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org/bc

Zeta Potential Mediated Reaction Monitoring on Nano and Microparticles

Frank Thielbeer, Ken Donaldson, and Mark Bradley*,

University of Edinburgh, School of Chemistry, Kings Buildings, Joseph Black Building, West Mains Road, EH9 3JJ, Edinburgh, United Kingdom ELEGI Colt Laboratory, Queens Medical Research Institute, University of Edinburgh, 47 Little France Crescent, EH16 4TJ, Edinburgh, United Kingdom
S b Supporting Information

ABSTRACT: Nano and microparticles are widely used across the life science interface, with applications ranging from chemical probes of biological function to uorescent particles for ow cytometry and cellular tracking. Increasingly, particles are modied with a variety of chemistries to boost their functionality and broaden their biological applicability. However, although particle modication has become standard laboratory practice, the ability to determine the extent and eciency of chemical modication is often very limited and empirical in nature. Herein, we report the use of zeta potential analysis as a simple and rapid direct-onparticle approach allowing levels of bead modication and derivatization to be evaluated. As a proof-of-concept, aminomethylfunctionalized nano and microparticles were derivatized to display a variety of surface functionalities and their zeta potentials measured, allowing verication of the applicability of the approach for particle analysis. We demonstrate that zeta potential measurement is a convenient approach which allows multistep reaction sequences to be followed, and show that this method can be used to verify and validate successful particle modication.

INTRODUCTION Nano and microparticles have gained great importance in chemical biology,1 with particles of various sizes, shapes, and compositions2 having demonstrated abilities in cellular tagging, intracellular sensing, and molecular imaging, to name but a few. Microparticles have been demonstrated to deliver proteins,3 nucleic acids,4 and drugs5 into cells and sense intracellular levels of calcium6 and pH7 and protein function.8,9 In addition, microparticles have been used as probes for ow cytometry10 and cellular tracking.11 Nanoparticles have also been used to attach molecules such as biological sensors12 and imaging complexes,13 and they have also been used as biomimetic enzymes.14 It is commonly anticipated that the developments in nanoparticle chemistry will lead to an increase in their use in medicine and biotechnology.15 However, although synthesis on micro and nanoparticles is nowadays routinely carried out, the monitoring and analysis of chemical modication remains a challenge. Currently, the most widely used analysis tools for particles are colorimetric tests which can identify and quantify dierent functional groups on solid support. For example, the Kaiser test can be used to determine the levels of free amino groups.16,17 Other colorimetric methods18 include the Ellman test for thiols19 and the Pomonis test for the detection of alcohols.20 However, in general, these methods are laborious and typically have high material demands. In addition to colorimetric methods, spectroscopic methods, such as NMR21-23 and FTIR,24 are used. Mass spectrometric analysis has also been applied to gain information regarding the chemical composition of the particles.25,26 Since various particles are commonly employed,
r XXXX American Chemical Society

there is an urgent need for a practical, inexpensive, and universal method to analyze chemical modications on them. Herein, we report the use of zeta potential measurements as a convenient tool which allows chemical reactions to be quickly monitored on particles with minimal sample preparation. The zeta potential is generated when charged particles are dispersed in a medium and results in the formation of an electric potential between the surface of the particle and the medium, due to the fact that the surface charge of the particle attracts counterions. A double layer is formed at the interface of the particle; the rst layer (the Stern-layer) contains immobilized ions which are directly adsorbed to the surface. This layer is surrounded by a second more diuse layer of mobile ions attracted by the surface charge. The potential between the diuse layer and the medium is called the zeta potential. The potential of the Stern-layer is extremely dicult to measure; however, the easily measured zeta potential can be used to derive information concerning the particle surface charge, and hence chemical composition.27 In this study, we synthesized aminomethyl-functionalized submicron polystyrene particles followed by conjugation to a variety of surface groups (basic, acidic, and neutral) and demonstrated that the respective surface chemistry corresponds to the measured zeta potential values. Furthermore, we demonstrate that zeta potential can be employed to follow multistep reaction sequences, as described herein.
Received: Revised: November 16, 2010 December 20, 2010

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Bioconjugate Chemistry

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MATERIALS AND METHODS

Chemicals. Adipic acid, 2,20 -azobis(2-amidinopropane) dihydrochloride (V-50), N,N0 -bis(tert-butoxycarbonyl)-S-methylurea, 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB), divinylbenzene (DVB), methanol, diisopropylcarbodiimide (DIC), 6-hydroxycaproic acid, piperidine, pyridine, sodium acetate, styrene, 4-sulfobenzoic acid potassium salt, and triethylamine were purchased from Sigma-Aldrich (Gillingham, UK). Acetic anhydride, magnesium sulfate (MgSO4), sodium hydroxide (NaOH), sodium chloride (NaCl), and tris(hydroxymethyl)aminomethane were purchased from Fisher Scientific (Loughborough, UK). Ethyl 2-cyano-2-(hydroxyimino) acetate (oxyma) was purchased from Apollo Scientific (Stockport, UK). Dimethylformamide was purchased from Rathburn (Walkerburn, UK). Fmoc-Glu(tBu)OH was purchased from GL Biochem (Shanghai, China). 5-Amino3-(bis(carboxymethyl) amino)pentanoic acid (NTA) was purchased from NBS Biologicals (Huntingdon, UK). N,N0 -Diisopropylethylamine (DIPEA) was purchased from Bioproducts (Hessle, UK). Phosphate buffered saline (PBS) was purchased from Oxoid (Basingstoke, UK). Trifluoroacetic acid (TFA) was purchased from Acros Organics (Geel, Belgium). Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Merck Bioscience (Nottingham, UK). 4-Vinylbenzylamine hydrochloride (VBAH)28 and Fmoc-4,7,10-trioxa-1,13-tridecaneediamine succinimic acid (FmocHN-PEG3-OH)29 were synthesized following literature procedures (see Supporting Information). 200 nm Aminomethyl-Functionalized Microparticles 1 10,28. Styrene (5.5 mL, 48 mmol, freshly washed with 4 25% NaOH and 4 brine), DVB (0.14 mL, 1 mmol, freshly washed with 4 25% NaOH and 4 brine), VBAH (265 mg, 1.5 mmol), and MgSO4 (19 mg, 0.16 mmol) were added to N2 purged H2O (90 mL) and stirred for 30 min under a N2 atmosphere. A solution of V-50 (70 mg, 0.26 mmol) in H2O (1 mL) was added, and the reaction mixture was stirred at 80 C for 15 h. The microparticles were collected by centrifugation (15 min at 30 000 g) and washed with MeOH (3 100 mL) and H2O (3 100 mL). The hydrodynamic diameter of the particles was determined to be 232 nm by dynamic light scattering (Malvern Zetasizer Nano-ZS). The amino loading of the particles was determined as 0.135 ( 0.002 mmol/g by a quantitative Kaiser test with a solid content of 2.16 ( 0.01%.17 Modification of Aminomethyl Particles . General Procedure for the Oxyma/DIC Coupling 4, 5, 6, and 7. Ethyl 2-cyano-2-(hydroxyimino) acetate (oxyma)/diisopropylcarbodiimide (DIC) couplings30 were carried out. The acid (4-sulfobenzoic acid potassium salt (12 mg, 50 mol, 5 equiv), FmocHNPEG3-COOH (27 mg, 50 mol, 5 equiv), 6-hydroxycaprioic acid (10 mg, 50 mol, 5 equiv), or Fmoc-Glu(tBu)-OH (22 mg, 50 mol, 5 equiv)) was preactivated in DMF (0.2 mL) by the addition of oxyma (7 mg, 50 mol, 5 equiv) and DIC (8 L, 50 mol, 5 equiv) for 10 min. The preactivated acid was added to the suspension of amino-functionalized microparticles in DMF (75 mg, 2 mL, 10 mol, 1 equiv), and the reaction mixture was heated to 60 C by microwave irradiation (Initiator 60 microwave, Biotage) for 20 min. The coupling was repeated three times. Upon completion, the microparticles were collected by centrifugation (Eppendorf, 5430R) and washed with DMF (3 2 mL). Carboxylated Microparticles 3 and 12. A solution of DIC (8 L, 50 mol, 5 equiv) in DMF (100 L) was added to adipic acid (7 mg, 50 mol, 5 equiv) or 3,30 -dithiopropionic acid (11 mg, 50 mol, 5 equiv) in DMF (1 mL), and the mixture was shaken
B

for 10 min and subsequently added to prewashed (DMF, 2 mL) microparticles (75 mg, 10 mol, 1 equiv) and N,N0 -diisopropylethylamine (DIPEA) (2 L, 10 mol, 1 equiv) in DMF (2 mL). The reaction mixture was heated to 60 C using microwave irradiation for 20 min. Finally, the microparticles were washed with DMF (3 2 mL) with centrifugation (30 000 g for 15 min). Coupling of NTA 14. Attachment of 5-amino-3-(bis(carboxymethyl)amino)pentanoic acid (NTA) was carried out by preactivation (oxyma (0.3 mg, 2 mol, 1 equiv) and DIC (0.3 L, 2 mol, 1 equiv) in DMF (0.2 mL)) of acid-functionalized microspheres (15 mg, 2 mol, 1 equiv) followed by addition of NTA (2 mg, 6 mol, 3 equiv) and heating to 60 C under microwave irradiation for 20 min. Afterward, the microparticles were washed, with centrifugation (30 000 g for 15 min) with DMF (3 2 mL). Guanidinium Microparticles 2. A solution of N0 ,N-bis(tert-butoxycarbonyl)-S-methylurea (15 mg, 50 mol, 5 equiv) in DMF (0.2 mL) and TEA (4 L, 50 mol, 5 equiv) was added to prewashed (DMF, 3 2 mL) aminomethyl microparticles (2 mL, 75 mg, 10 mol, 1 equiv) in DMF (2 mL) to 60 C under microwave irradiation for 30 min. The microparticles were washed with DMF (3 2 mL) with centrifugation (30 000 g for 15 min). N-Acetylated Microparticles 8. Microparticles (2 mL, 75 mg, 10 mol, 1 equiv) were washed with DMF (3 2 mL), and the supernatant was removed. A solution of acetic anhydride, pyridine, and DMF (2:3:15 (v/v/v), 2 mL) was added and the reaction mixture shaken for 10 min. The microparticles were centrifuged, the supernatant was removed, and the acetylation reaction was repeated. The reaction mixture was removed and washed with DMF (3 2 mL) with centrifugation (30 000 g for 15 min). Disulfide Bond Cleavage 13. Microparticles 12, previously coupled to 3,30 -dithiopropionic acid, were washed with PBS (3 2 mL, pH 7.4) and emulsified in a solution of 50 mM tris(2carboxyethyl)phosphine (TCEP) in PBS (pH 7.4, 2 mL). The reaction mixture was shaken for 3 h at 30 C. Finally, the particles were washed with PBS (3 2 mL, pH 7.4) with centrifugation (30 000 g for 15 min). Protecting Group Cleavage . Fmoc-Deprotection. The microparticles were washed in DMF (3 2 mL) and the supernatant removed. A solution of 20% piperidine in DMF (2 mL) was added to the microparticles and the reaction mixture was shaken for 20 min. The microparticles were centrifuged, the supernatant was removed, and the procedure was repeated. t Butylester and tButyloxycarbonyl carbamate Deprotection. Functionalized microparticles with tBu and Boc protecting groups on side chains of amino acids were washed in DMF (3 2 mL) and resuspended in a solution of 10% TFA in DMF (2 mL). The reaction mixture was shaken for 4 h followed by centrifugation of the microparticles, removal of the supernatant, and washing with DMF (3 2 mL) with centrifugation (30 000 g for 15 min). Colorimetric Tests . Kaiser Test. The qualitative and quantitative content of free amine groups was determined with the Kaiser test as reported previously.16,17 The sample was prepared by washing an emulsion of microparticles (3 mg) with MeOH (2 500 L) and removing the supernatant. Formation of a deep blue color indicated the presence of free primary amines. Ellman Test. Free thiol groups were detected using the Ellman test.19 A microparticle suspension (1 mg, 50 L) was added to 50 L of DTNB (5,50 -dithiobis-(2-nitrobenzoic acid)) solution (50 mM sodium acetate and 2 mM DTNB in water), 100 L Tris-buffer (1 M, pH 8), and 800 L water and incubated for 5 min at room temperature. Formation of a yellow color indicated the presence of free thiols.
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Bioconjugate Chemistry Scheme 1. Synthesis of Amino-Functionalized Microparticles 1 and Its Modicationsa

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a (i) V-50, MgSO4 in H2O at 80 C for 15 h. (ii) N0 ,N-Bis(tert-butoxycarbonyl)-S-methylurea, TEA in DMF at 60 C (microwave) for 30 min. (iii) Adipic acid, DIC in DMF at 60 C (microwave) for 20 min. (iv) 4-Sulfobenzoic acid, Oxyma, DIC in DMF at 60 C (microwave) for 20 min. (v) 6-Hydroxycaprioic acid, Oxyma, DIC in DMF at 60 C (microwave) for 20 min. (vi) Fmoc-Glu(tBu)-OH, Oxyma, DIC in DMF at 60 C (microwave) for 20 min followed by 5% TFA in DMF for 4 h and 20% piperidine in DMF for 2 20 min. (vii) Fmoc-4,7,10-trioxa-1,13-tridecaneediamine succinamic acid (FmocHN-PEG3-OH), Oxyma, DIC in DMF at 60 C (microwave) for 20 min. (viii) Acetic anhydride, Pyridine, DMF for 2 20 min.

Solid Content. The solid content of the microparticles was determined by drying the microparticle suspension (100 L) under vacuum (<20 bar) at 40 C overnight and weighing. Dynamic Light Scattering and Zeta Potential Measurement (Laser Doppler Velocimetry). The hydrodynamic diameter and zeta potential of the particles were determined with a Malvern Zetasizer Nano-ZS according to the manufacturers recommendations. A suspension of microparticles (20 g, 1 L, solid content 20 mg/mL) was diluted in 10% PBS (pH = 7.4, 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4) in H2O (1 mL), vortexed, and transferred into either a 4 mL polystyrene cuvette (FB55143, Fisher Scientific) or a 1 mL clear zeta potential cuvette (DTS1060, Malvern). The electrophoretic mobility of the sample was measured and converted into the zeta potential by applying the Henry equation. The data were collected and analyzed with the Dispersion Technology software 4.20 (Malvern) producing histograms for the particles size as a number distribution or diagrams for the zeta potential as a distribution versus total counts.

RESULTS AND DISCUSSION

Microparticle Preparation and Coupling Reactions. Nano and microparticles have been modified for numerous applications in life sciences. However, it is still very challenging to monitor and analyze chemical transformations on solid supports, in particular, when permanent functionalization takes place and when the particles are less than 100 nm. To address the problem of reaction monitoring on solid particles, the ability of the particles zeta potential to be used as a reaction indicator was investigated. First, submicrometer polystyrene particles 1 (200 nm), with an aminomethyl functionality, were synthesized by emulsifier-free emulsion polymerization (Scheme 1).10,28 The amino-functionalized particles were chosen since they allow subsequent modifications into various functionalities in a single reaction step. Initially, we investigated the zeta potential of particles bearing dierent surface functionalities and, in particular, analyzing the shift in
C

zeta potential following chemical reactions. Since the particles surface chemistry has a major eect on the zeta potential, the aminomethyl-functionalized microparticles 1 were conjugated to dierent molecules resulting in a variety of surface chemistries (Table 1, Figure 1). All the modications were also benchmarked with the Kaiser test,16 which demonstrated modication. Following the coupling reactions in DMF, the particle zeta potential was measured directly after standard washing steps in DMF. A small quantity of the particle suspension (20 g) was dispersed in PBS (pH 7.4) followed by zeta potential measurement. The zeta potential of the starting aminomethyl microparticles 1 was 25 mV and changed to 31 mV during conversion to the more basic guanidinium group 2. A strongly negative zeta potential was found after conversion to carboxylic acid 3 (-44 mV), and as expected, even stronger negative values were obtained after attachment of the sulfonic acid 4 (-65 mV). Hydroxy groups 5 on the surface resulted in a slightly negative zeta potential (-7 mV). Furthermore, conversion to a zwitterionic surface 6 by the attachment of glutamic acid was detected by a very broad zeta potential peak with a mean value of -10 mV. Conjugation of neutral groups to the amino-functionalized particles, such as via PEGylation 7 (3 mV) or acetylation 8 (-5 mV), produced zeta potential values close to 0 mV, thus demonstrating the lost charge on the particle surface. It should be noted that the use of the zeta potential for reaction monitoring is based on the comparison between the initial particle zeta potential and the reaction product zeta potential. We observed that dierent batches of polymeric nanoparticles 1 had slightly dierent initial zeta potentials (25 mV ( 3 mV); however, the shifts in zeta potential were signicantly greater than after surface modication. The results demonstrate that zeta potential allows information to be derived regarding the surface chemistry on the particle, though a quantitative relation between the number of surface groups and the zeta potential could not be achieved; however, it proves to be a tool able to distinguish between dierent strengths of acidity and basicity.
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Bioconjugate Chemistry Table 1. Functionalized Polystyrene Microparticles and Their Zeta Potentials

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Figure 1. Zeta potential of aminomethyl and subsequently functionalized polystyrene microparticles.

Monitoring Multistep Reaction Sequences with Zeta Potential. Nano and microparticles are increasingly used as biomedical devices in vitro and in vivo, or for applications in biopurifications. This often requires modifications of particles via multistep reaction sequences in order to attach the desired molecules be they linkers or imaging complexes. It can be difficult to follow such sequences on polymeric supports due to the unknown coupling efficiencies of different reaction steps. In addition, the reactions are often performed in aqueous media involving highly charged biomolecules. We wanted to investigate if the changes in particle zeta potential could be used to monitor multistep reactions on particles. A common coupling sequence on nano and microparticles includes addition of a spacer such as PEG or a carboxylic acid functionality. Scheme 2 represents the ability to use the zeta potential to follow such a reaction sequence directly on a particle. The initial charge-stabilized aminomethyl polystyrene microparticles 1 had a zeta potential of 26 mV. The amino groups were coupled with an Fmoc-protected PEG3-spacer. Treatment with acetic anhydride capped the remaining, nonaccessible amino groups 9. A reduction of the zeta potential to -2 mV (before
D

capping -1 mV) was observed. Fmoc deprotection gave particles with free amines on the particles surface 10 and therefore a strongly positive zeta potential (31 mV). Finally, the coupling of adipic acid 11 onto the particle surface resulted in a negative zeta potential (-39 mV) representing the carboxylic functionality. The three-step reaction sequence was easily monitored by notable changes in zeta potential without signicant sample preparation, loss of material, or chemical cleavage from the particles. This demonstrates that zeta potential is an excellent tool to follow direct-on-bead modications without necessary cleavage or introduction of specic spectroscopic groups for analysis. Reaction Control. Commonly used functionalities in nano and microparticle bioconjugates are thiol and transition metalbearing ligands. A thiol-containing molecule can be easily coupled to a variety of biomolecules such as small interfering RNAs31 or proteins;32 metal-bearing functionalities are also widely used due to their ability to bind the His-tag and therefore noncovalently link proteins to particles.33 Thiols were generated on the particles by coupling 3,30 dithiopropionic acid (DTPA) to the aminomethyl microparticles 1 followed by subsequent reduction with tris(2-carboxyethyl)phosphine (TCEP). The rst coupling resulted in a conversion from a positive zeta potential charge 1 (26 mV) to a negatively charged surface 12 (-29 mV). The following reduction with TCEP in PBS gave free thiol groups 13 on the particle surface with a zeta potential value of -18 mV (Scheme 3a), an expected value due to the relatively acidic nature of the thiol group (pKa 9). The result from the zeta potential analysis was also veried by a positive Ellman test proving the presence of free thiols, although not quantitative.19 Another reaction sequence, which is not easily monitored by current methods, is the synthesis of triacidic ligands, which are commonly used for transition metal bearing complexes. Initially, we followed the synthesis of carboxylic acid bearing microparticles 11 (Scheme 2) yielding particles with a zeta potential of -39 mV, as expected for negatively charged particles. The commonly used N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA)-linker, bearing three carboxylic acid moieties, was introduced by a further coupling step to give 14 with a notably decreased zeta potential of -48 mV.
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Bioconjugate Chemistry Scheme 2. Multistep Reaction Control by Zeta Potential Monitoring of Microparticles

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Scheme 3a

(a) Synthesis and zeta potential monitoring of thiol-functionalized (a) and tricarboxylated (b) microparticles.

These two experiments show the ability of zeta potential to verify reactions which are dicult to analyze with common techniques. A decrease in the strength of the surface acid, from carboxylic acid to thiol, was clearly observed as a change in the zeta potential. Similarly, a notable dierence in zeta potential values was detected when the number of carboxylic functionalities increased on the particle surface without a change in the surface functionality.
E

CONCLUSIONS The use of nano and microparticles has increased dramatically with novel applications in chemistry and life sciences. An increasing number of particles are being chemically modied by attachment of dierent molecules including proteins and nucleic acids leading to variety of functionalities. However, the chemical analysis of the modied particles has been dicult and remains a challenge; indeed, in many cases it is just assumed that
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Bioconjugate Chemistry the reaction took place. Herein, we demonstrated the ability of the zeta potential to be used as a practical and easy method to follow conjugations on nano and microparticles. We employed 200 nm amino-functionalized submicrometer particles, representing nano and microsized particles, to couple a variety of dierent acidic, basic, and neutral groups. The zeta potential values of the dierent surface-modied particles correlated to the nature of chemical modication and therefore demonstrated the strength of the technique. Furthermore, we successfully followed a multistep coupling sequence. Finally, zeta potential was used to analyze modications which are dicult to follow using conventional analysis techniques. Decreasing the potency of a coupled acid, from carboxylic acid to thiol, and increasing the number of presented acids, from a monoacidic to a triacidic linker, was also veried by measuring the shift in zeta potential values on the respective particles. The presented zeta potential-based method is universal, fast, and convenient, and simplies the analysis of reactions carried out on nano and microparticles. We have demonstrated that zeta potential is a very useful method with the ability to directly analyze nano and microparticles, which can easily be used to validate chemical transformations and multistep reaction sequences on solid particles.

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ASSOCIATED CONTENT
S b

Supporting Information. Synthesis of 4-vinylbenzylamine hydrochloride (VBAH) and Fmoc-4,7,10-trioxa-1,13-tridecanediamine succinamic acid (FmocHN-PEG3-OH). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION
Corresponding Author

*Mark Bradley, University of Edinburgh, School of Chemistry, Kings Buildings, Joseph Black Building, West Mains Road, EH9 3JJ Edinburgh, U.K. Tel.: 0044 131 650 4820. Fax: 0044 131 650 6453. E-mail: mark.bradley@ed.ac.uk.

ACKNOWLEDGMENT This research was supported by the MRC. REFERENCES

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