Elsevier

Biosensors and Bioelectronics

Volume 86, 15 December 2016, Pages 913-919
Biosensors and Bioelectronics

Fluorescent molecularly imprinted nanogels for the detection of anticancer drugs in human plasma

https://doi.org/10.1016/j.bios.2016.07.087Get rights and content

Highlights

  • We have designed and synthesized fluorescent imprinted nanogels capable to bind sunitinib, an anticancer drug.

  • Coumarin - containing nanogels form stable colloidal solutions in human plasma.

  • Quenching of their fluorescence by sunitnib allows its detection.

Abstract

Several fluorescent molecularly imprinted nanogels for the detection of the anticancer drug sunitinib were synthesized and characterized. A selection of functional monomers based on different aminoacids and coumarin allowed isolation of polymers with very good rebinding properties and sensitivities. The direct detection of sunitinib in human plasma was successfully demonstrated by fluorescence quenching of the coumarin-based nanogels. The plasma sample simply diluted in DMSO allowed the recovery of various amounts of sunitib, as determined by an averaged calibration curve. The LOD was 400 nM, with within-run variability <9%, day to day variability <5%, and good accuracy in the recovery of sunitinib from spiked samples.

Introduction

Keeping toxicity to a minimal level while ensuring optimal activity and minimal side effects are key priorities in cancer therapy, however, individual germ line mutations, in metabolizing enzymes, and other pharmacogenomics variations may vary pharmacokinetic and pharmacodynamic responses within a pool of individuals. Therapeutic drug monitoring, aimed at obtaining personalized medicines, is one of the main targets in current clinical oncology (Walko and McLeod, 2014; De Jonge et al. 2005; Alnaim, 2007). At present, therapeutic drug monitoring (TDM) heavily relies on drug extraction and quantification from blood or plasma samples by HPLC or LC-MS. The instrumentation required for these analyses is expensive and dependent on highly specialised personnel, therefore limiting the applicability. The development of a point of care device, that allows tailoring of the therapy protocol upon individual responses, with simple sample preparation, high sensitivity and good selectivity, remains a challenge.

In recent years molecular imprinting has consolidated its place as a viable approach for the generation of polymeric matrices with excellent molecular recognition characteristics. The templating approach, together with an appropriate choice of functional monomer and cross-linker, allows the formation of three-dimensional cavities that can rebind the target molecule, or its analogues, with high selectivity.

In the field of therapeutic drug monitoring, imprinted polymers (MIPs) have been developed for pre-concentration of samples, as purification cartridges for LC-MS analysis (Thibert et al., 2014, Yang et al., 2014), or as recognition elements for microbalances, plasmon resonance and electrochemical systems (Altintas et al., 2015; Blanco-López et al., 2004). Alternatively, MIPs have also been used as the sensing system, by embedding the signal-generating monomer in the polymeric matrix, such as in the case of optical/fluorimetric units (Manju et al., 2010, Awino and Zhao, 2014, Ton et al., 2013). In terms of polymer matrices, most work has been done with bulk polymers, acting as recognition elements for electrochemical, quantum dots or fiber optic based sensors. MIP-based electrochemical sensors have been used to detect uracil- (Prasad et al., 2012, Prasad et al., 2009) and anthraquinone-based anticancer drugs (Nezhadali et al., 2016). CdTe@SiO2 quantum dots coated with a MIP were used as fluorescent sensor for norepinephrine (Wei et al., 2014), and fiber optic array was developed to quantify enrofloxacin in sheep serum (Carrasco et al., 2015).

The main target of this work was to develop fluorescent imprinted nanogels, specific for anticancer drugs, that would form stable colloidal solutions when dispersed into human plasma, therefore allowing detection of the target with minimal sample preparation.

Plasma is a very complex matrix, containing thousands of different molecules and binding proteins such as albumins and immunoglobulins. A potential sensor for the quantification of drugs must overcome key issues like the possible cross-reactivity with plasma proteins and small molecules, competitive binding of the drug to albumin, stability issues leading to aggregation and precipitation of the nanoparticles.

For the purpose of this work sunitinib (SU11248, Sutent) 1 (Fig. 1) was selected as the target drug. Sunitinib is a tyrosine kinase inhibitor used for the treatment of renal cell carcinoma and of imatinib-resistant gastrointestinal stromal tumour since 2006 (Noble et al., 2004, Zhang et al., 2009). It is commonly administered to patients as sunitinib malate, with dosages ranging from 25 to 50 mg to 150 mg daily. Pharmacokinetic and pharmacodynamic preclinical studies demonstrated that although the therapeutic window of concentrations for sunitinib is between 50 ng/mL and 500 ng/mL, concentrations higher than 100 ng/mL result in a significant increase in drug toxicity (Faivre et al., 2006). Therefore, the TDM of this drug represents a useful system to develop personalized therapies for patients decreasing side effects and increasing therapy efficiency. As for other anticancer drugs, sunitinib quantification in plasma is currently performed by HPLC coupled with UV detector (Etienne-Grimaldi et al., 2009, Blanchet et al., 2009) or mass spectrometer (De Bruijn et al., 2010) or by LC/MS/MS methods (Andriamanana et al., 2013). Currently there are no rapid methods available for the therapeutic monitoring of sunitinib in alternative to such high specialised equipment, and point of care devices or immunoenzymatic assays for anticancer drugs have yet to be reported.

Section snippets

Materials

Sunitinib was purchased from Bepharm ltd. All the other reagents were from Sigma-Aldrich.

Instrumentation

HPLC analyses were run on an Agilent series 1100 liquid chromatograph equipped with a Phenomenex, Luna C18 5μ column with a column guard and a 20 μL loop. The flow was set to 1 mL/min. UV–visible spectra were recorded on a UV-1800 (Shimadzu) spectrometer. The fluorescence titrations were performed by a CARY Eclipse (Varian) spectrometer with a cuvette of 1 cm optical path, and by a Perkin Elmer LS 50B

Functional monomers

The formation of a stable complex between the functional monomer and the template in the prepolymerisation mixture is a key requirement in molecular imprinting for obtaining matrices with high rebinding characteristics. Four functional monomers were selected for their potential ability to interact with Sunitinib, the target drug, via a variety of non-covalent interactions. Sunitinib (Fig. 1) contains three hydrogen bond donor NH groups, three hydrogen bond acceptors (two carbonyls and a

Conclusions

In conclusion, we have designed and synthesized a set of fluorescent MIPS that bind sunitinib with good sensitivity. We have also developed a novel analytical protocol for the fluorimetric sensing of sunitinib in plasma samples exploiting the quenching of the MIP fluorescence by bound sunitinib. Simple dilution of human plasma with DMSO allows the detection of the drug with MIP 1.5. The encouraging results obtained with this proof of concept open the way to the possible use of a fluorescent MIP

Acknowledgements

We acknowledge the AIRC 5×1000 grant 12214 “Application of Advanced Nanotechnology in the Development of Innovative Cancer Diagnostics Tools”. We thank Dr. G. Mastroianni for the TEM data.

References (36)

  • I. Andriamanana et al.

    J. Chromotogr. B

    (2013)
  • Z. Altintas et al.

    Sens. Actuators B

    (2015)
  • U. Athikomrattanakul et al.

    Biosens. Bioelectron.

    (2009)
  • B. Blanchet et al.

    Clin. Chim. Acta

    (2009)
  • S. Carrasco et al.

    Chem. Sci.

    (2015)
  • P. De Bruijn et al.

    J. Pharm. Biomed. Anal.

    (2010)
  • M.C. Etienne-Grimaldi et al.

    J. Chromatogr. B

    (2009)
  • L. Gao et al.

    Food Chem.

    (2014)
  • S. Manju et al.

    Biosens. Bioelectron.

    (2010)
  • A. Nezhadali et al.

    Talanta

    (2016)
  • P. Pasetto et al.

    Anal. Chim. Acta

    (2005)
  • B.B. Prasad et al.

    Electrochim. Acta

    (2012)
  • V. Thibert et al.

    J. Chromatogr. B.

    (2014)
  • A. Tjernberg et al.

    J. Biomol. Screen.

    (2006)
  • S. Sarzehi et al.

    Int. J. Biol. Macromol.

    (2010)
  • L. Alnaim

    J. Oncol. Pharm. Pract.

    (2007)
  • J.K. Awino et al.

    Chem. Commun.

    (2014)
  • A. Bentolila et al.

    J. Med. Chem.

    (2000)
  • Cited by (21)

    • Molecularly imprinted polymers: Applications and challenges in biological and environmental sample analysis

      2023, Molecularly Imprinted Polymers (MIPs): Commercialization Prospects
    • Analysis of sunitinib malate, a multi-targeted tyrosine kinase inhibitor: A critical review

      2021, Microchemical Journal
      Citation Excerpt :

      Plasma and serum protein was' precipitated by acetonitrile. Looking at the environmental impact of the used reagents, the method developed by Kashani et al. [33] used a regent of lower toxicity than those reagents used in the process developed by Pellizzoni et al. [32]. On the other hand, sample pre-treatment steps used by Pellizzoni et al. [32] were elementary and the number of chemicals used for the removal of the plasma matrix was tiny.

    • Introduction to nanosensors

      2019, New Developments in Nanosensors for Pharmaceutical Analysis
    • Anticancer activity and pharmacokinetics of TanshinoneⅡA derivative supramolecular hydrogels

      2018, Journal of Drug Delivery Science and Technology
      Citation Excerpt :

      As shown in Fig. 3B and C, the storage moduli (G′) of hydrogels were obviously higher than loss moduli (G″), which demonstrated Nap-Ts hydrogels were solid-like materials. The above results indicated that we successfully prepared injectable hydrogels of Ts-OH as drug delivery [23]. The crystal structural analyses of the products powder were measured by XRD assays.

    View all citing articles on Scopus
    1

    Centro di Riferimento Oncologico, IRCCS, Via Franco Gallini 2, 33081 Aviano, Italy.

    View full text