Skip to main content

Advertisement

SpringerLink
Log in

Loading PEG-Catalase into Filamentous and Spherical Polymer Nanocarriers

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Based on a unique phase alignment that occurs during formulation, we postulated that PEG-ylation of the cargo enzyme would enhance its encapsulation within diblock copolymer nanocarriers and thus resistance to proteases.

Methods

A freeze–thaw modified double emulsion technique was utilized to encapsulate either the catalytically active enzyme catalase (MW ∼250 kDa) or PEG-catalase in PEG–PLA polymer nanocarriers (PNC). Spectrophotometer measurement of substrate depletion was utilized to monitor enzyme activity. Isotope labeling of the enzyme was used in conjunction with activity measurements to determine PNC loading efficiency and PNC-enzyme resistance to proteases. This labeling also enabled blood clearance measurements of PNC-loaded and non-loaded enzymes in mice.

Results

Non-loaded PEG-catalase exhibited longer circulation times than catalase, but was equally susceptible to proteolysis. Modulation of the ratio of relatively hydrophilic to hydrophobic domains in the diblock PEG–PLA copolymer provided either filamentous or spherical PNC loaded with PEG-catalase. For both PNC geometries, encapsulation and resistance to proteases of the resultant PNC-loaded enzyme were more effective for PEG-catalase than catalase. Isotope tracing showed similar blood levels of PNC-loaded and free PEG-catalase in mice.

Conclusions

PEGylation enhances active catalase loading within PNC and resistance to protease degradation, relative to unloaded PEG-catalase.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

DCM:

dichloromethane

MW:

molecular weight

PEG-cat:

Peg-Catalase

PEG:

poly(ethylene glycol)

PLA:

poly(lactic acid)

PLGA:

poly(lactic-co-glycolic acid)

PNC:

polymer nanocarrier

PVA:

poly(vinyl alcohol)

THF:

tetrahydrofuran

References

  1. S. M. Moghimi, A. C. Hunter, and J. C. Murray. Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol. Rev. 53:283–318 (2001).

    PubMed  CAS  Google Scholar 

  2. S. M. Moghimi, and J. Szebeni. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog. Lipid Res. 42:463–478 (2003) doi:10.1016/S0163-7827(03)00033-X.

    Article  PubMed  CAS  Google Scholar 

  3. E. Roux, M. Lafleur, E. Lataste, P. Moreau, and J. C. Leroux. On the characterization of pH-sensitive liposome/polymer complexes. Biomacromolecules. 4:240–248 (2003) doi:10.1021/bm025651x.

    Article  PubMed  CAS  Google Scholar 

  4. V. R. Muzykantov. Targeting of superoxide dismutase and catalase to vascular endothelium. J. Control. Release. 71:1–21 (2001) doi:10.1016/S0168-3659(01)00215-2.

    Article  PubMed  CAS  Google Scholar 

  5. M. Christofidou-Solomidou, A. Scherpereel, R. Wiewrodt, K. Ng, T. Sweitzer, E. Arguiri, V. Shuvaev, C. C. Solomides, S. M. Albelda, and V. R. Muzykantov. PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress. Am. J. Physiol. Lung Cell. Mol. Physiol. 285:L283–L292 (2003).

    PubMed  CAS  Google Scholar 

  6. B. D. Kozower, M. Christofidou-Solomidou, T. D. Sweitzer, S. Muro, D. G. Buerk, C. C. Solomides, S. M. Albelda, G. A. Patterson, and V. R. Muzykantov. Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury. Nat. Biotechnol. 21:392–398 (2003) doi:10.1038/nbt806.

    Article  PubMed  CAS  Google Scholar 

  7. K. Nowak, S. Weih, R. Metzger, R. F. Albrecht 2nd, S. Post, P. Hohenberger, M. M. Gebhard, and S. M. Danilov. Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 293:L162–L169 (2007) doi:10.1152/ajplung.00001.2007.

    Article  PubMed  CAS  Google Scholar 

  8. S. Muro, X. Cui, C. Gajewski, J. C. Murciano, V. R. Muzykantov, and M. Koval. Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress. Am. J. Physiol. Cell. Physiol. 285:C1339–C1147 (2003).

    PubMed  CAS  Google Scholar 

  9. A. M. Klibanov. Enzyme stabilization by immobilization. Anal. Biochem. 93:1–25 (1979) doi:10.1016/S0003-2697(79)80110-4.

    Article  PubMed  CAS  Google Scholar 

  10. V. P. Torchilin, E. G. Tischenko, V. N. Smirnov, and E. I. Chazov. Immobilization of enzymes on slowly soluble carriers. J. Biomed. Mater Res. 11:223–235 (1977) doi:10.1002/jbm.820110208.

    Article  PubMed  CAS  Google Scholar 

  11. T. D. Dziubla, A. Karim, and V. R. Muzykantov. Polymer nanocarriers protecting active enzyme cargo against proteolysis. J. Control. Release. 102:427–439 (2005) doi:10.1016/j.jconrel.2004.10.017.

    Article  PubMed  CAS  Google Scholar 

  12. R. Langer. Drug delivery and targeting. Nature. 392:5–10 (1998).

    PubMed  CAS  Google Scholar 

  13. T. D. Dziubla, V. V. Shuvaev, N. K. Hong, B. J. Hawkins, M. Madesh, H. Takano, E. A. Simone, M. T. Nakada, A. Fisher, S. M. Albelda, and V. R. Muzykantov. Endothelial targeting of semi-permeable polymer nanocarriers for enzyme therapies. Biomaterials. 29:215–227 (2008)doi:10.1016/j.biomaterials.2007.09.023.

    Article  PubMed  CAS  Google Scholar 

  14. L. C. Seaver, and J. A. Imlay. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J. Bacteriol. 183:7182–7189 (2001) doi:10.1128/JB.183.24.7182-7189.2001.

    Article  PubMed  CAS  Google Scholar 

  15. E. A. Simone, T. D. Dziubla, F. Colon-Gonzalez, D. E. Discher, and V. R. Muzykantov. Effect of polymer amphiphilicity on loading of a therapeutic enzyme into protective filamentous and spherical polymer nanocarriers. Biomacromolecules. 8:3914–3921 (2007) doi:10.1021/bm700888h.

    Article  PubMed  CAS  Google Scholar 

  16. A. Hillgren, and M. Alden. Differential scanning calorimetry investigation of formation of poly(ethylene glycol) hydrate with controlled freeze-thawing of aqueous protein solution. J. Appl. Polym. Sci. 91:1626–1634 (2004) doi:10.1002/app.13249.

    Article  CAS  Google Scholar 

  17. C. Branca, S. Magazu, G. Maisano, F. Migliardo, P. Migliardo, and G. Romeo. Hydration study of PEG/water mixtures by quasi elastic light scattering, acoustic and rheological measurements. J. Phys. Chem. B. 106:10272–10276 (2002) doi:10.1021/jp014345v.

    Article  CAS  Google Scholar 

  18. A. M. Bellocq. Phase equilibria of polymer-containing microemulsions. Langmuir. 14:3730–3739 (1998) doi:10.1021/la970821q.

    Article  CAS  Google Scholar 

  19. C. L. Bell, and N. A. Peppas. Water, solute and protein diffusion in physiologically responsive hydrogels of poly (methacrylic acid-g-ethylene glycol). Biomaterials. 17:1203–1218 (1996) doi:10.1016/0142-9612(96)84941-6.

    Article  PubMed  CAS  Google Scholar 

  20. V. V. Shuvaev, T. Dziubla, R. Wiewrodt, and V. R. Muzykantov. Streptavidin-biotin cross linking of therapeutic enzymes with carrier antibodies: nanoconjugates for protection against endothelial oxidative stress. Methods Mol. Biol. 283:3–19 (2004).

    PubMed  CAS  Google Scholar 

  21. R. F. Beers Jr., and I. W. Sizer. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:133–140 (1952).

    PubMed  CAS  Google Scholar 

  22. S. B. Barker, and W. H. Summerson. The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138:535–554 (1941).

    CAS  Google Scholar 

  23. P. Dalhaimer, F. S. Bates, and D. E. Discher. Single molecule visualization of stable, stiffness-tunable, flow-conforming worm micelles. Macromolecules. 36:6873–6877 (2003) doi:10.1021/ma034120d.

    Article  CAS  Google Scholar 

  24. J. S. Beckman, R. L. Minor Jr., C. W. White, J. E. Repine, G. M. Rosen, and B. A. Freeman. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance. J. Biol. Chem. 263:6884–6892 (1988).

    PubMed  CAS  Google Scholar 

  25. A. Abuchowski, J. R. McCoy, N. C. Palczuk, T. van Es, and F. F. Davis. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252:3582–3586 (1977).

    PubMed  CAS  Google Scholar 

  26. P. S. Pyatak, A. Abuchowski, and F. F. Davis. Preparation of a polyethylene glycol: superoxide dismutase adduct, and an examination of its blood circulation life and anti-inflammatory activity. Res. Commun. Chem. Pathol. Pharmacol. 29:113–127 (1980).

    PubMed  CAS  Google Scholar 

  27. J. S. Beckman, R. L. Minor Jr., and B. A. Freeman. Augmentation of antioxidant enzymes in vascular endothelium. J. Free Radic. Biol. Med. 2:359–365 (1986) doi:10.1016/S0748-5514(86)80036-8.

    Article  PubMed  CAS  Google Scholar 

  28. T. Minko, P. V. Paranjpe, B. Qiu, A. Lalloo, R. Won, S. Stein, and P. J. Sinko. Enhancing the anticancer efficacy of camptothecin using biotinylated poly(ethylene glycol) conjugates in sensitive and multidrug-resistant human ovarian carcinoma cells. Cancer Chemother. Pharmacol. 50:143–150 (2002) doi:10.1007/s00280-002-0463-1.

    Article  PubMed  CAS  Google Scholar 

  29. S. V. Vinogradov, T. K. Bronich, and A. V. Kabanov. Self-assembly of polyamine-poly(ethylene glycol) copolymers with phosphorothioate oligonucleotides. Bioconjug. Chem. 9:805–812 (1998) doi:10.1021/bc980048q.

    Article  PubMed  CAS  Google Scholar 

  30. I. J. Castellanos, R. Crespo, and K. Griebenow. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J. Control. Release. 88:135–145 (2003) doi:10.1016/S0168-3659(02)00488-1.

    Article  PubMed  CAS  Google Scholar 

  31. M. Diwan, and T. G. Park. Pegylation enhances protein stability during encapsulation in PLGA microspheres. J. Control. Release. 73:233–244 (2001) doi:10.1016/S0168-3659(01)00292-9.

    Article  PubMed  CAS  Google Scholar 

  32. M. Diwan, and T. G. Park. Stabilization of recombinant interferon-alpha by pegylation for encapsulation in PLGA microspheres. Int. J. Pharm. 252:111–122 (2003) doi:10.1016/S0378-5173(02)00636-1.

    Article  PubMed  CAS  Google Scholar 

  33. W. Wasfi Al-Azzam, E. A. Pastranna, B. King, J. Méndez, and K. Griebenow. Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres. J. Pharm. Sci. 94:1808–1819 (2005) doi:10.1002/jps.20407.

    Article  PubMed  Google Scholar 

  34. T. H. Kim, H. Lee, and T. G. Park. Pegylated recombinant human epidermal growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres. Biomaterials. 23:2311–2317 (2002) doi:10.1016/S0142-9612(01)00365-9.

    Article  PubMed  CAS  Google Scholar 

  35. A. Abuchowski, T. van Es, N. C. Palczuk, and F. F. Davis. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J. Biol. Chem. 252:3578–3581 (1977).

    PubMed  CAS  Google Scholar 

  36. D. J. McClements. Protein-stabilized emulsions. Curr. Opin. Colloid Interface Sci. 9:305–313 (2004) doi:10.1016/j.cocis.2004.09.003.

    Article  CAS  Google Scholar 

  37. I. Fuke, T. Hayashi, Y. Tabata, and Y. Ikada. Synthesis of poly(ethylene glycol) derivatives with different branchings and their use for protein modification. J. Control. Release. 30:27–34 (1994) doi:10.1016/0168-3659(94)90041-8.

    Article  CAS  Google Scholar 

  38. R. Federico, A. Cona, P. Caliceti, and F. M. Veronese. Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application. J. Control. Release. 115:168–174 (2006) doi:10.1016/j.jconrel.2006.07.020.

    Article  PubMed  CAS  Google Scholar 

  39. F. M. Veronese, C. Monfardini, P. Caliceti, O. Schiavon, M. D. Scrawen, and D. Beer. Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly (ethylene glycol). J. Control. Release. 40:199–209 (1996) doi:10.1016/0168-3659(95)00185-9.

    Article  CAS  Google Scholar 

  40. F. M. Veronese, P. Caliceti, A. Pastorino, O. Schiavon, L. Sartore, L. Banci, and L. M. Scolaro. Preparation, physico-chemical and pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase. J. Control. Release. 10:145–154 (1989) doi:10.1016/0168-3659(89)90025-4.

    Article  CAS  Google Scholar 

  41. F. Fuertges, and A. Abuchowski. The clinical efficacy of poly(ethylene glycol)-modified proteins. J. Control. Release. 11:139–148 (1990) doi:10.1016/0168-3659(90)90127-F.

    Article  CAS  Google Scholar 

  42. D. Dikovsky, H. Bianco-Peled, and D. Seliktar. Defining the role of matrix compliance and proteolysis in three-dimensional cell spreading and remodeling. Biophys. J. 94:2914–2525 (2008).

    Article  PubMed  CAS  Google Scholar 

  43. M. Gonen-Wadmany, L. Oss-Ronen, and D. Seliktar. Protein-polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering. Biomaterials. 28:3876–3886 (2007) doi:10.1016/j.biomaterials.2007.05.005.

    Article  PubMed  CAS  Google Scholar 

  44. D. Dikovsky, H. Bianco-Peled, and D. Seliktar. The effect of structural alterations of PEG-fibrinogen hydrogel scaffolds on 3-D cellular morphology and cellular migration. Biomaterials. 27:1496–1506 (2006) doi:10.1016/j.biomaterials.2005.09.038.

    Article  PubMed  CAS  Google Scholar 

  45. H.-C. Chiu, C. Konak, P. Kopeckova, and J. Kopecek. Enzymatic degradation of poly(ethylene glycol) modified dextrans. J. Bioact. Compat. Polym. 9:388–410 (1994) doi:10.1177/088391159400900403.

    Article  CAS  Google Scholar 

  46. X. Y. Xiong, K. C. Tam, and L. H. Gan. Effect of enzymatic degradation on the release kinetics of model drug from Pluronic F127/poly(lactic acid) nano-particles. J. Control. Release. 108:263–270 (2005) doi:10.1016/j.jconrel.2005.08.005.

    Article  PubMed  CAS  Google Scholar 

  47. O. Schiavon, P. Caliceti, P. Ferruti, and F. M. Veronese. Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine). Farmaco. 55:264–269 (2000) doi:10.1016/S0014-827X(00)00031-8.

    Article  PubMed  CAS  Google Scholar 

  48. C. Monfardini, O. Schiavon, P. Caliceti, M. Morpurgo, J. M. Harris, and F. M. Veronese. A branched monomethoxypoly(ethylene glycol) for protein modification. Bioconjug. Chem. 6:62–69 (1995) doi:10.1021/bc00031a006.

    Article  PubMed  CAS  Google Scholar 

  49. Y. Ren, H. Zhang, and J. Huang. Synthesis and cytotoxic activity of platinum complex immobilized by branched polyethylene glycol. Bioorg. Med. Chem. Lett. 15:4479–4483 (2005) doi:10.1016/j.bmcl.2005.07.019.

    Article  PubMed  CAS  Google Scholar 

  50. Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko, and D. E. Discher. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat. Nano. 2:249–255 (2007) doi:10.1038/nnano.2007.70.

    Article  CAS  Google Scholar 

  51. S. Cai, K. Vijayan, D. Cheng, E. M. Lima, and D. E. Discher. Micelles of different morphologies-advantages of worm-like filomicelles of PEO-PCL in paclitaxel delivery. Pharm. Res. 24:2099–2109 (2007).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This work was supported by grants from the National Institutes of Health (NIH #’s HL007954, HL073940-01-A1, HL087036 and PO1-HL079063). We thank Tony Lowman and the Centralized Materials Characterization Facility and Industry Consortium of Drexel University for assistance with NMR and SEM studies and Dennis Discher of the University of Pennsylvania for GPC studies.

Author information

Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA

    Eric A. Simone

  2. Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 1 John Morgan Building, Philadelphia, Pennsylvania, 19104, USA

    Eric A. Simone, Vladimir V. Shuvaev & Vladimir R. Muzykantov

  3. Department of Chemical and Materials Engineering, University of Kentucky, Lexington, Kentucky, 40506, USA

    Thomas D. Dziubla

  4. Pulmonary Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104, USA

    Evguenia Arguiri & Melpo Christofidou-Solomidou

  5. Department of Chemical Engineering, Drexel University, Philadelphia, Pennsylvania, 19104, USA

    Vanessa Vardon

  6. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104, USA

    Vladimir R. Muzykantov

Authors
  1. Eric A. Simone
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas D. Dziubla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evguenia Arguiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vanessa Vardon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vladimir V. Shuvaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Melpo Christofidou-Solomidou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vladimir R. Muzykantov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eric A. Simone.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Simone, E.A., Dziubla, T.D., Arguiri, E. et al. Loading PEG-Catalase into Filamentous and Spherical Polymer Nanocarriers. Pharm Res 26, 250–260 (2009). https://doi.org/10.1007/s11095-008-9744-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-008-9744-7

KEY WORDS

Search

Navigation