid (C4H6O4, Succ) or the alkali glutaraldehyde (C5H8O2, GA).
The protein streptavidin, commonly used to attach biomolecules to one another, was then added and some of the samples labelled with one of two fluorophores: Streptavidin Alexa Fluor 555 conjugate (Invitrogen Molecular Probes) or Streptavidin DyLight 549 fluorophore (Pierce Biotechnology, Rockford).9 The fluorphore was used to enable the molecules to be tracked as they move through the body.
Particle size using dynamic light scattering (DLS) and zeta potential measurements were performed using a Zetasizer Nano ZS particle characterization system at 25°C.
The surface charge of nanoparticles in suspension can be reduced to zero by controlling solution pH to suppress surface ionization. The resulting isoelectric point (IEP) is normally the point at which the colloidal system is least stable. This zeta potential/pH response was measured for all sample variations in both water and a saline solution.
Confocal fluorescence microscopy was used to verify the flocculation behaviour of the various labelled streptavidin particles in water and results were compared with those predicted by their measured zeta potentials. The two fluorophore variations provide comparative results that distinguish the influence of the fluorescing molecule itself.
Figure 1 shows the response of zeta potential to pH for the PEI-functionalized silica particles (PEI-SiO2) measured in water. The IEP of 10.3 is very different from the value of around 2 for non-functionalized silica particles. This increase originates from the presence of amino groups on the surface, which are virtually fully protonated at pH values below 9. The zeta potential of these particles is around +60mV in water at neutral pH making them easily dispersible.
The results of adding acidic or alkaline surface groups to the PEI-SiO2 foundation particles are also shown in Figure 1.
Modification with glutaraldehyde (GA-PEI-SiO2) resulted in a reduction in the IEP to pH9.0. Attaching GA decreases the number of effective amino groups thereby increasing the relative number of negatively charged silanol (silicon compound) groups. In comparison, the introduction of proton donating carboxylic acid groups (-COOH) to produce Succ-PEI-SiO2 gave an even greater drop in the IEP, to pH5.4. This IEP is however still higher than expected; succinic acid has a pKa of 4.2, suggesting some amine groups remain in the PEI layer. This is also evidenced by the high zeta potential at lower pH values.
Linker molecule effects
The influence of pH on the zeta potential for all other samples is summarized in Table 1.
Streptavidin itself has an IEP between pH5 and 6. Data therefore suggest that linking streptavidin to the PEI-SiO2 particles consumes amino groups, reducing the IEP of both samples, while streptavidin amino groups are used when linking to GA-PEI-SiO2 nanoparticles. Additionally, the pH-dependent charging of the Alexa555 fluorophore could also influence zeta potential.
The closer a sample’s IEP gets to zero zeta potential, the less its ability to form a stable suspension. Any sample with an IEP close to the pH of pure water (~pH6) would be expected to flocculate. Results from the confocal fluorescence microscopy confirmed this to be the case. Virtually no flocculation was observed for the Alexa555-streptavidin-PEI-SiO2 suspensions (IEP=9.3) while Alexa555-streptavidin-GA-PEI-SiO2 and Alexa555-Succ-PEI-SiO2 nanoparticles displayed strong agglomeration, in agreement with their low zeta potential values (IEP=6.1 and 5.9 respectively).
PEI-functionalized silica particles are promising candidates for biological applications, providing highly positive zeta potentials even under high ionic strength conditions, as long as surface functionalising additions do not decrease the particle IEP to values close to zero at neutral pH.
Effective characterization of these behaviours can be quantified using the Zetasizer Nano ZS by combining DLS and zeta potential measurements.
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This article is based on work available as an application note on the Malvern Instruments website Electrokinetic Characterization of Functionalized Silica Nanoparticles. Prepared by L Bergman1, J Rosenholm1, A-B Öst2, A Duchanoy1, P Kankaanpää2, J Heino2 and M Lindén1
1. Center for Functional Materials, Department of Physical Chemistry, Abo Akademi University, Porthansgatan 3-5, FIN-20500 Turku, Finland.
2. Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland. Malvern ref. MRK1267
This article originally ran in the February 2012 issue of Lab Product News.