Nucleolin aptamer conjugated MSNPs-PLR-PEG multifunctional nanoconstructs for targeted co-delivery of anticancer drug and siRNA to counter drug resistance in TNBC

Highlights

• First report of targeted co-delivery of drug and siRNA in drug-resistant TNBC cells.

• DOX-resistant TNBC cells effectively internalized T-MSNPs owing to presence of AS1411.

• Significant silencing of apoptotic gene achieved via BCL-2 siRNA and T-MSNPs.

• 40-fold reduction in IC₅₀ achieved for DOX and BCL-2 siRNA co-delivered via T-MSNPs.

• T-MSNPs showed significantly higher cytotoxicity via targeted co-delivery in 3D scaffold.

Abstract

The emergence of drug resistance in cancer cells is among the major challenges for treating cancer. In the last few years, the co-delivery of drug and siRNA has shown promising results against drug-resistant cancers. In the present study, we developed mesoporous silica-based multifunctional nanocarrier for co-delivery against drug-resistant triple-negative breast cancer (TNBC) cells. We synthesized the nanocarrier by modifying mesoporous silica nanoparticles with poly-L-arginine, polyethylene glycol and AS1411 aptamer to impart siRNA binding ability, biocompatibility, and cancer cell specificity, respectively. We optimized the loading of doxorubicin (DOX) within the developed nanocarrier to avoid interference with siRNA binding. We ascertained the target specificity by performing a receptor blockade assay during cellular uptake studies. The cytotoxic efficacy of DOX and siRNA co-delivered using the developed nanocarrier was assessed using DOX-resistant MDA-MB-231 TNBC cells. The nanocarrier exhibited >10-fold and 40-fold reduction in the IC₅₀ values of DOX due to co-delivery with BCl-xL and BCL-2 siRNA, respectively. The results were further validated using a 3-D in vitro cell culture system. This study demonstrates that the targeted co-delivery of drug and siRNA has a strong potential to overcome drug resistance in TNBC cells.

Introduction

Triple-negative breast cancer (TNBCs), arguably, is the most aggressive form of breast cancer and is characterized by lack of hormone receptors (ERs and PRs) and excessive expression of HER2 [1]. The majority of TNBC patients demonstrate an inadequate response to treatment strategies. Only ∼35–40 % of patients suffering from TNBCs show a complete pathological response (pCR) with similar survival rates. Approximately 15–20 % of the young female (<40 years) population diagnosed with breast cancer are found to be suffering from TNBCs [2]. Patients suffering from TNBCs are expected to demonstrate a first 5-year survival rate of 70 %, i.e., 10 % lower than other breast cancer subtypes [3]. Currently, a significant hurdle in developing an appropriate treatment strategy for TNBCs is the heterogeneity of TNBCs and their resistance to various treatment strategies [4], [5], [6]. The primary treatment strategy for TNBCs involves a chemotherapeutic approach comprising the regulated use of anthracyclines, taxanes, or platinum compounds for disrupting cancer cells. Since most TNBC patients receiving chemotherapy do not reach pCR, a discussion regarding whether chemotherapy choices need to be distinct for TNBCs is ongoing [7]. Various treatment strategies presently used clinically for TNBCs are not highly effective due to the lack of targets known to be frequently involved in driving oncogenic transformation [4]. Further, other therapies, like hormone therapy, immunotherapy, radiation therapy, surgery, etc., are conventionally employed for the treatment. However, the usage of these therapies is limited by significant side effects, reduced efficacy, and resistance to anticancer drugs. The development of resistance to chemotherapy could be ascribed to the genetically highly diverse population of tumor cells in TNBCs, which can overcome treatment by enhanced expression of anti-apoptotic genes or genes involved in drug-metabolizing pathways [8], [9]. In such a scenario, siRNAs hold tremendous potential as targeted therapeutics. The ability of small interfering RNAs (siRNA) to silence desired genes selectively has generated massive awareness of their use as therapeutic agents. These siRNAs can be delivered to cancer cells using nanocarriers [10]. The inclusion of siRNA in nanocarrier systems may be through physical entrapment, electrostatic interaction, or chemical conjugation [11]. However, the use of either siRNA or drug is insufficient to combat the emerging threat of drug resistance. One approach to overcome the issue is to co-deliver the drug and siRNA, where siRNA can modulate the expression level of the molecules responsible for resistance, thus enhancing the drug's chemotherapeutic effect. However, due to various limitations, delivering drug and siRNA together is a formidable task. Nanotechnology is at the forefront in offering multiple options to co-deliver different bioactive molecules. One such nanocarrier, MSNP, has shown great potential for the co-delivery of cargo.

MSNPs have emerged as promising drug carriers because of tunable pore sizes and high pore volume [12], [13], [14]. The possibility of diverse surface functionalization allows MSNPs to interact with a range of agents like polymers, dendrimers, bio-macromolecules, nanoparticles, and even therapeutic drugs. Considering these facts, few researchers have shown effective co-delivery of drugs and other biomacromolecules (e.g., siRNAs) using MSNPs. These reports have primarily utilized passive targeting of the nanocarrier to the target site. Passive targeting (based on the EPR effect) forms the basis of various cancer treatment strategies, but it has a few drawbacks [15], [16], [17]. Variation in the diffusion efficiency of drug molecules makes it increasingly harder to target all the cells within the tumor. Furthermore, lack of specificity in passive targeting leads to difficulty in process control, ultimately resulting in drug resistance. On the other hand, active targeting utilizes molecules on the nanocarrier, which can bind specifically to a receptor on cancer cells. This approach provides a greater degree of control than passive targeting wherein, post extravasation, the nanocarriers bind to cancer cells through specific interaction. Such binding specificity could be achieved by modifying nanocarriers with cancer-targeting antigens through various conjugation chemistries [18], [19]. These nanocarriers utilize receptor-ligand interactions for recognizing tumor cells, and the bound nanocarriers are effectively internalized by tumor cells, wherein the therapeutic molecules are released. A pre-requisite for such an approach is the overexpression of the receptors on cancer cells allowing targeting of these cells [12], [20].

Nucleolin is one such antigen localized on the surface of tumor cells on nucleolus dissolution. The dissolution of the nucleolus is one of the hallmarks of cancers, which also results in the dysregulation of nucleolar activities [21]. The disintegration of the nucleolus results in the release of the protein machinery [including nucleoplasmin and nucleolin (NCL)] in the cytoplasm [22]. The released NCL localizes to the membrane of the cancer cells, and thus, cancer cells pose an enhanced presence of NCL on their surface. The enhanced representation of NCL on cancer cell membranes makes them promising molecules for targeting cancer cells. To achieve nucleolin targeting, an aptamer, AS1411, has been developed against it. It is a 26-base, G-rich DNA oligonucleotide that functions as the NCL-binding aptamer [23]. AS1411 can be used to develop a platform that can effectively target different types of cancers, owing to the presence of NCL on the surface of various cancer cells. Hence, AS1411 holds tremendous potential to target nanocarriers to a wide range of cancer cells.

The present study aimed to develop an MSNPs-based nanocarrier for targeted co-delivery of drug and siRNA to drug-resistant TNBCs. To achieve this, MSNPs were sequentially modified with poly-L-arginine (PLR) and PEG. To develop a targeted nanocarrier, AS1411 aptamer has been conjugated to MSNPs-PLR-PEG in the present study. In parallel, TNBC cells (MDA MB-231) were made resistant to DOX. The developed nanocarrier protected siRNA against serum-nuclease for up to 24 h and was found to be biocompatible. The co-delivery of DOX and siRNA against BCL-xL and BCL-2 showed >10- and 40-fold reduction in IC₅₀ of DOX, respectively. These results were further validated on the cells grown on 3D scaffolds. Thus, the present work highlights the potential of a developed nanocarrier against highly aggressive TNBC.