Summary:

Glioblastoma multiform (GBM) is one of the most malignant forms of brain tumors with very few treatment options other than aggressive surgical removal and radiotherapy. While most conventional chemotherapeutics fail when it comes to GBM, it is anticipated that modern gene therapy approaches can potentially improve prognosis. Macromolecules such as RNA and DNA will not be challenged by the drug resistance phenomenon since they bypass the cell efflux pumps that are responsible for chemoresistance. Unmodified nucleic acids on their own however, are not potent therapeutics. Since they can be immunogenic and unstable in biological fluids and due to their inefficient cellular uptake, they require a delivery vehicle. In the recent years, nanomedicine has been used to increase drug potency for a diverse collection of drug molecules. For conditions such as GBM, gene therapy approaches such as small interference RNA (siRNA) encapsulated in nanoparticles can be used to improve prognosis.  siRNA can knockdown and regulate genes of interest and when encapsulated in lipid nanoparticles (LNPs) it has proven to be highly safe and efficacious. In their article featured in ACS Nano in 2015, the Peer lab describes an siRNA-LNP system that can specifically target and get taken up by the GBM cells when injected directly in the tumor vicinity. The targeting moiety, hyaluronan (HA), is a naturally occurring glycosaminoglycan which is the ligand of the CD44 receptor. CD44 is over-expressed on many cancer cell types and HA binds only activated CD44, which is the predominant form in cancerous cells. HA conjugated siRNA-LNPs can therefore effectively target and knockdown proteins in tumors and not healthy tissue.

The Peer lab first confirmed the over-expression of CD44 on GBM cell lines and primary tumors. The high expression of CD44 on both cell types suggested that using HA as targeting moiety can be an effective approach. The authors then formulated siRNA-LNPs according to a previously established method using the NanoAssemblr^TM Benchtop tool and chemically conjugated HA to LNPs. In vitro, the HA-LNPs bound to patient glioma cells whereas the control LNPs (missing the HA) did not. The authors then encapsulated an siRNA sequence against Polo-like kinase1 (PLK1) which has been reported to play an important role in malignant transformation and at high levels, correlates to poor prognosis in glioblastoma patients. They then mimicked the cerebrospinal fluid flow for cell transfection conditions by introducing a shear flow for 10 minutes followed by static incubation to determine whether HA-LNPs are better retained by glioma cells compared to non-HA-LNPs. The robust knockdown of PLK1 in glioma cells for 96 hours after transfection was observed only for the HA-LNPs containing PLK1-siRNA and not controls. This implies that HA efficaciously binds CD44 receptors on glioma cells and facilitates internalization of LNPs.

The HA-LNPs were then tested in a glioma orthotopic xenograph model. HA-LNPs containing a fluorescently labeled siRNA were injected directly into the tumor vicinity. At different time points post-injection, brain sections were prepared and tested for the presence of a fluorescent probe incorporated into the LNPs and only animals injected with HA-LNP had detectable signal levels in all brain sections. The in vivo efficacy of HA-LNP was further assessed by 2 local administrations of PLK1 siRNA-HA-LNP at the tumor site of the GBM-bearing mice. Tumor cells were then isolated and sorted by flow cytometry for CD44 expression. The CD44 positive cells had an 80% knockdown of PLK1 as a result of HA-LNP treatment, whereas the control group which received a luciferase siRNA HA-LNP showed no knockdown of PLK1.  Additionally, analysis of the isolated primary brain cells treated with HA-LNP indicated no significant changes in proinflammatory cytokines such as TNF-α and IL-6. Moreover, the GBM mice that received four local injections of PLK1 siRNA-HA-LNP had a prolonged survival rate (95 days) compared to both mock treated (33 days) and luciferase siRNA HA-LNP treated (34.5 days) mice.

In brief, the Peer lab has developed a nanomedicine approach that could potentially battle chemoresistant glioblastoma multiform. By using HA as the targeting ligand, the siRNA-LNPs described in this study specifically target and internalize into CD44 positive cells which is a known marker for glioma cells. The Peer group also successfully knocked down PLK1, a gene correlated with GBM patient prognosis, using the same HA-LNP system. HA-LNP can be used to knockdown other target genes or a combination of them that’s personalized based on the patients’ needs and the GBM subtype and gene expression profile. The findings in this paper open new doors into treatment of drug resistant malignancies and introduces nanomedicine for delivery of siRNA as a new solution to treat brain tumors.

Abstract:

Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood–brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNP’s surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.