Optimizing cisplatin delivery to triple-negative breast cancer through novel EGFR aptamer-conjugated polymeric nanovectors

Background Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC. Methods Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses. Results We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs. Conclusions Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-021-02039-w.

. Quantification of entrapped Cisplatin through MP-AES determination of Pt. The calibration line has been obtained with the intensity measured for different molar concentrations of Pt (here is reported the λ=265.945 nm line).

Purification and characterization of BODIPY@PNPs, BODIPY@PNPs-CL4 and BODIPY@PNPs-SCR
The BODIPY containing PNPs, conjugated or not with CL4 and SCR, were washed and concentrated by using centrifugal filter devices (Amicon Ultra, Ultracell membrane with 100.000 NMWL, Millipore) to a final volume of 5 ml and finally filtered by using a syringe filters of nylon (Ø = 13 mm, 0.22 μm, Nazionale, Italy). Dynamic light scattering (DLS) analysis and ζ-potential values were obtained with a Zetasizer Nano-S (Malvern) instrument, working with a 532 nm laser beam at 25 °C, using standard cuvettes or DTS1070 Clear Disposable zeta cells, and the results expressed as average of three measurements. The DLS results reported that the BODIPY@PNPs were characterized by a diameter equal to 97.6 ± 0.3 nm, polydispersity index (PDI) of 0.142 and ζ-potential value of 0.08 mV. BODIPY@PNPs-CL4 and BODIPY@PNPs-SCR were characterized by a diameter equal to 131.9 ± 0.2 nm and 142.2 ± 1.5 nm respectively, PDI of 0.185 and negative ζ-potential value of -17.9 and -23.4 mV each.
The final concentrations of the suspensions were determined by gravimetric analysis by drying 100 µL of solution at 120 °C for 24 h then accurately weighting the residual dry matter amount. The results showed a final concentration of 8 mg/ml for BODIPY@PNPs and 5 mg/ml for both BODIPY@PNPs-CL4 and BODIPY@PNPs-SCR.

Quantitative determination of BODIPY®505-515 in BODIPY@PNPs, BODIPY@PNPs-CL4 and BODIPY@PNPs-SCR
The amount of BODIPY®505-515 entrapped in the polymeric nanoparticles was assessed by fluorescence quantitative determination using an Edinburgh FLSP920 spectrofluorometer equipped with a 450 W Xenon arc Lamp. A volume of 100 µl of each kind of sample was diluted 50 times in Dimethyl sulfoxide (DMSO) and put in an ultrasonic bath for 30 min. This process was aimed to destroy the pre-formed polymeric micelles due to the presence of organic solvent resulting in the BODIPY release. The obtained solutions have been further diluted 50 times in DMSO and the The concentration of the entrapped BODIPY®505-515 in BODIPY@PNPs resulted to be 166 µM, while the concentration in BODIPY@PNPs-CL4 and BODIPY@PNPs-SCR resulted to be respectively 13.6 µM and 9.7 µM. The higher amount of dye in the first sample is due to the different centrifugal filter steps undertaken by the aptamers-conjugated samples.

Purification and characterization of Cy7@PNPs, Cy7@PNPs-CL4 and Cy7@PNPs-SCR
The Cy7 labelled PNPs, conjugated or not with CL4 and SCR, were washed and concentrated by using centrifugal filter devices (Amicon Ultra, Ultracell membrane with 100.000 NMWL, Millipore) to a final volume of 5 ml and finally filtered by using a syringe filters of nylon (Ø = 13 mm, 0.22 μm, Nazionale, Italy). Dynamic light scattering (DLS) analysis and ζ-potential values were obtained with a Zetasizer Nano-S (Malvern) instrument, working with a 532 nm laser beam at 25 °C, using standard cuvettes or DTS1070 Clear Disposable zeta cells, and the results expressed as average of three measurements. The DLS results reported that the Cy7@PNPs were characterized by a diameter equal to 90.5 ± 0.5 nm, polydispersity index (PDI) of 0.218 and ζ-potential value of -24.9 mV. Cy7@PNPs-CL4 and Cy7@PNPs-SCR were characterized by a diameter equal to 107.9 ± 0.3 nm and 104.2 ± 2.7 nm respectively, PDI of 0.350 and negative ζ-potential value of -20.6 mV.
The final concentrations of the suspensions were determined by gravimetric analysis by drying 100 µl of solution at 120°C for 24 h then accurately weighting the residual dry matter amount. The results showed a final concentration of 4 mg/ml for Cy7@PNPs and Cy7@PNPs-CL4 and 6 mg/ml for Cy7@PNPs-SCR.

Quantitative determination of Cy7@PNPs, Cy7@PNPs-CL4 and Cy7@PNPs-SCR
The amount of Cy7 conjugated to the carboxylic residues onto the polymeric nanoparticles' surface was assessed by absorbance quantification using an Agilent Cary 3500 Multicell UV-Vis spectrophotometer equipped with a Xenon flash lamp.
A volume of 100 µl of each kind of sample was diluted 20 times in H2O and the spectrophotometric analysis was performed after a preliminary scan (λ range from 1100 to 400 nm): 745 nm was chosen as max absorption wavelength. Cy7 standards solution at different concentrations (0.5 to 5 µM) were prepared in H2O ( Figure S3). Figure S3. Determination of Cy7 by UV-Vis absorbance measurement (λabs max = 745 nm).