Synthesis of magnetic molecularly imprinted polymers with excellent biocompatibility for the selective separation and inhibition of testosterone in prostate cancer cells

Figures & data

Figure 1 Schematic diagram of the synthetic route for Fe₃O₄@TSTO-MIPs.

Abbreviations: APTES, aminopropyltriethoxysilane; MIPs, molecularly imprinted polymers; TSTO, testosterone.

Figure 2 Effect of the amount of gelation on the imprinting performance of Fe₃O₄@ TSTO-MIPs and Fe₃O₄@NIPs.

Abbreviations: IF, imprinting factor; MIPs, molecularly imprinted polymers; NIPs, nonimprinted polymers; TSTO, testosterone.

Figure 3 TEM images of Fe₃O₄ (A) and Fe₃O₄@TSTO-MIPs (B).

Abbreviations: MIPs, molecularly imprinted polymers; TEM, transmission electron microscopy; TSTO, testosterone.

Figure 4 FT-IR spectra (A), XRD (B), and VSM (C) of Fe₃O₄ (a), Fe₃O₄@TSTO-MIPs (b), Fe₃O₄@NH₂ (c), and Fe₃O₄@CHO (d).

Abbreviations: FT-IR, Fourier transform infrared; MIPs, molecularly imprinted polymers; TSTO, testosterone; VSM, vibrating sample magnetometry; XRD, X-ray diffraction.

Figure 5 Adsorption isotherms (A) and kinetics (B) of Fe₃O₄@TSTO-MIPs and Fe₃O₄@NIPs toward TSTO.

Abbreviations: MIPs, molecularly imprinted polymers; NIPs, nonimprinted polymers; TSTO, testosterone.

Table 1 The adsorption capacity, imprinting factor, and selectivity coefficient of TSTO, DHT, MTSTO, PROG, and TSTOP for Fe₃O₄@TSTO-MIPs and Fe₃O₄@NIPsa

Targets	QMIP (mg g^−1)	QNIP (mg g^−1)	IF	SC
TSTO	5.24	1.51	3.47	–
DHT	3.61	1.43	2.52	1.38
MTSTO	2.96	1.34	2.21	1.57
PROG	2.11	1.49	1.42	2.44
TSTOP	1.43	1.37	1.04	3.34

Notes:

a In this experiment, 10 mg of Fe₃O₄@TSTO-MIPs and Fe₃O₄@NIPs was incubated in the mixture of TSTO, DHT, PROG, MTSTO, and TSTOP with a concentration of 24 μg mL⁻¹ in 5 mL of ethanol at room temperature for 35 min. n=5.

Abbreviations: DHT, dihydrotestosterone; IF, imprinting factor; MIPs, molecularly imprinted polymers; MTSTO, methyltestosterone; NIPs, nonimprinted polymers; PROG, progesterone; SC, selectivity coefficient; TSTO, testosterone; TSTOP, testosterone propionate.

Table 2 Comparison with other published methods for the determination of TSTO

Analyte	Extractant	Analytical system	LOD	References
TSTO	MISPE	HPLC-DAD	0.47 ng mL^−1	^Citation23
TSTO	MISPE	LC–MS–MS	0.30 ng mL^−1	^Citation9
TSTO	SPE C18 sorbents	HPLC–MS–MS	2.0 ng L^−1	^Citation24
TSTO	Immunoaffinity capillary column	LIF	20 ng mL^−1	^Citation10
TSTO	Fe3O4@TSTO-MIPs	HPLC-UV	0.05 ng mL^−1	This study

Abbreviations: HPLC-DAD, high-performance liquid chromatography system equipped with a diode array detector; LC, liquid chromatography; LIF, laser-induced fluorescence detection; LOD, limit of detection; MIPs, molecularly imprinted polymers; MISPE, molecularly imprinted solid-phase extraction; MS, mass spectrometry; SPE, solid-phase extraction; TSTO, testosterone; UV, ultraviolet.

Figure 6 Chromatograms of the human prostate cancer LNCaP cell spiked with TSTO at the concentration of 5.0 ng mL⁻¹ (a) and elution of absorbed Fe₃O₄@TSTO-MIPs (b).

Abbreviations: MIPs, molecularly imprinted polymers; TSTO, testosterone.

Figure 7 The state of prostate cancer cells after adding the MMIPs, observed under high-power microscope.

Notes: MMIPs could freely enter into the LNCaP prostate cancer cells with different concentrations of MMIPs after 24 h. Arrows indicate the locations of MMIPs inside cells.

Abbreviation: MMIPs, magnetic molecularly imprinted polymers.

Figure 8 MMIPs inhibit the translocation of AR into nucleus of LNCaP (A) and C4-2 (B) prostate cancer cells, as detected by immunofluorescence assay.

Notes: AR was stained by fluorescein isothiocyanate (green). Control: normal cell culture medium. 40× magnification.

Abbreviations: AR, androgen receptor; MMIPs, magnetic molecularly imprinted polymers.

Figure 9 MMIPs inhibit the expression of AR downstream target genes in LNCaP (A) and C4-2 (B) prostate cancer cells, as detected by real-time quantitative polymerase chain reaction assay.

Notes: Cells were treated with normal cell culture medium (control), medium without androgen (no androgen), MDV3100, and different concentration of MMIPs for 24 h, and then 10 ng mL⁻¹ DHT was added and made to react for 12 h.

Abbreviations: AR, androgen receptor; DHT, dihydrotestosterone; MMIPs, magnetic molecularly imprinted polymers.

Figure 10 MMIPs inhibit cell growth of LNCaP cells (A), but not C4-2 cells (B), as detected by MTT assay.

Notes: Cells were treated with 160 μg mL⁻¹ of MMIPs for 0, 24, 48, and 72 h, respectively. ***P<0.001.

Abbreviations: MMIPs, magnetic molecularly imprinted polymers; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.

Figure 11 MMIPs induce cell cycle arrest in LNCaP cells (A), but not C4-2 cells (B), as detected by cell flow cytometric analysis.

Abbreviation: MMIPs, magnetic molecularly imprinted polymers.

Figure S1 Molecular structures of template and other analogs.

Abbreviations: DHT, dihydrotestosterone; MTSTO, methyltestosterone; PROG, progesterone; TSTO, testosterone; TSTOP, testosterone propionate.

Figure S2 The reusability of Fe₃O₄@TSTO-MIPs and Fe₃O₄@NIPs toward TSTO.

Abbreviations: MIPs, molecularly imprinted polymers; NIPs, nonimprinted polymers; TSTO, testosterone.

RicanyovaJGadzala-KopciuchRReiffovaKBazelYBuszewskiBMolecularly imprinted adsorbents for preconcentration and isolation of progesterone and testosterone by solid phase extraction combined with HPLCAdsorpt J Int Adsorpt Soc2010164–5473483

 Google Scholar

BuiBTSMerlierFHauptKToward the use of a molecularly imprinted polymer in doping analysis: selective preconcentration and analysis of testosterone and epitestosterone in human urineAnal Chem201082114420442720438053

 PubMedGoogle Scholar

DanaceauJPMorrisonMSSlawsonMHQuantitative confirmation of testosterone and epitestosterone in human urine by LC/Q-ToF mass spectrometry for doping controlJ Mass Spectrom2008437993100018563866

 PubMedGoogle Scholar

ChenHXHuangTZhangXXImmunoaffinity extraction of testosterone by antibody immobilized monolithic capillary with on-line laser-induced fluorescence detectionTalanta200978125926419174235

 PubMedGoogle Scholar