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Lipid–Saporin Nanoparticles for the Intracellular Delivery of Cytotoxic Protein to Overcome ABC Transporter-Mediated Multidrug Resistance in Vitro and in Vivo

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Lipid–Saporin Nanoparticles for the Intracellular Delivery of Cytotoxic Protein to Overcome ABC Transporter-Mediated Multidrug Resistance in Vitro and in Vivo cancers Article Lipid–Saporin Nanoparticles for the Intracellular Delivery of Cytotoxic Protein to Overcome ABC Transporter-Mediated Multidrug Resistance In Vitro and In Vivo 1, 1, 2 1 1 Guan-Nan Zhang y, Pranav Gupta y, Ming Wang , Anna Maria Barbuti , Charles R. Ashby Jr. , Yun-Kai Zhang 1, Leli Zeng 1,3, Qiaobing Xu 2, Ying-Fang Fan 1,4,* and Zhe-Sheng Chen 1,* 1 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY 11439, USA; [email protected] (G.-N.Z.); [email protected] (P.G.); [email protected] (A.M.B.); [email protected] (C.R.A.J.); [email protected] (Y.-K.Z.); [email protected] (L.Z.) 2 Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA; [email protected] (M.W.); [email protected] (Q.X.) 3 Tomas Lindahl Nobel Laureate Laboratory, Research Centre, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518107, China 4 Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China * Correspondence: [email protected] (Y.-F.F.); [email protected] (Z.-S.C.); Tel.: +86-131-8909-7816 (Y.-F.F.); +1-718-990-1432 (Z.-S.C.); Fax: +1-718-990-1877 (Z.-S.C.) These authors contributed equally to this paper. y Received: 5 January 2020; Accepted: 16 February 2020; Published: 21 February 2020 Abstract: Although the judicious use of anticancer drugs that target one or more receptor tyrosine kinases constitutes an effective strategy to attenuate tumor growth, drug resistance is commonly encountered in cancer patients. The ATP-binding cassette transporters are one of the major contributors to the development of multidrug resistance as their overexpression significantly decreases the intracellular concentration and thus, the efficacy of certain anticancer drugs. Therefore, the development of treatment strategies that would not be susceptible to efflux or excretion by specific ABC transporters could overcome resistance to treatment. Here, we investigated the anticancer efficacy of saporin, a ribosome-inactivating protein. Since saporin has poor permeability across the cell membrane, it was encapsulated in a lipid-based nanoparticle system (EC16-1) that effectively delivered the formulation (EC16-1/saporin) intracellularly and produced anti-cancer efficacy. EC16-1/saporin, at nanomolar concentrations, significantly inhibited the cellular proliferation of parental and ABCB1- and ABCG2-overexpressing cancer cells. EC16-1/saporin did not significantly alter the subcellular localization of ABCB1 and ABCG2. In addition, EC16-1/saporin induced apoptosis in parental and ABCB1- and ABCG2-overexpressing cancer cells. In a murine model system, EC16-1/saporin significantly inhibited the tumor growth in mice xenografted with parental and ABCB1- and ABCG2-overexpressing cancer cells. Our findings suggest that the EC16-1/saporin combination could potentially be a novel therapeutic treatment in patients with parental or ABCB1- and ABCG2-positive drug-resistant cancers. Keywords: cancer; multidrug resistance; ATP-binding cassette transporter; saporin; EC16-1; nanoparticles Cancers 2020, 12, 498; doi:10.3390/cancers12020498 www.mdpi.com/journal/cancers Cancers 2020, 12, 498 2 of 17 1. Introduction Multidrug resistance (MDR) is a phenomenon in which cancer cells develop resistance to structurally and mechanistically unrelated anticancer drugs [1]. MDR is one of the major causes of chemotherapy failure and increased cancer patient mortality. In the last decades of research into the cause of MDR, several mechanisms have been identified. However, the overexpression of the ATP-binding cassette (ABC) transporters in cancer continues to be the primary mediator of MDR. The ABC transporters are a family of proteins that utilize the energy derived from the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) [2–4]. The hydrolysis of ATP by the ABC transporters catalyzes the efflux of the substrate drugs from the cancer cells, reducing their intracellular drug concentration and their efficacy [5,6]. In normal cells, the ABC transporters prevent the intracellular accumulation of potentially toxic endogenous and xenobiotic compounds; however, in cancer cells, the overexpression of these transporters significantly reduces the intracellular concentration of the substrate anticancer drug, producing desensitization, and eventually, resistance to drugs with distinct structures and mechanisms of action. To date, 49 human ABC transporters have been identified, which have been further subdivided into 7 subfamilies (ABC A through G). The two ABC transporters most commonly involved with the development of MDR in cancer cells are the ABC transporter subfamily B member 1 (ABCB1/P-gp/MDR1) and the ABC transporter subfamily G member 2 (ABCG2/BCRP/MXR) [7–10]. These two transporters are structurally distinct in that the ABCB1 transporter contains two nucleotide-binding domains (NBDs) and two transmembrane-binding domains (TMDs) [11] whereas the ABCG2 transporter, also known as a “half transporter”, contains only one TMD and one NBD, which requires a dimerization step for functional transport activity. [12,13] The ABCB1 transporter protein encoded by the ABCB1 gene located on chromosome 7p21 was the first identified ABC transporter [14,15]. The ABCB1 transporter is present on the apical membrane in a variety of tissues, including the blood–brain barrier, intestines, adrenal glands, placenta, and kidney. The transport function is vital for these tissues as it plays an important role in protecting cells from the accumulation of xenobiotics and cellular toxicants [16–18]. The ABCB1 transporter catalyzes the transport of a variety of classes of anticancer drugs, including taxanes (e.g., paclitaxel, doxetaxel), epipodophyllotoxins (e.g., etoposide and teniposide), vinca alkaloids (e.g., vinblastine and vincristine), and anthracyclines (e.g., doxorubicin and daunorubcin) [19–21]. The ABCG2 transporter is expressed in placental syncytiotrophoblasts, the apical surface of small intestines, colon epithelium, luminal surfaces of microvessels, endothelium of human brain, and in the veins and capillaries of blood vessels [22–24]. The expression of ABCG2 transporters in tumor tissues is associated with the development of resistance to chemotherapeutic drugs, such as nucleoside analogs, anthracyclines, camptothecin-derived topoisomerase I inhibitors, methotrexate, and flavopiridols [25,26]. Furthermore, clinical studies conducted in patients with adult and childhood leukemia indicate that the overexpression of the ABCG2 transporter is significantly correlated with a poor clinical prognosis [27,28]. Several recent studies have been conducted to investigate the potential of surmounting MDR by blocking the function of ABC transporters or designing novel anticancer compounds that can bypass the efflux function of these transporters. In the search for novel inhibitors of ABC transporters, the first- and second-generation tyrosine kinase inhibitors (TKIs) were considered unacceptable due to their pharmacokinetic interactions with other chemotherapeutic drugs. Although third-generation TKIs have less toxicity than the first two generations, the third-generation inhibitors did not produce beneficial results in clinical trials. Thus, it is pertinent to develop new strategies that are effective and safe while avoiding ABC transporter-mediated efflux, such as anticancer prodrugs, which can bypass adverse pharmacokinetic interactions as well as circumvent the ABC transporter function. Novel formulations can deliver anticancer drugs directly to the target site while safely bypassing the efflux transporters. One approach involves nanoparticle systems that encapsulate a protein-based toxin, allowing for a reduced concentration of anticancer drug compared to conventional small molecule-based drugs [29]. Certain nanoparticles can enter the cell by endocytosis across the cell membrane, and the loaded drug would be transported away from the ABC transporters [30,31]. This approach can be used to co-deliver Cancers 2020, 12, x FOR PEER REVIEW 3 of 17 Cancers 2020, 12, 498 3 of 17 can be used to co-deliver an anticancer drug in combination with an inhibitor of the ABC transporters, inan drug-resistant anticancer drug tumors, in combination avoiding withthe efflux an inhibitor effect on of thethe ABCintracellular transporters, drug inconcentration drug-resistant and tumors, thus re-sensitizingavoiding the ethefflux cancer effect cells on theto the intracellular anticancer drugdrug. concentration and thus re-sensitizing the cancer cellsSince to the the anticancer 1980s, protein drug. therapy, such as insulin-based formulations, have been used as a safe and effectiveSince the method 1980s, proteinto treat various therapy, diseases such as [32 insulin-based–34]. The majority formulations, of the protein-based have been used drugs as aexert safe theirand etherapeuticffective method efficacy to treat by binding various to diseases targets [ 32lo–cated34]. Theon the majority cell surface of the protein-basedor extracellular drugs domains. exert Althoughtheir therapeutic studies ehavefficacy shown by binding that the to therapeutic targets located benefits on theof protein-based cell surface or drugs extracellular are greater domains. than conventionalAlthough studies drug have delivery shown techniques, that the therapeutic the deliver benefitsy of ofprotein protein-based drug intracellularly drugs are greater is still than a considerableconventional
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