Skip to main content
SpringerLink
Log in

Molar-Mass Characterization of Cationic Polymers for Gene Delivery by Aqueous Size-Exclusion Chromatography

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

Purpose

This study was performed to develop a reliable aqueous size-exclusion chromatography (SEC) method to obtain the absolute molar masses and distributions of various cationic polymers used in gene delivery.

Methods

Water-soluble cationic [2-(dimethylamino) ethyl methacrylate] polymers (PDEs) with different molar masses and low polydispersities were synthesized by living polymerization and these were used to optimize the SEC conditions. Online coupled multiangle light scattering (MALS) detection was applied to obtain the absolute molar masses. Narrow fractions of high molar mass were obtained by semipreparative SEC.

Results

It was found that 0.3 M NaAc (pH 4.4) is a suitable eluent in combination with Shodex OHpak SB columns for SEC analysis of PDEs and other cationic polymers, such as poly(l-lysine) and poly(ethylene imine). The absolute molar masses of different PDEs were determined directly using SEC–MALS. A calibration curve was established using narrow PDEs.

Conclusions

A reliable routine method for molar-mass characterization of cationic polymers was established. Because standards of known molar masses with narrow distributions are not commercially available for most polymers used in pharmaceutics and biotechnology, the procedure described in this work can also be applied for molar-mass characterization of other water-soluble polymers.

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
Fig. 6

Similar content being viewed by others

References

  1. S. C. Smedt ParticleDe J. Demeester W. E. Hennink (2000) ArticleTitleCationic polymer based gene delivery systems Pharm. Res. 17 113–126 Occurrence Handle10751024

    PubMed  Google Scholar 

  2. D. G. Anderson D. M. Lynn R. Langer (2003) ArticleTitleSemi-automated synthesis and screening of a large library of degradable cationic polymers for gene delivery Angew. Chem. Int. Ed. 42 3153–3158 Occurrence Handle10.1002/anie.200351244 Occurrence Handle1:CAS:528:DC%2BD3sXlvF2hsb0%3D

    Article  CAS  Google Scholar 

  3. E. Wagner (2004) ArticleTitleStrategies to improve DNA polyplexes for in vivo gene transfer—will “artificial viruses” be the answer? Pharm. Res. 21 8–14 Occurrence Handle10.1023/B:PHAM.0000012146.04068.56 Occurrence Handle1:CAS:528:DC%2BD2cXkt1OmsQ%3D%3D Occurrence Handle14984252

    Article  CAS  PubMed  Google Scholar 

  4. D. T. Curiel E. Wagner M. Cotton M. L. Birnstiel S. Agarwal C. M. Li et al. (1992) ArticleTitleHigh-efficiency gene transfer mediated by adenovirus coupled to DNA-complexes Hum. Gene Ther. 3 147–154 Occurrence Handle1:STN:280:ByyD3czltVw%3D Occurrence Handle1391034

    CAS  PubMed  Google Scholar 

  5. O. Boussif F. Lezoualc’h M. A. Zanta M .D. Mergny D. Scherman B. Demeneix et al. (1995) ArticleTitleA versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine Proc. Natl. Acad. Sci. USA 92 7297–7301 Occurrence Handle1:CAS:528:DyaK2MXntlKmt7s%3D Occurrence Handle7638184

    CAS  PubMed  Google Scholar 

  6. J. Y. Cherng P. Wetering ParticleVan de H. Talsma D. J. A. Crommelin W. E. Hennink (1996) ArticleTitleEffect of size and serum proteins on transfection efficiency of poly((2-dimethylamino) ethyl methacrylate)-plasmid nanoparticles Pharm. Res. 13 1038–1042 Occurrence Handle10.1023/A:1016054623543 Occurrence Handle1:CAS:528:DyaK28Xks1Sqsr4%3D Occurrence Handle8842041

    Article  CAS  PubMed  Google Scholar 

  7. S. Pirotton C. Muller N. Pantoustier F. Botterman S. Collinet C. Grandfils G. Dandrifosse P. Degee P. Dubois M. Raes (2004) ArticleTitleEnhancement of transfection efficiency through rapid and noncovalent post-PEGylation of poly(dimethylaminothyl methacrylate)/DNA complexes Pharm. Res. 21 1471–1479 Occurrence Handle10.1023/B:PHAM.0000036923.25772.97 Occurrence Handle1:CAS:528:DC%2BD2cXmtlGntbk%3D Occurrence Handle15359584

    Article  CAS  PubMed  Google Scholar 

  8. G. Borchard (2001) ArticleTitleChitosans for gene delivery Adv. Drug Deliv. Rev. 52 145–150 Occurrence Handle10.1016/S0169-409X(01)00198-3 Occurrence Handle1:CAS:528:DC%2BD3MXosFKltbY%3D Occurrence Handle11718938

    Article  CAS  PubMed  Google Scholar 

  9. T. Kean S. Roth M. Thanou (2005) ArticleTitleTrimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency J. Control. Release 103 643–653 Occurrence Handle10.1016/j.jconrel.2005.01.001 Occurrence Handle1:CAS:528:DC%2BD2MXjt1aht7k%3D Occurrence Handle15820411

    Article  CAS  PubMed  Google Scholar 

  10. J. Luten J. H. Steenis Particlevan R. Someren Particlevan J. Kemmink N. M. E. Schuurmans-Nieuwenbroek G. A. Koning D. J. A. Crommelin C. F. Nostrum Particlevan W. E. Hennink (2003) ArticleTitleWater-soluble biodegradable cationic polyphosphazenes for gene delivery J. Control. Release 89 483–497 Occurrence Handle10.1016/S0168-3659(03)00127-5 Occurrence Handle1:CAS:528:DC%2BD3sXjsVSjsbY%3D Occurrence Handle12737850

    Article  CAS  PubMed  Google Scholar 

  11. Y. B. Lim S. O. Han H. U. Kong Y. Lee J. S. Park B. Jeong S. W. Kim (2000) ArticleTitleBiodegradable polyester, poly[alpha-(4-aminobutyl)-l-glycolic acid], as a non-toxic gene carrier Pharm. Res. 17 811–816 Occurrence Handle10.1023/A:1007552007765 Occurrence Handle1:CAS:528:DC%2BD3cXmsFGqtL8%3D Occurrence Handle10990199

    Article  CAS  PubMed  Google Scholar 

  12. A. M. Funhoff C. F. Nostrum Particlevan A. P. C. A. Janssen M. H. A. M. Fens D. J. A. Crommelin W. E. Hennink (2004) ArticleTitlePolymer side-chain degradation as a tool to control the destabilization of polyplexes Pharm. Res. 21 170–176 Occurrence Handle10.1023/B:PHAM.0000012165.68765.e6 Occurrence Handle1:CAS:528:DC%2BD2cXkt1OgtA%3D%3D Occurrence Handle14984272

    Article  CAS  PubMed  Google Scholar 

  13. P. Wetering ParticleVan de J. Y. Cherng H. Talsma W. E. Hennink (1997) ArticleTitleRelation between transfection efficiency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes J. Control. Release 49 59–71

    Google Scholar 

  14. C. Arigita N. J. Zuidam D. J. A. Crommelin W. E. Hennink (1999) ArticleTitleAssociation and dissociation of polymer/DNA complexes used for gene delivery Pharm. Res. 16 1534–1541 Occurrence Handle10.1023/A:1015096302720 Occurrence Handle1:CAS:528:DyaK1MXntVOhtbo%3D Occurrence Handle10554094

    Article  CAS  PubMed  Google Scholar 

  15. S. Choksakulnimitr S. Masuda H. Tokuda Y. Takakura M. Hashida (1995) ArticleTitleIn vitro cytotoxicity of macromolecules in different cell culture systems J. Control. Release 34 233–241 Occurrence Handle10.1016/0168-3659(95)00007-U Occurrence Handle1:CAS:528:DyaK2MXlvVWjtbw%3D

    Article  CAS  Google Scholar 

  16. K. Kunath A. Harpe Particlevon D. Fischer H. Petersen U. Bickel K. Voigt T. Kissel (2003) ArticleTitleLow-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine J. Control. Release 89 113–125 Occurrence Handle10.1016/S0168-3659(03)00076-2 Occurrence Handle1:CAS:528:DC%2BD3sXivVCqur8%3D Occurrence Handle12695067

    Article  CAS  PubMed  Google Scholar 

  17. D. Fischer T. Bieber Y.-X. Li H.-P. Elsasser T. Kissel (1999) ArticleTitleA novel non-viral vector for gene delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity Pharm. Res. 16 1273–1279 Occurrence Handle10.1023/A:1014861900478 Occurrence Handle1:CAS:528:DyaK1MXlsVyntb4%3D Occurrence Handle10468031

    Article  CAS  PubMed  Google Scholar 

  18. W. T. Godbey K. K. Wu A. G. Mikos (1999) ArticleTitleSize matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle J. Biomed. Mater. Res. 45 268–275 Occurrence Handle10.1002/(SICI)1097-4636(19990605)45:3<268::AID-JBM15>3.0.CO;2-Q Occurrence Handle1:CAS:528:DyaK1MXisVequrw%3D Occurrence Handle10397985

    Article  CAS  PubMed  Google Scholar 

  19. C. L. Gebhart A. V. Kabanov (2001) ArticleTitleEvaluation of polyplexes as gene transfer agents J. Control. Release 73 401–416 Occurrence Handle10.1016/S0168-3659(01)00357-1 Occurrence Handle1:CAS:528:DC%2BD3MXksVSqu7s%3D Occurrence Handle11516515

    Article  CAS  PubMed  Google Scholar 

  20. X.-L. Jiang. Separation and Characterization of functional polymers, Ph.D. thesis, University of Amsterdam, 2004, Chap. 1. ISBN 90 5776 126 6.

  21. E. Pérez-Payá L. Braco A. Campos V. Soria C. Abad (1989) ArticleTitleSolution properties of polyelectrolytes: IV. Use of a new hydrophilic size-exclusion chromatographic packing for the separation of anionic and cationic polyions J. Chromatogr. A 461 229–242 Occurrence Handle10.1016/S0021-9673(00)94289-4

    Article  Google Scholar 

  22. A. Harpe Particlevon H. Petersen Y.-X. Li T. Kissel (2000) ArticleTitleCharacterization of commercially available and synthesized polyethylenimines for gene delivery J. Control. Release 69 309–322

    Google Scholar 

  23. X. Guo M. Condra K. Kimura G. Berth H. Dautzenberg P. L. Dubin (2003) ArticleTitleDetermination of molecular weight of heparin by size-exclusion chromatography with universal calibration Anal. Biochem. 312 33–39 Occurrence Handle10.1016/S0003-2697(02)00428-1 Occurrence Handle1:CAS:528:DC%2BD38XpsF2rt70%3D Occurrence Handle12479832

    Article  CAS  PubMed  Google Scholar 

  24. B. Wittgren A. Welinder B. Porsch (2003) ArticleTitleMolar mass characterization of cationic methacrylate–ethyl acrylate copolymers using size-exclusion chromatography with online multi-angle light scattering and refractometric detection J. Chromatogr. A 1002 101–109 Occurrence Handle10.1016/S0021-9673(03)00729-5 Occurrence Handle1:CAS:528:DC%2BD3sXksF2gsL0%3D Occurrence Handle12885083

    Article  CAS  PubMed  Google Scholar 

  25. D. J. Nagy D. A. Terwillger (1989) ArticleTitleSize-exclusion chromatography/differential viscometry of cationic polymers J. Liq. Chromatogr. 12 1431–1449 Occurrence Handle1:CAS:528:DyaK3cXitFGmtg%3D%3D

    CAS  Google Scholar 

  26. G. Volet J. Lesec (1994) ArticleTitleNon-exclusion effects in aqueous SEC: behaviour of some polyelectrolytes using on-line mass detectors J. Liq. Chromatogr. 17 559–577 Occurrence Handle1:CAS:528:DyaK2cXhvFaku7Y%3D

    CAS  Google Scholar 

  27. R. D. Ricker L. A. Sandoval (1996) ArticleTitleFast, reproducible size-exclusion chromatography of biological macromolecules J. Chromatogr. A 743 43–50 Occurrence Handle10.1016/0021-9673(96)00283-X Occurrence Handle1:CAS:528:DyaK28XlsFWrsLY%3D Occurrence Handle8817873

    Article  CAS  PubMed  Google Scholar 

  28. M. Adler, H. Pasch, C. Meier, R. Senger, H.-G. Koban, M. Augenstein, and G. Reinhold. Molar mass characterization of hydrophilic copolymers, 2 Size exclusion chromatography of cationic (meth)acrylate copolymers. e-Polymers no. 057 (2005).

  29. N. J. Zuidam G. Posthuma E. T. Vries Particlede D. J. A. Crommelin W. E. Hennink G. Storm (2000) ArticleTitleEffects of physicochemical characteristics of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes on cellular association and internalisation J. Drug Target 8 51–66 Occurrence Handle1:CAS:528:DC%2BD3cXit1Knsbo%3D Occurrence Handle10761645

    CAS  PubMed  Google Scholar 

  30. F. J. Verbaan C. Oussoren I. M. Dam ParticleVan Y. Takakura M. Hashida D. J. A. Crommelin et al. (2001) ArticleTitleThe fate of poly(2-dimethyl amino ethyl)-methacrylate-based polyplexes after intravenous administration Int. J. Pharm. 214 99–101 Occurrence Handle10.1016/S0378-5173(00)00642-6 Occurrence Handle1:CAS:528:DC%2BD3MXitFWgsLY%3D Occurrence Handle11282245

    Article  CAS  PubMed  Google Scholar 

  31. F. J. Verbaan C. Oussoren C. J. Snel D. J. A. Crommelin W. E. Hennink G. Storm (2004) ArticleTitleSteric stabilization of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes mediates prolonged circulation and tumor targeting in mice J. Gene Med. 6 64–75 Occurrence Handle10.1002/jgm.475 Occurrence Handle1:CAS:528:DC%2BD2cXhvVGgtrg%3D Occurrence Handle14716678

    Article  CAS  PubMed  Google Scholar 

  32. G. W. Bos T. Kanellos D. J. A. Crommelin W. E. Hennink C. R. Howard (2004) ArticleTitleCationic polymers that enhance the performance of HbsA9 DNA in vivo Vaccine 23 460–469 Occurrence Handle10.1016/j.vaccine.2004.06.020 Occurrence Handle1:CAS:528:DC%2BD2cXpsFemtLo%3D Occurrence Handle15530694

    Article  CAS  PubMed  Google Scholar 

  33. S. Creutz P. Teyssié R. Jérôme (1997) ArticleTitleLiving anionic homopolymerization and block copolymerization of (dimethylamino)ethyl methacrylate Macromolecules 30 6–9 Occurrence Handle1:CAS:528:DyaK2sXksVCjtw%3D%3D

    CAS  Google Scholar 

  34. Y. K. Chong T. P. T. Le G. Moad E. Rizzardo S. H. Thang (1999) ArticleTitleA more versatile route to block copolymers and other polymers of complex architecture by living radical polymerization: the RAFT process Macromolecules 32 2071–2074 Occurrence Handle1:CAS:528:DyaK1MXht1art7k%3D

    CAS  Google Scholar 

  35. Y. Mitsukami M. S. Donovan A. B. Lowe C. L. McCormick (2001) ArticleTitleWater-soluble polymers. 81. Direct synthesis of hydrophilic styrenic-based homopolymers and block copolymers in aqueous solution via RAFT Macromolecules 34 2248–2256 Occurrence Handle10.1021/ma0018087 Occurrence Handle1:CAS:528:DC%2BD3MXhtFymtLw%3D

    Article  CAS  Google Scholar 

  36. H. de Brouwer. RAFT memorabilia, living radical polymerization in homogeneous and heterogenous media, Ph.D. thesis, Eindhoven University of Technology, 2001, Chap. 3. ISBN 90 3862 802 1.

  37. A. B. Sieval M. Thanou A. F. Kotze J. C. Verhoef J. Brussee H. E. Junginger (1998) ArticleTitlePreparation and NMR-characterization of highly substituted N-trimethyl chitosan chloride Carbohydr. Polym. 36 157–165 Occurrence Handle10.1016/S0144-8617(98)00009-5 Occurrence Handle1:CAS:528:DyaK1cXmtlSkurw%3D

    Article  CAS  Google Scholar 

  38. D. Snyman J. H. Hamman J. S. Kotze J. E. Rollings A. F. Kotze (2002) ArticleTitleThe relationship between the absolute molecular weight and the degree of quaternisation of N-trimethyl chitosan chloride Carbohydr. Polym. 50 145–150 Occurrence Handle10.1016/S0144-8617(02)00008-5 Occurrence Handle1:CAS:528:DC%2BD38Xksl2gt70%3D

    Article  CAS  Google Scholar 

  39. W. W. Yau J. J. Kirkland D. D. Bly (1979) Modern Size-Exclusion Liquid Chromatography Wiley New York

    Google Scholar 

  40. M.-S. Song G.-X. Hu X.-Y. Li B. Zhao (2002) ArticleTitleStudy on the concentration effects in size exclusion chromatography VII. A quantitative verification for the model theory of concentration and molecular mass dependences of hydrodynamic volumes for polydisperse polymers J. Chromatogr. A 961 155–170 Occurrence Handle10.1016/S0021-9673(02)00347-3 Occurrence Handle1:CAS:528:DC%2BD38Xlt1Onu7o%3D Occurrence Handle12184617

    Article  CAS  PubMed  Google Scholar 

  41. M. Andersson B. Wittgren K.-G. Wahlund (2003) ArticleTitleAccuracy in multiangle light scattering measurements for molar mass and radius estimations. Model calculations and experiments Anal. Chem. 75 4279–4291 Occurrence Handle10.1021/ac030128+ Occurrence Handle1:CAS:528:DC%2BD3sXltVWltb8%3D Occurrence Handle14632147

    Article  CAS  PubMed  Google Scholar 

  42. M. Netopilík S. Podzimek P. Kratochvíl (2001) ArticleTitleEstimation of width of narrow molecular-weight distributions by size-exclusion chromatography with concentration and light scattering detection J. Chromatogr. A 922 25–36 Occurrence Handle10.1016/S0021-9673(01)00952-9 Occurrence Handle11486870

    Article  PubMed  Google Scholar 

  43. P. J. Wyatt D. N. Villapando P. Alden (1997) ArticleTitlePolydispersity study of narrow polystyrene standards using light scattering J. Liq. Chromatogr. Relat. Technol. 20 2169–2180 Occurrence Handle1:CAS:528:DyaK2sXkslOqurw%3D

    CAS  Google Scholar 

  44. M. Netopilik (2003) ArticleTitleProblems connected with band-broadening in size-exclusion chromatography with dual detection J. Biochem. Biophys. Methods 56 79–93 Occurrence Handle1:CAS:528:DC%2BD3sXkvF2jtr8%3D Occurrence Handle12834970

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge Maryam Amidi and Jordy Luten for the synthesis of TMC and polyphosphazene, respectively. Helpful discussions on SEC–MALS with Wybren Frankema (University of Amsterdam) were appreciated. We also thank Dr. Bert Klumperman (Eindhoven University of Technology) for his guidance on the synthesis of the RAFT agent and on the RAFT polymerization.

Author information

Authors and Affiliations

  1. Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80 082, 3508 TB, Utrecht, The Netherlands

    Xulin Jiang, Mies J. van Steenbergen, Niels Akeroyd, Cornelus F. van Nostrum & Wim E. Hennink

  2. Polymer-Analysis Group, van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands

    Aschwin van der Horst & Peter J. Schoenmakers

Authors
  1. Xulin Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aschwin van der Horst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mies J. van Steenbergen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Niels Akeroyd
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cornelus F. van Nostrum
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter J. Schoenmakers
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wim E. Hennink
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wim E. Hennink.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jiang, X., van der Horst, A., van Steenbergen, M.J. et al. Molar-Mass Characterization of Cationic Polymers for Gene Delivery by Aqueous Size-Exclusion Chromatography. Pharm Res 23, 595–603 (2006). https://doi.org/10.1007/s11095-006-9574-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-006-9574-4

Key Words

Search

Navigation