[Advances in Protein Chemistry and Structural Biology] Volume 80 || Sonochemically born proteinaceous micro- and nanocapsules

SONOCHEMICALLY BORN PROTEINACEOUS MICRO- ANDNANOCAPSULES

By ELENA D. VASSILEVA* AND NELI S. KOSEVA†

*Faculty of Chemistry, St. Kliment Ohridsky University of Sofia, Sofia, Bulgaria†Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria

I. In

ADVANSTRUCDOI: 1

troduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CES IN PROTEIN CHEMISTRY AND 205 Copyright 2010, ETURAL BIOLOGY, Vol. 80 All righ0.1016/S1876-1623(10)80006-9

2

lseviets rese

06
II. M ethods for Protein Particle Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 08 III. U ltrasound Sonochemistry and Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 12
A
. C avitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 12 B . S onochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 14 C . U ltrasound and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 15
IV. P
roteinaceous Microspheres Obtained Through Sonochemistry . . . . . . . . . . . . . . . . 2 21 A . M echanism of Proteinaceous Microspheres Formation
Through Sonochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
222 B . B ioactivity of Proteinaceous Microspheres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 29 C . E ffect of Experimental Parameters on the Characteristics of the
Proteinaceous Microcapsules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
232 V. A pplications of the Proteinaceous Microcapsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 37 VI. C onclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 44
R
eferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 44
Abstract

The use of proteins as a substrate in the fabrication of micro- andnanoparticulate systems has attracted the interest of scientists, manufac-tures, and consumers. Albumin-derived particles were commercialized ascontrast agents or anticancer therapeutics. Food proteins are widely usedin formulated dietary products. The potential benefits of proteinaceousmicro- and nanoparticles in a wide range of biomedical applications areindisputable. Protein-based particles are highly biocompatible and biode-gradable structures that can impart bioadhesive properties or mediateparticle uptake by specific interactions with the target cells. Currently,protein microparticles are engineered as vehicles for covalent attachmentand/or encapsulation of bioactive compounds, contrast agents for mag-netic resonance imaging, thermometric and oximetric imaging, sonogra-phy and optical coherence tomography, etc.

r Inc.rved.

206 VASSILEVA AND KOSEVA

Ultrasound irradiation is a versatile technique which is widely used inmany and different fields as biology, biochemistry, dentistry, geography,geology, medicine, etc. It is generally recognized as an environmentalfriendly, cost-effective method which is easy to be scaled up. Currently, itis mainly applied for homogenization, drilling, cleaning, etc. in industry, aswell for noninvasive scanning of the human body, treatment of musclestrains, dissolution of blood clots, and cancer therapy.

Proteinaceous micro- and nanocapsules could be easily produced in aone-step process by applying ultrasound to an aqueous protein solution.The origin of this process is in the chemical changes, for example, sulfhy-dryl groups oxidation, that takes place as a result of acoustically generatedcavitation. Partial denaturation of the protein most probably occurs whichmakes the hydrophobic interactions dominant and also responsible for theformation of stable capsules.

This chapter aims to present the current state-of-the-art in the field ofsonochemically produced protein micro- and nanocapsules, paying specialattention to the proposed mechanisms for their formation, the factors thatinfluence the capsules characteristics as well to the current applications ofthese particles. Current challenges in the field are also outlined as, forexample, the ultrasound–protein interaction and other possible aspects ofthe mechanism of their formation.

I. Introduction

Polymer micro- and sub-microparticles usually designate both capsulesand spheres having size less than 1000 mm. Generally, such particles arefabricated as carriers of liquid, gaseous, or solid materials encapsulated/embedded within their polymeric matrix, adsorbed or conjugated ontothe surface (Fig. 1).

Microparticles have wide applicability in medicine (Barratt et al., 2002;Finne-Wistrand and Albertsson, 2006; Oh et al., 2008), agriculture (Stoilovaet al., 2001; Puoci et al., 2008), food and cosmetics (Brannon-Peppas, 1993;Gouin, 2000), electronics (Farah et al., 2008; Liu et al., 2009; Lee et al.,2010), etc. Microparticulate systems are developed to meet the great varietyof formulation needs in pharmaceutical production for oral and pulmonarydelivery, intramuscular and subcutaneous injection, controlled drug deliv-ery, masking the taste and odor of drugs, storage convenience, and protec-tion of the agents from degradation both during storage and in an in vivo

(A) (B) (C)

FIG. 1. Different microparticle structures: (A) a microcapsule composed of a coreand a shell/envelope which distinctly differ from each other, the core serves as areservoir for the active ingredient(s); (B) a microcapsule with more domains containingthe active ingredient(s); and (C) a microsphere made of a continuous phase of one ormore miscible polymers incorporating dispersed active agent(s).

PROTEINACEOUS MICRO- AND NANOCAPSULES 207

environment (Uchegbu and Schatzlein, 2006). By decreasing thematrix sizefrom micrometers to nanometers, new vehicles with enhanced deliveryproperties can be developed. Due to their subcellular size, nanoparticlescan penetrate deeply into tissues through fine capillaries and are efficientlytaken up by cells (Schafer et al., 1992; Desai et al., 1997; Lamprecht et al.,2004). Engineered particles are expected to result in ‘‘smart’’ therapeuticscapable of active targeting and programmed delivery thus allowing efficientdrug action and minimized side effects.Particle design for a desired application exploits the advances in parti-

cle-processing methods and the utilization of new substrates. Particularly,colloidal systems based on proteins may be very promising since proteinspossess unique functional properties including their ability to form gelsand emulsions, which make them an ideal material for the encapsulationof bioactive compounds. Because of the defined primary structure ofproteins, various possibilities for surface modification and covalent drugattachment to the proteinaceous micro- and nanoparticles may beexploited. Protein-enriched surfaces can impart bioadhesive properties(Goldstein et al., 1980) or mediate particle uptake by specific target-cellpopulations (Goppert and Muller, 2005). The combination of nanoparti-cles with specific biomolecules offers opportunities for the design ofefficient medicines against cancer and immunological diseases (Drexler,1981; Alonso, 1996; Rajagopal and Schneider, 2004; Sinha et al., 2006).


Moreover, proteins are biodegradable and nonantigenic (Rubino et al.,1993). Specifically, food proteins are widely used in formulated foodsbecause they have high nutritional value and are generally recognized assafe (Chen et al., 2006).

II. Methods for Protein Particle Preparation

Different approaches and methods have been developed for fabricationof micro- and nanoparticulate systems. Recent technologies provide avariety of possibilities to control over critical particle design features,such as particle size and distribution, particle density, surface energy,surface area, porosity in order to tune system performance—the degrada-tion rate of the polymer matrix, the compound release rate, uptake, anddistribution in the body.

Spray drying is the most widely used industrial process involving particleformation and drying. Spray drying is an economic continuous processthat includes an atomization step (bulk liquid is converted into droplets bya nozzle), followed by solvent evaporation and subsequent powder collec-tion. The particle size of the dried powder directly correlates with the sizeof the droplets controlled not only by the formulation variables such asviscosity, surface tension, and density, but also by the atomizing pressure.During the spray drying process, proteins are exposed to heat and me-chanical stress, as well as to adsorption at the air–water interface (Maa andPrestrelski, 2000; Webb et al., 2002) often recognized as the dominantsource for protein denaturation and aggregation in the process( Jalalipour et al., 2008). Proteins such as gelatin, sodium caseinate,whey, and soy proteins have been used as wall material in spray dryingencapsulation in food industry (Kim et al., 1996; Bruschi et al., 2003).Gomez et al. (1998) explored the applicability of the electrospray drying asa nanoscale equivalent technique for protein particle production. Thedispersion of a liquid with sufficient electric conductivity is driven byelectric forces resulting in a tight control of the droplets size distributionof the obtained aerosol. Relatively monodispersed insulin particles with anaverage diameter of approximately 110 nm and doughnut shape wereproduced. The electrospray-processed insulin displayed the same recep-tor-binding properties as the control insulin, implying that electrospraydrying is a sufficiently ‘‘gentle’’ processing technique not to hinder


biomacromolecule activity. The authors pointed the low production ratefrom a single cone-jet (about 0.23 mg/h) as a limitation of the methodwhich can be increased by multiplexing the device (Gomez et al., 1998).Coacervation is widely exploited for preparing microcapsules with very

high payloads and controlled release of the active components such aspharmaceuticals, flavors, and fragrances. This technique is based on phaseseparation of one or more hydrocolloids from the aqueous solution andformation of a coating, or shell of the newly formed phase around thedispersed active ingredients. The coacervation of the polymer coating canbe accomplished by different mechanisms. In the simplest system, coacer-vation is induced by changes in temperature, pH or salt concentration, oraddition of a nonsolvent, while polyelectrolyte complex coacervation isdriven by electrostatic attractive forces between complementary chargedmacromolecules, with secondary stabilization by hydrogen bonding.A large number of hydrocolloid systems have been evaluated for coacerva-tion microencapsulation. Microspheres of corn protein zein for ivermectindelivery (Liu et al., 2005) and albumin nanocapsules loaded with aspirin(Das et al., 2005) were obtained by a simple coacervation process. Bothformulations released drug in a sustained manner. Gelatin is often used asa partner in complex coacervation systems with gum acacia (Burgess andCarless, 1984; Planas et al., 1990; Mauguet et al., 1999), pectin, alginate(Saravanan and Rao, 2010), and synthetic polymers (Rong et al., 2004).In 1978, Marty et al. (1978) described for the first time the preparation

of gelatin nanoparticles using desolvating or desalting agents. This method isappropriate for the preparation of nanoparticles based on protein orsynthetic macromolecules with defined molecular weight (Chen et al.,1994; Weber et al., 2000; Nguyen and Ko, 2010; Trzebicka, et al., 2010).Due to the molecular heterogeneity of gelatin, the preparation of homo-geneous micro- and especially nanoparticulate formulations is challeng-ing. The new protocol of a two-step desolvation technique proposed byCoester et al. (2000) enabled the production of homogeneous colloidalgelatin spheres. After the first desolvation step, the low molecular gelatinfractions present in the supernatant were removed and particles wereprepared from the high molecular fractions. The process was optimizedand the specific conditions for the preparation of homogeneous (polydis-persity index<0.15) nanoparticles with a diameter within the range of100–300 nm were defined (Azarmia et al., 2006; Zwiorek, 2006). However,the one-step desolvation method is the preferred one in terms of


technological and regulatory considerations. Further investigationrevealed that a mean molecular weight of �500 kDa and a threshold ofmaximum 20% (w/w) of low molecular weight fraction (<65 kDa) couldbe defined as a prerequisite for the successful manufacturing of gelatinnanoparticles by a one-step desolvation procedure (Zillies, 2007).

Emulsion-based methods for the preparation of micro- or nanoparticlesallow obtaining a homogeneous dispersion of the active ingredients withinthe polymeric matrix and thereby an optimized control over the release ofthe encapsulated material (approaching the classical zero-ordered releasekinetics) to be achieved (Kreitz et al., 1999). The first step of the processinvolves the preparation of two separate phases: a first phase, whichgenerally consists of a dispersion or solution of an active agent in asolution of polymer dissolved in a first solvent, and a second phase,which generally consists of a solution of surfactant and a second solventthat is at least partially immiscible with the first solvent. The two phases arecombined, and after applying dynamic or static mixing, an emulsion isformed, in which microdroplets of the first phase are dispersed in thesecond, that is, continuous, phase. The droplet formation step determinesthe size and size distribution of the resulting microspheres. The extent ofdroplet size reduction depends on the viscosity of the dispersed andcontinuous phases, the interfacial tension between the two phases, theirvolume ratio, and the geometry of the device (Sansdrap and Moes, 1993).A suitable surfactant or viscosity-enhancing stabilizers such as PVA andpolysorbates are generally added to produce a stable emulsion. Stirring isthe simplest and straightforward method used in laboratories to generatedroplets of the emulsion. For large-scale production of emulsions, manydevices have been designed (Gabor et al., 1999; Freitas et al., 2005).

Proteins such as gelatin, albumin, casein, and whey proteins are solublein water and have extremely good emulsifying properties; therefore, theyare suitable substrates for microsphere preparation. Single water-in-oil (W/O)-emulsions are used in the case of hydrophilic active ingredients. Theaqueous biopolymer solution containing the active substance is emulsifiedin a hydrophobic phase like vegetable oil or organic solvent. Then, thematrix material is stabilized by cross-linking and the particles are isolatedafter removal of the oil phase. Double oil–water–oil (O/W/O)-emulsionscan be used if the encapsulated substance is hydrophobic. It is first addedto an oil phase which is emulsified in the aqueous biopolymer phase toform an O/W-emulsion. Then, the O/W-emulsion is added to a


hydrophobic phase to form the double O/W/O-emulsion. The mainadvantages of the emulsion-based technologies are the flexibility incontrolling the degree of stabilization and the small particle size that canbe obtained. Disadvantages include the costs and effort related to removalof the oil phase and the loss of encapsulant during processing.Albumin is the most investigated protein in microparticle preparation

by emulsion techniques (Ishizaka et al., 1981; Gallo et al., 1984; Torradoet al., 1989; Arshady, 1990; Yang et al., 2007). Two methods are routinelyused to solidify albumin microspheres—heat denaturation and chemicalcross-linking. The W/O emulsion, where the inner phase contains dro-plets of aqueous albumin solution while the external phase is mostlycottonseed oil, is heated for some time at a temperature that may rangefrom 90 to 180 �C (Gupta et al., 1986; Dubey et al., 2003). Both heatingtemperature and heating time affect mean particle size, particle sizedistribution, and drug entrapment efficiency of albumin microspheres.Chemical cross-linking of albumin microspheres with glutaraldehyde

was used to develop sustained release forms (Chuo et al., 1996;Luftensteiner and Viernstein, 1998). Microcapsules were prepared fromhuman serum albumin (HSA) through an interfacial cross-linking processusing terephthaloyl chloride at various pH values (Edwards-Levy et al.,1993, 1994).Microparticles were produced from other proteins applying procedures

very similar to those used to make albumin microparticles. Fibrinogenmicrospheres containing doxorubicin or adriamycin were obtained andevaluated against Ehrlich ascites carcinoma (Miyazaki et al., 1986a,b).Collagen microparticles of diameters ranging from about 3 to 40 mmwere prepared by the method of emulsifying and cross-linking. The parti-cle size was mainly controlled by the molecular weight of the collagenused: an increase in denaturation of the collagen resulted in smallerparticle sizes. Spheres of 0.1 mm in size were obtained from gelatin.Collagen microparticles were thermally stable and allowed sterilization.They were tested as carriers for lipophilic drugs (Rossler et al., 1995).Micro- and nanoparticles with improved heat stability were obtained fromwhey protein that may enable novel biotechnological applications of wheyproteins (Picot and Lacroix, 2004; Zhang and Zhong, 2010).A variety of processes have been developed to prepare protein micro-

and sub-microparticles. The preparation method influences the interac-tions among the components in the formulation and thereby, it is very


important for the properties of the particulate systems such as type andsize of particles, colloidal stability on storage and/or in biological media,toxic effects, and loading efficiency. Most of the techniques briefly men-tioned above require heating, strong shear stress, or organic agents (sol-vents, cross-linkers, surfactants, or stabilizers) in at least one of theproduction steps. It can cause some destruction of sensitive-encapsulatedcompounds, toxicity problems associated with residual organic agents, aswell as protein denaturation. Coacervation, heat denaturation, and deso-lvation methods yield protein micro- or nanospheres with short storagestability.

An effective and feasible way to produce protein micro- and nanoparti-cles is the sonochemical method developed by Suslick and co-workers(Suslick and Grinstaff, 1990; Suslick et al., 1994). Microcapsules contain-ing gas or water-immiscible liquids are easily obtained in a short timeperiod, neither emulsifier nor stirrer is required, the equipment is sim-ple—only an ultrasonic probe and the materials are needed. This one-stepmethod affords stable proteinaceous micro- and sub-microcapsules whichmembrane is synthesized from various kinds of proteins. The accumulatedexperimental results in the field have been reviewed by Gedanken (2008)in an excellent article that discussed both fundamental and applicationaspects of the sonochemical synthesis of proteinaceous microspheres.Next sections of the present chapter will attempt to review the progressmade in the field, to mark the method advantages (in the context of somewidely used encapsulation techniques) and current challenges, for exam-ple, the ultrasound–protein interaction and other possible aspects of themechanism of the sonochemical preparation of protein-based micro- andnanoparticles.

III. Ultrasound Sonochemistry and Proteins

A. Cavitation

Ultrasound cannot be heard by the human ear as it goes beyond theaudio frequency limit of 20 kHz. Depending on its intensity, ultrasoundapplications fall in two categories—low-intensity ones related only totransmitting energy through a media by which one could obtain or conveyinformation (e.g., nondestructive testing, medical diagnostics, etc.) and


high-intensity ones which affect in some way the propagation media (e.g.,medical therapy and surgery, cleaning, welding of polymers and metals,homogenization of materials).Sound waves are made of high- and low pressure pulses traveling through

amedia.When a soundwave travels through a liquid, itmakes themoleculesof the liquid to oscillate around their mean position and thus, the meandistance between them increases and decreases alternately. If the soundwave intensity is high enough, it could result into breaking down of theliquid—the mean interparticle distance becomes higher than the criticalmolecular distance necessary to hold the liquid intact. This process is knownas acoustic cavitation and results in the formation of voids or cavities (orbubbles). The so produced bubbles (voids or cavities) usually increase andthen decrease in volume, some of them could disappear (Fig. 2). A similarprocess of ‘‘breaking down’’ water also takes place when water is boiling orwhen liquid is mechanically stirred (e.g., ship’s propeller).The acoustic pressure necessary to cause cavitation in water should be

approximately 1500 atm (Mason and Lorimer, 2002); however, the pres-ence of weak spots (e.g., gas molecules and/or solid particles dispersed inthe liquid) makes possible cavitation at pressures smaller than 20 atm. Ifthe liquid is degassed or purified (e.g., by ultrafiltration), the threshold forcavitation rises significantly.

100mm

FIG. 2. Photographic series of a trapped sonoluminescing bubble driven at21.4 kHz. (Photo # Reinhard Geisler. Reproduced with permission.) The bubble dy-namics are presented at an interframe time of approximately 2.5 ms (Domnitch andGelfand, 2004).


B. Sonochemistry

The bubbles could be divided into two categories—transient (whichcollapse and disappear, these are usually the empty bubbles) and stable(filled with gas and/or vapors that expand and collapse but do notdisappear).

Transient bubbles survive one or no more than a few acoustic cycles andcollapse, usually forming smaller bubbles which could be further nucleifor the formation of new bubbles or could simply dissolve in the media.Temperature and pressure within these bubbles at the total collapsemoment are very high. For example, a bubble containing nitrogen inwater at ambient temperature (20 �C) and pressure (1 atm) at the mo-ment of the final collapse has a within temperature of 4200 K and pressure975 atm (Mason and Lorimer, 2002). These extreme values could initiatethe formation of radicals when a liquid is sonicated (in case of water H

�

and OH�are obtained). When the bubble completely collapses, these

pressures are released as shock waves into the liquid (Mason andLorimer, 2002) and they are responsible for the increased chemical reactivity(because of the increased molecular collision) as well for degradation ofsubstances (e.g., contaminants, polymers, etc.). These high temperaturesand pressures are the base of the so-called sonochemistry which deals withhigh-energy chemical reactions that occur during ultrasonic irradiation ofliquids. The chemical effects of ultrasound are not a result from directmolecular interactions but arise from the effects of acoustic cavitation.Cavitation leads to concentrating the diffuse energy of sound, and bubblecollapse produces intense local heating and high pressures that are ex-tremely transient. Ultrasonic chemical effects can provide dramaticimprovements in stoichiometric and catalytic reactions; sometimes, thereactivity could increase a million-fold.

The chemical effects of ultrasound can be categorized as follows: (a)homogeneous sonochemistry of liquids, (b) heterogeneous sonochemistryof liquid–liquid or liquid–solid systems, and (c) sonocatalysis (an overlapof the first two categories). Chemical reactions have not been observedwhen solids and solid–gas systems were irradiated by ultrasound (Raichel,2006).

Stable bubbles contain gas and/or vapors and are produced at irradiationwith lower intensity ultrasound (1–3 W cm�2). Bubbles stay stable formany acoustic cycles, oscillating around an equilibrium size. The long


life of these bubbles allows for some processes to take place inside them as,for example, mass diffusion of gas which could result in bubble sizeincrease.The influence of some parameters on the acoustic cavitation stages

(nucleation, bubble growth, and collapse) will be outlined:

(i) The increase in frequency results in decreased bubble production andcavitation intensity.

(ii) Cavitation will be more difficult to take place in viscous liquids or inliquids with high surface tension where the forces between moleculesare stronger.

(iii) The higher the temperature, the lower the acoustic intensity necessaryto start cavitation. However, the high temperature of the liquid leadsto lower temperature and pressure developed into the collapsingbubbles, thus decreasing the sonochemical benefit. Also, the lowvapor pressure of the solvent increases the sonochemical benefitsso the influence of temperature is not unambiguous.

(iv) High gas content in the liquid results in a lower cavitation threshold;however, it also lowers the intensity of the shock wave released whenbubbles collapse (Fig. 3). The high gas content increases the weakpoints that could be nuclei for bubble formation. If gases with highersolubility are used, the number of nuclei is increased as well as theintensity of cavitation is lowered. The greater the gas solubility, thegreater the amount which penetrates into the cavitation bubble andthe smaller the intensity of the shock wave created on bubble collapse.

(v) Increase in the external pressure leads to an increase in cavitationthreshold as well as in the intensity of bubble collapse.

(vi) Ultrasound intensity increase will have a positive impact on sonochem-ical effects. The formation of bubbles will be easier, but if theintensity is too high, the bubbles could grow so large that they willnot collapse at all.

C. Ultrasound and Proteins

Ultrasound has tremendously increasing applications inmedicine duringthe past years. Despite the intensive exploitation of ultrasonics in health-related issues, the effects of sonication on biomolecules, especially proteins,remain poorly understood and characterized. Indeed, the problem of ultra-sound-induced damage in biomolecules (in particular, in proteins) is

0.02

Equilibrium gas pressure (mPa)

0.2

Aco

ustic

cav

itatio

n th

resh

old

(mP

a)

0.6

1.0

1.4

1.8

0.04 0.06 0.08 0.10

B

C

A

FIG. 3. Variationof acoustic thresholdofwaterwithdissolvedgas content: (A)distilledwater, s¼7.2�10�2 Nm�1; (B) aqueous guar gum (100 ppm), s¼6.2�10�2 Nm�1;(C) aqueous photoflow (80 ppm), s¼4.0�10�2 Nm�1 (Mason and Lorimer, 2002).Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.


complex and hard to be evaluated as the mechanism is still unclear.However, some attempts for characterization of this phenomenon havebeen recently done.

The fact that ultrasound causes protein denaturation has been demon-strated in many studies. Ultrasound can cause the breakdown of the hydro-gen bonding and Van der Waals interactions in the polypeptide chains,leading to modifications of the secondary and tertiary structure of theproteins, and the oxidation of the SH-group has often been observed.

This problem has been recently recognized because of one of thecurrent trends in controlled delivery of protein drugs—their encapsula-tion into polymer microspheres. Many authors observed loss of biologicalactivity of proteins after their encapsulation (Tabata et al., 1993; Blancoand Alonso, 1998; Pean et al., 1998). The initial step in protein encapsu-lation is usually the ultrasound-induced emulsification, as sonication gen-erates more homogeneous dispersion with high encapsulation efficiency.After a careful evaluation of protein denaturation at different stages of the


encapsulation processes, some authors have clearly shown that sonicationis partially responsible for protein denaturation. Tian et al. (2004) showedthat the activity of trypsin decreased with increasing ultrasound power orat prolonged irradiation time. They suggested two possible mechanismsresponsible for the enzyme deactivation: (1) the large interfacial areabetween water and air created after ultrasound cavitation which disturbsthe conditions around the trypsin molecules, for example, hydrogenbonds and hydrophobic interactions, and leads to conformational alter-ation of trypsin molecules and (2) the free radicals and the shock wavepropagation outside the bubble, resulting in high shear stresses—theresult of ultrasound cavitation—caused the modification or damage ofthe trypsin molecular structure. It has been already outlined in the previ-ous section that ultrasonic cavitation generates bubbles into liquids, and inthe case of protein solutions, it results in more protein molecules exposedto the water–air interface which in its turn causes disruption of hydrogenbonds and hydrophobic interactions. This could be a good reason for theattenuation of hydrophobic interaction and the denaturation of proteinafter ultrasonic irradiation. In order to prove this suggestion, Tian et al.(2004) aerated the trypsin solution and then applied ultrasound. Theresult was enhanced trypsin inactivation, which supported the first ofthe proposed by the authors’ mechanisms. The second cause for proteindenaturation, namely the ultrasound generated free radicals, was alsoproved to be valid. Tian et al. (2004) detected the presence of fragmentsof the trypsin molecule in the sample solution, and this was a key indicatorthat the trypsin molecular structure was damaged by ultrasound. This wasone of the rare cases when such a disruption of protein molecules wasobserved, as in most studies, denaturation but not degradation of proteinmolecules has been reported.Krishnamurthy et al. (2000) showed that even a stable protein as the

lysozyme could be denatured by ultrasound, but there was no evidence forfragmentation or aggregation of lysozyme upon sonication. The authorsdid not observe any changes in the electrophoretic mobility of native andsonicated lysozyme which suggested that the loss in the enzyme activityupon sonication was not attributable to fragmentation, aggregation, or achange in the surface charge of lysozyme. However, the significance of air–water interface for protein denaturation was again proved—reducing theair–water interface prevented protein precipitation and even the loss ofenzyme activity.


For some of the authors, the way ultrasound affects the protein is closelyrelated to protein structure. According to Barteri et al. (1996), the effects ofultrasound on the chemical and conformational modification of proteins,enzymes, and nucleic acids may be associated with the mechanical stress ofcavitation, while deactivation of enzymes is caused by molecular damage oftheir active site geometries which results in a loss of enzyme–substrateaffinity. These authors suggested that the larger the a-helix fraction of theprotein is, thehigher its rigidity and the greater the rate and the extensionofconformational damage induced by the mechanical stress of cavitation.According to them, proteins containing a large amount of ordered second-ary structure have more ‘‘fragile’’ tertiary structure, which is more suscepti-ble to mechanical stresses. On the contrary, when proteins have a ‘‘moreflexible’’ molecule (as e.g., cytochrome c), they are capable of attenuatingthe mechanical effects caused by ultrasound, most probably through‘‘local’’ reversible modification of their disordered structure.

Satheeshkumar and Jayakumar (2002) observed that upon sonication,protein solution containing large amounts of random coil and smallamounts of b-sheet structures increases significantly the b-sheet conforma-tion content. This, according to the authors, may be due to the fact thatthe acoustic cavitation results in newly created air–water interface at whicha stable b-sheet-enriched state similar to the amyloid is formed. The reasonfor this structural transition may be due to the intrinsic hydrophobicity ofthe air–water interfaces, which form a hydrophobic–hydrophilic system,with air being the hydrophobic component (Satheeshkumar andJayakumar, 2002). El-Agnaf et al. (1997) have also reported that sonicationof protein solution promoted the irreversible formation of the sheetstructure, which according to them, was due to oxidation of the methio-nine residue.

Similar results were reported in a recent work by Stathopulos et al.(2004). Sonication of a range of structurally diverse proteins resulted inthe formation of aggregates that have similarities to amyloid aggregates.For example, circular dichroism (CD) revealed that sonication-inducedaggregates had high b-content, and proteins with significant nativeb-helical structure showed increased b-structure in the aggregates. Theauthors concluded that most probably the amyloid-like structure was aconsequence of the protein denaturation after sonication. It is known thatall proteins under conditions where the native state is destabilized canadopt the amyloid structure (Fandrich et al., 2001). Moreover, partial or


complete unfolding of the native state of proteins is generally believed tobe required for amyloid formation (Fink, 1998; Kelly, 1998; Rochet andLansbury, 2000; Fandrich et al., 2001). Stathopulos et al. (2004) suggestedthat the cause of protein aggregation was not the intermolecular disulfidebonds but rather noncovalent linkages.Recently, a very detailed study on changes that take place with bovine

serum albumin (BSA) upon sonication has been carried out by Gulserenet al. (2007). They found that the surface activity of BSA increases as theprotein molecules were exposed to ultrasound, that is, ultrasonication ofBSA lowered the surface tension and ultrasonicated BSA adsorbed morerapidly than native BSA. As a reason for this behavior of sonicated proteins,the authors pointedout the change in the initial state of theprotein (proteinwas partially or completely denatured) or the change of the adsorbed state ofthe protein. The data from calorimetric investigation onBSA irradiated withultrasound showed no significant variations in denaturation temperaturebetween native and ultrasonicated samples. The transition temperatures forboth nontreated and sonicated BSA solutions were approximately 71 �Cwhich is very close to the reported denaturation temperature of commer-cially available BSA (73 �C). However, the denaturation enthalpy of nativeBSA (893 kJ/mol) decreased to 625, 611, and763 kJ/mol for 15-, 30-, and 45-min sonicated samples. Thatmeans that less energy was required to cause anunfolding of ultrasonicated BSA samples than of the native one. So, onecould conclude that the treatment of protein solutions with high-intensityultrasounddid not result in full denaturation of proteins but rather proteinsbecame more susceptible to the heat treatment, as evidenced by the de-crease in the denaturation enthalpy.The other BSA characteristic that has changed because of sonication was

the hydrophobicity measured by using the relative fluorescence intensity(RFI) of two different fluorescent dyes. The hydrophobicity of BSA mole-cules increased with increasing sonication time—24% increase in surfacehydrophobicity (i.e., more hydrophobic groups are exposed to the solventphase) upon a 45-min sonication was measured. However, a 24% increasein hydrophobicity is relatively small compared to typical increases ob-served upon thermal denaturation of proteins. For example, the hydro-phobicity of lysozyme increased 14-fold after the protein was heated to90 �C (Ibrahim et al., 1996), while a-lactalbumin surface hydrophobicityincreased by a factor of 15 compared to the native form of the protein(Cornec et al., 2001).


The application of high-intensity ultrasound resulted also in an increasein the magnitude of BSA zeta potential, with the difference between thezeta potential of native and sonicated protein solutions becoming larger athigher pH values. Such an increase is most probably related to an increasein the number of charged residues at the surface of the protein moleculewhich are exposed to solvent molecules, but the exact cause of theincreased exposure is not clear.

BSA particle sizes increased at longer sonication times (>40 min), whichthe authors attributed to the formation of small aggregates that may havebeen formed.However, no dimerization was observed which proved that theformation of aggregates was not due to the formation of covalent bondsbetween protein molecules, for example, intermolecular disulfide bridges.Instead, the aggregation wasmost probably due to noncovalent interactionssuch as electrostatic and hydrophobic interactions.

The sonication of BSA resulted also in a decreased amount of freesulfhydryl groups as the sonication time increased; for example, the numberof the initially present sulfhydryl groups decreased by 31% after sonicationfor 90 min. This could be explained by the oxidizing of free sulfhydryl groupcaused by the cavitation-generated hydrogen peroxide. Oxidation of thiolgroups typically results in the formation of disulfides, while the presence ofactivated oxygen radicals may lead to the formation of sulfinic and sulfonicacid (Cecil and McPhee, 1959). Furthermore, disulfide bonds may bedirectly oxidized with peroxide to yield sulfonic acid.

The authors also followed the influence of the ultrasound on the second-ary structure of BSA by two methods—infrared adsorption and CD. It wasshown that thea-helical content increases as a consequenceof high-intensityultrasound. For example, after 45 min of ultrasonication, the percentage ofa-helices increased from 61.1% to 74.5%, while the percentage of b-sheetand b-turns decreased by 2.8% and 1.6%, respectively. These results wereopposite to those reported by Satheeshkumar and Jayakumar (2002) andStathopulos et al. (2004) and also in contrast to those observed for thethermal denaturation of BSA loss of ordered structure (Giacomelli andNorde, 2001; Militello et al., 2003). This ultrasonically induced conversionof the secondary structure was proved to be irreversible, as over time, theproteins did not regain their former structure or function.

Although we do not pretend to present a comprehensive picture ofprotein–ultrasound interactions, we could draw the following conclusions:


(i) The ultrasound causes partial denaturation of proteins throughincreased air–water interface and due to high shear forces developedin the media after the shock waves were released as a result of cavitationand bubbles’ total collapse. These shear forces disrupt the hydrophobic,van der Waals, and H-bonds that hold up the native protein conforma-tion and in this way make proteins more susceptible to further denatur-ation caused by other factors (temperature, chemical reactions, etc.).

(ii) This partial denaturation could result in protein aggregation, but thelatter is due exceptionally to noncovalent bonds and not to disulfidebridges.

(iii) In very a few studies, degradation of protein molecules under ultra-sound has been detected, with most of the studies reporting mainlyconformational changes in protein molecules.

(iv) The influence of the ultrasound on the protein secondary structure isambiguous. While more data exist for the preferable formation ofb-structures after ultrasound irradiation, some new data show thata-helices content increases with sonication time at the expense of theb-structures.

(v) While the effects of ultrasound on the conformational characteristicsof proteins, enzymes, and nucleic acids are mainly associated with themechanical stress of cavitation, deactivation of enzymes is most prob-ably caused by molecular damage of their active site geometries whichresults in a loss of enzyme–substrate affinity.

It is clear that much more studies on the topic are necessary in order todraw more reliable conclusions. It is indisputable that these investigationswill have a significant impact on health-related issues as ultrasound isprogressively used in medicine.

IV. Proteinaceous Microspheres Obtained Through

Sonochemistry

At the end of the 1980s, Keller et al. (1986, 1988) demonstrated thathuman albumin might be an ideal material for the production of micro-bubbles to be used as echo contrast agents because it did not producesignificant changes in coronary blood flow, left ventricular function, orsystemic hemodynamics. The authors sonicated a heated 5% (w/v)human albumin for 40 s and produced air-filled albumin microspheres


which ranged in diameter from 1 to 15 mm, with less than 5% being largerthan10 mm.During the sonicationprocess,microbubbles of air were formedand encapsulated in a thin shell of aggregated albumin about 15 nm inthickness. Due to the stabilizing effect of the albumin shell, the air-filledmicrosphere suspension was stable about 2 years under refrigeration. Aftersonication in the protein solution, the microspheres represented about1.5% of the total protein, the remaining protein being soluble albuminmolecules (Christiansen et al., 1994). These HSA microspheres were com-mercialized under the trademark Albunex (by Molecular Biosystems Inc.(MBI), San Diego, USA and Nycomed Imaging AS, Oslo, Norway) andwere the first ultrasound contrast agent that was stable enough to showtranspulmonary passage (Sponheim et al., 1993). This means that thecontrast agent can be injected intravenously and still give contrast in theleft side of the heart. Albunex was the first FDA-approved contrast agent. It isno longer in production, because although it is capable of transpulmonarypassage, it often failed to produce adequate imaging of the left heart.

The sonochemically born protein microspheres were further developedby Suslick and co-workers in an extensive and detailed long years lastingresearch. As a further step, they applied ultrasound to make aqueoussuspensions of proteinaceous microcapsules filled with water-immiscibleliquids. The proteinaceous microcapsules were synthesized from BSA aswall material that encapsulated n-dodecane, n-decane, n-hexane, cyclohex-ane, or toluene. Spherical microcapsules were obtained as seen by scan-ning electron microscopy (Fig. 4) (Suslick and Grinstaff, 1990). Whensonication was carried out under air or O2, quite a high yield of1.5�109 microcapsules/mL with an average diameter of 2.5 mm (Gaussiandistribution, s¼� l.0 mm) was obtained. This high yield could beexplained by the increased quantity of gas that resulted in an increasednumber of ‘‘weak spots’’ in water media. The size distribution was similarfor all nonaqueous liquids examined and was quite narrow.

A. Mechanism of Proteinaceous Microspheres FormationThrough Sonochemistry

Suslick and Grinstaff (1990) were the first to give an answer to thequestion ‘‘How the microcapsules are formed and what holds them to-gether?’’ Ultrasonic emulsification occurred in this biphasic ‘‘protein

FIG. 4. Scanning electron micrograph of a dodecane-filled proteinaceous macro-capsules. Themicrocapsules were prepared for SEM by cross-linking with glutaraldehydeand coating with Au/Pd. Volatile nonaqueous liquids produced deformed microcap-sules due to evaporation during sample preparation. Reprinted with permission fromSuslick and Grinstaff (1990). Copyright (1990), American Chemical Society.


aqueous solution—nonaqueous liquid’’ systems but it was not enough,according to the authors, for the formation of stable microcapsules. If onlyvortex was used for emulsification, no microcapsules were obtained. Theauthors also showed that when the sonication of the biphasic system wasrun under an inert atmosphere (He, Ar, or N2), microcapsules were alsonot formed. Only in the case of air or O2 sparged systems, a high yield ofmicrospheres was gained.It is known from sonochemistry that under sonication, water molecules

can produce OH�and H

�radicals. The latter can further form H2, H2O2

and in the presence of O2, superoxide (HO2). Among these species,hydroxyl, superoxide, and peroxide are the potential protein cross-linkingagents. To identify which one is involved in the process of microspheresformation, Suslick and Grinstaff (1990) used three radical traps—glutathi-one (nonspecific trap), catalase (which decomposes hydrogen peroxide tooxygen and water), and superoxide dismutase (which decomposes super-oxide to oxygen and hydrogen peroxide). The microcapsule formationwas inhibited by glutathione and superoxide dismutase, but not by catalase(Fig. 5). Thus, most probably, the sonochemically generated superoxide isinvolved in the cross-linking reaction through disulfide bond formation.The authors concluded that the protein microcapsules were stable withtime because they were held together via disulfide bonds formed between

700

600

500

400

300

200

100

0

Mic

roca

psul

e co

ncen

trat

ion

(mill

ions

/ml/m

m)

0 2 4 6 8

Diameter (mm)

10 12 14 16 18 20

0.09% w/v catalase

0.1% w/v superoxide dismutase

0.1 M glutathione

FIG. 5. The effect of radical traps on microcapsule formation. Aqueous solutions(5%, w/v) of BSA and toluene were irradiated in the presence of catalase, glutathione, orsuperoxide dismutase. Inhibition of microcapsule formation also occurred with 2,6-di-tert-butyl-4-methylphenol. Reprinted with permission from Suslick and Grinstaff (1990).Copyright (1990), American Chemical Society.


protein cysteine residues, and that superoxide, sonochemically producedin the aqueous medium, was the cross-linking agent. The absence of O2

and the lack of free cysteine residues in the protein molecule had anegative influence on the cross-linking process and consequently, on theformation of protein microspheres.

The importance of cysteine residues for protein microsphere formationwas investigated at length in other works of the research group (Grinstaffand Suslick, 1991; Suslick et al., 1994). The authors found that BSA, HSA,and hemoglobin (Hb) (all of which have cysteine residues) formed micro-bubbles, whereas myoglobin (which has no cysteine residues) did not,although it has very similar sequences and monomeric three-dimensionalstructure to Hb. Moreover, the alkylation of the BSA and Hb cysteineresidues with N-ethylmaleimide prevented formation of disulfide bonds,and a dramatic decrease in microbubble formation was observed.

Dithioerythritol is known to be a cleavage reagent for the disulfide bond(Jocelym, 1976). The addition of dithioerythritol rapidly destroyed


Hb–toluene or BSA–toluene microcapsules (Suslick et al., 1994). Thus, theauthors concluded that the cysteine residues were oxidized during ultra-sound irradiation and subsequently, interprotein disulfide bonds thatcross-linked the proteins and held the protein microspheres togetherwere formed. These results confirmed the significance of disulfide bondformation in microsphere formation.Interestingly, Suslick et al. (1994) did not observe a decrease of protein

functions as a consequence of the ultrasound treatment. The heme heldwithin the Hb wall was fully retained. Deoxy-Hb microspheres wereobtained by using an enzyme-reducing system to reduce the heme to Fe(II). These microspheres were able to bind O2 when exposed to O2 andformed oxy-Hb microspheres. The IR spectrum of the latter was identicalto normal oxy-Hb solutions. The reverse process of O2 release wasachieved by flushing with Ar, and as a consequence, again deoxy-Hbmicrospheres were obtained. The transition deoxy-Hb microspheres tooxy-Hb microspheres could be cycled more than 10 times without signifi-cant degradation. It means that the ultrasonic irradiation did not signifi-cantly alter the environment surrounding the active heme site (Wong andSuslick, 1995). The partial pressure of O2 at which half of the availablebinding sites on Hb were bound by O2 was measured to be similar for thenative and sonicated Hb. This fact, along with the reversibility of O2

binding process, makes the Hb microspheres appropriate to be used asblood substitute. An interesting result obtained for Hb microspheres wasthat the maximum Hill coefficient, which indicates the level of coopera-tivity between oxygen binding sites, was significantly higher for Hb micro-spheres than for native Hb. This fact was explained by Wong and Suslick(1995) with the formation of disulfide bridges between adjacent Hbtetramers in the shell of one microsphere.The effect of microsphere formation on the BSA structure was moni-

tored by means of CD (Suslick et al., 1994). BSA contains a high percent-age of a-helix in a native form. The normalized CD spectrum of air-filledBSA microbubbles revealed minor changes in the a-helix content, thusindicating the absence of an extensive denaturation of the protein. This isin agreement with the observations of Gulseren et al. (2007) who haveshown (also by CD) that the amount of a-helices is almost unchanged until15 min ultrasonication time and then it increases. For comparison, theultrasonication time that Suslick et al. (1994) used was 3 min.

0.25mm

FIG. 6. Transmission electronmicrograph of air-filled proteinaceous microbibblesmade from hemoglobin. Reprinted from Suslick et al. (1994). Copyright (1994), withpermission from Elsevier.


Transmission electron microscopy reveals that the Hb microspheres arereally empty (clear central region in Fig. 6). The thickness of the proteinmicrosphere shell ranges from 25 to 35 nm and having in mind that Hb isa roughly spherical protein with a diameter of 5.5 nm. Suslick et al. (1994)calculated that this thickness of the protein shell corresponds to a thick-ness of roughly 4–7 protein molecules. That means a microsphere with adiameter of 3 mm would contain about 106 Hb molecules.

In summary, the suggested mechanism responsible for forming theproteinaceous microbubbles is a combination of two acoustic phenomena:emulsification and cavitation (Grinstaff and Suslick, 1991; Suslick et al.,1994; Wong and Suslick, 1995). Dispersion of gas into the protein solutiontogether with disulfide bridges formation between protein molecules(chemical cross-linking in Fig. 7) at the bubble interface results in theformation of stable microspheres that could be filled with air or nonaque-ous liquids.

COOH

COOH

COOH

COOHNH2

NH2

NH2

NH2

S S

S S

S S

SS

S S

S S

SS

SS

FIG. 7. Disulfide cross-linking holds the proteinmicrospheres together. Reprintedfrom (Suslick et al., 1999). Permission granted by The Royal Society.


In 2002, Gedanken et al. (Avivi and Gedanken, 2002) obtained sono-chemically proteinaceous microspheres from protein that does not containany sulfur-containing residues. Streptavidin microspheres, in which mor-phology was very similar to theHb or BSAmicrospheres prepared by Suslickand collaborators, showed abroad sizedistributionwith an averagediameterof 5 mm. The authors were able to obtain these microspheres only at pHlower than 6.0. The lack of cysteine residues in streptavidin means that themechanism suggested by Suslick and collaborators about protein disulfidecross-linking as a result of cysteine oxidation after ultrasonical irradiation isnot applicable here. Moreover, Gedanken et al. succeeded in obtainingstreptavidin microspheres even when the solution was sonicated underargon. A new mechanism for streptavidin microspheres formation underultrasound treatment was proposed (Avivi andGedanken, 2002). Accordingto this, hydrophobic or thermal denaturation of the protein as a result of the


ultrasonic irradiation takes place and assists the microsphere formation.The number of the hydrophobic residues exposed to water increases as aresult of the denaturation, and as they tend to interact between themselvesrather than with water molecules, a more condensed structure of streptavi-din macromolecules is obtained. The low pH contributes to this process byneutralizing the carboxyl groups, which makes hydrophobic interactionsdominant, that is, creates amore favorable hydrophobic environment (Aviviand Gedanken, 2002). As a proof of this new mechanism, the authorssucceeded to obtain poly(glutamic acid) microspheres upon sonication.Poly(glutamic acid) has only carboxy groups, as pendant groups andmicro-spheres were formed only at pH lower than 4.5 similar to the streptavidincase. As pKa of the pendant carboxyl groups of aspartic acid and glutamicacid residues is 4–4.8, the authors confirm the significance of the hydropho-bic interactions for the production of microspheres from poly(glutamicacid) as well from streptavidin. The authors explicitly mentioned the highstability of the microspheres, that is, the hydrophobic interactions werestrong enough to produce so stable microspheres.

Another mechanism for microsphere formation from proteins withoutSH groups but containing disulfide bridges was suggested by Avivi (Levi)and Gedanken (2005). These authors used avidin in the sonochemicalsynthesis of microspheres. Avidin, as it is known, contains one disulfidebridge and no free sulfide residue. Thus, the authors assume a two-stepmechanism for the avidin microspheres formation. In the first step, open-ing of the disulfide bridge takes place, while in the second step, ‘‘a newintermolecular disulfide bonding in a required geometry’’ occurs. Theauthors were able to vary the size of the avidin microspheres by changingavidin concentration. While for an avidin concentration of 4% (w/v),microspheres with an average diameter of 3.4 mm were obtained, whenconcentration was reduced to 3% (w/v), the average diameter dropped to1.8 mm. At the same time, the yield of the microspheres also dropped. Theavidin microspheres still were able to bind biotin after the ultrasoundirradiation, although their biological activity was reduced compared to thenative avidin. The authors concluded that ‘‘the same binding schemeoperates for native and microspherical avidin biotin complexes.’’

The synthesis of stable lysozyme microbubbles was reported for the firsttime by Cavalieri et al. (2008). Aqueous solutions of chemically reducedlysozyme were irradiated with high-intensity ultrasound to induce emulsi-fication and cross-linking of the protein shell. Egg white lysozyme is a small


globular polypeptide (Mw 14,000) chain that contains 129 amino acids inthe primary sequence and 4 intrachain disulfide bridges buried inside thehydrophobic core of the protein. Sonication of native or thermally dena-tured lysozyme did not produce stable microbubbles. The possible reasonwas the absence of free thiol groups. Therefore, prior to sonication,lysozyme was treated with dl-dithiothreitol (DTT) to disrupt the disulfidebonds and generate thiol moieties. The chemical denaturation resulted inimproving lysozyme foaming and cross-linking properties, and in prepara-tion of stable microbubbles. It was found that, besides sonochemicaltreatment parameters, DTT concentration and denaturation time alsoaffected the yield and the size of microbubbles. Denaturation treatmentranging from 2 to 5 min coupled to 30 s of sonication was identified as theexperimental conditions to obtain microbubbles with an optimal sizedistribution and a good yield. A possible mechanism of stabilization oflysozyme microbubbles at the interface of the cavitation bubbles wasproposed. It involved formation of lysozyme clusters by protein aggrega-tion due to hydrophobic interaction resulting from the chemical denatur-ation. These aggregates adsorbed to the air–water interface generated byemulsification and formed a shell that was stabilized via cross-linking.Therefore, the efficient cross-linking between lysozyme clusters at thebubble–solution interface was pointed as one of the key factors to preparestable lysozyme microbubbles. The SEM and AFM images (Fig. 8) visualizea bumpy surface generated by protein clusters that formed the wall.Compared to air-filled BSA microbubbles, lysozyme microbubbles exhibita longer shelf life (months). Their wall is 130-nm thick with a compactstructure, which reduced gas permeability of the protein membrane.

B. Bioactivity of Proteinaceous Microspheres

Thefirst authors thatusedultrasound toobtainmicrospheres fromenzymeswere Avivi (Levi) and Gedanken (2007). The enzymes allow for an easy andmore quantitative estimation of the biological activity. Themicrospheres wereproduced by the sonochemical method from two enzymes—a-amylase (withhigh thermal stability) and a-chymotrypsin (less thermostable enzyme). Amy-lase solutions with two concentrations were treated with high-intensity ultra-sound: from 0.05% (w/v) a-amylase solution, microspheres with an averagediameter of about 2 mm were synthesized, while at a lower concentration

mm

mm

mm

1 mm

0.00.0

0.0

0.0

0.4

0.4

0.20.40.6

0.8

0.8

0.8

1.2

1.2

1.2 1.0

1.6

1.6

2.0

2.0

700.0 nm

100.0 nm

0.0 nm

(A) (B)

(C) (D)

FIG. 8. (A, B) AFM and (C, D) SEM images of lysozyme microbubbles (15 mindenaturation, 30 s sonication). Reprinted with permission from Cavalieri et al. (2008).Copyright (2008), American Chemical Society.


(0.017%, w/v), microspheres with an average diameter of about 300 nmwereproduced. In both cases, very broad size distributions were detected (Fig. 9).The yield of a-amylase microspheres was estimated to be 70%; 30% of theenzyme remained unreacted (Avivi (Levi) and Gedanken, 2007).

The activity of a-amylase microspheres was determined by measuringthe released reducing sugar using starch as the substrate. The enzymaticactivity of the amylase microspheres obtained at the higher concentrationwas 27% of that of the native enzyme for 3 min reaction time andincreased to 56% when the reaction time increased to 1 h. According tothe authors (Avivi (Levi) and Gedanken, 2007), the observed decrease inthe enzyme activity could be explained by the fact that only the amylasemolecules on the microsphere surface react, while some of the enzyme

70

60

50

40

30

20Wei

ght (

%)

10

00 1000 2000 3000

Size (nm)

4000 5000

(A)

70

80

60

50

40

30

20

Wei

ght (

%)

10

00 100 200 300 400 500 600 700

Size (nm)

(B)

FIG. 9. Particle distribution of (A) the high concentrated solution (0.05 vol.% ofamylase in water) and (B) the low concentrated solution (0.017 vol.% of amylase inwater). Determined with Coulter particle analyzer (N4). Reprinted from (Avivi (Levi),and Gedanken, 2007). Copyright (2007), with permission from Elsevier.


active centers are buried into the microsphere shell (its thickness wasestimated to be 30 nm; Suslick et al., 1994) and could not take part inthe reaction. In general, the amylase microspheres stay catalytically activeafter sonication and the process does not destroy the enzyme active sites.The reaction slows down, while some of the reactive centers cannot bereached at all, and the approach to the active sites by the reactantsbecomes more difficult.


Microspheres from a less thermostable enzyme, a-chymotrypsin werealso prepared by the sonochemical method. The enzyme preserved itscatalytic activity in 51% compared to the native protein after 1 min reac-tion time and 65% after 10 min reaction time. The authors (Avivi (Levi)and Gedanken, 2007) stated that the sonication used to prepare enzymemicrospheres is not a ‘‘denaturation process. The proteinaceous micro-spheres are catalytically active but their reactivity is reduced as comparedto the native protein.’’

The enzymatic activity of lysozyme microbubbles obtained by Cavalieriet al. (2008) was estimated by applying a turbidometric Micrococcus leuteustest (Ibrahim et al., 1996). At first, microbubbles were extensively washedbefore testing to remove free lysozyme from the solution. The authors didnot consider a comparison of enzymatic activity of lysozyme in solutionwith lysozyme microbubbles to be meaningful since most of the protein inthe wall is not available to exhibit its activity. Therefore, lysozyme solutionswere obtained after 5 min sonication of the microbubbles. The treatedenzyme showed antimicrobial activity comparable to the native protein.The authors claimed that they reported the first example of microbubblesexhibiting enzymatic and antimicrobial activity.

Avivi and Gedanken (2002) investigated the influence of the ultrasoundtreatment on the streptavidin activity by testing the ability of streptavidinmicrospheres to bind biotin. They found that streptavidin microspheresbound approximately 50% of the amount of biotin bound to the nativeprotein, that is, the biological activity of proteinaceous microspheres wasreduced by 30–50% compared to the native protein.

C. Effect of Experimental Parameters on the Characteristics of the ProteinaceousMicrocapsules

The effect of various experimental parameters, such as frequency of theultrasound, the time period of ultrasound irradiation, albumin concentra-tion, the kind of organic solvents and their volume fractions on themicroencapsulation yield of the organic solvents (toluene, chloroform,and soybean oil), and the size distribution of the BSA microcapsules wasdetermined by Makino et al. (1991). The authors found that ultrasoundirradiation between 5 and 10 min was required to obtain microcapsules.The acoustic frequency affected both the microencapsulation yield and


size distribution of the microcapsules. Capsules with a narrower sizedistribution were obtained at 45 kHz irradiation than at 28 kHz, whilehigher yield was achieved at the lower frequency applied. No microcap-sules were obtained at 100 kHz. The microencapsulation yield increasedwith the increase of the BSA concentration from 0.005% to 0.02% (w/v),and on the contrary, the yield decreased considerably when the volumefraction of toluene was over 0.5. The size of the microcapsules was depen-dent on the kind of liquids that were microencapsulated. Microcapsuleswith a more viscous core liquid, that is, soybean oil, were smaller thanthose containing toluene or chloroform.Zhou et al. (2010) also found out that the size and the stability of the

microcapsules were dependent on the nature of the encapsulated materi-als. They achieved effective encapsulation of liquid materials within lyso-zyme microspheres. Four different liquids, namely, sunflower oil,tetradecane, dodecane, and perfluorohexane, were used in the experi-ments. Among them, the perfluorohexane-filled microspheres showedsmaller mean sizes and narrow size distributions, as well as the greateststability on drying. The authors suggested that lysozyme adsorption at theperfluorohexane–water interface (when an emulsion is produced by soni-cation) is very effective due to the lower surface tension of this liquid,which leads to the formation of a strong/thick shell wall of the micro-spheres. Using an oil-soluble fluorescent dye, the potential use of lysozymemicrocapsules as suitable reservoirs for lipophilic compounds, that is,water-insoluble drugs or food ingredients, has been demonstrated.Another recent study (Han et al., 2008a) was focused on the stability and

size dependence of protein microspheres prepared by ultrasonication.Such microspheres are able to deliver many types of pharmaceuticals,and via surface modification, drug targeting to specific tissues and cellsis possible (for examples and references, see the section below). In thisrespect, the size control of the protein microspheres and its correlation tostability of the formulation, especially, the stability against aging andaggregation, are a major issue in the development of drug delivery systems.Han et al. (2008a) used HSA solution—silicon oil biphasic system in theirstudy on the influence of the acoustic variables and solution characteristicson the size distribution of the prepared protein microcapsules. It wasfound that the mean size of the particles decreased with sonication timeuntil equilibrium was reached. That required longer time at lower poweramplitudes, for instance, 4 min at 25% amplitude (125 W) and only 2 min


at 100% amplitude. The authors emphasized the fact that the sonicationtime needed to reach equilibrium was strongly dependent upon thesolution volume subjected to sonication. The acoustic power also influ-enced the equilibrium sizes of microspheres; higher amplitudes led to theformation of smaller microspheres and vice versa.

The protein microspheres prepared by sonication usually have a broadsize distribution with diameters between 100 nm and 50 mm. Han et al.(2008a) also monitored a broad size distribution for the HSA micro-spheres obtained at different power amplitudes. They measured signifi-cant size differences for the microcapsules formed in different zones ofthe ultrasonic vessel (Fig. 10) as a result of the uneven acoustic powerdistribution in the vessel. Therefore, the authors advised that to improvepower distribution, it would be preferable to use a slender vessel with adiameter close to that of the ultrasonic probe. It was observed that thesmall microspheres precipitated since they had a dense protein shell anddisplayed a higher density than that of water. These microspheres had anaverage diameter of 1 mm with a narrow size distribution and were sepa-rated by natural sedimentation. The microcapsules were easily loaded bysimply dissolving the drug in the oil phase before sonication. Moreover,they kept the drug inside for several weeks affording excellent protectionfor the loaded agent.

We have recently applied the sonochemical method to obtain gelatinnanocapsules as drug carriers for hydrophobic, water-insoluble drugs(Yankova and Vassileva, 2010). In Fig. 11, the scanning electron microsco-py image of the obtained gelatin capsules is presented. For better qualityof the SEM pictures, the gelatin capsules have been filtered and that is whyonly capsules with a diameter around 1 mm are seen in the figure, thesmaller not being retained by the filter.

By transmission electron microscopy, it is clearly seen the core-shellstructure of the gelatin particles, that is, they are really capsules and notdense particles (Fig. 12). A rough estimate of the gelatin shell thicknessshows a value of 10–20 nm, much smaller than the BSA shell estimated bySuslick and collaborators (25–40 nm). This value coincides well with thethickness of nanocapsules obtained through the coacervation method.

In contrast to the results reported by Gedanken and Suslick groups, wehave obtained bimodal size distribution for all gelatin nanocapsules synthe-sized through ultrasound. An example of a size distribution curve for gelatinnanocapsules measured by dynamic laser scattering is presented in Fig. 13.

A

B

B C

A

C D

E

20mm

20mm20mm

FIG. 10. The partitioned regions in the vessel used for sonication and the micro-spheres formed in each region. Reprinted from Han et al. (2008a). Permission grantedby The Royal Society of Chemistry.


The bimodal size distribution could originate from the very wide molecularweight distribution of gelatin or could also be a result of two independentmechanisms of gelatin particles formation that takes place simultaneouslyduring ultrasound irradiation (e.g., both mechanisms suggested by Suslickand collaborators and Gedanken and collaborators). However, more inves-tigations are necessary to clarify the origin of the bimodal size distribution.Besides, most of the authors until now reported a wide size distribution,while in this case, both peaks are quite narrow.Clearly, nanosized gelatin capsules were obtained from gelatin through

the sonochemical method in contrast to the reported until now microsizedprotein capsules. The influence of three parameters—pH, temperature,and duration of ultrasound irradiation—on gelatin nanocapsules size was

FIG. 11. SEM image of gelatin capsules obtained by ultrasound.

FIG. 12. Transmission electron microscopy of gelatin nanocapsules obtained byultrasound.


0

1

2

3

4

5

0.001 0.01 0.1 1 10 100 1000 10,000

Inte

nsity

(%

)

Size (r. nm)

Size distribution by intensity

FIG. 13. Dynamic light scattering of aqueous solutions of gelatin nanocapsulesobtained by the sonochemical method.


investigated. As the protein properties in aqueous solutions strongly de-pend on pH of the solution, we have followed how the pH influences thenanocapsules size. Similar detailed investigation on the pH influence onproteinaceous microspheres has not been performed until now, althoughsome data have been already reported from both groups of Gedanken andSuslick, for example, for poly(glutamic acid) and streptavidin. It appearedthat at any pH in the range of 2.5–6.5, gelatin could form nanocapsuleswhen irradiated by ultrasound. By increasing pH, the size of gelatinparticles decreased and the lowest size was observed in the range of theisoionic pH of gelatin (by viscosity measurements, it was estimated to be4.95 (unpublished results)). Some more investigations at higher pH valuesare in progress.Increase in temperature resulted in an increase of the gelatin nanocap-

sules size, this dependence was observed for both peaks of the bimodal sizedistribution. The increase in ultrasound irradiation time also resulted inan increase of gelatin nanocapsules size, in contrast to that reported byHan et al. (2008a).

V. Applications of the Proteinaceous Microcapsules

In a number of papers, Gedanken and co-workers have explored theBSA microspheres obtained through the sonochemical method for differ-ent applications. The BSA choice is based on its availability in pure form


and its biodegradability, nontoxicity, and nonimmunogenicity (Grinberget al., 2007). Another strong advantage of BSA is that it accumulates insolid tumors (Matsumura and Maeda, 1986; Takakura et al., 1990) whichmakes it a very good candidate for site-targeted delivery of antitumordrugs.

The outer surface of the protein microsphere contains a number ofchemically active moieties that could be used for chemical attachment ofmodifying molecules. For example, polyethylene glycol (PEG) chains wereattached to the BSA microcapsules via coupling of the terminal hydroxylgroup of the polyether chain with the primary amine groups from the sidechains of the lysine residues in BSA molecules (Webb et al., 1996). PEGwas chosen as it is well known that the nonspecific protein adsorption onPEGylated surfaces is very low (Wattendorf and Merkle, 2008). This factdetermines the property of PEG-coated particles to avoid phagocytosis bythe RES (reticuloendothelial system) and thus, the blood circulation timeof the microspheres increases.

Besides conjugation, multilayer deposition of polyelectrolytes onto themicrocapsule surface is an alternative approach for imparting new proper-ties and functions. The coating of microbubbles with polyelectrolytes isstraightforward and a mild procedure. Lysozyme microbubbles are posi-tively charged colloidal particles and provide a good template for poly-electrolyte adhesion. Cavalieri et al. (2008) demonstrated that thelysozyme air-filled microbubbles can be modified by stepwise layer-by-layer (LbL) deposition of complementary charged polymers. Two layersof sodium poly(styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) were assembled on the lysozyme shell, and layer deposition wasmonitored by electrophoresis and confirmed by fluorescein labeling.

Toublan et al. (2006) used the noncovalent, electrostatic LbL modifica-tion for successful targeting of protein microspheres to the integrinreceptors that are overexpressed in several tumor types, and the RGD(arginine–glycine–aspartic acid) tripeptide is one of the most often recog-nized. Three different peptides were synthesized with the motifincorporated at the ends or in the middle of a polylysine sequence. BSAmacromolecule has a net charge of �17 at pH 7. In this way, the BSAmicrospheres are negatively charged and interact electrostatically with thepositive lysine-containing peptides to form a layer over the microspheresurface. Thus, an easy method for the labeling of microspheres withpeptide ligands to important cell membrane receptors was developed.


The RGD-modified BSA microspheres showed increased binding to tumorcells than the unmodified ones, the best results obtained with BSA micro-spheres complexed with the peptide containing RGD motif at the end,that is, RGDKKKKKK exhibit better binding to the tumor cells thanKKKKRGDKKK.Avivi (Levi) et al. (2001) found an easy way to fabricate magnetic

microspheres with BSA shell by ultrasound. Magnetic microspheres are asubject of strong research interest as they could be easily brought in vivo tothe target site by an externally applied magnetic field. When they arepreferentially accumulated in cancer cells, they could be used for selectivedeath of the latter by hyperthermia. Two precursors, iron acetate and ironpentacarbonyl, were used for the preparation of the iron oxide nanopar-ticles with BSA shell. The thickness of the protein shell was estimated to be25–40 nm which is in the range of the Hb microsphere thickness estimatedby Wong and Suslick (1995). Different mean size of the BSA-coatedmagnetic microspheres was obtained depending on the iron oxide pre-cursor—from iron pentacarbonyl, the average diameter was approximately4200 nm, while from iron acetate, smaller spheres of about 1900 nm wereobserved.A different approach was used by Han et al. (2008b) to synthesize

magnetic protein containers. HSA microcapsules with a silicon oil corewere prepared sonochemically. The microcapsules with a size less than3 mm were functionalized to achieve the proper surface charge by poly-electrolyte PSS/PAH multilayer coating. Then, magnetic nanoparticleswere deposited on the surface and an outer layer of PAH was added toprevent the flocculation of the magnetic containers. A hydrophobic dye(5,10,15,20-tetraphenylporphin) was loaded in the capsules by dissolutionin the oil phase before sonication. The loaded microcontainers were stableand sustained the surface modification without any loss of dye. Themagnetic particles moved under the action of an external magnet in adesired direction and subsequently, they were redispersed in the solutionby a gentle agitation after removal of the magnet. The successful magneticmodification of the containers demonstrated their potential for magnetic-driven drug targeting.BSA was used for encapsulation of an antitumor drug, Taxol (paclitax-

el), and the BSA–Taxol composite was examined for its anticancer activity(Grinberg et al., 2007). Taxol is mostly being used for the treatment ofovarian cancer, breast cancer, nonsmall cell lung carcinomas, and Kaposi’s


sarcoma. The agent is known for its short half-life and extensive systemictoxicity. The authors have made an attempt to avoid cell exposure to thedrug by its encapsulation in a protein that is friendly to the cells. Interest-ingly, Taxol-loaded BSA microspheres were obtained only at a low drugconcentration—5–100 ml of Taxol injection (6 mg/ml) added to the or-ganic solvent used to fill the BSA microspheres. No proteinaceous micro-spheres were produced for higher Taxol concentrations (e.g., uponaddition of 100–300 ml).

The encapsulating efficiency of the BSA microspheres increased as theconcentration of Taxol in the initial solution increased and maximumloading capacity of 90% for Taxol was achieved. The size distribution ofthe Taxol-filled BSA microspheres was quite wide, ranging between 300and 2500 nm. Their average diameter was smaller compared to the filledwith various nonaqueous liquids BSA microspheres without drug obtainedby Suslick and Grinstaff (1990) which mean size was 2500 nm.

Taxol was released from the protein microspheres due to the presenceof proteases in the medium. It appeared that the number of dead cellsafter microsphere treatment was almost the same as the number of deadcells after applying the same concentration of freshly prepared Taxol.However, the organic solvent (mesytylene) used for dissolving of Taxolcaused the death of some of the cancer cells.

A similar approach was used for encapsulation in BSA microspheres ofanother anticancer drug, namely, Gemzar (Gemcitabine HCl) (Grinberget al., 2009). Gemzar is a drug with a wide spectrum of antitumor activityused for the treatment of various types of kidney cancer in humans.Because of the toxic effect of mesytylene used for the preparation ofTaxol-loaded microcapsules (Grinberg et al., 2007), a more biocompatibleorganic solvent, dodecane, was used for the encapsulation of Gemzar.Compared to the method or BSA microspheres production developedby Suslick and collaborators, these authors reduced the concentration ofBSA solution (0.005%, w/v). Under these conditions, the loading capacityof Gemzar did not exceed 30% because of the limited solubility of thedrug in the solvent.

The size of Gemzar-filled BSA microspheres ranged between 400 and2800 nm, falling more sharply between 500 and 1500 nm (Grinberg et al.,2009). The same trend of decreasing the size of drug-BSA microspherescompared to the BSA microspheres without an encapsulating drug wasobserved as already reported for the case of Taxol.


The release of Gemzar from BSA microspheres was due, according tothe authors, to the action of proteases available at the target environmentrather than to the acidic pH inside the tumor cells (early endosomalpH¼6.1–6.2 and late endosomal pH¼5.4–5.6). Concerning the antican-cer activity tests, the Gemzar encapsulated in BSA microspheres appearedto be more active than pristine Gemzar.Besides for anticancer therapy, the potential of sonochemically born

BSA microspheres as carriers of an antibiotic (tetracycline) was tested(Avivi (Levi) et al., 2003). Tetracycline (TTCL) antibiotics have a broadspectrum of activity; they are relatively safe and can be administrated bymany routes. The size distribution of BSA-encapsulated TTCL rangedbetween 400 and 2800 nm, with an average diameter of 2.5 mm. Thatmeans that BSA microspheres size was not significantly influenced bythe TTCL encapsulation in contrast to the cases of encapsulation of thetwo anticancer drugs, Gemzar and Taxol. The BSA microspheres loadingcapacity for TTCL was estimated to be 65%. Again as in the case ofGemzar, the saturation at this moderately high drug loading is due tothe limited solubility of TTCL in the organic solvent (mesitylene). Abovecertain TTCL concentration, the TTCL molecules leaving the micro-spheres via the walls are in equilibrium with the ones entering the micro-spheres, thus determining the saturation level of drug loading. Theantimicrobial activity of the TTCL-loaded BSA microspheres was testedon two bacterial strains that are sensitive to TTCL—Staphylococcus aureus(Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria).Microspheres loaded with TTCL and TTCL disk that contain the samequantity of the antibiotic and was used in clinical diagnostic showed thesame inhibition zone.The drug release behavior of protein microspheres has been recently

investigated and reported for the first time (Han et al., 2010). Rifampicin,a semisynthetic antibiotic widely used for the chemotherapy of tuberculo-sis, was chosen as a model drug. Two different drug formulations werecompared. HSA microspheres were prepared and loaded with the modeldrug, as described in a previous publication (Han et al., 2008a). A newcontainer composed of an outer protein shell and an inner gel core wasdesigned and sonochemically synthesized. 12-Hydroxystearic acid was usedas gelator. It was dissolved in the silicon oil phase before sonication andencapsulated in the oil core applying a similar sonication procedure at45 �C. When the sample is cooled down to room temperature, the gelator


transforms the oil core to a gel. The protein microspheres had an averagediameter of approximately 1 mm, while that of the new containers was0.6 mm. The reduction of the size was attributed to the increased oilviscosity after the gelator addition. Protein microspheres released thedrug slowly—more than 90 wt% of the loaded drug over a period of2500 min at 25 �C. The drug-release profile of the new containers followeda different pattern. The release saturation level was reached after 300 minand only 16 wt% of drug was released at 25 �C. This release was ascribed tothe diffusion of the drug located in the outer region of the gel core. Theremaining drug was completely released at 40 �C, when the gel core wastransferred to an oil core. Hence, the new container displayed a tempera-ture-responsive drug release behavior, and the authors stated a purpose todevelop a multifunctional delivery system on the basis of the obtainedresults (Han et al., 2010).

We have successfully used the sonochemically prepared gelatin nano-capsules for encapsulation of two drugs with low water solubility—acet-ylsalicylic acid and a-tocopherol (vitamin E) (Yankova, 2010). In bothcases, a modulated drug-release profile was observed due to the proteinnanocapsules, for example, vitamin E was not released after 2 h at pH¼1.2(stomach media) but started to diffuse out from the capsules and wascompletely released at pH¼7.4 (the intestine pH). All these investigationsconfirm that the sonochemical method allows an easy, one-step encapsu-lation of hydrophobic drugs into protein capsules and it has a greatpotential for controlled drug delivery.

Recently, for the first time, mixed microspheres from three proteinswere synthesized by using ultrasound irradiation (Angel (Shimanovich)et al., 2010). The authors used three proteins—GFP (recombinant greenfluorescent protein), CFP–GBP–YFP (cyan fluorescent protein, glucosebinding protein, and yellow fluorescent fused protein), and BSA, thefirst two being fluorescent. The aim was to synthesize fluorescent micro-spheres with reduced price by combining a small amount of the expensivefluorescent proteins and a large amount of the less expensive nonfluores-cent protein BSA. Two kinds of microspheres were synthesized sonochemi-cally: one-protein microspheres, made from BSA, (CFP–GBP–YFP) andGFP, and mixed protein microspheres made from the pairs BSA-(CFP–GBP–YFP) and BSA–GFP proteins. BSA-(CFP–GBP–YFP) microsphereswere the largest, followed by the BSA spheres (with an average size2.34 mm), and the BSA–GFP particles were the smallest. The authors


explain this order by the molecular weight of GFP and (CFP–GBP–YFP)proteins. The molecular weight of GFP is the smallest and GFP micro-spheres have an average size of 244 nm. As (CFP–GBP–YFP) has a highermolecular weight than GFP, BSA-(CFP–GBP–YFP) microspheres are big-ger (3.52 mm) than BSA–GFP spheres (1.40 mm). Both mixed micro-spheres (BSA-(CFP–GBP–YFP) and BSA–GFP MPMs) emittednonhomogeneously spread light from the microspheres walls, while one-protein GFP or CFP–GBP–YFP microspheres emitted homogeneouslyspread green or blue light, respectively, from their walls.The sonication method was used for fabrication of protein-based hybrid

nano- and microparticles. Bioceramics, such as calcium phosphates (CaP),represent another class of materials with excellent biocompatibility, bioac-tivity, and high affinity to proteins. Therefore, CaP nanoparticles havebeen studied in advance applications, that is, gene delivery and tissueengineering (Bisht et al., 2005; Duan et al., 2008). The CaP/BSA colloidalparticles were obtained in a short time from aqueous solutions of Ca(H2PO4)2 and Ca(OH)2 in the presence of BSA by high intense ultrasonicirradiation (Han et al., 2005). The netlike morphology of CaP/BSAcolloidal particles was attributed to the sonochemically induced cross-linking of BSA molecules (Suslick et al., 1994). The presence of BSAplayed a key role in the stability of the colloidal particles. The proteinconcentration slightly influenced the size distribution and the zeta poten-tial of the colloidal particles. These CaP/BSA colloidal particles weresuccessfully used as precursors in the preparation of hydroxyapatite(HAP) rod-like crystals by thermolysis (Han et al., 2007). While BSAdisintegrated and burned out with increasing sintering temperature,rod-like HAP crystals formed at about 600 �C, possessing a diameter ofabout 60–160 nm and a length of about 0.5 mm. By increasing the BSAconcentration (from 2 to 4 g/L) in the colloidal precursor, the phasecomposition of products did not change, only HAP rod-like crystalsbecame more uniform and smaller.Shiomi et al. (2005) proposed a new approach for the synthesis of

protein–silica hybrid hollow microparticles. The paper reports a novelcatalytic activity of commercially available lysozyme for polysiloxane for-mation from tetraethoxyorthosilicate (TEOS). The authors combined thesonochemical method for microsphere fabrication with the catalytic activi-ty of the enzyme and the result was hollow spherical particles with alysozyme–siloxane hybrid shell structure and a particle diameter ranging


from 500 nm to 15 mm. The shell of hollow spheres was visualized by SEMas a well-defined and flexible wall, the thickness of which was estimated tobe approximately 100 nm. When stirring was applied to a lysozyme–TEOSmixture instead of sonication, granular particles 250–1000 nm in sizewithout any hollow structure were obtained. Moreover, no spherical par-ticles were observed without lysozyme in the reacting solution, suggestingthat the biomimetic patterning of silica was catalyzed by lysozyme.

VI. Conclusions

The sonochemical method for preparation of proteinaceous micro- andnanocapsules has received great attention over the last few years as it isevidenced by the increased number of papers and research groupsinvolved in this topic. Great advantages are the one-step procedure, simpleequipment required, and high yields gained when choosing the appropri-ate combination of parameters (ultrasound characteristics, irradiationtime, components, temperature, pH, etc.). Moreover, it appears to be anefficient microencapsulation technique for producing core-shell vehiclesof bioactive agents. The successful application of microbubbles in medicaldiagnostics encourages the design of protein-based microcapsules for drugtargeting. The proteinaceous colloid systems seem to be promising candi-dates as multifunctional platforms in the emerging field of theranostics.The extension of the application area of sonochemistry as a tool forfabricating nanomaterials makes the method more universal and signifi-cantly increases its potential for practical implementation.

Acknowledgment

The support by the NSF of Bulgaria (Contract NoDO 02-198/2008) is highly acknowledged.

References

Alonso, M. J. (1996). Nanoparticulate drug carrier technology. In: MicroparticulateSystems for the Delivery of Proteins and Vaccines, Cohen, S., Bernstein, H. (Eds.),pp. 203–242. Marcel Dekker, New York, NY.

Angel (Shimanovich), U., Matas, D., Michaeli, S., Cavaco-Paulo, A., Gedanken, A.(2010). Microspheres of mixed proteins. Chem. Eur. J. 16, 2108–2114.


Arshady, R. (1990). Albumin microspheres and microcapsules: methodology ofmanufacturing techniques. J. Control. Release 14, 111–131.

Avivi (Levi), S., Gedanken, A. (2005). The preparation of avidin microspheres usingthe sonochemical method and the interaction of the microspheres with biotin.Ultrason. Sonochem. 12, 405–409.

Avivi (Levi), S., Gedanken, A. (2007). Are sonochemically prepared a-amylase proteinmicrospheres biologically active? Ultrason. Sonochem. 14, 1–5.

Avivi (Levi), S., Felner, I., Novik, I., Gedanken, A. (2001). The preparation of magneticproteinaceous microspheres using the sonochemical method. Biochim. Biophys. Acta1527, 123–129.

Avivi (Levi), S., Nitzan, Y., Dror, R., Gedanken, A. (2003). An easy sonochemical routefor the encapsulation of tetracycline in bovine serum albumin microspheres. J. Am.Chem. Soc. 125, 15712–15713.

Avivi, S., Gedanken, A. (2002). S–S bonds are not required for the sonochemicalformation of proteinaceous microspheres: the case of streptavidin. Biochem. J.366, 705–707.

Azarmia, S., Huanga, Y., Chend, H., McQuarriea, S., Abramse, D., Road, W., et al.(2006). Optimization of a two-step desolvation method for preparing gelatinnanoparticles and cell uptake studies in 143B osteosarcoma cancer cells.J. Pharm. Pharm. Sci. 9, 124–132.

Barratt, G., Couarraze, G., Couvreur, P., Dubernet, C., Fattal, E., Gref, R., et al. (2002).Polymeric micro- and nanoparticles as drug carriers. In: Polymeric Biomaterials,Dumitriu, S. (Ed.). second ed. Marcel Dekker, Inc, New York.

Barteri, M., Fioroni, M., Gaudiano, M. C. (1996). Oxidation of Fe(II) horse heartcytochrome c by ultrasound waves. Biochim. Biophys. Acta 1296, 35–40.

Bisht, S., Bhakta, G., Mitra, S., Maitra, A. (2005). pDNA loaded calcium phosphatenanoparticles: highly efficient non-viral vector for gene delivery. Int. J. Pharm. 288,157–168.

Blanco, D., Alonso, M. J. (1998). Protein encapsulation and release from poly(lactide-co-glycolide) microspheres: effect of protein and polymer properties and of the co-encapsulation of surfactants. Eur. J. Pharm. Biopharm. 45, 285–294.

Brannon-Peppas, L. (1993). Controlled Release in the Food and Cosmetics Industries,Polymeric Delivery Systems. In: ACS Symposium Series, El-Nokaly, M. A.,Piatt, D. M., Charpentier, B. A. (Eds.), Vol. 520, pp. 42–52 (Chapter 3).

Bruschi, M. L., Cardoso, M. L., Lucchesi, M. B., Gremiao, M. P. (2003). Gelatinmicroparticles containing propolis obtained by spray-drying technique: prepara-tion and characterization. Int. J. Pharm. 264, 45–55.

Burgess, D. J., Carless, J. E. (1984). Microelectrophoretic studies of gelatin and acaciafor the prediction of complex coacervation. J. Colloid Interface Sci. 98, 1–8.

Cavalieri, F., Ashokkumar, M., Grieser, F., Caruso, F. (2008). Ultrasonic synthesis ofstable, functional lysozyme microbubbles. Langmuir 24, 10078–10083.

Cecil, R., McPhee, J. (1959). The sulfur chemistry of proteins. In: Advances in ProteinChemistry, Anfinsen, C., Anson, M., Bailey, K., Edsall, J. (Eds.), Vol. 14,pp. 255–389. Academic Press, New York.


Chen, C. Q., Lin, W., Coombes, A. G., Davis, S. S., Illum, L. (1994). Preparation ofhuman serum albumin microspheres by a novel acetone-heat denaturation method.J. Microencapsul. 11, 395–407.

Chen, L., Remondetto, G. E., Subirade, M. (2006). Food protein-based materials asnutraceutical delivery systems. Trends Food Sci. Technol. 17, 272–283.

Christiansen, C., Kryvi, H., Sontum, P. C., Skotland, T. (1994). Physical and biochemi-cal characterization of Albunex, a new ultrasound contrast agent consisting of air-filled albumin microspheres suspended in a solution of human albumin. Biotechnol.Appl. Biochem. 19, 307–320.

Chuo, W. -H., Tsai, T. -R., Hsu, Sh. -H., Cham, Th. -M. (1996). Preparation and in-vitroevaluation of nifedipine loaded albumin microspheres cross-linked by differentglutaraldehyde concentrations. Int. J. Pharm. 144, 241–245.

Coester, C., von Briesen, H., Langer, K., Kreuter, J. (2000). Gelatin nanoparticles bytwo step desolvation—a new preparation method, surface modifications and celluptake. J. Microencapsul. 17, 187–194.

Cornec, M., Kim, D., Narsimhan, G. (2001). Adsorption dynamics and interfacialproperties of a-lactalbumin in native and molten globule state conformation atair–water interfaces. Food Hydrocolloids 15, 303–313.

Das, S., Banerjee, R., Bellare, J. (2005). Aspirin loaded albumin nanoparticles bycoacervation: implications in drug delivery. Trends Biomater. Artif. Organs 18,203–212.

Desai, M. P., Labhasetwar, V., Walter, E., Levy, R. J., Amidon, G. L. (1997). Themechanism of uptake of biodegradable microparticles in Caco-2 cells is size depen-dent. Pharm. Res. 14, 1568–1573.

Domnitch, E., Gelfand, D. (2004). Camera lucida: a three-dimensional sonochemicalobservatory. Leonardo 37, 391–396.

Drexler, K. E. (1981). Molecular engineering: an approach to the development ofgeneral capabilities for molecular manipulation. Proc. Natl. Acad. Sci. USA 78,5275–5278.

Duan, B., Wang, M., Zhou, W. Y., Cheung, W. L. (2008). Synthesis of Ca–P nanoparti-cles and fabrication of Ca–P/PHBV nanocomposite microspheres for bone tissueengineering applications. Appl. Surf. Sci. 255, 529–533.

Dubey, R. R., Parikh, J. R., Parikh, R. R. (2003). Effect of heating temperature and timeon pharmaceutical characteristics of albumin microspheres containing 5-fluoroura-cil. AAPS PharmSciTech 4, 1–6.

El-Agnaf, O. M. A., Irvine, G. B., Gulthrie, J. S. (1997). Conformations of beta-amyloidin solution. J. Neurochem. 68, 437–439.

Edwards-Levy, F., Andry, M. C., Levy, M. C. (1993). Determination of free amino groupcontent of serum albumin microcapsules using trinitrobezenesulfonic acid: effectof variations in polycondensation pH. Int. J. Pharm. 96, 85–90.

Edwards-Levy, F., Andry, M. C., Levy, M. C. (1994). Determination of free amino groupcontent of serum albumin microcapsules: II. Effect of variations in reaction timeand in terephthaloyl chloride concentration. Int. J. Pharm. 103, 253–257.

Fandrich, M., Fletcher, M. A., Dobson, C. M. (2001). Amyloid fibrils from musclemyoglobin. Nature 410, 165–166.


Farah, A., Alvarez-Puebla, R. A., Fenniri, H. (2008). Chemically stable silver nanoparti-cle-crosslinked polymer microspheres. J. Colloid Interface Sci. 319, 572–576.

Fink, A. L. (1998). Protein aggregation: folding aggregates, inclusion bodies andamyloid. Fold. Des. 3, R9–R23.

Finne-Wistrand, A., Albertsson, A. -C. (2006). The use of polymer design in resorbablecolloids. Annu. Rev. Mater. Res. 36, 369–395.

Freitas, S., Merkle, H. P., Gander, B. (2005). Microencapsulation by solvent extraction/evaporation reviewing the state of the art of microsphere preparation processtechnology. J. Control. Release 102, 313–332.

Gabor, F., Ertl, B., Wirth, M., Mallinger, R. (1999). Ketoprofen-poly(D, Llactic-co-glycolic acid) microspheres: influence of manufacturing parameters and type ofpolymer on the release characteristics. J. Microencapsul. 16, 1–12.

Gallo, J. M., Hung, C. T., Perrier, D. G. (1984). Analysis of albumin micro-spherepreparation. Int. J. Pharm. 22, 63–74.

Gedanken, A. (2008). Preparation and properties of proteinaceous microspheres madesonochemically. Chem. Eur. J. 14, 3840–3853.

Giacomelli, C., Norde, W. (2001). The adsorption–desorption cycle. Reversibility of theBSA-silica system. J. Colloid Interface Sci. 233, 234–240.

Goldstein, I. J., Hughes, R. C., Monsigny, M., Osawa, T., Sharon, N. (1980). Whatshould be called lectin? Nature 285, 66–69.

Gomez, A., Bingham, D., Tang, K. (1998). Production of protein nanoparticles byelectrospray drying. J. Aerosol Sci. 29, 561–574.

Goppert, T. M., Muller, R. H. (2005). Adsorption kinetics of plasma proteins on solidlipid nanoparticles for drug targeting. Int. J. Pharm. 302, 172–186.

Gouin, S. (2000). Microencapsulation: industrial appraisal of existing technologies andtrends. Trends Food Sci. Technol. 15, 330–347.

Grinberg, O., Hayun, M., Sredni, B., Gedanken, A. (2007). Characterization and activityof sonochemically-prepared BSA microspheres containing Taxol—an anticancerdrug. Ultrason. Sonochem. 14, 661–666.

Grinberg, O., Gedanken, A., Patra, C. R., Patra, S., Mukherjee, P., Mukhopadhyay, D.(2009). Sonochemically prepared BSA microspheres containing Gemcitabine, andtheir potential application in renal cancer therapeutics. Acta Biomater. 5,3031–3037.

Grinstaff, M. W., Suslick, K. S. (1991). Air-filled proteinaceous microbubbles: synthesisof an echo-contrast agent. Proc. Natl. Acad. Sci. USA 88, 7708–7710.

Gulseren, I., Guzey, D., Bruce, B. D., Weiss, J. (2007). Structural and functionalchanges in ultrasonicated bovine serum albumin solutions. Ultrason. Sonochem.14, 173–183.

Gupta, P. K., Hung, C. T., Perrier, D. G. (1986). Albumin microspheres, II: effect ofstabilization temperature on the release of adriamycin. Int. J. Pharm. 33, 147–153.

Han, Y., Li, S., Wang, X., Cao, X., Jia, L., Li, J. (2005). Preparation and characterizationof calcium phosphate–albumin colloidal particles by high ultrasonic irradiation.Colloid Polym. Sci. 284, 203–207.

Han, Y., Li, S., Wang, X., Jia, L., He, J. (2007). Preparation of hydroxyapatite rod-likecrystals by protein precursor method. Mater. Res. Bull. 42, 1169–1177.


Han, Y., Radziuk, D., Shchukin, D., Moehwald, H. (2008a). Stability and size depen-dence of protein microspheres prepared by ultrasonication. J. Mater. Chem. 18,5162–5166.

Han, Y., Radziuk, D., Shchukin, D., Moehwald, H. (2008b). Sonochemical synthesis ofmagnetic protein container for targeted delivery. Macromol. Rapid Commun. 29,1203–1207.

Han, Y., Shchukin, D., Mohwald, H. (2010). Drug release of sonochemical proteincontainers. Chem. Lett. 39, 502–503.

Ibrahim, H., Higashiguchi, S., Koketsu, M., Juneja, L., Kim, M., Yamamoto, T., et al.(1996). Partially unfolded lysozyme at neutral pH agglutinates and kills gram-negative and gram-positive bacteria through membrane damage mechanisms.J. Agric. Food Chem. 44, 3799–3806.

Ishizaka, T., Endo, K., Koishi, M. (1981). Preparation of egg albumin microcapsulesand microspheres. J. Pharm. Sci. 70, 358–363.

Jalalipour, M., Gilani, K., Tajerzadeh, H., Najafabadi, A. R., Barghi, M. (2008). Charac-terization and aerodynamic evaluation of spray dried recombinant human growthhormone using protein stabilizing agents. Int. J. Pharm. 352, 209–216.

Jocelym, P. C. (1976). Biochemistry of SH Group. Academic Press, New York.Keller, M. W., Feinstein, S. B., Briller, R. A., Powsner, S. M. (1986). Automated

production and analysis of echo contrast agents. J. Ultrasound Med. 5, 493–498.Keller, M. W., Glasheen, W., Teja, K., Gear, A., Kaul, S. (1988). Myocardial contrast

echocardiography without hyperemic response or hemodynamic effects. J. Am. Coll.Cardiol. 12, 1039–1047.

Kelly, J. W. (1998). The alternative conformations of amyloidogenic proteins and theirmulti-step assembly pathways. Curr. Opin. Struct. Biol. 8, 101–106.

Kim, Y. D., Morr, C. V., Schenz, T. W. (1996). Microencapsulation properties of gumArabic and several food proteins: liquid orange oil emulsion particles. J. Agric. FoodChem. 44, 1308–1313.

Kreitz, M., Brannon-Peppas, L., Mathiowitz, E. (1999). Microencapsulation. Encyclope-dia of Controlled Drug Delivery. Wiley, New York, pp.493–553.

Krishnamurthy, R., Lumpkin, J. A., Sridhar, R. (2000). Inactivation of lysozyme bysonication under conditions relevant to microencapsulation. Int. J. Pharm. 205,23–34.

Lamprecht, A., Saumet, J.-L., Roux, J., Benoit, J.-P. (2004). Lipid nanocarriers as drugdelivery system for ibuprofen in pain treatment. Int. J. Pharm. 278, 407–414.

Lee, S. J., Jung, S. Y., Ahn, S. (2010). Flow tracing microparticle sensors designed forenhanced X-ray contrast. Biosens. Bioelectron. 25, 1571–1578.

Liu, X., Sun, Q., Wang, H., Zhang, L., Wang, J.-Y. (2005). Microspheres of corn protein,zein, for an ivermectin drug delivery system. Biomaterials 26, 109–115.

Liu, G., Wang, H., Yang, X., Li, L. (2009). Synthesis of tri-layer hybrid microsphereswith magnetic core and functional polymer shell. Eur. Polym. J. 45, 2023–2032.

Luftensteiner, Ch.P., Viernstein, H. (1998). Statistical experimental design based stud-ies on placebo and mitoxantrone-loaded albumin microspheres. Int. J. Pharm. 171,87–99.


Maa, Y. F., Prestrelski, S. J. (2000). Biopharmaceutical powders: particle formation andformulation considerations. Curr. Pharm. Biotechnol. 1, 283–302.

Makino, K., Mizorogi, T., Ando, S., Tsukamoto, T., Ohshima, H. (1991). Sonochemi-cally prepared bovine serum albuminmicrocapsules: factors affecting the size dis-tribution and the microencapsulation yield. Colloids Surf. B Biointerfaces 22,251–255.

Marty, J. J., Oppenheimer, R. C., Speiser, P. (1978). Nanoparticles—a new colloidaldrug delivery system. Pharm. Acta Helv. 53, 17–23.

Mason, T. J., Lorimer, J. P. (2002). Applied Sonochemistry. Wiley-VCH Verlag, Wein-heim (Chapter 2).

Matsumura, Y., Maeda, H. (1986). A new concept for macromolecular therapeutics incancer chemotherapy: mechanism of tumoritropic accumulation of proteins andthe antitumor agent. Cancer Res. 46, 6387–6392.

Mauguet, M. C., Hirech, K., Brujes, L., Carnelle, G., Legrand, J. (1999). Microcapsulesprepared from gelatin/gum arabic systems by complex coacervation. Recents ProgresGenie Procedes 13, 183–190.

Militello, V., Vetri, V., Leone, M. (2003). Conformational changes involved in thermalaggregation processes of bovine serum albumin. Biophys. Chem. 105, 133–141.

Miyazaki, S., Hashiguchi, N., Yokouchi, C., Takada, M., Hou, W. M. (1986a). Antitu-mour effect of fibrinogen microspheres containing doxorubicin on Ehrlich ascitescarcinoma. J. Pharm. Pharmacol. 38, 618–620.

Miyazaki, S., Hashiguchi, N., Hou, W. M., Yokouchi, C., Takada, M. (1986b). Antitumoreffect of fibrinogen microparticles containing adriamycin on Ehrlich ascites carci-noma in mice. Chem. Pharm. Bull. (Tokyo) 34, 2632–2636.

Nguyen, H. H., Ko, S. (2010). Preparation of size-controlled BSA nanoparticlesby intermittent addition of desolvating agent. IFMBE Proceedings LNCSE,Vol. 27, pp. 231–234.

Oh, J. K., Drumright, R., Siegwart, D. J., Matyjaszewski, K. (2008). The development ofmicrogels/nanogels for drug delivery applications. Prog. Polym. Sci. 33, 448–477.

Pean, J. M., Venier-Julienne, M. C., Boury, F., Menei, P., Denizot, B., Benoit, J. P.(1998). NGF release from poly(D, L-lactide-co-glycolide) microspheres. Effect ofsome formulation parameters on encapsulated NGF stability. J. Control. Release 56,175–187.

Picot, A., Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein-basedmicrocapsules and survival in simulated gastrointestinal conditions and in yoghurt.Int. Dairy J. 14, 505–515.

Planas, M., Fernandez-Reiriz, M. J., Ferreiro, M. J., Labarta, U. (1990). Effect of selectedvariables on the preparation of gelatin-acacia microcapsules for aquaculture.Aquac. Eng. 9, 329–341.

Puoci, F., Iemma, F., Spizzirri, U. G., Cirillo, G., Curcio, M., Picci, N. (2005). Polymer inAgriculture: a Review. Am. J. Agri. Biol. Sci. 3, 299–314.

Raichel, D. R. (2006). The Science and Applications of Acoustics second ed. SpringerScience þ Bussines Meduia Inc., New York (Chapter 16).

Rajagopal, K., Schneider, J. P. (2004). Self-assembling peptides and proteins for nano-technological applications. Curr. Opin. Struct. Biol. 14, 480–486.


Rochet, J. C., Lansbury, P. T. Jr., (2000). Amyloid fibrillogenesis: themes and variations.Curr. Opin. Struct. Biol. 10, 60–68.

Rong, Y., Chen, H.-Zh., Wei, D.-Ch., Sun, J.-Zh., Wang, M. (2004). Microcapsules withcompact membrane structure from gelatin and styrene–maleic anhydride copoly-mer by complex coacervation. Colloids Surf. A Physicochem. Eng. Asp. 242, 17–20.

Rossler, B., Kreuter, J., Scherer, D. (1995). Collagen microparticles: preparation andproperties. J. Microencapsul. 12, 49–57.

Rubino, O. P., Kowalsky, R., Swarbrick, J. (1993). Albumin microspheres as a drugdelivery system: relation among turbidity ratio, degree of cross-linking, and drugrelease. Pharm. Res. 10, 1059–1065.

Sansdrap, P., Moes, A. J. (1993). Influence of manufacturing parameters on the sizecharacteristics and the release profiles of nifedipine from poly(DL-lactide-co-glyco-lide) microspheres. Int. J. Pharm. 98, 157–164.

Saravanan, M., Rao, K. P. (2010). Pectin–gelatin and alginate–gelatin complex coacer-vation for controlled drug delivery: influence of anionic polysaccharides and drugsbeing encapsulated on physicochemical properties of microcapsules. Carbohydr.Polym. 80, 808–816.

Satheeshkumar, K. S., Jayakumar, R. (2002). Sonication induced sheet formation at theair–water interface. Chem. Commun. 19, 2244–2245.

Schafer, V., von Briesen, H., Andreesen, R., Steffan, A. M., Royer, C., Troster, S., et al.(1992). Phagocytosis of nanoparticles by human immunodeficiency virus (HIV)-infected macrophages: a possibility for antiviral drug targeting. Pharm. Res. 9,541–546.

Shiomi, T., Tsunoda, T., Kawai, A., Chiku, H., Mizukami, F., Sakaguchi, K. (2005).Synthesis of protein–silica hybrid hollow particles through the combination ofprotein catalysts and sonochemical treatment. Chem. Commun. 42, 5325–5327.

Sinha, R., Kim, G. J., Nie, S., Shin, D. M. (2006). Nanotechnology in cancer therapeu-tics: bioconjugated nanoparticles for drug delivery. Mol. Cancer Ther. 5, 1909–1917.

Sponheim, N., Hoff, L., Waaler, A., Muan, B., Morris, H., Holm, S., et al. (1993).Albunex-a new ultrasound contrast agent. Acoustic Sensing and Imagingpp. 103–108, International Conference on Acoustic Sensing and Imaging, PrintISBN: 0-85296-575-3.

Stathopulos, P. B., Scholz, G. A., Hwang, Y.-M., Rumfeldt, J. A. O., Lepock, J. R.,Meiering, E. M. (2004). Sonication of proteins causes formation of aggregatesthat resemble amyloid. Protein Sci. 13, 3017–3027.

Stoilova, O., Koseva, N., Petrova, Ts., Manolova, N., Rashkov, I., Naydenov, M. (2001).Hydrolysis of chitosan, chitosan-polyoxyethylene and chitosan-poly(2-acryloylamido-2-methylpropanesulfonic acid) by a crude enzyme complex from Trichoderma viride.J. Bioact. Compat. Polym. 16, 379–392.

Suslick, K. S., Grinstaff, M. W. (1990). Protein microencapsulation of nonaqueousliquids. J. Am. Chem. Soc. 112, 7807–7809.

Suslick, K. S., Grinstaff, M. W., Kolbeck, K. J., Wong, M. (1994). Characterization ofsonochemically prepared proteinaceous microspheres. Ultrason. Sonochem. 1,S65–S68.


Suslick, K. S., Didenko, Y., Fang, M. M., Hyeon, T., Kolbeck, K. J., McNamara, W. B., III,et al. (1999). Acoustic cavitation and its chemical consequences. Philos. Transact.Math. Phys. Eng. Sci. 357, 335–353.

Tabata, Y., Gupta, S., Langer, R. (1993). Controlled delivery systems for proteins usingpolyanhydride microspheres. Pharm. Res. 10, 487–496.

Takakura, Y., Fujita, T., Hashida, M., Sezaki, H. (1990). Disposition characteristics ofmacromolecules in tumor-bearing mice. Pharm. Res. 7, 339–346.

Tian, Zh.M., Wan, M. X., Wang, S. P., Kang, J. Q. (2004). Effects of ultrasound andadditives on the function and structure of trypsin. Ultrason. Sonochem. 11, 399–404.

Torrado, J. J., Illum, L., Davis, S. S. (1989). Particle size and size distribution ofalbumin microspheres produced by heat and chemical stabilization. Int. J. Pharm.51, 85–93.

Toublan, F. J.-J., Boppart, S., Suslick, K. S. (2006). Tumor targeting by surface-modifiedprotein microspheres. J. Am. Chem. Soc. 128, 3472–3473.

Trzebicka, B., Koseva, N., Mitova, V., Dworak, A. (2010). Organization of poly(2-ethyl-2-oxazoline)-block-poly(2-phenyl-2-oxazoline) copolymers in water solution. Polymer51, 2486–2493.

Uchegbu, I. F., Schatzlein, A. G. (2006). Polymers in Drug Delivery. Taylor & FrancisGroup, Boca Raton.

Wattendorf, U., Merkle, H. P. (2008). PEGylation as a tool for the biomedical engi-neering of surface modified microparticles. J. Pharm. Sci. 97, 4655–4669.

Webb, A. G., Wong, M., Kolbeck, K. J., Magin, R. L., Suslick, K. S. (1996). Sonochem-icaly produced fluorocarbon microspheres: a new class of magnetic resonanceimaging agent. J. Magn. Reson. Imaging 6, 676–683.

Webb, S. D., Golledge, S. L., Cleland, J. L., Carpenter, J. F., Randolph, T. W. (2002).Surface adsorption of recombinant human interferon-gamma in lyophilized andspray-lyophilized formulations. J. Pharm. Sci. 91, 1474–1487.

Weber, C., Coester, C., Kreuter, J., Langer, K. (2000). Desolvation process and surfacecharacterisation of protein nanoparticles. Int. J. Pharm. 194, 91–102.

Wong, M., Suslick, K. S. (1995). Sonochemically produced haemoglobin microbubbles.Mat. Res. Soc. Symp. Proc. 372, 89–95.

Yang, L., Cui, F., Cun, D., Tao, A., Shi, K., Lin, W. (2007). Preparation, characterizationand biodistribution of the lactone form of 10-hydroxycamptothecin (HCPT)-load-ed bovine serum albumin (BSA) nanoparticles. Int. J. Pharm. 340, 163–172.

Yankova, I. (2010). Sonochemically obtained gelatin micro- and nanocapsules and theirapplication for the controlled drug delivery of hydrophibic drugs. Diploma Thesis,Faculty of Chemistry, Sofia University.

Yankova, I., Vassileva, E. (2010). Gelatin Nanocapsules via Sonochemical Method.submitted for publication.

Zhang, W., Zhong, Q. (2010). Microemulsions as nanoreactors to produce whey pro-tein nanoparticles with enhanced heat stability by thermal pretreatment. FoodChem. 119, 1318–1325.

Zhou, M., Leong, T. S. H., Melino, S., Cavalieri, F., Kentish, S., Ashokkumar, M. (2010).Sonochemical synthesis of liquid-encapsulated lysozyme microspheres. Ultrason.Sonochem. 17, 333–337.


Zillies, J. C. (2007). Gelatin Nanoparticles for Targeted Oligonucleotide Delivery toKupffer Cells. Analytics, Formulation Development, Practical Application. http://edoc.ub.uni-muenchen.de/6616/1/Zillies_Jan.pdf, PhD Thesis, Hamburg, p. 46.

Zwiorek, K. (2006). Gelatin Nanoparticles as Delivery System for Nucleotide-BasedDrugs. http://edoc.ub.uni-muenchen.de/6356/1/zwiorek_klaus.pdf, Dissertation,Verlag Dr. Hut, Munich, p. 40.

http://edoc.ub.uni-muenchen.de/6616/1/Zillies_Jan.pdf

http://edoc.ub.uni-muenchen.de/6616/1/Zillies_Jan.pdf

http://edoc.ub.uni-muenchen.de/6356/1/zwiorek_klaus.pdf

Documents

[Advances in Protein Chemistry and Structural Biology] Volume 80 || Sonochemically born proteinaceous micro- and nanocapsules