[ACS Symposium Series] Macromolecular Assemblies in Polymeric Systems Volume 493 || Proteinaceous...

Chapter 18

Proteinaceous Microspheres

Mark W. Grinstaff and Kenneth S. Suslick

School of Chemical Sciences, University of Illinois at Urbana—Champaign, Urbana, IL 61801

Using high-intensity ultrasound, we have synthesized aqueous suspensions of proteinaceous microspheres. Microspheres filled with nonaqueous liquids (i.e., microcapsules) or air-filled (i.e., microbubbles) were examined by optical microscopy, scanning electron microscopy, and particle counting. High concentrations of microspheres were observed with Gaussian size distributions. Microsphere formation is strongly inhibited by the absence of O2, by free radical traps, by superoxide dismutase (but not by catalase), and by the lack of free cysteine residues in the protein. It is proposed that superoxide, which is produced by acoustic cavitation, cross-links the microcapsule protein by oxidizing cysteine residues to form disulfide bonds.

The organization of macromolecules into even larger structures determines the physical properties of much of the macroscopic world. The cellular structure of life itself is an obvious example of the general case of the formation of micrometer-sized structures from nanometer-sized macromolecules. As a class of macromolecular assemblies, such microencapsulation has proved to be a valuable technique in modern science and has found numerous technological applications. Some important uses include encapsulation of active metals, deodorants, dyes, perfumes, cosmetic ointments, and pesticides. The pharmaceutical industry has found this technology especially valuable with applications ranging from encapsulated drugs and vitamins (1-6) to contrast agents for sonography (7-10) and magnetic resonance imaging (11).

The most common microspheres are composed of liposomes (i.e., lipid bilayer microspheres) or synthetic polymers. Specific compositions, however, are usually complex formulations with proteins often added to increase

0097-6156/92/0493-0218$06.00/0 © 1992 American Chemical Society

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In Macromolecular Assemblies in Polymeric Systems; Stroeve, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

18. GRINSTAFF & SUSLICK Proteinaceous Microspheres 219

biocompatibility or to modify the microsphere's properties. For use in vivo, the ideal material would have a long shelf life, low toxicity and micron size.

A sonochemical technique has been developed for the synthesis of nonaqueous liquid-filled microcapsules and air-filled microbubbles entirely composed of proteins that meet these criteria (12-15). These materials are an assembly of protein molecules linked together by disulfide bonds. The chemical crosslinking responsible for these microspheres is a direct result of the chemical effects of ultrasound on aqueous media.

Morphology of Protein Microspheres

The morphology of these materials was determined using scanning electron microscopy and light microscopy. A scanning electron micrograph of dodecane-filled proteinaceous microcapsules shows their morphology and size (Figure 1). The air-filled and liquid-filled (dodecane, decane, toluene) microspheres both have Gaussian size distributions with an average diameter of approximately 5.5 μπι and 2.3 /xm, respectively (Figure 2). Proteinaceous microspheres have been synthesized with a high intensity ultrasonic probe (Heat Systems, W375, 20 kHz, 0.5 in. T i horn) from various proteins including bovine serum albumin (BSA), human serum albumin (HSA) and hemoglobin (Hb) as describe in detail elsewhere (12-15). A typical experiment to synthesize proteinaceous microcapsules involves irradiating a toluene and 5 % w/v BSA solutions for three minutes at an acoustic power of « 150 W/cm 2 , with an initial reaction cell temperature of 2 3 e C , and at pH 7.0. We find that more microcapsules and microbubbles are produced with increased acoustic power starting from the same initial temperature (Figure 3).

Mechanism of the Sonochemical Synthesis of Microspheres

How are the microspheres formed and what holds them together? Ultrasonic irradiation of liquids is well known to produce both emulsification (16) and cavitation (17-19). In forming the microspheres, the nonaqueous liquid or air is dispersed into the aqueous protein solution. Ultrasonic emulsification does occur in this bi-phasic system. Emulsification alone, however, is insufficient: emulsions produced by vortex mixing, instead of ultrasonic irradiation, produced no long-lived microspheres (Figure 2). Furthermore, the vortex emulsions are not stable and phase separation occurs immediately. In contrast, the proteinaceous microspheres are stable for many hours at room temperature and for several months at 4 e C (Figure 4). Preliminary experiments on leakage from interior liquids indicates that the stability of the microsphere content is on the order of hours at 38 " C .

Hydrophobic or thermal denaturation of the protein after the initial ultrasonic emulsification might be responsible for microsphere formation. High concentrations of microspheres, however, are only observed when the mixture

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220 MACROMOLECULAR ASSEMBLIES IN POLYMERIC SYSTEMS

Fig . 1 Scanning electron micrograph of dodecane-filled proteinaceous microcapsules. The microcapsules were prepared for S E M by secondary cross-linking with glutaraldehyde and coating with Au/Pd. Volatile nonaqueous liquids gave non-spherical microcapsules due to evaporation during sample preparation.

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GRINSTAFF & SUSLICK Proteinaceous Microspheres 221

80

80

70

I 6 0

S 50

I 40

I 30

20

10

0

3 2 4 β 8 10 12 14 16 18 20

Diameter (microns)

Β

16 20 4 8 12

Diameter (microns)

Fig . 2 Particle distribution of an aqueous suspension of proteinaceous microspheres, determined with an Elzone particle counter (Model 180XY).

(A) • Toluene-filled microcapsules were synthesized from ultrasonic irradiation toluene and of 5 % w/v BSA solutions for three minutes at an acoustic power of » 150 W/cm 2 , with an initial cell temperature of 2 3 e C a t p H 7 . 0 .

Toluene and 5 % w/v BSA solution vortexed for three minutes at pH 7.0.

(B) Air-filled microbubbles were synthesized from ultrasonic

irradiations of 5 % w/v BSA solutions for three minutes at an acoustic power of « 1 5 0 W/cm 2 , with an initial cell temperature of 50"C at pH 7.0.

5% w/v BSA solution vortexed for three minutes at pH 7.0.

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c

Fig . 3 The effect of acoustic power on microcapsule and microbubble formation. Acoustic powers were taken from the manufacturer supplied nomograph and are only approximate.

(A) Microcapsule formation. « 150 W/cm 2

« 70 W/cm 2

« 40 W/cm 2

(B) Microbubble formation. « 150 W/cm 2

« 40 W/cm 2

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is sparged with air or 0 2 . If the reaction is run under an inert atmosphere (He, Ar , or N 2 ) microcapsules are not formed. Thus, thermal or solvent denaturation (for which 0 2 , N 2 , and Ar should give similar results) cannot explain the microsphere permanence.

Another, chemical process must be involved. As mentioned above, ultrasonic irradiation of liquids generates acoustic cavitation: the formation, growth and collapse of bubbles in a liquid (17-19). The collapse of these bubbles produces high energy chemistry. Specifically, aqueous sonochemistry produces OH* and H* (20-22). The radicals so produced by ultrasound form H 2 , H 2 0 2 , and (in the presence of 0 2 ) superoxide, H 0 2 (22-24). Hydroxyl radicals, superoxide, and peroxide are all potential protein cross-linking agents.

To identify the specific oxidant involved, the formation of microspheres was examined in the presence of radical traps (Figure 5). The addition of nonspecific traps (e.g., 2,6-di-t-butyl-4-methylphenol or glutathione), catalase (which decomposes hydrogen peroxide to oxygen and water (25)) and superoxide dismutase (which decomposes superoxide to oxygen and hydrogen peroxide (26)) were tested. Both microcapsule and microbubble formation were inhibited by nonspecific traps and by superoxide dismutase, but not by catalase. Catalase activity was confirmed after ultrasonic irradiation; ultrasonic irradiation did not, therefore, destroy the functioning of this enzyme. We propose that the important oxidant involved in microsphere formation is superoxide.

To determine the specific effect of superoxide on the proteins, several experiments were performed. Cysteine is easily oxidized by superoxide (27) and is present in BSA, HSA, and Hb. In fact, ultrasonic irradiation of proteins has been reported to oxidize cysteine residues (28). If the microspheres are held together by protein cross-linking through disulfide linkages from cysteine oxidation, Hb and myoglobin (Mb) provide an interesting test: they have very similar sequences and monomelic structures, but Mb has no cysteine amino acid residues. Ultrasonic irradiation of Mb solutions does not form microspheres; Hb does. In another set of tests, the addition of a disulfide cleavage reagent, dithioerythritol (29), destroys Hb-toluene or BSA-toluene microcapsules. Finally, the oxidation of cysteine residues can be inhibited by alkylation with N -ethylmaleimide (30), and microsphere formation from Hb solutions so treated is greatly reduced. These results confirm the importance of disulfide bond formation in microsphere formation.

Summary

In summary, ultrasound can produce proteinaceous microcapsules and microbubbles of a few microns diameter at high concentrations with narrow size distributions. The process involves two separate acoustic phenomena: emulsification and cavitation. The dispersion of gas or nonaqueous liquid into the protein solution, coupled with the chemical cross-linking of the protein molecules, produces stable protein microspheres.

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800

Diameter (microns)

F ig . 4 The stability of toluene-filled proteinaceous microcapsules at 4 ' C .

— 20 Minutes 42 Days 83 Days

S 700

Diameter (microns) F i g . 5 The effect of radical traps on microcapsule formation. 5 % w/v aqueous solution of B S A and toluene were irradiated in the presence of catalase, glutathione or superoxide dismutase. Inhibition of microcapsule formation also occurred with 2,6-di-t-butyl-4-methylphenol.

0.09% Catalase 0.1 M Glutathione 0.1% Superoxide Dismutase

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Acknowledgments

This work was supported by N . S . F . and N . I . H . M W G gratefully acknowledges receipt of the Procter and Gamble Graduate Fellowship of the Colloid and Surface Chemistry Division of the American Chemical Society.

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