Saporin-loaded CD44 and EGFR dual-targeted nanogels for potent inhibition of metastatic breast cancer in vivo
Graphical abstract
Introduction
Cancer metastasis presents a main challenge for cancer therapy (Chaffer and Weinberg, 2011, Mehlen and Puisieux, 2006), and over 90% of breast cancer patients died from distant metastasis to different organs (Obenauf and Massague, 2015, Su et al., 2016). The small sized metastatic nodules often have poor vasculature and broad dissemination in invaded organs, thus may not be accessible by many current therapeutic agents (Schroeder et al., 2012). Nano-drugs that are able to target and release therapeutic agents to metastatic sites have emerged as a potent strategy to treat metastatic breast cancers (Landesman-Milo et al., 2015, Schroeder et al., 2012). For example, an albumin-bound paclitaxel (Abraxane®) has been approved to treat metastatic breast cancer (Lluch et al., 2014, Wicki et al., 2015). Nevertheless, the nanomedicines in the clinic are often restricted by relatively low tumor targetability and slow drug release at targeted metastatic sites (Yu et al., 2016b). To boost drug release in cancer cells, pH-sensitive nanoparticles as well as redox-sensitive nanogels were developed to achieve fast release of anticancer agents including paclitaxel, cabazitaxel, doxorubicin, and siRNA upon internalization into cancer cells, resulting in an obvious reduction of primary tumor growth and lung metastasis incidence in 4T1 breast tumor model (Chen et al., 2018, Tang et al., 2017, Xu et al., 2016, Yu et al., 2016a). Meanwhile, some targeting peptides like LHRH, iRGD, and tLyP-1 have been selected and decorated on nanoparticles to promote the cellular uptake of anticancer agents (cisplatin, doxorubicin, docetaxel, etc.) in metastatic breast cancer cells (Hamilton et al., 2015, Han et al., 2017, Li et al., 2015, Liang et al., 2017, Morshed et al., 2016). These active tumor-targeted nano-drugs demonstrated elevated suppression on tumor metastasis in mice bearing 4T1 and MDA-MB-231 tumors in comparison with the counterparts without targeting ligands. Although dual-targeted nano-drugs often provide better tumor selectivity and more efficient target cell uptake (Nan et al., 2017, Qiao et al., 2018, Zhao et al., 2017, Zhu et al., 2018), few of them have been employed to treat tumor metastasis. In addition, chemotherapeutic agents that used for cancer treatment often cause notorious side effects to healthy tissue and organs, and thus largely reduce their therapeutic windows and efficacy. Conversely, protein drugs possess potent anticancer effect, high specificity, and low toxicity, and have recently received growing interests for treatment of various cancers (Dutta et al., 2017, Qiu et al., 2018, Walsh, 2014, Wang et al., 2014). We previously showed that a therapeutic protein (granzyme B) could be efficiently deliver by EGFR and CD44 dual-targeting hyaluronic acid nanogels (EGFR/CD44-NGs), and resulted in nearly complete growth suppression of primary SKOV-3 and MDA-MB-231 tumors in mice, without causing obvious side effects (Chen et al., 2017b).
Here, we report on inhibiting metastatic breast cancer in mice using saporin (Sap)-loaded EGFR/CD44-NGs (Fig. 1). Saporin can irreversibly block protein synthesis in eukaryotic cells and has been advanced into clinical trials to treat different cancers including leukemia that are refractory to traditional chemotherapy (Chang et al., 2017, Kreitman et al., 2001). Nanogels possess watery atmosphere that provides high loading and excellent compatibility with proteins, are thus extremely appealing for protein delivery (Jiang et al., 2014, Li et al., 2017, Vermonden et al., 2012, Zhang et al., 2015). Hyaluronic acid (HA) and GE11 peptide (YHWYGYTPQNVI) are known to target CD44 and EGFR receptors of cancer cells, respectively (Chen et al., 2017a, Hosseinzadeh et al., 2017, Liang et al., 2016, Lim et al., 2018, Rao et al., 2016, Wang et al., 2015). Many metastatic cancer cells like 4T1 and MDA-MB-231 cells have been reported to overexpress CD44 and EGFR receptors (Liu et al., 2012, Morishige et al., 2008, Ravar et al., 2016, Yae et al., 2012, Yang et al., 2009, Yang et al., 2013). The cystamine moieties affords nanogels with reduction-sensitivity, facilitating the swift release of encapsulated Sap in cancer cells. We hypothesized that EGFR/CD44-NGs following the encapsulation of therapeutic proteins could effectively inhibit the metastasis of breast cancers in vivo.
Section snippets
Preparation of saporin-loaded dual-targeting hyaluronic acid nanogels (Sap-EGFR/CD44-NGs)
Sap-EGFR/CD44-NGs were developed by forming nanodroplets via inverse nanoprecipitation technique, followed by covalent crosslinking using catalyst-free “tetrazole-alkene” click chemistry. Briefly, HA grafted with tetrazole (HA-g-Tet), cystamine-methacrylmide (HA-g-Cys-MA), tetrazole plus GE11 (HA-g-GE11/Tet), and saporin (theoretical loading content: 2 wt%) were completely dissolved in PB (pH 7.4, 10 mM) to obtain an aqueous solution (polymer concentration = 1.25 mg/mL). The molar ratios of
Preparation of Sap-EGFR/CD44-NGs
Sap-EGFR/CD44-NGs were easily prepared from HA-g-(Cys-MA), HA-g-tetrazole, and HA-g-GE11/tetrazole by combining nanoprecipitation and photo-click-crosslinking (Fig. 2), as previously reported for granzyme B-loaded NGs (Chen et al., 2017b). Sap is much less expensive than granzyme B and currently tested for treating refractory leukemia patients (Chang et al., 2017, Kreitman et al., 2001). The loading levels were assessed by microBCA assays, and the results exhibited that almost quantitative
Conclusions
We have demonstrated that CD44 and EGFR dual-targeted nanogels (EGFR/CD44-NGs) afford enhanced targetability and protein therapy for metastatic breast cancer in vivo. Notably, EGFR/CD44-NGs display more than 6-fold higher cellular uptake than CD44 mono-targeting nanogels (CD44-NGs) in CD44 and EGFR-positive metastatic 4T1 breast cancer cells. Saporin-loaded EGFR/CD44-NGs show obviously better antitumor effect and inhibition of cell migration as compared with saporin-loaded CD44-NGs. The
Conflict of interest
None.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (NSFC 51773145, 51473110, and 51633005), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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