Skip to main content
SpringerLink
Log in

Automated and enhanced extraction of a small molecule-drug conjugate using an enzyme-inhibitor interaction based SPME tool followed by direct analysis by ESI-MS

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 μg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 μg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range.

Graphical abstract

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
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  1. Krall N, Scheuermann J, Neri D. Small targeted cytotoxics: current state and promises from DNA-encoded chemical libraries. Angew Chem Int Ed Engl. 2013;52:1384–402. https://doi.org/10.1002/anie.201204631.

    Article  CAS  PubMed  Google Scholar 

  2. Srinivasarao M, Galliford CV, Low PS. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nat Rev Drug Discov. 2015;14:203–19. https://doi.org/10.1038/nrd4519.

    Article  CAS  PubMed  Google Scholar 

  3. van der Veldt AAM, Hendrikse NH, Smit EF, Mooijer MPJ, Rijnders AY, Gerritsen WR, et al. Biodistribution and radiation dosimetry of 11C-labelled docetaxel in cancer patients. Eur J Nucl Med Mol Imaging. 2010;37:1950–8. https://doi.org/10.1007/s00259-010-1489-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van der Veldt AA, Lubberink M, Mathijssen RH, Loos W, Herder GJ, Greuter HN, Comans EF, Rutten H, Eriksson J, Windhorst AD, Hendrikse H, Postmus PE, Smit EF, Lammertsma AA. Towards prediction of efficacy of chemotherapy: a proof of concept study in lung cancer patients using [11C] docetaxel and positron emission tomography. Clin Cancer Res clincanres.3779.2012. 2013 https://doi.org/10.1158/1078-0432.CCR-12-3779.

    Article  CAS  Google Scholar 

  5. van der Meel R, Vehmeijer LJC, Kok RJ, Storm G, van Gaal EVB. Ligand-targeted particulate nanomedicines undergoing clinical evaluation: current status. In: Prokop A, Weissig V, editors. Intracellular delivery III: market entry barriers of Nanomedicines. Cham: Springer International Publishing; 2016. p. 163–200.

    Chapter  Google Scholar 

  6. Ravi VJ, Miller ML, Widdison WC. Antibody–drug conjugates: an emerging concept in cancer therapy. Angew Chem Int Ed Engl. 2014;53:3796–827. https://doi.org/10.1002/anie.201307628.

    Article  CAS  Google Scholar 

  7. Wang AZ, Farokhzad OC. Current progress of aptamer-based molecular imaging. J Nucl Med. 2014;55:353–6. https://doi.org/10.2967/jnumed.113.126144.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang X, Zhang J, Ma Y, Pei X, Liu Q, Lu B, et al. A cell-based single-stranded DNA aptamer specifically targets gastric cancer. Int J Biochem Cell Biol. 2014;46:1–8. https://doi.org/10.1016/j.biocel.2013.10.006.

    Article  CAS  PubMed  Google Scholar 

  9. Yu B, Tai HC, Xue W, Lee LJ, Lee RJ. Receptor-targeted nanocarriers for therapeutic delivery to cancer. Mol Membr Biol. 2010;27:286–98. https://doi.org/10.3109/09687688.2010.521200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kurzrock R, Gabrail N, Chandhasin C, Moulder S, Smith C, Brenner A, et al. Safety, pharmacokinetics, and activity of GRN1005, a novel conjugate of angiopep-2, a peptide facilitating brain penetration, and paclitaxel, in patients with advanced solid tumors. Mol Cancer Ther. 2012;11:308–16. https://doi.org/10.1158/1535-7163.MCT-11-0566.

    Article  CAS  PubMed  Google Scholar 

  11. Zhang X-X, Eden HS, Chen X. Peptides in cancer nanomedicine: drug carriers, targeting ligands and protease substrates. J Control Release. 2012;159:2–13. https://doi.org/10.1016/j.jconrel.2011.10.023.

    Article  CAS  PubMed  Google Scholar 

  12. Rana S, Nissen F, Lindner T, Altmann A, Mier W, Debus J, et al. Screening of a novel peptide targeting the proteoglycan-like region of human carbonic anhydrase IX. Mol Imaging. 2013;12:7290.2013.00066. https://doi.org/10.2310/7290.2013.00066.

    Article  CAS  Google Scholar 

  13. McGuire MJ, Gray BP, Li S, Cupka D, Byers LA, Wu L, et al. Identification and characterization of a suite of tumor targeting peptides for non-small cell lung cancer. Sci Rep. 2014;4:4480. https://doi.org/10.1038/srep04480.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xia W, Low PS. Folate-targeted therapies for cancer. J Med Chem. 2010;53:6811–24. https://doi.org/10.1021/jm100509v.

    Article  CAS  PubMed  Google Scholar 

  15. Varghese B, Vlashi E, Xia W, Ayala Lopez W, Paulos CM, Reddy J, et al. Folate receptor-β in activated macrophages: ligand binding and receptor recycling kinetics. Mol Pharm. 2014;11:3609–16. https://doi.org/10.1021/mp500348e.

    Article  CAS  PubMed  Google Scholar 

  16. Thomas M, Kularatne SA, Qi L, Kleindl P, Leamon CP, Hansen MJ, et al. Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues. Ann N Y Acad Sci. 2009;1175:32–9. https://doi.org/10.1111/j.1749-6632.2009.04977.x.

    Article  CAS  Google Scholar 

  17. Shen J, Chelvam V, Cresswell G, Low PS. Use of Folate-conjugated imaging agents to target alternatively activated macrophages in a murine model of asthma. Mol Pharm. 2013;10:1918–27. https://doi.org/10.1021/mp3006962.

    Article  CAS  PubMed  Google Scholar 

  18. Dennis MS, Jin H, Dugger D, Yang R, McFarland L, Ogasawara A, et al. Imaging tumors with an albumin-binding fab, a novel tumor-targeting agent. Cancer Res. 2007;67:254–61. https://doi.org/10.1158/0008-5472.CAN-06-2531.

    Article  CAS  PubMed  Google Scholar 

  19. Borsi L, Balza E, Bestagno M, Castellani P, Carnemolla B, Biro A, et al. Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int J Cancer. 2002;102:75–85. https://doi.org/10.1002/ijc.10662.

    Article  CAS  PubMed  Google Scholar 

  20. Adem YT, Schwarz KA, Duenas E, Patapoff TW, Galush WJ, Esue O. Auristatin antibody drug conjugate physical instability and the role of drug payload. Bioconjug Chem. 2014;25:656–64. https://doi.org/10.1021/bc400439x.

    Article  CAS  PubMed  Google Scholar 

  21. Liu X, Guo J, Han S, Yao L, Chen A, Yang Q, et al. Enhanced immune response induced by a potential influenza a vaccine based on branched M2e polypeptides linked to tuftsin. Vaccine. 2012;30:6527–33. https://doi.org/10.1016/j.vaccine.2012.08.054.

    Article  CAS  PubMed  Google Scholar 

  22. Jeannin P, Delneste Y, Buisine E, Le Mao J, Didierlaurent A, Stewart GA, et al. Immunogenecity and antigenicity of synthetic peptides derived from the mite allergen Der p I. Mol Immunol. 1993;30:1511–8. https://doi.org/10.1016/0161-5890(93)90459-O.

    Article  CAS  PubMed  Google Scholar 

  23. Low PS, Henne WA, Doorneweerd DD. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res. 2008;41:120–9. https://doi.org/10.1021/ar7000815.

    Article  CAS  PubMed  Google Scholar 

  24. Hillier SM, Maresca KP, Lu G, Merkin RD, Marquis JC, Zimmerman CN, et al. 99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer. J Nucl Med. 2013;54:1369–76. https://doi.org/10.2967/jnumed.112.116624.

    Article  CAS  PubMed  Google Scholar 

  25. Ginj M, Zhang H, Waser B, Cescato R, Wild D, Wang X, et al. Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proc Natl Acad Sci U S A. 2006;103:16436–41. https://doi.org/10.1073/pnas.0607761103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Krall N, Pretto F, Decurtins W, Bernardes GJL, Supuran CT, Neri D. A small-molecule drug conjugate for the treatment of carbonic anhydrase IX expressing tumors. Angew Chem Int Ed Engl. 2014;53:4231–5. https://doi.org/10.1002/anie.201310709.

    Article  CAS  PubMed  Google Scholar 

  27. Cazzamalli S, Dal Corso A, Widmayer F, Neri D. Chemically defined antibody– and small molecule–drug conjugates for in vivo tumor targeting applications: a comparative analysis. J Am Chem Soc. 2018;140:1617–21. https://doi.org/10.1021/jacs.7b13361.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cazzamalli S, Corso AD, Neri D. Acetazolamide serves as selective delivery vehicle for dipeptide-linked drugs to renal cell carcinoma. Mol Cancer Ther. 2016;15:2926–35. https://doi.org/10.1158/1535-7163.MCT-16-0283.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ghiasikhou S, Marchand A, Zenobi R. A comparative study between a miniaturized liquid junction built in a capillary gap and semi-open capillaries for nL sample infusion to mass spectrometry. Microfluid Nanofluid. 2019;23:60. https://doi.org/10.1007/s10404-019-2229-7.

    Article  CAS  Google Scholar 

  30. Neu V, Steiner R, Müller S, Fattinger C, Zenobi R. Development and characterization of a capillary gap sampler as new microfluidic device for fast and direct analysis of Low sample amounts by ESI-MS. Anal Chem. 2013;85:4628–35. https://doi.org/10.1021/ac400186t.

    Article  CAS  PubMed  Google Scholar 

  31. Ghiasikhou S, da Silva MF, Zhu Y, Zenobi R. The capillary gap sampler, a new microfluidic platform for direct coupling of automated solid-phase microextraction with ESI-MS. Anal Bioanal Chem. 2017. https://doi.org/10.1007/s00216-017-0652-8.

    Article  CAS  Google Scholar 

  32. Gasymov OK, Glasgow BJ. ANS fluorescence: potential to augment the identification of the external binding sites of proteins. Biochim Biophys Acta. 2007;1774:403–11. https://doi.org/10.1016/j.bbapap.2007.01.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Friess SD, Zenobi R. Protein structure information from mass spectrometry? Selective titration of arginine residues by sulfonates. J AM SOC MASS SPECTR. 2001;12:810–8. https://doi.org/10.1016/S1044-0305(01)00257-4.

    Article  CAS  Google Scholar 

  34. Slavík J. Anilinonaphthalene sulfonate as a probe of membrane composition and function. Biochim Biophys Acta. 1982;694:1–25. https://doi.org/10.1016/0304-4157(82)90012-0.

    Article  PubMed  Google Scholar 

  35. Almstedt K, Lundqvist M, Carlsson J, Karlsson M, Persson B, Jonsson B-H, et al. Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II. J Mol Biol. 2004;342:619–33. https://doi.org/10.1016/j.jmb.2004.07.024.

    Article  CAS  PubMed  Google Scholar 

  36. Pawliszyn J. Sample preparation: Quo Vadis. Anal Chem. 2003;75:2543–58. https://doi.org/10.1021/ac034094h.

    Article  CAS  PubMed  Google Scholar 

  37. Motlagh S, Pawliszyn J. On-line monitoring of flowing samples using solid phase microextraction-gas chromatography. Anal Chim Acta. 1993;284:265–73. https://doi.org/10.1016/0003-2670(93)85310-G.

    Article  CAS  Google Scholar 

  38. Górecki T, Yu X, Pawliszyn J. Theory of analyte extraction by selected porous polymer SPME fibres†. Analyst. 1999;124:643–9. https://doi.org/10.1039/A808487D.

    Article  Google Scholar 

  39. Chapron DJ, Gomolin IH, Sweeney KR. Acetazolamide blood concentrations are excessive in the elderly: propensity for acidosis and relationship to renal function. J Clin Pharmacol. 1989;29:348–53. https://doi.org/10.1002/j.1552-4604.1989.tb03340.x.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We gratefully thank Dr. Christof Fattinger (Roche) for his support in sampler development. Moreover, we thank the Scientific Center for Optical and Electron Microscopy (ScopeM), a central technology platform of ETH Zurich, for providing us with resources and services in electron microscopy. Finally, we thank the Swiss National Science Foundation (SNSF) for funding this project (Grant numbers 200020-159929 & 200020-178765).

Associated content

The original data used in this publication are made available in a curated data archive at ETH Zurich (https://www.researchcollection.ethz.ch) under the https://doi.org/10.3929/ethz-b-000334909.

Author information

Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, ETH Zurich, HCI E 329, 8093, Zurich, Switzerland

    Sahar Ghiasikhou, Samuele Cazzamalli, Jörg Scheuermann, Dario Neri & Renato Zenobi

Authors
  1. Sahar Ghiasikhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samuele Cazzamalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jörg Scheuermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dario Neri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Renato Zenobi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Renato Zenobi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 354 kb).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ghiasikhou, S., Cazzamalli, S., Scheuermann, J. et al. Automated and enhanced extraction of a small molecule-drug conjugate using an enzyme-inhibitor interaction based SPME tool followed by direct analysis by ESI-MS. Anal Bioanal Chem 411, 7387–7398 (2019). https://doi.org/10.1007/s00216-019-02165-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-02165-7

Keywords

Search

Navigation