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Ultrasonic Synthesis of Stable, Functional Lysozyme Microbubbles Francesca Cavalieri,*,†,‡ Muthupandian Ashokkumar,*,§ Franz Grieser,§ and Frank Caruso† Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular, Engineering, The UniVersity of Melbourne, Melbourne, Victoria 3010, Australia, Dipartimento di Scienze e Tecnologie Chimiche, UniVersita` di Roma Tor Vergata, 00173 Roma, Italy, and Particulate Fluids Processing Centre, School of Chemistry, The UniVersity of Melbourne, Melbourne, Victoria 3010, Australia ReceiVed April 7, 2008. ReVised Manuscript ReceiVed July 16, 2008 High-intensity ultrasound induces emulsification and cross-linking of protein molecules in aqueous medium. The stability and the functionality of the resultant protein-coated microbubbles are crucial in many of their applications. For example, the stability of drug-loaded microbubbles should be sufficiently long enough, in vivo, so that they can be ruptured only at specific sites for release of the drugs. In this study, we report the synthesis of stable and functional microbubbles, coated with chemically reduced lysozyme, using high-intensity ultrasound in aqueous solution. In the absence of chemical reduction, stable microbubbles were not produced with native lysozyme, indicating the importance of free -SH functional groups for protein cross-linking. The degree of cross-linking between lysozyme molecules was controlled by manipulating both the extent of chemical reduction of the intramolecular disulfide bonds and sonication time. The lysozyme-coated microbubbles are stable for several months and retain the enzymatic (antimicrobial) activity of lysozyme. The layer-by-layer (LbL) deposition of polyelectrolytes onto the protein-shell air-core template has been used as a versatile procedure to modify the surface properties of the microbubbles, indicating the possibility of adsorbing potential drugs and/or biolabels on the surface of these microbubbles for therapeutic and diagnostic applications.
* To whom correspondence should be addressed. E-mail: masho@ unimelb.edu.au (M.A.);
[email protected] (F.C.). † Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular, Engineering, The University of Melbourne. ‡ Universita` di Roma Tor Vergata. § Particulate Fluids Processing Centre, School of Chemistry, The University of Melbourne.
ultrasound triggered bubble disruption and subsequent drug release is a concept successfully applied in preclinical studies.10-12 One emerging application that is currently being investigated is ultrasound-targeted microbubble destruction (UTMD) for efficient DNA transfection in gene therapy. There is a wealth of literature on BSA and lipid-shelled microbubbles for gene delivery applications,13,14 however, the safe and efficient delivery of therapeutic levels of drugs remains a central goal. The engineering of polymer-coated microbubbles with therapeutic features is challenging, as many parameters spanning from shell materials, targeting ligands, response to ultrasound and drug loading capacity need to be addressed. A shell encapsulating the gas bubble is essential for the longevity and circulation time of the contrast agent. Although the elimination half-life (the time it takes for microbubbles to disappear in vivo) of the commercialized ultrasound contrast agents has increased in the past,3,4 it is still a primary concern for ultrasound drug delivery applications. For the potential use in therapeutic applications, the chemical nature of the shell and its mechanical properties are crucial, and require a tailored synthetic approach. It can be advantageous to select a biopolymer as the shell material, since it is generally acknowledged that polymer-based contrast agents should be biodegradable in order to facilitate their ultimate elimination after injection into the body. The ultrasound contrast agent should
(1) Grinstaff, M. W.; Suslick, K. S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7708–7710. (2) Grinstaff, M. W.; Suslick, K. S; Kolbeck, K. J.; Wong, M. Ultrason. Sonochem. 1994, 1, S65–S68. (3) Kim, J-H; Park, K.; Nam, H. Y.; Lee, S.; Kim, K.; Kwon, I. C. Prog. Polym. Sci. 2007, 32, 1031–1053. (4) Schutt, E. G.; Klein, D. H.; Mattrey, R. M.; Riess, J. G. Angew. Chem., Int. Ed. 2003, 42, 3218–3235. (5) Tu, J.; Matula, T. J.; Brayman, A. A.; Crum, L. A. Ultrasound Med. Biol. 2006, 2, 281–288. (6) Tu, J.; Hwang, J. H.; Matula, T. J.; Brayman, A. A.; Crum, L. A. Ultrasound Med. Biol. 2006, 10, 1601–1609. (7) Klibanov, L. AdV. Drug DeliV. ReV. 1999, 37, 139–157. (8) Unger, E. C.; Porter, T.; Culp, W.; Labell, R.; Matsinaga, T.; Zutshi, R. AdV. Drug DeliV. ReV. 2004, 56, 1291–1314. (9) Ferrara, K.; Pollard, R.; Borden, M. Annu. ReV. Biomed. Eng. 2007, 9, 415–447.
(10) Xie, F.; Tsutsui, J. M.; Lof, J.; Unger, E. C.; Johanning, J.; et al. Ultrasound Med. Biol. 2005, 31, 979–985. (11) Weller, G. E. R.; Villanueva, F. S.; Klibanov, A. L.; Wagner, W. R. Ann. Biomed. Eng. 2002, 30, 1012–1019. (12) Leong-Poi, H.; Christiansen, J.; Klibanov, A. L.; Kaul, S.; Lindner, J. R. Circulation 2003, 107, 455–460. (13) (a) Frenkel, P. A.; Chen, S.; Thai, T.; Shohet, R. V.; Grayburn, P. A. Ultrasound Med. Biol. 2002, 28, 817–822. (b) Lentacker, I.; De Geest, B. G.; Vandenbroucke, R. E.; Peeters, L.; Demeester, J.; De Smedt, S. C.; Sanders, N. N. Langmuir 2006, 22, 7273–7278. (14) (a) Borden, M. A.; Caskey, C. F.; Little, E.; Gillies, R. J.; Ferrara, K. W. Langmuir 2007, 23, 9401–9408. (b) Lentacker, I.; De Smedt, S. C.; Demeester, J.; Van Marck, V.; Bracke, M.; Sanders, N. N AdV. Funct. Mater. 2007, 17, 1910–1916. (c) Suzuki, R.; Takizawa, T.; Negishi, Y.; Utoguchi, N.; Maruyama, K. J. Drug Targ. 2007, 17, 531–537.
Introduction Gas-filled polymer-coated microbubbles are intrinsically ultrasound responsive systems: ultrasonically generated gas-filled proteinshelled microbubbles respond to the pressure fluctuations caused by an ultrasonic field. At reasonable high acoustic pressure levels, the gas/shell wall can be expanded during the expansion phase of the acoustic cycle leading to the breakage of the shell. Thus, when tailored with targeting features, the protein-shelled microspheres are promising candidates for ultrasound triggered drug delivery applications. Air- and perfluoropropane-filled serum albumin microspheres with a 50 nm thick shell have been widely used as ultrasound contrast agents.1,2 In recent years, lipid-, surfactantand polymer-shelled microbubbles have been introduced in biomedical imaging.3-6 The successful use of microbubbles in medical diagnostics as ultrasound contrast agents opens new opportunities for the application of such vehicles in targeted drug delivery.7-9 The combination of bubbles targeted at specific tissues, including thrombi, atheroma plaques and tumors with
10.1021/la801093q CCC: $40.75 2008 American Chemical Society Published on Web 08/19/2008
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by Suslick et al.18 Avivi and Gedanken recently reported sonochemically produced oil-filled streptavidin, avidin and amylase microspheres.19,20 Previous studies1,19,20 indicate that the involvement of free thiols in the protein cross-linking process depends on the nature of the protein. Also, controlling the stability of the gas core has not been addressed in these studies and it is well-known that the lifetime of air-filled albumin microparticles is very short since the air diffuses rapidly out of the microbubbles.21 In this manuscript, we report for the first time, the synthesis of stable and versatile lysozyme microbubbles, which have been prepared by high intensity ultrasound-induced emulsification and crosslinking of chemically reduced lysozyme in aqueous solutions. A mechanism for the formation of the microbubbles is proposed by taking into consideration the structural and functional properties that have been determined using different analytical approaches. We have also investigated the secondary structure and the enzymatic activity of the lysozyme-coated microbubbles. These microbubbles retain the enzymatic activity of lysozyme and represent the first example of ultrasound-responsive microparticles with enzymatic (antimicrobial) activities.
Experimental Section Figure 1. Microbubble size distribution: (a) 15 min denaturation, 30s sonication; (b) 2 min denaturation 30 s sonication; (c) 15 min denaturation, 15 s sonication.
also be effective at low doses, and this implies a high drugloading capacity when used as drug delivery agents. Thus, the key features to engineering formulations of therapeutic microbubbles are the loading capacity of drugs onto the shell, the functionalization of surface ligands, and their biodegradability. Generally, lipid and protein monolayer shells (5-50 nm) are considered to be too thin to hold a sufficient amount of drug cargo9,10 and only microbubbles with potential for effective drug delivery should be considered. A multilayer construction onto lipid-coated microbubbles was recently reported14 to be effective in increasing DNA loading but in vivo circulation has not been studied. Several attempts have been made to stabilize and modify surface properties of BSA and surfactant microbubbles by using the LbL deposition of polyelectrolytes.14,15 Although the deposition/coating of polyelectrolytes prolongs the lifetime of the microbubbles compared to the uncoated bubbles, the stability of these microbubbles is still only several hours, which is inadequate for use since there is insufficient time for further chemical conjugation or manipulation. A thicker polymer shell endowed with a tunable mechanical strength compared to the monolayers of proteins, lipids or surfactants may provide a greatly increased circulation time and a higher loading capacity. Protein-coated oil emulsion16-18 and gas bubbles still raise interest in drug delivery applications because of their biodegradability, and immunogenic issues can be easily overcome by grafting a surface brush of a hydrophilic polymer, such as poly(ethylene glycol) (PEG) on the microparticle surface. Core-shell BSA vesicles, surface modified by LbL electrostatic adhesion of small tripeptide arginine-glycine-aspatric acid (RGD) were recently used as effective systems for targeting colon tumors (15) Shchunkin, D. G.; Ko¨hler, K.; Mo¨hwald, H.; Sukhorukov, G. B. Angew. Chem., Int. Ed 2005, 44, 3310–3314. (16) Dibbern, E. M.; Toublan, F. J-J.; Suslick, K. S. J. Am. Chem. Soc. 2006, 128, 6540–6541. (17) Suslick, K. S.; Toublan, F. J.J.; Boppart, S. A.; Marks, D. L. U.S. Patent. , 2007, 7,217,410 B2. (18) Toublan, F. J.J.; Boppart, S. A.; Suslick, K. S. J. Am. Chem. Soc. 2006, 128, 3472–3473.
Materials. Hen Egg white lysozyme and DL-dithiothreitol (DTT) were purchased from Fluka. L-cysteamine, Micrococcus lysodeikticus cells, sodium poly(styrene sulfonate), (PSS, Mw ) 70 000), and poly(allylamine hydrochloride) (PAH, Mw ) 70 000) were purchased from Sigma-Aldrich. High-purity water was from an inline Millipore RiOs/Origin System (MilliQ water). Microsphere Preparation. Fluorescently labeled PAH at 0.05% was prepared by using tetramethylrhodamine isocyanate (TRITC) (Sigma-Aldrich). Lysozyme (5% w/v) was denatured in 50 mM of trishydroxymethylaminomethane-Tris-HCl buffer (pH 8.3) containing 200 mM of DTT for 2, 5, 10, and 15 min. Twenty kHz ultrasound was applied at the air-water interface with a Sonics and Materials ultrasound instrument for 30 s with a 1 cm diameter horn at an acoustic power of 200 W cm-2. Microbubbles were separated from the remaining protein and broken microparticles by flotation and repeated washing. We have independently verified the operating conditions of sonication for the successful formation of disulfide bonds (see later). An aqueous solution containing cysteamine was sonicated for 30 s at an acoustic intensity of 200 W cm-2. The effectiveness of disulfide formation during sonication was evaluated by measuring the residual concentration of thiols. A significant decrease of thiol content (from the initial concentration of 5.9 mM to 1.8 mM and 1.2 mM after 1 and 3 min ultrasound treatment, respectively) was observed not only immediately after sonication but also for several minutes after the treatment, since hydrogen peroxide produced during sonication continues to oxidize thiol functional groups. Methods. The free thiol groups were determined by using Ellman’s reagent, DTNB 5-5′-dithiobis(2-nitrobenzoic acid). Lysozyme activity was determined according to a turbidometric method by monitoring the decrease of absorbance of a suspension of M. lysodeikticus cells at 450 nm as a fnction of time, using a spectrophotometer (Agilent 8453 UV-vis). The assembly of multilayers on 1 mg mL-1 lysozyme microbubbles was performed by alternately adsorbing PSS and PAH (1 mg mL-1 in 0.5 M NaCl) with 10 min of incubation. After adsorption, the microbubbles were centrifuged at 800g and washed 3 times with Milli-Q water. An inverted Olympus IX71 wide field fluorescence microscope with a 60x objective lens and green filter cube was used to view the microbubbles. A CCD camera (Cool SNAP fx, Photometrics, Tucson, (19) Avivi, S.; Gedanken, A. Biochem. J. 2002, 366, 705–707. (20) (a) Avivi, S.; Gedanken, A Ultrason. Sonochem. 2005, 12, 405–409. (b) Avivi, S.; Gedanken, A. Ultrason. Sonochem. 2007, 14, 1–5. (21) Kalbanov, A.; Bradley, J.; Flaim, S.; Klein, D.; Pelura, T.; Peters, B.; Otto, S.; Reynolds, J.; Schutt, E. J. Ultrasound Med. Biol. 1998, 24, 751.
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Scheme 1. Schematic Illustration of Ultrasound-Induced Crosslinking of Lysozyme Clusters at the Air-Water Interfacea
a
Acoustic cavitation in water generates oxidants, which promote interprotein disulfide crosslinking of cysteine residues.1
Figure 2. Confocal laser scanning microscopy (CLSM) image of lysozyme microbubbles (15 min denaturation, 30 s sonication). The inset shows clusters of proteins adsorbed on the surface of microbubbles.
AZ) was mounted on the left-hand port of the microscope. Confocal microscopy experiments were performed on a Leica TCS-SP2 confocal scanning microscope using an excitation wavelength of 480 nm. Zeta (ξ) potential measurements were carried out on a Malvern Zetasizer. SEM images were recorded on air-dried microcaspules sputter-coated with a thin gold film using a XL 30 FEG (Philips) operated at an acceleration voltage of 5 kV. AFM images were taken using an MFP-3D Asylum Research Instrument in tapping mode on air-dried microcapsules. The scan area was 2 µm × 2 µm. Circular dichroism, CD, spectra (typically in the range 200-260 nm) were recorded by using a quartz cell (1 mm) with a JASCO J600 spectrometer equipped with a thermoregulated cell compartment.
Results and Discussion Synthesis of Lysozyme Microbubbles. Previous studies1,22 on serum albumin and hemoglobin have suggested that the mechanism responsible for the formation of air-filled microbubbles is a combination of two ultrasound-induced phenomena: emulsification and cavitation. Although hydrophobic interaction between the protein subunits is one of the requirements for the formation of microbubbles, it is not the main mechanism responsible for the stabilization of microbubbles. The protein shell is stabilized by interprotein disulfide cross-linking of cysteine (22) Suslick, K. S.; Grinstaff, M. W. J. Am. Chem. Soc. 1990, 112, 7807–7809.
residues by the radicals and superoxides generated during the sonolysis of water.22 In principle, a number of polymers containing sulfhydryl groups or disulfide bonds may be employed for the preparation of disulfide cross-linked shells. The sulfhydryl groups may preexist within the polymer structure or they may be introduced by a chemical reaction. Controlling the balance between the hydrophobicity and the number of sulfhydryl units in order to increase the stability of the microbubbles as well as to prevent the dissolution of the bubbles is experimentally demanding. Sealing a gas phase into a microcavity by crosslinking a biopolymer at the air-water interphase requires fine control over the chemical and physical properties of both the polymer and gas. In this study, we have used lysozyme for synthesizing stable microspheres. Egg white lysozyme is a small globular polypeptide (Mw 14 000) chain that contains 129 amino acids in the primary sequence and 4 intrachain disulfide bridges buried inside the hydrophobic core of the protein. In our preliminary experiments, stable microbubbles were not obtained by either native or thermally denatured lysozyme. This might be due to the absence of free thiol groups. Thermal denaturation is less efficient in generating active thiols.23 The disulfide bonds, although quite stable to heat treatment, are easily disrupted by reducing agents, such as DTT. In our system, thiol groups were introduced by chemical reduction of lysozyme prior to sonication by using a high concentration of DTT. The denaturation treatment results in lysozyme exposing its surface hydrophobicity, thus improving its foaming as well as cross-linking properties. The importance of intermolecular disulfide bond formation for the preparation of microspheres was determined by analyzing the light microscopy images of lysozyme microbubbles prepared at different denaturation times ranging from 2 (Figure 1b) to 15 min (Figure 1a) and the corresponding size distributions (Figure 1). It can also be observed that larger size microbubbles (Figure 1c) are obtained with shorter sonication times (15 s). We observed that the sonochemical treatment parameters, such as exposure time, DTT concentration, and denaturation time significantly affected the yield and the size of microbubbles. For a constant sonication time (30 s), a denaturation time of 2 and 15 min resulted in the formation of microbubbles with diameters of 6 ( 2 and 4 ( 1 µm, respectively, whereas 15 s of sonication (and 15 min denaturation) produced microbubbles of diameter 10 ( 2 µm. Since larger size microbubbles are usually formed at shorter chemical denaturation and ultrasound exposure times, we speculate that the degree of cross-linking is less under these
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Figure 3. (a, b) AFM and (c, d) SEM images of lysozyme microbubbles (15 min denaturation, 30 s sonication).
Figure 4. Changes in CD spectra of lysozyme after microbubble preparation. Lysozyme concentration of 0.06 mg mL-1, pH 6, 20 °C. (0) Native lysozyme, (O) lysozyme microbubbles (2 min denaturation and 30 s sonication).
experimental conditions. A lower cross-linking degree may lead to a loosely (chemically) cross-linked network of lysozyme at the interface that can still sustain gas expansion and compression (in an ultrasonic field) and reach a larger microbubble size stabilized by further protein stratification. It is possible that the larger microbubbles are susceptible to the physical effects generated during cavitation that may lead to complete disintegration and consequently, a time-dependent refinement of microbubble size distribution may occur. A denaturation treatment ranging from 2 to 5 min coupled to 30 s of sonication was identified as the best operative condition to obtain an optimal size distribution and a good yield (∼15%).
Figure 5. Antimicrobial activity of (O) native lysozyme, (0) lysozyme microbubbles (2 min denaturation and 30 s sonication), (•) resuspended lysozyme microbubbles after 5 min sonication at 200 W cm-2.
It is noted that one of the key factors to prepare stable lysozyme microbubbles is the efficient cross-linking between lysozyme clusters at the bubble-solution interface. Stable microbubbles were only formed when lysozyme aggregates were close to the condition of coagulation induced by denaturation. A schematic illustration of the possible mechanism of stabilization of lysozyme microbubbles at the interface of the cavitation bubbles is shown in Scheme 1. The lysozyme clusters are produced by protein aggregation caused by the hydrophobic interaction resulting from chemical denaturation. Lysozyme aggregates of nanoscale dimensions adsorb to the air-water interface generated by emulsification and because of the Laplace overpressure and ultrasound oscillation, the gas core shrinks, leading the protein
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Figure 6. Fluorescence microscopy images of (PSS/FITC-PAH)-coated microbubbles.
nanoparticles to form a cross-linked shell due to disulfide crosslinking. Optical and electron microscopy characterization provide further insight into the mechanism of formation and stabilization of the microbubbles. The composite structure of the microbubble shell can be observed by confocal microscopy (Figure 2), where small protein particles on the surface are visualized. An average thickness of the lysozyme shell of about 130 ( 90 and 800 ( 100 nm was obtained from the height profile of the AFM images for samples with denaturation times of 15 and 2 min, respectively (30 s sonication time). Both AFM and SEM (Figure 3) images indicate a bumpy surface generated by protein clusters at the gas-water interface. The lifetime of air-filled BSA microbubbles is short (a few hours) because air diffuses rapidly out of the microbubbles. Compared to air-filled BSA microbubbles, lysozyme microbubbles exhibit a longer shelf life (months). The cross-linked shell of BSA (Mw 67 kDa) is roughly 50 nm thick, corresponding to ∼6 protein molecules.24 Considering a hydrodynamic radius of 2.32 nm for an unfolded lysozyme molecule, the measured wall thickness of the microbubble of 130 nm corresponds to ∼26 molecules and is consistent with a more compact structure, which would reduce gas permeability of the protein membrane. Although disulfide bonds significantly enhance the stability of microbubbles, physical and covalent cross-linking in the polymer shell are known to dampen the oscillation of the gas core9 until the shell is fractured and jets of gas escape. This complex behavior is not desirable for biomedical applications because higher amplitudes of ultrasound are required to break the shell. We are currently investigating how the extent of shell cross-linking can affect the mechanism of shell rupture upon sonication. Antimicrobial Properties of Lysozyme Microbubbles. Unfolded lysozyme is a potent bactericidal agent against both gram-negative and gram-positive bacteria regardless of its enzyme activity.25 It is well-known that lysozyme unfolding is a reversible process.23 During the ultrasound-induced cross-linking process, lysozyme may recover, in part, some of its structural integrity for molecular recognition. To determine conformational changes (23) Touch, V.; Hayakawa, S.; Saito, K. Food Chem. 2004, 84, 421–428. (24) Shchukin, D. G.; Ko¨hler, K.; Mo¨hwald, H. Phys. Chem. Chem. Phys. 2006, 8, 3496–3506. (25) Ibrahim, H.; Higashiguchi, S.; Koketsu, M.; Lekh, R.; Juneja, L. R.; Mujo, K. J. Agric. Food Chem. 1996, 44, 3799–3806.
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associated with the microbubble formation process, we investigated both the secondary structure and antimicrobial activity of lysozyme microbubbles. Microbubbles were extensively washed before testing the enzymatic activity and spectroscopic investigation to remove free lysozyme that may be present in the solution. Figure 4 illustrates the CD spectra of a lysozyme microbubble suspension relative to the native protein. Microbubbles still exhibit a second peak at 207 nm, which is characteristic of the lysozyme secondary structure, although the intensity profile is slightly different compared to the native protein. The circular dichroism spectra of native protein and microbubbles are very similar and suggest either the lysozyme partially recovers its secondary structure or that the microbubble fabrication process does not significantly alter the secondary structure of the protein that comprises the cross-linked shell. This finding was also confirmed by a turbidometric Micrococcus leuteus test25 of enzymatic activity carried out on both the microbubble and lysozyme solutions obtained after 5 min sonication of the microbubbles (Figure 5). Both samples showed antimicrobial activity, particularly for the lysozyme solution obtained by the sonication of microbubbbles′ catalytic activity comparable to the native protein was observed. It is worth noting that a comparison of enzymatic activity of lysozyme in solution with lysozyme microbubbles is not considered meaningful since most of the protein contained in the shell of the microbubbles is buried within the shell and is therefore not available to exert binding to the substrate. However, microbubble formation using ultrasound-assisted cross-linking of partially denatured lysozyme is compatible with protein structure and functionality and herein represents the first example of microbubbles exhibiting enzymatic and antimicrobial activity. Layer-by-layer (LbL) Assembly of Polyelectrolytes on Lysozyme Microbubbles. The polymer shell of microbubbles can be modified to introduce ligands26 that are selectively recognized by cell surface receptors or neoplasm specific markers. The availability of numerous exploitable side groups in proteins such as amine, hydroxyl, thiol and carboxyl groups, makes it possible to use conjugation routes on prefabricated microbubbles. However, coupling chemistry to introduce specific ligands sometimes requires the use of harsh conditions that can be detrimental to the colloidal stability of microbubbles. Multilayer deposition of polyelectrolytes onto air-filled microbubbles may be an attractive strategy to design targeted microparticles. In order to demonstrate the possibility of surface functionalization of lysozyme microbubbles, which is of importance in targeted drug delivery, we adsorbed a polyelectrolyte on the surface of the microbubbles. The coating of microbubbles with polyelectrolytes is a rather straightforward and mild approach. Lysozyme air-filled microbubbles are positively charged colloidal particles and provide a good template for assembly of polyelectrolyte multilayer (PEMs) using the LbL approach.27 Recently, multilayers deposition of polyelectrolytes onto air-filled microbubbles was reported as method to stabilize short lifetime-microbubbles, since polyelectrolytes act as a further barrier to prevent gas diffusion.13,15 Lysozyme microbubbles are, however, stable for months and stepwise LbL deposition of PSS and PAH onto the protein aircore template was accomplished to demonstrate the effectiveness of this approach in modifying the microbubble surface properties. Two layers of PSS/PAH were assembled on the lysozyme shell and layer deposition was monitored by electrophoresis. Lysozyme (26) Cavalieri, F.; Al Hamassi, A.; Chiessi, E.; Paradossi, G.; Villa, R.; Zaffaroni, N. Biomacromolecules 2006, 7, 604–611. (27) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111–1114.
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is a positively charged protein at pH 7 and the uncoated microbubbles had a positive potential of +40 ( 3 mV. Upon PSS and PAH adsorption the surface potential of the coated particles was reversed to -30 and 40 mV, respectively. Additionally, FITC-labeled PAH was used to confirm the presence of multilayers on the surface of the microbubbles (Figure 6). This clearly demonstrates that the microbubbles can be surface functionalized. Subsequent tethering of specific drugs and/or biolabels to the surface functionalized microbubbles is currently underway, as such systems are of importance in diagnostic and therapeutic applications.
Conclusions Ultrasound-induced physical and chemical effects have been used for synthesizing lysozyme microspheres. Our experimental data suggests that the hydrophobic nature of the enzyme is important to provide foaming properties, which is one of the requirements to produce microbubbles. However, the formation of disulfide bonds is the primary requirement for the formation
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of stable microbubbles. Stable microbubbles could not be produced using native lysozyme since they lack free thiol functional groups. The size and size distribution of the microbubbles are found to be dependent upon the length of sonication and denaturation times. A shorter sonication and/or denaturation time yielded larger microbubbles. A denaturation treatment ranging from 2 to 5 min coupled to 30 s of sonication were identified to yield an optimal size distribution. The lysozyme microbubbles partly retained the enzymatic (antimicrobial) activity of lysozyme despite denaturation and sonication treatments. Surface functionalization of the microbubbles was demonstrated by LbL deposition of polyelectrolytes on the surface of the microbubbles. Acknowledgment. We thank Almar Postma for providing SEM analysis. This work was funded by the ARC Linkage International and Federation Fellowship Schemes. LA801093Q
