(M1330-07-43) Biodegradable Nanoparticles for the Treatment of Triple-Negative Breast Cancer Fabricated by the Dispersion Polymerization Technique

Purpose: DNA damage activates BRCA1, and mutations in BRCA1 impair DNA repair, linking it to breast cancer pathogenesis¹. Around 70% of breast cancer cases in Triple-Negative Breast Cancer (TNBC) show BRCA1 involvement, which accounts for 15% of invasive breast cancers². TNBC is aggressive, with poor prognosis, resistance, visceral metastases, and early recurrence. TNBC lacks expression of estrogen receptor (ER), progesterone receptor (PR), and HER2, making it unresponsive to hormonal receptor-targeted therapies and HER2-targeted therapies^3,4. Chemotherapy, primarily with paclitaxel and doxorubicin, exploits the tumor’s high proliferative capacity but faces limitations such as toxicities and drug efflux, which leads to resistance⁵. Despite standard treatments, early-stage TNBC often progresses to metastatic disease, highlighting the need for improved therapies⁶. Platinum-based drugs like cisplatin, in combination with taxanes such as paclitaxel, have shown promise by inducing DNA crosslinking, inhibiting repair, and triggering apoptosis⁷. In this work, we synthesized dual-loaded stealth nanoparticles using two biodegradable polymeric carriers (polylactide and polycaprolactone) and their 1:1 blend to deliver paclitaxel and cisplatin. This approach aims to assess the synergistic effects of the drugs against TNBC cells, using a nanotechnology platform for targeted delivery with reduced toxicity, offering a more effective treatment option for TNBC patients.

Methods: Synthesis and characterization of pH-sensitive Crosslinkers and Macromonomers: The pH-sensitive crosslinker, polylactide macromonomer, and polycaprolactone macromonomer were synthesized. Characterization was by proton nuclear magnetic resonance (¹H NMR) spectroscopy, Fourier transform-infrared spectroscopy (FT-IR), Gel Permeation Chromatography (GPC), and Liquid chromatography-mass spectrometry (LC-MS). Fabrication and characterization of three types of stealth nanoparticles containing cisplatin and paclitaxel in the core: Polyethylene glycol (PEG)-surface-tagged multifunctional nanoparticles were synthesized by the dispersion polymerization method at ambient temperature. The nanoparticles fabricated were further characterized for surface morphology (Scanning electron micrograph (SEM)), Transmission electron micrograph (TEM (structure)), particle size distribution, zeta potential, and drug loading. Drug release was quantified by high-performance liquid chromatography (HPLC) (paclitaxel) and atomic absorption spectrometer (AAS) (cisplatin). In vitro cytotoxicity or antiproliferative studies and nanoparticles’ cellular uptake studies were carried out using TNBC cancer cell lines (MDA-MB-231) for various drug-loaded nanoparticle formulations.

Results: Three types of dual-loaded (paclitaxel and cisplatin) nanoparticles were successfully fabricated with polymer cores made of biodegradable polyesters (polylactide, polycaprolactone, and a 1:1 blend of both polymers) using a pH-sensitive cross-linking agent capable of degradation in the acidic tumor environment. The surface of the nanoparticles was covered with polyethylene glycol to provide stealth properties. The nanoparticles have optimum particle sizes (180-300nm), and drug release was for 6 days. The nanoparticles’ cellular uptake results indicate that discrete particle uptake was visible within 1 hour, with significantly increased internalization at 6, 24, and 48 hours. Cytotoxicity assays on the MDA-MB-231 TNBC cell line demonstrated synergistic effects of paclitaxel and cisplatin, achieving comparable cytotoxicity at reduced (half) drug concentrations.

Conclusion: Dual-loaded (paclitaxel and cisplatin) nanoparticles were developed by a dispersion polymerization process at ambient temperature, suitable for thermolabile bioactive agents. The nanoparticles were characterized for drug loading, encapsulation efficiency, in vitro release isotherms, and antiproliferative effects. The in vitro release study showed sustained drug release profiles from the nanoparticles, indicating that both hydrophilic and hydrophobic drugs can be delivered efficiently to TNBC. These multifunctional nanoparticles provide a promising drug delivery strategy for treating triple-negative breast cancer.

References: 1. Schneider BP, Winer EP, Foulkes WD, et al. Triple-Negative Breast Cancer: Risk Factors to Potential Targets. Clin Cancer Res. 2008;14(24):8010-8018. doi:10.1158/1078-0432.CCR-08-1208
2. Rouzier R, Perou CM, Symmans WF, et al. Breast Cancer Molecular Subtypes Respond Differently to Preoperative Chemotherapy. Clin Cancer Res. 2005;11(16):5678-5685. doi:10.1158/1078-0432.CCR-04-2421
3. Zhang L, Mu C, Zhang T, et al. Development of targeted therapy therapeutics to sensitize triple-negative breast cancer chemosensitivity utilizing bacteriophage phi29 derived packaging RNA. J Nanobiotechnology. 2021;19(1):13. doi:10.1186/s12951-020-00758-4
4. Jing X, Shao S, Zhang Y, et al. BRD4 inhibition suppresses PD-L1 expression in triple-negative breast cancer. Exp Cell Res. 2020;392(2):112034. doi:10.1016/j.yexcr.2020.112034
5. Yin L, Duan JJ, Bian XW, Yu S cang. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res BCR. 2020;22:61. doi:10.1186/s13058-020-01296-5
6. Lee J, Cho YJ, Lee JW, Ahn HJ. KSP siRNA/paclitaxel-loaded PEGylated cationic liposomes for overcoming resistance to KSP inhibitors: Synergistic antitumor effects in drug-resistant ovarian cancer. J Control Release Off J Control Release Soc. 2020;321:184-197. doi:10.1016/j.jconrel.2020.02.013
7. Wan X, Beaudoin JJ, Vinod N, et al. Co-delivery of paclitaxel and cisplatin in poly(2-oxazoline) polymeric micelles: Implications for drug loading, release, pharmacokinetics and outcome of ovarian and breast cancer treatments. Biomaterials. 2019;192:1-14. doi:10.1016/j.biomaterials.2018.10.032

Acknowledgements: The authors acknowledge with thanks funding support from NIH/NIGMS Grant #1 R16GM145483-01 awarded to Emmanuel O. Akala. This work was carried out in facilities supported by NCRR/NIH Grants #1 C06 RR 020608-01 and #1 C06 RR 14469-01.


Figure 1. Synthesis and Characterization of pH-sensitive Crosslinker and macromonomers. (A) Synthesis of pH-sensitive Crosslinker (di(2-methacryloyloxyethoxy)-[2,4 dimethoxyphenyl] methane), (B) 1H NMR of pH-sensitive crosslinker, (C) Synthesis of polylactide macromonomer by ring-opening polymerization (a similar ring-opening polymerization method was carried out to obtain polycaprolactone macromonomer), (D) FT-IR of polylactide macromonomer (polycaprolactone macromonomer was also analyzed by FT-IR).


Figure 2. Synthesis and Characterization of dual-drug loaded nanoparticles (a) In vitro drug release profile of dual-loaded nanoparticles, (b) SEM of drug-loaded nanoparticles, (c) TEM of drug-loaded nanoparticles


Figure 3. In vitro cellular studies in TNBC cell line (MDA-MB 231) (A) Cellular uptake images of MDA-MB 231 cells after 1 hour of incubation with rhodamine-123 loaded nanoparticles, (B) Concentration-dependent decrease in cell viability with increasing drug concentration