pubs.acs.org/Langmuir © 2010 American Chemical Society
Size Matters: Influence of the Size of Nanoparticles on Their Interactions with Ligands Immobilized on the Solid Surface Elena V. Piletska* and Sergey A. Piletsky Cranfield Biotechnology Centre, Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, United Kingdom Received December 22, 2009. Revised Manuscript Received February 9, 2010 The correlation between the size of biotinylated nanoparticles and their affinity in relation to interactions with the solid surface was investigated. The silica particles with a diameter of 50-200 nm containing amino groups on the surface were labeled with different quantities of biotin. The affinity properties of biotinylated nanoparticles were studied using a Biacore 3000 instrument equipped with a streptavidin-coated sensor chip (SA chip). It was shown that the increase in the particle size from 50 to 200 nm reduced the affinity (KD) of biotin-streptavidin interactions from 1.2 10-12 to 1.2 10-10 M. It was found that the particles with higher concentrations of immobilized biotin on particle surfaces demonstrated stronger binding with streptavidin.
The most promising applications areas for nanoparticles are in theranostics, in targeted drug delivery, in in vivo optical imaging, and in bioanalysis and diagnostics.1-3 Some of the applications (e.g., immuno-diagnostics, targeted drug delivery, or specific labels in electron microscopy) require the ability of nanoparticles to bind specifically to the protein or polysaccharide ligands on the cell surface. Although the biological elements in these systems are relatively well studied and documented, there is no systematic information on how the size of nanoparticles affects the affinity interaction in such systems. Due to the high affinity and wellunderstood mechanism of biotin binding to avidin and streptavidin (KD ∼ 1 10-15 and 1 10-13 M, respectively),4 these compounds are often used as a model system in the study of biomolecular interactions. In one such study, the biotin labeling of G5 polyamidoamine (PAMAM) dendrimers (generation 5, G5) was used to enhance their uptake into cancer HeLa cells.5 This work provided evidence that nanoparticles with diameter 5.4 nm did not have a noticeable effect on the affinity binding of the corresponding ligands.6 It has also been shown that affinity interactions were still possible after conjugation with 13 nm gold nanoparticles7 and antibodies (MW 150 kDa and size ∼ 20 nm).8,9 However, the conjugation of biotin with large endothelial cells (8-12 μm) had a big impact on their binding to the streptavidin immobilized on the surface, reducing the strength of streptavidin-biotin binding by 3 orders of magnitude.10 It is clear that the size of the particles and associated steric factors had an impact on their affinity. *To whom correspondence should be addressed. E-mail: e.piletska@ cranfield.ac.uk.
(1) Penn, S. G; He, L.; Natan, M. J. Curr. Opin. Chem. Biol. 2003, 7, 609–615. (2) Fortina, P.; Kricka, L. J.; Graves, D. J.; Park, J.; Hyslop, T.; Tam, F.; Halas, N.; Surrey, S.; Waldman, S. A. Trends Biotechnol. 2007, 25, 145–152. (3) H€arm€a, H.; Soukka, T.; L€ovgren, T. Clin. Chem. 2001, 47, 561–568. (4) Green, N. M. Methods Enzymol. 1990, 184, 51–67. (5) Yang, W.; Cheng, Y.; Xu, T.; Wang, X.; Wen, L. Eur. J. Med. Chem. 2009, 44, 862–868. (6) Islam, M. T.; Majoros, I. J.; Baker, J. R., Jr. J. Chromatogr., B 2005, 822, 21– 26. (7) Li, T.; Guo, L.; Wang, Z. Biosens. Bioelecron. 2008, 23, 1125–1130. (8) Henry, N.; Parce, J. W.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 3933–3937. (9) N€areoja, T.; M€a€att€anen, A.; Peltonen, J.; H€anninen, P. E.; H€arm€a, H. J. Immunol. Methods 2009, 347, 24–30. (10) Chan, B. P.; Chilkoti, A.; Reichert, W. M.; Truskey, G. A. Biomaterials 2003, 24, 559–570.
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Unfortunately, publications on this subject are nonsystematic and big questions remain as to what should be the optimum size of particles for efficient affinity interactions with corresponding immobilized ligands, and cell and tissue surfaces. The present work is an attempt to answer this question. As a model system for this study, we have chosen the affinity interactions between biotinylated silica nanoparticles with various sizes and streptavidin immobilized on the gold surface of Biacore chips. Silica nanoparticles with a diameter of 50, 100, and 200 nm were selected as models due to their rigidity and lack of undesirable swelling. The physical parameters of the tested nanoparticles are shown in the Table 1. Nanoparticles with a diameter larger than 200 nm were not considered for these experiments to prevent the possibility of the blockage of the fluidic system of the Biacore instrument. Nanoparticles functionalized with amino groups on the surface were chosen for sulfo-NHS-biotin immobilization. The idea was to assess whether the biotin-streptavidin interactions will be affected by the size of the carrier nanoparticles and by the density of the immobilized biotin on their surface. Different levels of biotinylation of 50, 100, and 200 nm silica nanoparticles with water-soluble sulfo-NHS-biotin were achieved by using two different biotin concentrations for the labeling: 15 μg 3 mL-1 (27 μM) and 150 μg 3 mL-1 (270 μM). The quantity of the biotin immobilized on the silica particles was measured using the HABA assay. According to the HABA assay, two fractions of silica particles with a diameter of 50 nm contained 57 and 97 nmol 3 mg-1 biotin, two fractions of 100 nm particles contained 64 and 128 nmol 3 mg-1 biotin, and two fractions of silica particles with a diameter of 200 nm contained 49 and 89 nmol 3 mg-1 biotin. The series of dilutions of 50, 100, and 200 nm particles with the immobilized biotin as well as the nonmodified particles were sequentially injected, and their binding to the streptavidin surface was recorded. It was found that the 50, 100, and 200 nm particles without biotin did not have any affinity toward the streptavidin surface which resulted in very low response values (RU) registered by the Biacore sensor: 27, 5, and 0.7 RU, correspondingly (nonmodified 50 nm particles are shown in Figure 1). It could be considered as evidence to the phenomenon of low nonspecific
Published on Web 02/12/2010
DOI: 10.1021/la904834y
3783
Letter
Piletska and Piletsky Table 1. Physical Parameters of the Tested Silica Particles
diameter (nm)
surface of the particle (m2)
volume of the particle (m3)
mass of the particle (kg)
total surface of the particles (m2 3 kg-1)
50 100 200
7.9 10-15 3.1 10-14 1.3 10-13
6.6 10-23 5.2 10-22 4.2 10-18
1.3 10-19 7.3 10-19 6.7 10-18
0.3 0.25 0.09
Table 2. Parameters of the Biotin-Streptavidin Interactions from the Biacore Study particle size (nm)
biotin surface density (biotin molecules concentration range (nM) per 10 nm2)
Rmax (RU)
KD (M)a
50 1 0.0028-280 2000 2.7 10-12 50 2 0.0048-480 4250 1.2 10-12 100 1.5 0.0038-380 1500 5 10-11 100 3 0.0077-770 2200 5.8 10-12 200 3 0.0024-240 70 1.2 10-10 200 6 0.0044-440 580 2.7 10-11 a The calculated value is an average result of three measurements. The STD is below 5%.
Figure 1. Sensorgram of time-dependent binding of 50 nm silica nanoparticles functionalized with biotin to the streptavidin-coated surface of the Biacore sensor chip. Particle concentrations are 5 ng mL-1, 50 ng mL-1, 0.5 μg mL-1, 5 μg 3 mL-1, 50 μg 3 mL-1, and 500 μg mL-1. The corresponding biotin concentrations for biotinylated particles are 0.005, 0.05, 0.49, 4.85, 48.5, and 485 nM.
binding between tested biotinylated silica particles and the surface of the SA chip. The kinetic data for the interactions between the streptavidin surface of the Biacore chip and the nanoparticles which contained different quantities of immobilized biotin were calculated using BIAevaluation software (Table 2). It is possible to make several conclusions based on these results. They show that bigger nanoparticles have lower affinities, possibly as a result of steric constrains and diffusion limitation affecting ligand-receptor interactions (Table 2). When comparing the similar concentration ranges of immobilized biotin (for example, 0.0028-280 nM for 50 nm particles and 0.0024-240 nM for 200 nm particles), the binding was 2 orders of magnitude lower for the larger particles in comparison with the smaller particles. It was observed that when interactions happened between particles and the solid surface, each increase of the size of nanoparticles by order of magnitude resulted in roughly 1 order of magnitude decrease in the strength of affinity interactions (Figure 2). This tendency is in agreement with data published on biotinstreptavidin and biotin-avidin interaction in different systems and can be followed in systems other than silica nanoparticles10,12 (see corresponding reference points in Figure 2). It is well-known that free biotin has the highest affinity toward free streptavidin and avidin (KD = 10-13 and 10-15 M, correspondingly).4,13 The size of an avidin molecule is reported as 3.6 nm,14 and the size of streptavidin is 5 nm in diameter.15 Although the immobilization on a solid support could be considered as a valuable analytical approach due to the improvement (11) Huang, S.-C.; Stump, M. D.; Weiss, R.; Caldwell, K. D. Anal. Biochem. 1996, 237, 115–122. (12) Lee, C.-S.; Lee, S.-H.; Kim, Y.-G.; Lee, J.-H.; Kim, Y.-K.; Kim, B. G. Biosens. Bioelectron. 2007, 22, 891–898. (13) Green, N. M. Adv. Protein Chem. 1975, 29, 85–133. (14) Chan, B. P.; Bhat, V. D.; Yegnasubramanian, S.; Reichert, W. M.; Truskey, G. A. Biomaterials 1999, 20, 2395–2403. (15) Kuzuya, A.; Numajiri, K.; Kimura, M.; Komiyama, M. Nucleic Acids Symp. Ser. 2008, 52, 681–682.
3784 DOI: 10.1021/la904834y
Figure 2. Dependence between the particle size and affinity of the biotin-streptavidin interactions. For all shown silica nanoparticles (50, 100, and 200 nm PSi) only the fractions with higher biotin density were used (see Table 2). The measured dissociating constants of other particles (3.6 nm free avidin, 944 nm PS (944 nm polystyrene particles), and 8 μm endothelial cells) refer to corresponding references.
of the efficiency of the separation of bound ligands from unbound ligands, it is necessary to remember that it significantly affects the kinetics of the reaction due to diffusion limitation and steric hindrance.16 It was reported that diffusion limitation was stronger for 0.65-1.25 μm diameter particles than for those in the 0.31-0.65 μm diameter size range.17 The samples in our study containing particles of smaller diameters have a larger total surface area and therefore contain a larger amount of immobilized ligand. The diffusion of the soluble reactant to the surface of the smaller particle is also much quicker and easier than that to the larger particle. A comprehensive study was published for the “liquid - surface” system where different sizes of biotinylated DNA fragments (range from 100 to 5000 base pairs) were interacting with polystyrene particles with a size range from 90 to 944 nm, which were coated with streptavidin.11 The authors observed that the experimental binding constants of the reaction were several orders of magnitude lower compared to the homogeneous reaction and that they depended on the size of both ligand and substrate. The results of the present study suggest that the interaction between biotin and streptavidin, immobilized on (16) Huang, S.-C.; Swerdlow, H.; Caldwell, K. D. Anal. Biochem. 1994, 222, 441–449. (17) Ku, C.-A.; Lentrichia, B. B. J. Colloid Interface Sci. 1989, 132, 578–584.
Langmuir 2010, 26(6), 3783–3785
Piletska and Piletsky
the solid surfaces (“surface-surface” system), is also governed and restricted by the factors described for the “liquid-surface” system such as steric hindrance and diffusion limitation. The conclusion could be drawn that the size of the nanoparticles suitable for the bioanalytical applications should not exceed the 50-100 nm range. A similar conclusion was also drawn in the work of H€arm€a et al. who tested the interaction of streptavidinlabeled polystyrene nanoparticles with biotinylated protein.3 In this case, nanoparticles would benefit from their features as a solid-state support material which has a high surface area but would not create significant problems for molecular interactions. The larger particle dimensions could be considered as beneficial in the case of preferred weaker binding for purification or other separation purposes. In this case, the particle size could be an important tool for the optimization and prediction of the binding rates and binding capacity of nanoparticles. Curiously, the increase in the density of the immobilized ligand (biotin) on the surface of the nanoparticles had no negative impact on their affinity. It was found that the particles with higher concentrations of immobilized biotin on the particle surface demonstrated stronger binding with streptavidin (Table 2). This was in close agreement with observations of Soukka et al. who stated that the binding affinity of the analyte was linearly related to the number of binding sites on the surface of the nanoparticles.18 This is counterintuitive, since it was expected that closely immobilized biotin molecules would prevent binding to (18) Soukka, T.; Paukkunen, J.; H€arm€a, H.; L€onnberg, S.; Lindroos, H.; L€ovgren, T. Clin. Chem. 2001, 47, 1269–1278.
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Letter
large streptavidin molecules. Since this is not the case, we can conclude that randomly immobilized ligands do not provide homogeneous coatings and even in the case of high immobilization density a number of steric accessible ligands would be available for affinity interactions. There is also no evidence of cooperative interactions between several biotin molecules on the surface of the same particle with the streptavidin-coated surface. This would lead to a substantial increase in the affinity of nanoparticles which was never observed in our experiments. Nanoparticles are very promising materials for biomedical applications which rely on affinity interactions. The optimization of their properties can have a profound effect on their performance. It is necessary to remember when developing new nanosized materials that the size of the particles should be considered not only from a delivery point of view but also as an important factor which affects their affinity and their interactions with biological receptors. Acknowledgment. E.V.P. would like to acknowledge with gratitude financial support from Leverhulme Trust and English Literature student Alexandra Piletska for her editorial input. Supporting Information Available: Details about materials and experimental procedures of biotin labeling and biotin quantification; details of Biacore experiments and kinetics curves for studied biotinylated particles. This material is available free of charge via the Internet at http:// pubs.acs.org.
DOI: 10.1021/la904834y
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