Skip to main content

Advertisement

Log in

Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas–liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Scheme 1
Fig. 3
Scheme 2
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

DNase I:

Deoxyribonuclease I

DTT:

Dithiothreitol

EtBr:

Ethidium bromide

FITC:

Fluorescein isothiocyanate

IPTG:

Isopropyl-β-d-thiogalactoside

Lf:

Lactoferrin

MBs:

Microbubbles

OD:

Oxidation degree

pWWf :

pQE30-wwf

RP-HPLC:

Reversed-phase chromatography

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SEM:

Scanning electron microscopy

References

  • Aceto A, Dragani B, Melino S, Principato G, Saccucci F, Gualtieri G, Petruzzelli R (1998) Structural characterization of human glyoxalase II as probed by limited proteolysis. Biochem Mol Biol Int 44:761–769

    PubMed  CAS  Google Scholar 

  • Bekeredjian R, Chen S, Frenkel PA, Grayburn PA, Shohet RV (2003) Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart. Circulation 108:1022–1026

    Article  PubMed  Google Scholar 

  • Bellamy W, Wakabayashi H, Takase M, Kawase K, Shimamura S, Tomita M (1993) Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin. Med Microbiol Immunol 182:97–105

    Article  PubMed  CAS  Google Scholar 

  • Bennett RM, Bagby GC, Davis J (1981) Calcium-dependent polymerization of lactoferrin. Biochem Biophys Res Commun 101:88–95

    Article  PubMed  CAS  Google Scholar 

  • Bezault J, Bhimani R, Wiprovnick J, Furmanski P (1994) Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice. Cancer Res 54:2310–2312

    PubMed  CAS  Google Scholar 

  • Bhimani RS, Vendrov Y, Furmanski P (1999) Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice. J Appl Microbiol 86:135–144

    Article  PubMed  CAS  Google Scholar 

  • Bielinska AU, Kukowska-Latallo JF, Baker JR Jr (1997) The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA. Biochim Biophys Acta 1353:180–190

    Article  PubMed  CAS  Google Scholar 

  • Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301

    Article  PubMed  CAS  Google Scholar 

  • Browning RJ, Mulvana H, Tang M, Hajnal JV, Wells DJ, Eckersley RJ (2011) Influence of needle gauge on in vivo ultrasound and microbubble-mediated gene transfection. Ultrasound Med Biol 37:1531–1537

    Article  PubMed  Google Scholar 

  • Caliceti P, Veronese FM (2003) Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261–1277

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Cavalieri F, Zhou M, Ashokkumar M (2010) The design of multifunctional microbubbles for ultrasound image-guided cancer therapy. Curr Top Med Chem 10:1198–1210

    Article  PubMed  CAS  Google Scholar 

  • Dang SP, Wang RX, Qin MD, Zhang Y, Gu YZ, Wang MY, Yang QL, Li XR, Zhang XG (2011) A novel transfection method for eukaryotic cells using polyethylenimine coated albumin microbubbles. Plasmid 66:19–25

    Google Scholar 

  • de Jong N, Frinking PJ, Bouakaz A, Goorden M, Schourmans T, Jingping X, Mastik F (2000) Optical imaging of contrast agent microbubbles in an ultrasound field with a 100-MHz camera. Ultrasound Med Biol 26:487–492

    Article  PubMed  Google Scholar 

  • Deelman LE, Decleves AE, Rychak JJ, Sharma K (2010) Targeted renal therapies through microbubbles and ultrasound. Adv Drug Deliv Rev 62:1369–1377

    Article  PubMed  CAS  Google Scholar 

  • Deng CX, Sieling F, Pan H, Cui J (2004) Ultrasound-induced cell membrane porosity. Ultrasound Med Biol 30:519–526

    Article  PubMed  Google Scholar 

  • Dial EJ, Romero JJ, Headon DR, Lichtenberger LM (2000) Recombinant human lactoferrin is effective in the treatment of Helicobacter felis-infected mice. J Pharm Pharmacol 52:1541–1546

    Article  PubMed  CAS  Google Scholar 

  • Dobson CM (2003) Protein folding and disease: a view from the first horizon symposium. Nat Rev Drug Discov 2:154–160

    Article  PubMed  CAS  Google Scholar 

  • During K, Porsch P, Mahn A, Brinkmann O, Gieffers W (1999) The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 449:93–100

    Article  PubMed  CAS  Google Scholar 

  • Elrod KC, Moore WR, Abraham WM, Tanaka RD (1997) Lactoferrin, a potent tryptase inhibitor, abolishes late-phase airway responses in allergic sheep. Am J Respir Crit Care Med 156:375–381

    PubMed  CAS  Google Scholar 

  • Ferrara KW (2008) Driving delivery vehicles with ultrasound. Adv Drug Deliv Rev 60:1097–1102

    Article  PubMed  CAS  Google Scholar 

  • Fink AL (1998) Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 3:R9–R23

    Article  PubMed  CAS  Google Scholar 

  • Fontana A, Fassina G, Vita C, Dalzoppo D, Zamai M, Zambonin M (1986) Correlation between sites of limited proteolysis and segmental mobility in thermolysin. Biochemistry 25:1847–1851

    Article  PubMed  CAS  Google Scholar 

  • Fontana A, Polverino de Laureto P, de Filippis V, Scaramella E, Zambonin M (1997) Probing the partly folded states of proteins by limited proteolysis. Fold Des 2:R17–R26

    Article  PubMed  CAS  Google Scholar 

  • Grinstaff MW, Suslick KS (1991) Air-filled proteinaceous microbubbles: synthesis of an echo-contrast agent. Proc Natl Acad Sci USA 88:7708–7710

    Article  PubMed  CAS  Google Scholar 

  • Hashiya N, Aoki M, Tachibana K, Taniyama Y, Yamasaki K, Hiraoka K, Makino H, Yasufumi K, Ogihara T, Morishita R (2004) Local delivery of E2F decoy oligodeoxynucleotides using ultrasound with microbubble agent (Optison) inhibits intimal hyperplasia after balloon injury in rat carotid artery model. Biochem Biophys Res Commun 317:508–514

    Article  PubMed  CAS  Google Scholar 

  • Hauff P, Seemann S, Reszka R, Schultze-Mosgau M, Reinhardt M, Buzasi T, Plath T, Rosewicz S, Schirner M (2005) Evaluation of gas-filled microparticles and sonoporation as gene delivery system: feasibility study in rodent tumor models. Radiology 236:572–578

    Article  PubMed  Google Scholar 

  • Hernot S, Klibanov AL (2008) Microbubbles in ultrasound-triggered drug and gene delivery. Adv Drug Deliv Rev 60:1153–1166

    Article  PubMed  CAS  Google Scholar 

  • Ikeda M, Nozaki A, Sugiyama K, Tanaka T, Naganuma A, Tanaka K, Sekihara H, Shimotohno K, Saito M, Kato N (2000) Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells. Virus Res 66:51–63

    Article  PubMed  CAS  Google Scholar 

  • Inoue M, Okamura T, Yasui M, Sakata N, Yagi K, Kawa K (2001) Lactoferrin for gut GVHD. Bone Marrow Transplant 28:1091–1092

    Article  PubMed  CAS  Google Scholar 

  • Kanyshkova TG, Buneva VN, Nevinsky GA (2001) Lactoferrin and its biological functions. Biochemistry (Mosc) 66:1–7

    Article  CAS  Google Scholar 

  • Kaufmann BA, Lindner JR (2007) Molecular imaging with targeted contrast ultrasound. Curr Opin Biotechnol 18:11–16

    Article  PubMed  CAS  Google Scholar 

  • Krebs MR, Wilkins DK, Chung EW, Pitkeathly MC, Chamberlain AK, Zurdo J, Robinson CV, Dobson CM (2000) Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain. J Mol Biol 300:541–549

    Article  PubMed  CAS  Google Scholar 

  • Kuhara T, Iigo M, Itoh T, Ushida Y, Sekine K, Terada N, Okamura H, Tsuda H (2000) Orally administered lactoferrin exerts an antimetastatic effect and enhances production of IL-18 in the intestinal epithelium. Nutr Cancer 38:192–199

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lentacker I, De Geest BG, Vandenbroucke RE, Peeters L, Demeester J, De Smedt SC, Sanders NN (2006) Ultrasound-responsive polymer-coated microbubbles that bind and protect DNA. Langmuir 22:7273–7278

    Article  PubMed  CAS  Google Scholar 

  • Li YS, Davidson E, Reid CN, McHale AP (2009) Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: potential applications for gene therapy of cancer. Cancer Lett 273:62–69

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Miyoshi H, Nakamura M (2006) Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery. J Control Release 114:89–99

    Article  PubMed  CAS  Google Scholar 

  • Mantel C, Miyazawa K, Broxmeyer HE (1994) Physical characteristics and polymerization during iron saturation of lactoferrin, a myelopoietic regulatory molecule with suppressor activity. Adv Exp Med Biol 357:121–132

    Article  PubMed  CAS  Google Scholar 

  • Martwiset S, Koh AE, Chen W (2006) Nonfouling characteristics of dextran-containing surfaces. Langmuir 22:8192–8196

    Article  PubMed  CAS  Google Scholar 

  • Mayer S, Grayburn PA (2001) Myocardial contrast agents: recent advances and future directions. Prog Cardiovasc Dis 44:33–44

    Article  PubMed  CAS  Google Scholar 

  • Mehier-Humbert S, Guy RH (2005) Physical methods for gene transfer: improving the kinetics of gene delivery into cells. Adv Drug Deliv Rev 57:733–753

    Article  PubMed  CAS  Google Scholar 

  • Meijering BD, Juffermans LJ, van Wamel A, Henning RH, Zuhorn IS, Emmer M, Versteilen AM, Paulus WJ, van Gilst WH, Kooiman K, de Jong N, Musters RJ, Deelman LE, Kamp O (2009) Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res 104:679–687

    Article  PubMed  CAS  Google Scholar 

  • Mela I, Aumaitre E, Williamson AM, Yakubov GE (2010) Charge reversal by salt-induced aggregation in aqueous lactoferrin solutions. Colloids Surf B Biointerfaces 78:53–60

    Article  PubMed  CAS  Google Scholar 

  • Melino S, Cicero DO, Forlani F, Pagani S, Paci M (2004) The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain. FEBS Lett 577:403–408

    Article  PubMed  CAS  Google Scholar 

  • Midoux P, Breuzard G, Gomez JP, Pichon C (2008) Polymer-based gene delivery: a current review on the uptake and intracellular trafficking of polyplexes. Curr Gene Ther 8:335–352

    Article  PubMed  CAS  Google Scholar 

  • Mine Y, Ma F, Lauriau S (2004) Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. J Agric Food Chem 52:1088–1094

    Article  PubMed  CAS  Google Scholar 

  • Mitra S, Gaur U, Ghosh PC, Maitra AN (2001) Tumour targeted delivery of encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles as carrier. J Control Release 74:317–323

    Article  PubMed  CAS  Google Scholar 

  • Moseley ME, Wendland MF, Rampil I, Barnhart J (1991) Microbubbles: a novel MR susceptibility con- trast agent In: Proceedings of the 10th annual meeting of the ISMRM. San Francisco, California USA, p 1020

  • Muller FM, Lyman CA, Walsh TJ (1999) Antimicrobial peptides as potential new antifungals. Mycoses 42(Suppl 2):77–82

    PubMed  CAS  Google Scholar 

  • Nair A, Thevenot P, Dey J, Shen J, Sun MW, Yang J, Tang L (2010) Novel polymeric scaffolds using protein microbubbles as porogen and growth factor carriers. Tissue Eng Part C Methods 16:23–32

    Article  PubMed  CAS  Google Scholar 

  • Ng KY, Liu Y (2002) Therapeutic ultrasound: its application in drug delivery. Med Res Rev 22:204–223

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa M, Huang L (2001) Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 12:861–870

    Article  PubMed  CAS  Google Scholar 

  • Porter TR, Xie F, Kilzer K (1995a) Intravenous perfluoropropane-exposed sonicated dextrose albumin produces myocardial ultrasound contrast that correlates with coronary blood flow. J Am Soc Echocardiogr 8:710–718

    Article  PubMed  CAS  Google Scholar 

  • Porter TR, Xie F, Kilzer K (1995b) Intravenous perfluoropropane-exposed sonicated dextrose albumin produces myocardial ultrasound contrast that correlates with coronary blood flow. J Am Soc Echocardiogr 8:710–718

    Article  PubMed  CAS  Google Scholar 

  • Porter TR, Hiser WL, Kricsfeld D, Deligonul U, Xie F, Iversen P, Radio S (2001) Inhibition of carotid artery neointimal formation with intravenous microbubbles. Ultrasound Med Biol 27:259–265

    Article  PubMed  CAS  Google Scholar 

  • Sacchettini JC, Kelly JW (2002) Therapeutic strategies for human amyloid diseases. Nat Rev Drug Discov 1:267–275

    Article  PubMed  CAS  Google Scholar 

  • Selkoe DJ (2003) Folding proteins in fatal ways. Nature 426:900–904

    Article  PubMed  CAS  Google Scholar 

  • Semba RD, Miotti PG, Lan Y, Chiphangwi JD, Hoover DR, Dallabetta GA, Yang LP, Saah AJ (1998) Maternal serum lactoferrin and vertical transmission of HIV. Aids 12:331–332

    PubMed  CAS  Google Scholar 

  • Shimamura M, Sato N, Taniyama Y, Yamamoto S, Endoh M, Kurinami H, Aoki M, Ogihara T, Kaneda Y, Morishita R (2004) Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound. Gene Ther 11:1532–1539

    Article  PubMed  CAS  Google Scholar 

  • Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 81:678–699

    Article  PubMed  CAS  Google Scholar 

  • Sunde M, Blake C (1997) The structure of amyloid fibrils by electron microscopy and X-ray diffraction. Adv Protein Chem 50:123–159

    Article  PubMed  CAS  Google Scholar 

  • Superti F, Ammendolia MG, Valenti P, Seganti L (1997) Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29. Med Microbiol Immunol 186:83–91

    Article  PubMed  CAS  Google Scholar 

  • Swart PJ, Kuipers EM, Smit C, Van Der Strate BW, Harmsen MC, Meijer DK (1998) Lactoferrin. Antiviral activity of lactoferrin. Adv Exp Med Biol 443:205–213

    PubMed  CAS  Google Scholar 

  • Tanaka T, Omata Y, Isamida T, Saito A, Shimazaki K, Yamauchi K, Suzuki N (1998) Growth inhibitory effect of bovine lactoferrin to Toxoplasma gondii tachyzoites in murine macrophages: tyrosine phosphorylation in murine macrophages induced by bovine lactoferrin. J Vet Med Sci 60:369–371

    Article  PubMed  CAS  Google Scholar 

  • Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R (2002) Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:372–380

    Article  PubMed  CAS  Google Scholar 

  • Tsunoda S, Mazda O, Oda Y, Iida Y, Akabame S, Kishida T, Shin-Ya M, Asada H, Gojo S, Imanishi J, Matsubara H, Yoshikawa T (2005) Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart. Biochem Biophys Res Commun 336:118–127

    Article  PubMed  CAS  Google Scholar 

  • Unger EC, Hersh E, Vannan M, Matsunaga TO, McCreery T (2001a) Local drug and gene delivery through microbubbles. Prog Cardiovasc Dis 44:45–54

    Article  PubMed  CAS  Google Scholar 

  • Unger EC, Hersh E, Vannan M, McCreery T (2001b) Gene delivery using ultrasound contrast agents. Echocardiography 18:355–361

    Article  PubMed  CAS  Google Scholar 

  • Unger EC, Matsunaga TO, McCreery T, Schumann P, Sweitzer R, Quigley R (2002) Therapeutic applications of microbubbles. Eur J Radiol 42:160–168

    Article  PubMed  Google Scholar 

  • Unger EC, Porter T, Culp W, Labell R, Matsunaga T, Zutshi R (2004) Therapeutic applications of lipid-coated microbubbles. Adv Drug Deliv Rev 56:1291–1314

    Article  PubMed  CAS  Google Scholar 

  • Villanueva FS, Jankowski RJ, Manaugh C, Wagner WR (1997) Albumin microbubble adherence to human coronary endothelium: implications for assessment of endothelial function using myocardial contrast echocardiography. J Am Coll Cardiol 30:689–693

    Article  PubMed  CAS  Google Scholar 

  • Wakabayashi H, Takakura N, Yamauchi K, Teraguchi S, Uchida K, Yamaguchi H, Tamura Y (2002) Effect of lactoferrin feeding on the host antifungal response in guinea-pigs infected or immunised with Trichophyton mentagrophytes. J Med Microbiol 51:844–850

    PubMed  CAS  Google Scholar 

  • Wong KK, Huang I, Kim YR, Tang H, Yang ES, Kwong KK, Wu EX (2004) In vivo study of microbubbles as an MR susceptibility contrast agent. Magn Reson Med 52:445–452

    Article  PubMed  CAS  Google Scholar 

  • Wu J, Pepe J, Rincon M (2006) Sonoporation, anti-cancer drug and antibody delivery using ultrasound. Ultrasonics 44(Suppl 1):21–25

    Article  CAS  Google Scholar 

  • Yuan QY, Huang J, Chu BC, Li XS, Si LY (2011) A visible, targeted high-efficiency gene delivery and transfection strategy. BMC Biotechnol 11:56

    Article  PubMed  CAS  Google Scholar 

  • Zhou M, Cavalieri F, Ashokkumar M (2011) Instrumentation science and technology (in press)

  • Zhou M, Cavalieri F, Ashokkumar M (2011b) Tailoring the properties of ultrasonically synthesised microbubbles. Soft Matter 7:623–630

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work has been supported by FP7-PEOPLE-IRSES-2008 grant.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Sonia Melino or Francesca Cavalieri.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supporting information (PDF 152 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Melino, S., Zhou, M., Tortora, M. et al. Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery. Amino Acids 43, 885–896 (2012). https://doi.org/10.1007/s00726-011-1148-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-011-1148-z

Keywords

Navigation