Novel Cytochrome P450-3A4 Enzymatic Nanobiosensor for Lapatinib (a Breast Cancer Drug) Developed on a Poly(anilino-co-4-aminobenzoic Acid-Green-Synthesised Indium Nanoparticle) Platform

Abstract

Breast cancer (BC) is one of the most common types of cancer disease worldwide and it accounts for thousands of deaths annually. Lapatinib is among the preferred drugs for the treatment of breast cancer. Possible drug toxicity effects of lapatinib can be controlled by real-time determination of the appropriate dose for a patient at the point of care. In this study, a novel highly sensitive polymeric nanobiosensor for lapatinib is presented. A composite of poly(anilino-co-4-aminobenzoic acid) co-polymer {poly(ANI-co-4-ABA)} and coffee extract-based green-synthesized indium nanoparticles (InNPs) was used to develop the sensor platform on a screen-printed carbon electrode (SPCE), i.e., SPCE||poly(ANI-co-4-ABA-InNPs). Cytochrome P450-3A4 (CYP3A4) enzyme and polyethylene glycol (PEG) were incorporated on the modified platform to produce the SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG lapatinib nanobiosensor. Experiments for the determination of the electrochemical response characteristics of the nanobiosensor were performed with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The nanobiosensor calibration for 0-100 ng/mL lapatinib was linear and gave limit of detection (LOD) values of 13.21 ng/mL lapatinib and 18.6 ng/mL lapatinib in physiological buffer and human serum, respectively. The LOD values are much lower than the peak plasma concentration (C_max) of lapatinib (2.43 µg/mL), which is attained 4 h after the administration of a daily dose of 1250 mg lapatinib. The electrochemical nanobiosensor also exhibited excellent anti-interference performance and stability.

Keywords: biosensor; breast cancer drug (lapatinib); cytochrome P450-3A4; green-synthesized indium nanoparticles; polyaniline.

Scheme 1

Schematic representation of the preparation of SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG nanobiosensor.

Figure 1

Typical FTIR spectra for (a) green-synthesized InNPs at a temperature of 600 $°\mathrm{C}$ and 800 $°\mathrm{C}$ and (b) the components, co-polymer and the co-polymer nanocomposite.

Figure 2

UV-vis absorption spectra of (a) green-synthesized InNPs at a temperature of 600 $°\mathrm{C}$ and 800 $°\mathrm{C}$ and (b) PANI alone, co-polymer, and the co-polymer nanocomposite. Tauc plot of (c) co-polymer (poly(ANI-co-4-ABA)) and (d) co-polymer nanocomposite (poly(ANI-co-4-ABA-InNPs)).

Figure 3

Transmission electron microscopy (TEM) images of green-synthesized InNPs at (a) 5 nm and (b) 50 nm magnification. The SEAD pattern for (c) green-synthesized InNPs, where the spherical dark zones correspond to InNPs in the bright amorphous matrix. XRD spectrum of (d) green-synthesized InNPs, recorded at room temperature. Debye–Scherrer relations and the 400-line width of XRD peak were used to the determine the average particle size (~27 nm).

Figure 4

Scanning electron microscope (SEM) image (a), and SEM-based energy-dispersive X-ray (EDX) spectrum (b), of green-synthesized InNPs in the time of 2 h.

Figure 5

SEM images of the electrode platforms on SPCEs: (a) bare SPCE, (b) electro-grafted 4-ABA, (c) electropolymerized PANI, (d) electrodeposited indium nanoparticles, (e) electropolymerized poly(ANI-co-4-ABA), and (f) electropolymerized poly(ANI-co-4-ABA-InNPs).

Figure 6

(a) CVs of electrode materials at 100 mV/s on SPCE. (b) The scan rate (ν) dependence CVs of poly(ANI-co-4-ABA-InNPs) modified electrode at 10 mV/s intervals (arrow refers to the CVs from inside outwards). 0.1 M PBS (pH 7.4) was used for the experiments under anaerobic conditions. (c) The Randles-Sevčik plots for the anodic Peak A (black line) and the cathodic Peak B (red line) of the CVs in (b).

Figure 7

Differential pulse voltammograms (DPVs) for the optimization of: (a) CYP3A4 enzyme concentration and (b) blocking buffer (BSA, MCH and PEG) for SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG nanobiosensor preparation. The measurements were performed in 10 mM DPBS buffer pH 7.4 under anaerobic conditions.

Figure 8

(a) CVs of the electrode systems at 50 mV/s scan rate, for the various stages of the nanobiosensor development. (b) Scan rate (ν) dependence CVs of SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG nanobiosensor at 10 mV/s intervals (arrow refers to the CVs from inside outwards). (c) The Randles-Sevčik plots of the anodic Peak A (black line) and the cathodic Peak B (red line) of the CVs in (b). Conditions: 100 µM CYP3A4; 5 mg/mL PEG; and argon-degassed 10 mM DPBS buffer (pH 7.4). (For the voltammograms in (a,b), A represents the anodic peak, while B and C are the cathodic peaks of the redox processes.)

Scheme 2

Schematics for the hydroxylation reaction of the SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG LAPA biosensor. Broken line (-----) separates aerobic condition (blue colour) and anaerobic condition (purple colour) sections of the reaction scheme.

Figure 9

DPVs (at 50 mV/s) and the calibration plots for SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG nanobiosensor responses to lapatinib in: (a,b) 10 mM DPBS buffer pH 7.4 and (c,d) human serum (diluted 10-fold in 10 mM DPBS buffer, pH 7.4). Each value in the calibration plots (i.e., (b,d)) corresponds to replicated experiments (n = 3) performed at −0.1 V under aerobic conditions, and the error bars indicate the RSD.

Figure 10

DPVs (at 50 mV/s) of SPCE||poly(ANI-co-4-ABA-InNPs) nanobiosensor responses to lapatinib in 10 mM DPBS buffer pH 7.4.

Figure 11

Selectivity of the SPCE||poly(ANI-co-4-ABA-InNPs)|CYP3A4|PEG nanobiosensor in the presence of 5 ng/mL LAPA and 50 ng/mL interferents: uric acid (UA), ascorbic acid (AA), glucose, and dopamine (DA) in DPBS (pH 7.4).