Van der Waals force-driven indomethacin-ss-paclitaxel nanodrugs for reversing multidrug resistance and enhancing NSCLC therapy

Abstract

The high expression of multidrug resistance-associated protein 1 (MRP1) in cancer cells caused serious multidrug resistance (MDR), which limited the effectiveness of paclitaxel (PTX) in non-small cell lung cancer (NSCLC) chemotherapy. Indomethacin (IND), a kind of non-steroidal anti-inflammatory drugs (NSAIDs), which has been confirmed to be a potential MRP1 inhibitor. Taking into account the advantages of old drugs without extra controversial biosafety issue, in this manuscript, the disulfide bond (-S-S-) was employed for connecting IND and PTX to construct conjugate IND-S-S-PTX, which was further self-assembled and formed nanodrug (IND-S-S-PTX NPs). The particle size of IND-S-S-PTX NPs was ~160 nm with a narrow PDI value of 0.099, which distributed well in water and also exhibited a stable characteristic. Moreover, due to the existence of disulfide bond, the NPs were sensitive to the high level of glutathione (GSH) in tumor microenvironment. Molecular dynamics (MD) simulation presented the process of self-assembly in detail. Density functional theory (DFT) calculations revealed that the main driving force in self-assembly process was originated from the van der waals force. In addition, this carrier-free nano drug delivery systems (nDDs) could reverse the MDR by downregulating the expression of MRP1 protein in A549/taxol.

Introduction

Cancer has been regarded as an urgent public health issue worldwide. In America, lung cancer was responsible for a quarter of cancer deaths and had the highest estimated mortality rate in 2020. Meanwhile, the 5-year relative survival rate of lung cancer was barely 19%, which is just higher than that of pancreatic cancer (Siegel et al., 2020). Unfortunately, the typical type of lung cancer belongs to NSCLC, accounting for almost 85% (Duan et al., 2019). First-line chemotherapy was still the priority choice for NSCLC treatment (NSCLC Meta-analysis Collaborative Group, 2014). Paclitaxel (PTX), a tubulin inhibitor with excellent anti-tumor potential, which has been applied to some cancer therapies (such as breast cancer (Diéras et al., 2020, Tolaney et al., 2015), lung cancer (Herbst et al., 2018), etc.). However, poor aqueous solubility and multidrug resistance (MDR) limited its clinical application and chemotherapeutic efficacy (Szakacs et al., 2014). In addition, there are non-negligible side effects including peripheral neuropathy (Hertz et al., 2018), cardiotoxicity (Herrmann, 2020), nephrotoxicity (Mitin et al., 2013), serious hemolysis effect (Namgung et al., 2014), etc. Formed by high-pressure homogenization, Albumin-bound paclitaxel (Abraxane®, approved by FDA), with a mean diameter of 130 nm, has shown superb therapeutic index and greater rapid clearance than solvent-based (sb-) paclitaxel (Yardley, 2013). Encouraged by the promising performance of nano-scaled medicine, a variety of multifunctional, novel and greatly potential nano drug delivery systems (nDDs) has been constructed over the past few decades. Moreover, more and more nDDs have presented the characteristics of well-defined structure (Wang et al., 2016), high drug loading (Zhang et al., 2019), stimulating response to tumor microenvironment (Cheng and Ji, 2020, Wang et al., 2019a), controlled drug release (Jin et al., 2019, Zhen et al., 2018), prolonged blood circulation and synergistic treatment (Cheng et al., 2020, Yang et al., 2018). For example, inorganic nDDs - BP-DcF@sPL, which was based on black phosphorus, achieved the effect of chemotherapy (apoptosis/necrosis), photothermal therapy (local heat and reactive oxygen species (ROS) generation) and synergistic immunotherapy by increasing the secretion of interferon (IFN)-γ and promoting dendritic cell (DC) maturity (Ou et al., 2018). Ang‐3I‐NM@(siPLK1 + siVEGFR2), a kind of Polymeric nDDs, could overcome the blood–brain barrier and showed massive tumor accumulation and unique ROS responsiveness (Zheng et al., 2019). PTX-LH2(M) of peptide nDDs possessed cell penetrating activity and showed great in-vivo efficacy (Nam et al., 2020). Clofarabine - Raltitrexed (CA:RT) of carrier-free nDDs exhibited a high drug loading, which obtained superior bioavailability, therapeutic effect and extremely reduced side effects in comparison to free drugs (Wang et al., 2018). In order to avoid the controversial biosafety issue of carrier materials, carrier-free nDDs was constructed for NSCLC therapy in this work, which possesses the nice performances of stability, appropriate solubility, sensitive release, well-defined structure and synergistic treatment.

Nano precipitation has been ubiquitous applied to prepare self-assembly nanoparticles (Xing et al., 2019). Even though many compounds or complicated materials have been reported to be successfully self-assembled into nanoparticles, it remained a great challenge for describing or explaining the mechanisms of self-assembly process in detail (Ianiro et al., 2019, Kim et al., 2017). However, molecular simulation could provide a unique angle to explore the mechanism of self-assembly process (Shao et al., 2020, Tavakkoli et al., 2016). Firstly, molecular dynamics (MD) simulations can visualize the self-assembly process in a vivid way by simplifying the model (Eslami et al., 2019b, Jain et al., 2019). Furthermore, non-bonding forces in the system, such as hydrogen bonding, electrostatic interactions, van der Waals forces and π-π stacking, can be captured dexterously, which has been widely regarded as driving forces in the self-assembly process (Chen et al., 2020, He et al., 2020, Shen et al., 2016, Wang et al., 2019b, Xiong et al., 2020, Zhang et al., 2020b). In addition, molecular simulation can also predict morphology of self-assembly micelle in specific ways (De Nicola et al., 2015, Li et al., 2019, Nie et al., 2015). More and more multiscale self-assembly process will be simulated properly and precisely with the development of algorithm model (Eslami et al., 2019a, Rolland et al., 2020, Wu et al., 2020). Therefore, for describing the self-assembly process of nDDs that we designed, MD simulations were utilized to visualize the formation of nanoparticles. The density functional calculations were employed to explore the driving forces.

A tricky problem for clinical chemotherapy of paclitaxel is inducing tumor immunosuppressive environment (TIME) (Chang et al., 2017). Furthermore, inflammation is often involved in the whole process (Palucka and Coussens, 2016). Paclitaxel can upregulate inflammatory ligands and receptors by activating toll-like receptor-4 (TLR4), which may cause systemic inflammation via inflammatory signaling pathways (NF-κB, PI3K, MAPK) (Ran, 2015, Volk-Draper et al., 2014). Hence, there are several inflammatory mediators over secretion such as tumor necrosis factor-α (TNF-α), vascular endothelial growth factor-A (VEGF-A), interleukin(IL)-1β, IL-6, IL-8 (Coussens et al., 2013, Nguyen et al., 2017, Shalapour and Karin, 2019). Those circulating inflammatory cytokines will promote the maturation, differentiation and recruitment of immunosuppressive cells (regulatory T cells (T_reg), myeloid-derived suppressor cells (MDSC), etc.), and further contribute to the TIME (Garner and de Visser, 2020). Multidrug resistance (MDR) is another obstacle which extremely restricts the clinical usage of PTX (Vaidyanathan et al., 2016). Multidrug resistance-associated protein 1 (MRP1), a kind of ABC transporter proteins, has tremendous ability to efflux massive anticancer drugs from cancer cells, which causes the low accumulation of anticancer drugs and the failing outcome of chemotherapy (Robey et al., 2018). Recently, immense evidence suggested that indomethacin (IND) not only applied to pain relief, fever recovery and anti-inflammatory but also could synergize antitumor activities. More importantly, IND was a great candidate for MRP1 inhibition (Lolli et al., 2019). Several in vivo models have confirmed that IND could downregulate the expression of MRP1 protein and increase the intracellular accumulation of chemotherapy drugs (Lee et al., 2017, Zeng et al., 2020). Meanwhile, IND greatly improved efficacy on revising TIME and promoting immune response (Zhang et al., 2019). Extensive research has shown that IND can inhibit the synthesis of prostaglandin E2 and increase the polarization of M1 phenotype macrophages, which may help to rebalance the immunity and inhibit the tumor immune escape (Martinez-Colon and Moore, 2018). Therefore, indomethacin has excellent potential to diminish several intractable problems of PTX in clinical usage, and it contributes to synergetic NSCLCL therapy as a part of carrier-free nDDs that we constructed.

Considering the synergistic chemotherapy effect of IND, the superior toxicity towards tumor of PTX and the multifunctional feature of nDDs, novel carrier-free nDDs of indomethacin-S-S-paclitaxel (IND-S-S-PTX) was designed and synthesized, where PTX and IND were bridged by a disulfide bond. The prepared nanoparticles (NPs) could be sensitive to the tumor microenvironment of high expression of GSH and realized stimulus release. NPs also overcame the poor solubility of PTX and IND. MD simulation was employed to describe the self-assembly process of NPs in detail. Meanwhile, due to the downregulation of intracellular MRP1, A549/taxol showed more sensitivity towards IND-S-S-PTX NPs.