On the Influence of Different Surfaces in Nano- and Submicrometer

Schematic Illustration of the Preparation of Biofunctional Fluorescent Labelsa ..... charge and to provide the best conditions for the adsorption of a...
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Langmuir 2004, 20, 9371-9379

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On the Influence of Different Surfaces in Nano- and Submicrometer Particle Based Fluorescence Immunoassays Yvonne Bruemmel,*,† Cangel Pui-yee Chan,‡ Reinhard Renneberg,‡ Andreas Thuenemann,§ and Matthias Seydack*,† 8sens.biognosticAG, Robert-Roessle-Strasse 10, D-13125 Berlin, Germany, Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, SAR Hong Kong, and Division 1.3 Structural Analysis, Federal Institute for Materials Research and Testing, Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany Received June 7, 2004. In Final Form: July 24, 2004 Recently, numerous attempts have been made to improve the performance of fluorescence immunoassays. One way pursued is the substitution of labeling molecules by micro- or nanocrystalline dyes. The surfaces of these particulate structures are typically engineered by a layerwise assembly of oppositely charged polyelectrolytes, the outer layer being constituted of biorecognition molecules, for example, immunoglobulins. In this study, we show that amphiphilic polymers such as alkylated poly(ethylene imine)s and 1,2-distearoylsn-glycero-3-phosphatoethanolamine-N-[amino(poly(ethylene glycol))] can fully substitute the more intricate layer-by-layer technique and evaluate the influence of surface charge and particle size on the overall performance of these assays.

Introduction Biochemical assays, especially immunoassays, play a pivotal role in clinical diagnostics, in food, and in environmental monitoring. Most common assay techniques require a label to detect the interaction of an affinity compound with the analyte to be detected. Labels may be enzymes, fluorescent compounds (including precursors of such), or radioisotopes which are used in enzyme-linked immunosorbent assays (ELISAs), in fluorescence immunoassays (FIAs), and in radio immunoassays (RIAs), respectively. The basic requirements for any immunoassay are high sensitivity, low limit of detection (LOD), and preserved biomolecule function. To avoid the obvious disadvantages of radioisotopes, most users now resort to enzymes and fluorescent molecules as labels. Enzyme and fluorescent labels offer good sensitivities and limits of detection but still fall short of those obtained with RIAs. Therefore, various efforts have been made to further improve the performance of ELISAs and FIAs. As far as FIAs are concerned, their sensitivity is generally limited by the ratio of fluorescent molecules per biomolecule (F/P ratio). The F/P ratio is typically 4-8 for a conventional, covalently coupled fluorescent immunolabel, for example, an immunoglobulin G labeled with fluorescein isothiocyanate (IgG-FITC) conjugate. A higher F/P may lead to a decrease of the specific binding affinity of the biomolecule and additionally cause self-quenching effects. Increasing the effective dye/biomolecule ratio while minimizing dye self-quenching and maintaining the biomolecule’s binding properties is thus highly desirable in assay development. Three different approaches have been pursued. * To whom correspondence should be addressed. E-mail: [email protected], [email protected]. Tel: +49309 4892120. Fax: +493094892117. † 8sens.biognosticAG. ‡ Department of Chemistry, The Hong Kong University of Science and Technology. § Division 1.3 Structural Analysis, Federal Institute for Materials Research and Testing.

The most straightforward one is the tailoring of the dyes to the spectroscopic demands (i.e., high extinction at the excitation wavelength and fluorescence quantum yields close to unity). Although the widely used FITC provides LODs of 400 pg/mL or above for the detection of biotinylated GtRMIgG, the utilization of the extremely absorbent R-phycoerythrin (R-PE) and SensiLightPL3 was shown to lower the detection limit down to 22 and 1.3 pg/mL, respectively.1 The application of quantum dot labels will be the next promising step in this field.2 Another different way to improve FIA performance is in using fluorescent rare-earth chelates, and europium chelates in particular, in time-resolved fluorometric assays. Due to their narrow band emissions, large Stokes shifts, and long lifetimes, rare-earth chelates allow for efficient background separation resulting in very high signal-to-noise (S/N) ratios.3 In general, this method provides very low LODs as well, but signal amplification and sensitivity are rather limited and in the presence of miniscule amounts of quencher molecules the results may easily be distorted. The third and most promising approach seems to be the utilization of extended labeling systems incorporating 103-108 fluorescent molecules. In these high-load systems, the mean dye-to-dye distances are large enough to preclude or diminish quenching effects. This may be accomplished either by the very structure of the labeling system4,5 or by (1) MicroPlate Detection comparison between SensiLight PBXL-3L, other fluorophores and enzymatic detection; Technical Bulletin 3; Martek Biosciences Corp.: Columbia, MD; www.martekbio.com/Fluorescent_Products/Technical_Literature.asp. (2) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41. (3) Vallarino, L. M.; Watson, B. D.; Hindman, D. H. K.; Jagodic, V.; Leif, R. C. Quantum Dyes, A New Tool for Cytology Automation. In The Automation of Cancer Cytology and Cell Image Analysis; Pressman, H. J., Wied, G. L., Eds.; Tutorials of Cytology: Chicago, 1979; pp 53-62. (4) Hall, M.; Kazakova, I.; Yao, Y. M. Anal. Biochem. 1999, 272, 165170. (5) Goeran, K.; Kroeger, D. Affinitaetssenoren und -assays. DE Patent 19703718, 1997.

10.1021/la0486032 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/01/2004

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Bruemmel et al.

Scheme 1. Schematic Illustration of the Preparation of Biofunctional Fluorescent Labelsa

a Three different systems are compared: (a) FDA was milled into microcrystals with a mode diameter of 0.680 µm and stabilized with SDS as surfactant, followed by encapsulation with polyelectrolytes (layer-by-layer technique) with a total number of four layers: PAH and alginic acid (Alg). The specific immunoreagents were attached by covalent binding. (b) FDA was milled into nanocrystals using differently alkylated poly(ethylene imine)s as surfactants (PEI1, PEI2, and PEI3). Two fractions of different mode diameter are obtained, 160 nm for the small fraction and 270 nm for the big fraction. The biorecognition molecules were directly adsorbed to the colloid’s surface. (c) FDA was milled in DSPE-PEG(2000)-amine (mode diameter, 117 nm), and the biorecognition molecules were directly adsorbed to the surface.

washing out the dye molecules after the affinity reaction.6,7 Typical systems that were successfully applied as immunolabels are fluorophore-loaded latex beads,4 liposomeencapsulated fluorophores,6,7 fluorescent conjugated dendrimers,5 and other related systems.8-10 All these systems provide very high F/P ratios of 103 or above. The most extreme example in this category is a labeling system that was recently established by Trau et al. using particulate organic biolabels based on polyelectrolyteencapsulated microcrystals of a fluorogenic precursor, fluorescein diacetate (FDA).11 Water-insoluble FDA crys(6) Imai, K.; Namura, Y. Immunoassay with Antigen or Antibody Labeled Microcapsules Containing Fluorescent Substances. U.S. Patent 4916080, 1986. (7) Schott, H.; Von Cunow, D.; Langhals, H. Biochim. Biophys. Acta 1992, 1110, 151-157. (8) Fribnau, T. C. J.; Roeles, F.; Leuvering, J. H. W. Application of Water-Dispersible Hydrophobic Dyes or Pigments as Labels in Immunoassays. U.S. Patent 4,373,932, 1981. (9) Mandle, R. M.; Wong, Y. N. Chemical Luminescence Amplification Substrate System for Immunochemistry Involving Microencapsulated Fluorescer. U.S. Patent 4,372,745, 1983. (10) Kamyshny, A.; Magdassi, S. Colloids Surf., B 2000, 18, 13-17. (11) Trau, D.; Yang, W.; Seydack, M.; Caruso, F.; Yu, N.; Renneberg, R. Anal. Chem. 2002, 74 (21), 5480-5486.

tals of submicrometer size were encapsulated using the layer-by-layer method.12 The outermost of 4-8 polyelectrolyte layers provides a suitable interface for the subsequent attachment of biorecognition molecules. Since (a) the entire microcrystal core is composed of precursor molecules which can, after the affinity reaction, be dissolved and converted into fluorescein and (b) the biomolecules form a layer on the encapsulated crystal surface, the F/P ratio is ∼108 for a crystal with a mean diameter of 500 nm. This value refers to one successful binding, that is, P ) 1. Amplification rates of 70-2000 (depending on analyte concentration and detector dilution), compared to a corresponding immunoassay with direct fluorescently labeled antibodies, were found for these assays.11 As shown by Caruso et al., organic polyelectrolyte layers may be substituted by amphiphilic polymers.13 With this taken into account and in order to further simplify the preparation of the fluorescent conjugates and enhance (12) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (13) Caruso, F.; Trau, D.; Moehwald, H.; Renneberg, R. Langmuir 2000, 16, 1485-1488.

Influence of Surface in Fluorescence Immunoassays

the sensitivity, a simple two-step approach was suggested by Chan et al.14 Abandoning the layer-by-layer procedure, FDA crystals with a mean diameter of 107 nm were encapsulated with di-stearylphosphatidyl-ethanolamine grafted amino(poly(ethylene glycol)) (PEG-amine). Biorecognition molecules were finally adsorbed onto this surface. Grafted PEG chains have been of much interest due to their use as surface-passivating coatings on drug delivery vehicles.15-20 The limit of detection for a sandwich assay with GtRMIgG-FDA nanocrystal labels having a mean diameter of 107 nm was found to be around 0.06 µg/L with an amplification rate 400-2700 times higher (depending on the analyte concentration) than for conventional GtRMIgG-FITC labels.14 In this study, we strived to understand the influence of different surface modifications and particle sizes on the performance of these FDA-based particulate labels in fluorescence immunoassays (Scheme 1). The surfaces were expected to affect the affinity molecules mainly via their different charge densities, while bigger particles, once bound, will be more exposed to mechanical forces during the washing steps. The signal-to-noise ratio and the LOD will be affected in both cases. To investigate these influences, FDA was milled in three different surfactants: sodium dodecyl sulfate (SDS), DSPE-PEG(2000)amine, and alkylated poly(ethylene imine)s (PEIs) including PEI1, PEI2, and PEI3. All PEIs are hyperbranched polymers and were alkylated with dodecyl bromide and thus exhibit amphiphilic properties as shown by Noeding and Heitz.21 The PEI backbone of PEI1 has a lower molecular weight of Mw ) 5000 g/mol, while the PEI backbone of PEI2 and PEI3 has a higher molecular weight of Mw ) 25 000 g/mol. The degree of alkylated amino functions of the PEIs was 15% (PEI1 and PEI2) and 30% (PEI3). We assumed that the alkylation of the PEIs not only facilitates their applicability as amphiphiles for the stabilization of colloidal suspensions but also permits the attachment of biomolecules. Since milling was accomplished by means of different methods, the mean particle size varied between 107 nm for FDA-[DSPE-PEG(2000)-amine)], 160 and 270 nm for FDA-PEI1 and FDA-PEI2, 80 and 270 nm for FDA-PEI3, and 688 nm for FDA-SDS. The FDA-SDS particles were afterward encapsulated with four layers of polyelectrolyte in total. The biorecognition molecules were then covalently bound to the outermost layer, sodium alginate. FDA[DSPE-PEG(2000)-amine)]-GtRMIgG and FDA-PEIxGtRMIgG (x ) 1, 2, 3) were prepared in a two-step approach according to the procedure described by Chan et al.14 All labeling systems from this study are compared with data from the literature in terms of LOD, amplification, and signal-to-noise ratio. Experimental Section Materials. FDA, alginic acid (Alg; sodium salt, low viscosity), poly(allylamine hydrochloride) (PAH, Mw ) 15 000 g/mol), (14) Chan, C. P.-y.; Bruemmel, Y.; Seydack, M.; Sin, K.-k.; Wong, L.-w.; Merisko-Liversidge, E.; Trau, D.; Renneberg, R. Anal. Chem., submitted. (15) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Biophys. J. 1998, 74, 1371-1379. (16) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436. (17) Pertsin, A. J.; Grunze, M.; Garbuzova, I. A. J. Phys. Chem. B 1998, 102, 4918-4926. (18) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8399-8404. (19) Halperin, A. Eur. Phys. J. B 1998, 3, 359-364.531. (20) Use of PEG-Derivatized Lipids as Surface Stabilizers for Nanoparticle Compositions; WO00/51572; Nanosystems/Division of Elan Pharmaceutical Technologies: Gainesville, GA, Sep 2000. (21) Noeding, G.; Heitz, W. Macromol. Chem. Phys. 1998, 199, 16371644.

Langmuir, Vol. 20, No. 21, 2004 9373 Chart 1. Chemical Structures of the Four Surfactants Used to Introduce a Surface Charge to FDA for Colloidal Stabilization and Further Attachment of Biomoleculesa

a (b) SDS. (a) Alkylated poly(ethylene imine)s (ref 20), PEI1, PEI2, and PEI3, which are modified with dodecyl bromide. All PEIs are hyperbranched polymers. The PEI backbone of PEI1 has a lower molecular weight of Mw ) 5000 g/mol, while the PEI backbone of PEI2 and PEI3 has a higher molecular weight of Mw ) 25 000 g/mol. The degree of alkylated amino functions of the PEIs was 15% (PEI1 and PEI2) and 30% (PEI3). (c) DSPEPEG(2000)-amine.

dimethyl sulfoxide (DMSO), SDS, and 2-morpholinoethanesulfonic acid (MES) were purchased from Sigma-Aldrich (Steinheim, Germany). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was obtained from Pierce (Rockford, IL). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (ammonium salt) [DSPE-PEG(2000)amine] was obtained from Avanti Polar Lipids Inc. (Alabaster, AL). PEI1 was synthesized using hyperbranched poly(ethylene imine) with molar ratios of primary to secondary to tertiary amino groups of 34:40:26 (Mw ) 5000 g/mol, Polyimin G100, BASF AG) and alkylated with dodecyl bromide (alkylation degree, 15%) (Chart 1). PEI2 and PEI3 were synthesized by alkylation of high molecular weight hyperbranched poly(ethylene imine) (Mw ) 25 000 g/mol, BASF) with dodecyl bromide (alkylation degree, 15% and 30%, respectively). Details of the synthesis and the properties of the amphiphilic PEIs are described by Noeding and Heitz.21 Affinity purified polyclonal goat anti-mouse IgG (GtRMIgG, whole molecule), mouse IgG (MIgG), and rabbit IgG (RbIgG) were supplied by Arista Biologicals Inc. (Allentown, PA) and Sigma-Aldrich (Taufkirchen, Germany). Goat anti-mouse IgGFITC conjugates were purchased from Molecular Probes (Eugene, OR; protein concentration, 2 mg/mL; F/P ratio, 6.3) and from Sigma-Aldrich (protein concentration, 1.1 mg/mL; F/P ratio, 4.2). Bovine serum albumin (BSA, fraction V) was from Roth (Karlsruhe, Germany). All other chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany) and were of analytical grade. FDA Nanocrystals Stabilized with SDS Followed by Encapsulation. FDA (1% w/v) was milled by HAW Ingenieure (Dresden, Germany) in 1% (w/v) SDS solution for 8 h in total. The procedure was interrupted several times to prevent the dispersion from heating above 20 °C. The milling was accomplished in a Retsch S100 (Haan, Germany) ball mill, using a 50 mL agate milling beaker (Retsch Mahlbecher “S”) and a mixture of 10 mm agate and 2 mm titanium dioxide balls as the milling medium. Polyelectrolyte multilayers were then assembled onto the crystal’s surface by alternating deposition of PAH and alginic acid. Typically, 0.5 mL of FDA-SDS suspension (1% w/v) was added to 1 mL of PAH-polyelectrolyte solution (5 mg/mL in

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10 mM MES, pH 5.8). The suspension was then incubated for 15 min. The excess polyelectrolyte was removed by centrifugation at 3000g for 5 min. Following removal of the supernatant, the coated particles were washed twice with water. For assembling the second layer, 1 mL of negatively charged alginic acid (5 mg/ mL in 10 mM MES, pH 5.8) was added and incubated for 15 min. The above-described centrifugation washing steps and the sequential adsorption steps were repeated until a total number of four layers was reached. The encapsulated particles were finally stored for further use in 1 mL of MES buffer (10 mM, pH 5.8) at 4 °C in an overhead shaker. FDA Nanocrystals Stabilized with DSPE-PEG(2000)Amine. FDA nanocrystal suspensions were prepared by Elan Drug Delivery Inc. (King of Prussia, PA). An amount of 5% (w/v) FDA in 1.25% (w/v) DSPE-PEG(2000)-amine and 0.05% SDS was milled for 48-72 h. The temperature was kept at 20 °C to prevent the material from hydrolyzing. FDA Nanocrystals Stabilized with Alkylated Poly(ethylene imine)s. FDA was prepared by ball milling using small glass beads (0.25-0.50 mm; Roth, Karlsruhe, Germany). FDA (20 mg) was mixed in a glass tube with 2 g of glass particles and 2 mL of polymer solution containing 0.05, 0.10, or 0.30% (w/v) of the alkylated poly(ethylene imine)s PEI1, PEI2, and PEI3. This mixture was vortexed for 15 min at room temperature and exposed to ultrasound for another 15 min. This procedure was repeated once. The colloidal suspension was separated from the glass particles by filtration using a 604 filterpaper (Schleicher und Schuell, Germany) and centrifuged for 5 min at 6000g. The pellet was then resuspended in 1 mL of surfactant solution, and the size distribution was measured. The supernatant was centrifuged for 10 min at 16 000g, the pellet was finally resuspended in 1 mL surfactant solution, and the size distribution was determined. Measurement of Particle Sizes. The volume size distributions were measured based on the Mie theory of light scattering in combination with the polarization intensity differential scattering (PIDS) method. This PIDS technology allows reliable determinations of multimodal size distributions in a range of 0.04-2000 µm. These measurements were carried out by means of a Coulter LS230 (Beckmann Coulter Inc., Krefeld, Germany). Microelectrophoresis. The ζ-potential of the FDA nanocrystals was examined by electrophoresis using a Zetasizer 3000 HSA (Malvern Instruments, U.K.) and taking the average of three measurements at the stationary level. The mobility u was converted to the ζ-potential using the Helmholtz-Smoluchowski relation ζ ) 4πuη/, where η is the viscosity of the solution and  is the permittivity.22 Scanning Electron Microscopy (SEM). The morphology of milled FDA nanoparticles was examined with a JEOL 6300F ultrahigh-resolution scanning electron microscope operating at 10 kV. Conjugation of FDA Nanoparticles with Affinity Molecules by Adsorption. The FDA nanoparticles were conjugated to antibodies as follows: Particle suspensions (0.0626%, w/v) were incubated with 200 µg/mL of GtRMIgG in 10 mM phosphatebuffered saline (PBS, pH 7.4) at 20 °C for 1 h. After centrifugation at 16 000g for 10 min, the supernatant was removed and its UV absorption was measured at 280 nm (Cary 50 Bio UV/VisSpectrophotometer, Australia). The antibody surface coverage on the nanoparticles was determined by the difference of the absorption at 280 nm between the supernatant and the original protein solution. The IgG-coated particles were then separated from soluble IgG by three centrifugation/washing cycles. Covalent Binding of Affinity Molecules to the Capsule Surface. The covalent binding was performed according to a Bangs Laboratories protocol of covalent coupling using a carbodiimide, EDC.23 Carbodiimides are zero-length cross-linking reagents used to mediate the formation of an amide linkage between a carboxylate and an amine.24-26 They are called zerolength because in forming these bonds no additional chemical structure is introduced between the conjugated molecules. (22) Hunter, R. J. Zeta potential in colloid science: Principles and applications; Academic Press: London, 1981. (23) Covalent Coupling; TechNote 205; Bangs Laboratories, Inc.: Fishers, IN; www.bangslabs.com.

Bruemmel et al. Encapsulated FDA particles (an aliquot of 0.333 mL) were washed twice with 1 mL of 100 mM MES buffer, pH 5.8. After washing, the pellet was resuspended in 1 mL of 100 mM MES buffer. For preactivation, 10 mg of EDC was added and the solution was incubated for 15 min at room temperature under continuous rocking. The unreacted EDC was removed by three repeated washing steps with 1 mL of 100 mM MES buffer. The preactivated particles were then incubated with 500 µL of 150 µg/mL GtRMIgG (1.5 mg/mL) for 2 h at room temperature under shaking. Finally the IgG-coated particles were separated from soluble IgG by three centrifugation/washing steps and stored in 1 mL of 10 mM PBS (pH 7.4) for further use at 4 °C in an overhead shaker. Solid-Phase Sandwich Fluorescence Immunoassay.11 GtRMIgG (1 µg/mL) in 0.1 mol/L carbonate buffer (pH 9.6) (100 µL/well) was coated overnight on Nunc-Immuno Maxisorp 96 microwell plates (Nunc International, Rochester, NY) at 4 °C.11 After rinsing three times with washing buffer (10 mM PBS, 0.1% (w/v) BSA, 0.5% (w/v) Tween-20), the wells were blocked with 300 µL/well of 1.0% BSA solution for half an hour at 37 °C. The plate was then washed four times and incubated with 100 µL/ well of MIgG (0, 1, 2, 4, 6, 8, and 10 µg/L) for 1 h at 37 °C. Afterward, the plate was washed five times. Suspensions of antimouse-coated nanocrystals (0.0125% w/v) were dispensed into the wells (100 µL/well), and the microwell plate was incubated again at 37 °C for 1 h. For comparison, the soluble fluorescent labels GtRMIgG-FITC, diluted to 1:100 (Molecular Probes) and 1:64 (Sigma-Aldrich), were used while 1 µg/mL of GtRMIgG (100 µL/well) served as the coating in this case. After incubation, excess detector antibody conjugates were washed off with buffer in five washing cycles. An aliquot of 100 µL/well of release reagent (DMSO and 1 M NaOH in a 1:1 ratio) was added. The fluorescence intensity was measured by an MFX microplate fluorometer (Dynex Technologies Inc., Chantilly, VA) at excitation/emission wavelengths of 485/538 nm. The measurements were performed using a kinetic setup. After complete dissolution of the particles, the fluorescence signal remains stable and was then taken for the analysis. Negative controls were prepared by omitting the MIgG in the second layer, by omitting the conjugate addition in the third layer, or by adding RbIgG as “false” analyte. Samples were run in replicates of three except six wells which were run without the addition of analyte (background) to establish the LOD of the assay, which is defined as the concentration of MIgG corresponding to the mean fluorescence of the background signal plus twice the standard deviation (SD) of this measurement. This LOD value determines the sensitivity of the assay at a 95% confidence interval (P < 0.05).

Results and Discussion Milling of FDA Nanocrystals in Surfactants To Introduce a Surface Charge. In general, FDA crystals in an aqueous environment are not sufficiently charged to form stable dispersions and therefore tend to aggregate. When FDA crystals are milled in a suitable ionic surfactant or charged amphiphilic polymer, a surface charge is generated. The surfactants’ hydrophobic parts most likely associate with the hydrophobic crystal surface, and the hydrophilic parts are orientated toward the surrounding water dipoles. The hydrophobic interaction between the surface of the FDA crystals and the carbon chains of the surfactants is the driving force for the adsorption process, which leads to an entropic gain in the overall system.27 The adsorbed charged layer introduces a surface charge to the FDA crystals, making them dispersible in water and preventing their aggregation, hence conferring colloidal stability.28 This was verified by microelectrophoresis (24) Hoare, D. G.; Koshland, D. E. J. Am. Chem. Soc. 1966, 88 (9), 2057. (25) Chu, B. C. F.; Kramer, F. R.; Orgel, L. E. Nucleic Acids Res. 1986, 14, 5591. (26) Gosh, S. S.; Kao, P. M.; McCue, A. W.; Chapelle, H. L. Bioconjugate Chem. 1990, 1, 71. (27) Myers, K. R.; Nemirovsky, A. M.; Freed, K. F. J. Chem. Phys. 1992, 97. (28) Caruso, F.; Yang, W.; Trau, D.; Renneberg, R. Langmuir 2000, 16 (23), 8923-8936.

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Figure 2. Size distribution of FDA-SDS (s) after adding different numbers of polyelectrolyte layers. After deposition of the first PAH layer (- - -), the size distribution shifts to bigger sizes. Sonication entails a slight reversion (‚‚‚). The deposition of four layers, FDA-SDS-(PAH/Alg)2 (-‚‚-) in total is strongly multimodal due to the formation of big aggregates. Table 1. Alternating ζ-Potentials after the Deposition of Oppositely Charged Polyelectrolytes on FDA Nanocrystals

Figure 1. Particle size distribution of FDA milled in different surfactants: (a) SDS-stabilized FDA microcrystals with a mode diameter of 680 nm and DSPE-PEG(2000)-amine stabilized FDA nanocrystals with a mode diameter of 117 nm. (b) FDA milled in PEI1 results in two fractions, the big fraction with high polydispersity and a mode diameter of 270 nm and the small fraction with nearly monodisperse distribution and a mode diameter of 160 nm.

measurements (expressed as a ζ-potential), which is an effective method to characterize the surface charge of colloidal particles.29 The sign of the ζ-potential indicates whether the particle surface is positively or negatively charged, and the magnitude indicates the stability of the colloidal dispersion. Generally, the greater the absolute magnitude of the ζ-potential, the more stable the colloidal dispersion. FDA Nanocrystals Stabilized with SDS Followed by Encapsulation. The particle size distributions of FDASDS microcrystals were determined by light scattering measurements shown in Figure 1a. Ninety percent (d90) of the particles were smaller than 1067 nm. The mode (i.e., maximum of the volume distribution) was around 680 nm, corresponding to the data from scanning electron microscopy (not shown). The surface charge introduced to the FDA crystals by adsorbing SDS is characterized by measuring the ζ-potential. Following the ball milling process, the FDA microcrystals exhibited a value of -45 mV, indicating a negatively charged, stable particle dispersion. The subsequent alternate adsorption of PAHpolyelectrolyte and alginic acid layers onto the SDS-coated FDA microcrystals yielded alternating ζ-potentials of about +40 and -40 mV as shown in Table 1. As noted for other systems, the charge reversal is indicative of the polyelectrolyte deposition with each adsorption step.27,28,30,31 To investigate changes of the particle sizes during the encapsulation process, the volume size distributions were measured after deposition of every single layer and removal of excess polyelectrolyte. Figure 2 shows that in contrast to what was known so far, after adsorption of the (29) Caruso, F.; Lichtenfeld, H.; Donath, E.; Moehwald, H. Macromolecules 1999, 32, 2317-2328. (30) Caruso, F.; Moehwald, H. J. Am. Chem. Soc. 1999, 121, 60396046. (31) Joanny, J. F. Eur. Phys. J. B 1999, 9, 117-122.

layer no.

surface

ζ-potential [mV]

standard deviation

0 1 2 3 4

FDA-SDS PAH alginic acid PAH alginic acid

-38 37.5 -43.3 44.1 -53

2 1.5 1 1.5 4.2

first layer of PAH the particle size distribution drastically changed toward bigger structures. The distribution becomes strongly multimodal. After deposition of PAH, 90% of the particles were smaller than 9941 nm and 50% smaller than 5362 nm. The mode of the initial colloidal dispersion of 688 nm increased to 755 nm. The strong increase of the 90% value in contrast to the small change of the volume mode value indicated the formation of aggregates. Only a small number of particles contributed to this aggregation process as the thickness of one layer is known to be around 2 nm and the mode was hardly changing.32 Before addition of the second layer, alginic acid, the sample was sonicated three times for 10 s. Subsequent measurement of particle sizes showed a slight decrease in the distribution maximum, which was also observable for the d90 and the d50 values. The deposition of the second layer, alginic acid, and the third, PAH, showed no real change in the distribution. After adsorption of the fourth layer, again alginic acid, a strong shift of the size distribution toward bigger structures was measured. The mode increased to a maximum of 19 790 nm. Ninety percent of the particles were smaller than 24 600 nm, and 50% smaller than 4382 nm. Subsequent ultrasonic treatment did not improve the system. Further measurements showed that this phenomenon may have occurred as early as the second or third layer was adsorbed and could never be improved by ultrasound. One of the main disadvantages of the layer-by-layer method is the instability of the system caused by this aggregation process. Two different mechanisms may account for the observed aggregation: bridging, that is, the connection of particles by at least one polyelectrolyte chain, is favored by high particle concentrations and/or long polymer chains. Aggregation caused by bridging is not a reversible process.33 The second process of aggrega(32) Lvov, Y.; Decher, G.; Moehwald, H. Langmuir 1993, 9, 481486.

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tion may be caused by an incomplete charge compensation leading to differently charged domains on the particle surfaces. Due to the resulting electrostatic attraction between the particles, tightly bound aggregates are formed. With increasing polyelectrolyte concentration, the formation of such aggregates should become less probable. This hypothesis was corroborated by experiments comparing two different concentrations of polyelectrolytes (1 and 5 mg/mL, respectively) in which aggregation phenomena were slightly reduced in the case of the higher polyelectrolyte concentration. After encapsulation with four layers of PAH and alginic acid (both 1 mg/mL), the particle size distribution was strongly multimodal with a mode of 4047 nm. In contrast, the size distribution using polyelectrolyte concentrations of 5 mg/mL is less multimodal with a mode of 1592 nm (data not shown). Higher polyelectrolyte concentrations led to smaller aggregates, but aggregation could not be totally inhibited by increasing concentrations. FDA Nanocrystals Stabilized with DSPE-PEG(2000)-Amine. The particle size distribution of the FDA nanocrystals determined by light-scattering measurements is shown in Figure 1a. Approximately 90% of the particles were found to be smaller than 236 nm, and the mode was 117 nm. This result is in agreement with an SEM analysis of the particles which shows that the particles have a wide range of shapes (not shown). During the milling process in the presence of DSPEPEG(2000)-amine solution, newly generated FDA/water interfaces were immediately coated with a layer of this polymer. The phospholipid chain of the DSPE-PEG(2000)amine provides the hydrophobic part for the adsorption on the uncharged FDA crystal. Thus, the particle surfaces become hydrophilic by virtue of the PEG chain. Following the milling process, the FDA nanocrystals exhibited a ζ-potential around zero when measured in deionized H2O containing 0.01 M KCl with pH 7. It was shown that FDA nanocrystals coated with DSPE-PEG(2000)-amine exhibited positive ζ-potentials when the pH is