Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jul 22;40(1):239.
doi: 10.1186/s13046-021-02039-w.

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

Lisa Agnello  1   2 , Silvia Tortorella  1   3 , Annachiara d'Argenio  1 , Clarissa Carbone  1 , Simona Camorani  1 , Erica Locatelli  3 , Luigi Auletta  4 , Domenico Sorrentino  5 , Monica Fedele  1 , Antonella Zannetti  4 , Mauro Comes Franchini #  3 , Laura Cerchia #  6
Affiliations

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

Lisa Agnello et al. J Exp Clin Cancer Res. .

Abstract

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.

Keywords: Aptamer; Cancer targeting; EGFR; Enhanced therapeutic effects; Nanomedicine; TNBC; Targeted drug delivery.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Schematic representation of the Water-in-Oil-in-Water protocol for the obtainment of the final PNP. Encapsulated (Cis-Pt and BODIPY) and covalently labelled (Cy7 and CL4/SCR) molecules were used for multifunctional nanovectors loading/decoration. Above all, the PLGA-b-PEG-COOH schematic structure consisting in the lipo and hydro fragments
Fig. 2
Fig. 2
Selective cell uptake of BODIPY@PNPs-CL4 compared to BODIPY@PNPs-SCR. (a) Upper, Representative confocal images of MDA-MB-231 cells left untreated or treated with BODIPY@PNPs-CL4 or BODIPY@PNPs-SCR at 37 °C for different periods (from 30 to 60 min). After washing and fixation, cells were labelled with WGA (red) to visualize cell membrane and with DAPI (blue) to stain nuclei. BODIPY@PNPs are displayed in green. White squares indicate the area shown in insets in a magnified view obtained using Image J software. Lower, mean fluorescence intensity (MFI) was evaluated by Zeiss software on a minimum of 50 cells for each sample (n = 3). ****P < 0.0001; ***P < 0.001. (b) Representative confocal images of BT-549 cells treated with BODIPY@PNPs-CL4 or BODIPY@PNPs-SCR at 37 °C for 40 min. (c) Representative confocal images of MDA-MB-231 and MDA-MB-231 EGFR-KO cells treated with BODIPY@PNPs-CL4 at 37 °C for 40 min. (a-c) Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. All digital images were captured at the same setting to allow direct comparison of staining patterns
Fig. 3
Fig. 3
Cis-Pt@PNPs-CL4 are more cytotoxic than free Cis-Pt and untargeted Cis-Pt-loaded nanovectors. (a-c) Representative IC50 curves for free Cis-Pt, Cis-Pt@PNPs, Cis-Pt@PNPs-SCR and Cis-Pt@PNPs-CL4 on MDA-MB-231 and MDA-MB-231 EGFR-KO. Cells were incubated with increasing concentration of free Cis-Pt, Cis-Pt@PNPs, Cis-Pt@PNPs-SCR or Cis-Pt@PNPs-CL4 (from 0.1 to 30 µM Cis-Pt, free or encapsulated in nanoparticles). After continuous 72 h-incubation (a, b) and after 40 min incubation followed by washes (WO = washout) and 72 h recovery (c), cell viability for each sample was determined and expressed as percent of viable treated cells with respect to mock-treated controls. Data were plotted in GraphPad Prism v.8.4.3 software to draw dose-response curve and to calculate IC50 values (d). IC50 was estimated on the basis of at least three different experiments. (e) BT-549 cells were incubated with Cis-Pt, Cis-Pt@PNPs-SCR or Cis-Pt@PNPs-CL4 at the indicated concentration of Cis-Pt for 72 h and cell viability for each sample was analyzed and expressed as percent of viable treated cells with respect to mock-treated controls. Each determination represents the average of three individual experiments and error bars represent SD. ****P < 0.0001; ***P < 0.001; *P < 0.05. (f) The apoptosis of MDA-MB-231 cells receiving 72-h treatment with Cis-Pt, Cis-Pt@PNPs-CL4 or Cis-Pt@PNPs-SCR was measured by Annexin-V/PI staining and flow cytometric analysis. The cell apoptosis data are shown on the right histograms. Values are shown relative to mock treatment, arbitrarily set to 1 (n = 3). ***P < 0.001, **P < 0.01
Fig. 4
Fig. 4
Selective tumor targeting of Cy7@PNPs-CL4 compared to Cy7@PNPs-SCR and Cy7@PNPs. (a) Nude mice bearing subcutaneous MDA-MB-231 xenografts (three animals/group) were i.v. injected with Cy7@PNPs-CL4, Cy7@PNPs or Cy7@PNPs-SCR (5 nmol Cy7/100 µl) and analyzed by in vivo FRI imaging at the indicated time points (i.e., Pre: before injection, 30 min, 1 h, 3 and 24 h acquisitions). Representative images for Cy7@PNPs-CL4, Cy7@PNPs-SCR and Cy7@PNPs injected mice are shown. The scale bar is in arbitrary units and is a colorimetric representation of the minimum and maximum signals; all the depicted images are reconstructed with the same scale. (b) Graphical representation of in vivo corrected Signal Intensity (SI) at the selected time points in the three groups. ****P < 0.0001, **P < 0.01, *P < 0.05, relative to 24 h acquisition if not differently indicated by connecting lanes
Fig. 5
Fig. 5
Ex vivo FRI analysis. (a)Ex vivo FRI of representative tumors (left) and different organs (right) explanted from mice 24 h post-injection with Cy7@PNPs-CL4, Cy7@PNPs or Cy7@PNPs-SCR treatment groups. The scale bars are in arbitrary units and are a colorimetric representation of the minimum and maximum signals; for comparing the three different treatments, all the depicted images are reconstructed with the same scale. (b) Graphical representation of the normalized mean FRI Signal Intensity ± DS (n = 3) of tumors and organs in the three groups. **P ≤ 0.01 relative to both Cy7@PNPs and Cy7@PNPs-SCR
Fig. 6
Fig. 6
In vivo antitumor efficacy of Cis-Pt@PNPs-CL4 compared to free Cis-Pt and Cis-Pt@PNPs-SCR. Mice bearing MDA-MB-231 xenografts were injected intravenously with free Cis-Pt, Cis-Pt@PNPs-CL4 or Cis-Pt@PNPs-SCR (0.6 mg Cis-Pt/kg) at the times indicated by the head arrows. Day 0 marks the start of treatments. Mice treated with DPBS were used as the control group (Ctrl). (a) Tumor growth was monitored by calipers over time and experimental raw data (expressed as fold change) were interpolated with no curve fitting or regression analysis. Each point represents the mean ± SD of five mice. ****P < 0.0001, ***P < 0.001, *P < 0.05. (b) Body weight changes in mice of each group at the indicated days. The results represent the means ± SD (n = 5). (c-e) Immunoblot with anti-pERK1/2 and ERK1/2 (c), anti-caspase-3 (d) and anti-γH2AX (e) antibodies of pooled lysates from recovered tumors (n = 5). Vinculin was used as an internal control. The histograms indicate pERK1/2/vinculin (c), cleaved caspase-3/vinculin (d) and γH2AX/vinculin (e) ratio of densitometric signals. Depicted results represent one of three typical experiments performed. Molecular weights of indicated proteins are reported
See this image and copyright information in PMC

References

    1. Newman LA, Reis-Filho JS, Morrow M, Carey LA, King TA. The 2014 Society of Surgical Oncology Susan G. Komen for the Cure Symposium: triple-negative breast cancer. Ann Surg Oncol. 2015;22:874 – 82. - PubMed
    1. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429–34. doi: 10.1158/1078-0432.CCR-06-3045. - DOI - PubMed
    1. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. 2010;363:1938–48. doi: 10.1056/NEJMra1001389. - DOI - PubMed
    1. Yin L, Duan JJ, Bian XW, Yu SC. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res. 2020;22:61. doi: 10.1186/s13058-020-01296-5. - DOI - PMC - PubMed
    1. Keung MY, Wu Y, Badar F, Vadgama JV. Response of Breast Cancer Cells to PARP Inhibitors Is Independent of BRCA Status. J Clin Med. 2020;9:940. doi: 10.3390/jcm9040940. - DOI - PMC - PubMed