br Introduction br Glioblastoma GBM is the most malignant
Glioblastoma (GBM) is the most malignant primary human brain cancer, and improved treatments for this disease are needed [1–7]. Increasing evidence has shown that a subset of GBM cells have stem-like properties and the ability to initiate tumors [8,9]. These cells, being resistant to conventional treatments, can migrate away from the pri-mary tumor mass and form new tumors, which is thought to be re-sponsible, in part, for the nearly inevitable recurrence of GBM despite aggressive treatment [10,11]. A novel treatment modality with poten-tial for clinical success is the use of RNA interference (RNAi), a natural
cellular process that suppresses gene expression with high sequence specificity following introduction of short interfering RNA (siRNA) into cells . By knocking down the expression of genes that promote cancer cell survival, migration, and tumorigenicity, siRNA can be a powerful tool for treating GBM.
However, siRNA must reach the cytoplasm of cells to be eﬀective, and delivering these oligonucleotides into cells safely, eﬃciently, and in combination is challenging. Viruses have historically been used as very eﬃcient nucleic Erteberel delivery vectors, but the toxicity and im-munogenicity associated with viruses hinders their clinical use. Also, viral vectors are generally limited to the delivery of only one siRNA
∗ Corresponding author. Department of Biomedical Engineering, Translational Tissue Engineering Center, And Institute for NanoBioTechnology, Johns Hopkins School of Medicine, Baltimore, MD, 21231, USA.
∗∗ Corresponding author. Departments of Neurosurgery and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA., E-mail addresses: [email protected] (A. Quiñones-Hinojosa), [email protected] (J.J. Green).
sequence per virus particle due to size constraints on the nucleic acid cargo . In previous work, we have developed a synthetic bior-educible poly(beta-amino ester) (PBAE) that is highly eﬀective at de-livering siRNA to GBM cells . Because this PBAE nanoparticle is degradable by hydrolysis as well as by bioreduction in the relatively reducing cytoplasmic environment, it exhibits low non-specific toxicity. Importantly, the cationic PBAE forms electrostatic complexes with ne-gatively charged cargo like siRNA spontaneously in aqueous conditions. We have also found that each nanocomplex contains many copies of siRNA, thus allowing multiple siRNA molecules, including multiple diﬀerent siRNA sequences, to be delivered simultaneously in the same particle targeting various genes .
siRNA is promising as a therapeutic to treat brain cancer for several reasons: i) it can hit targets that are seen as “undruggable” by con-ventional medicinal approaches; ii) it has the potential of overcoming drug resistances by aﬀecting multiple disease-causing biochemical pathways in parallel; iii) it does not necessarily require reformulation of a drug delivery particle when the type of siRNA cargo changes; and, unlike DNA, iv) it does not risk introducing inheritable genetic changes. Protein targets of particular interest to us include Roundabout homolog 1 (Robo1), a protein identified as key to normal and cancer cell mi-gration [16–18]; yes-associated protein 1 (YAP1), which promotes growth of GBM cells [19,20]; sodium-potassium-chloride cotransporter (NKCC1), an ion transporter that aﬀects cancer cell migration [21,22]; survivin, an anti-apoptotic gene whose expression in GBM correlates with proliferation  and with poor prognosis ; and endothelial growth factor receptor (EGFR), an oncogene whose aberrant expression is among the most common mutations in GBM [25–27]. Other reports of knockdown of Robo1 , YAP1 , NKCC1 [21,29], survivin , or EGFR  often focus on knockdown of a single gene to isolate cellular mechanisms of GBM survival, growth, and invasion. Of the studies that have used RNAi against one of these targets as a therapeutic strategy, to our knowledge, this is the first report of a delivery system that causes knockdown of all five of these targets at once for GBM therapy.
In this study, we design and use bioreducible PBAE nanoparticles to deliver siRNA to primary human GBM cells in vitro and in vivo and show both eﬀective gene knockdown and also the functional eﬀect of redu-cing cancer cell viability. For the first time, the potential to deliver five separate siRNAs within a single type of PBAE nanoparticle has been validated, opening up new avenues for combination cancer therapy. We show the eﬃcacy of this treatment strategy in vivo by using the PBAE to intratumorally administer a combination of siRNAs to an orthotopic brain tumor, causing a reduction in the tumor burden compared to the control. This strategy may serve as a widely applicable platform to treat many diﬀerent types of cancer that are otherwise refractory to treat-ment.