The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy.

Clinical translation of polymer-based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA-mediated knockdown in both glioma and melanoma cells in high-serum conditions compared to non-crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue-mediated siRNA delivery beyond the liver, unlike conventional nanoparticle-based delivery. These attributes of XbNPs facilitate robust siRNA-mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid-based therapeutics.

Next generation material interfaces for neural engineering.

Neural implant technology is rapidly progressing, and gaining broad interest in research fields such as electrical engineering, materials science, neurobiology, and data science. As the potential applications of neural devices have increased, new technologies to make neural intervention longer-lasting and less invasive have brought attention to neural interface engineering. This review will focus on recent developments in materials for neural implants, highlighting new technologies in the fields of soft electrodes, mechanical and chemical engineering of interface coatings, and remotely powered devices. In this context, novel implantation strategies, manufacturing methods, and combinatorial device functions will also be discussed.

Translational considerations for the design of untethered nanomaterials in human neural stimulation.

Neural stimulation is a powerful tool to study brain physiology and an effective treatment for many neurological disorders. Conventional interfaces use electrodes implanted in the brain. As these are often invasive and have limited spatial targeting, they carry a potential risk of side-effects. Smaller neural devices may overcome these obstacles, and as such, the field of nanoscale and remotely powered neural stimulation devices is growing. This review will report on current untethered, injectable nanomaterial technologies intended for neural stimulation, with a focus on material-tissue interface engineering. We will review nanomaterials capable of wireless neural stimulation, and discuss their stimulation mechanisms. Taking cues from more established nanomaterial fields (e.g., cancer theranostics, drug delivery), we will then discuss methods to modify material interfaces with passive and bioactive coatings. We will discuss methods of delivery to a desired brain region, particularly in the context of how delivery and localization are affected by surface modification. We will also consider each of these aspects of nanoscale neurostimulators with a focus on their prospects for translation to clinical use.

Engineered nanoparticles for systemic siRNA delivery to malignant brain tumours.

Improved delivery materials are needed to enable siRNA transport across biological barriers, including the blood-brain barrier (BBB), to treat diseases like brain cancer. We engineered bioreducible nanoparticles for systemic siRNA delivery to patient-derived glioblastoma cells in an orthotopic mouse tumor model. We first utilized a newly developed biomimetic in vitro model to evaluate and optimize the performance of the engineered bioreducible nanoparticles at crossing the brain microvascular endothelium. We performed transmission electron microscopy imaging which indicated that the engineered nanoparticles are able to cross the BBB endothelium via a vesicular mechanism. The nanoparticle formulation engineered to best cross the BBB model in vitro led to safe delivery across the BBB to the brain in vivo. The nanoparticles were internalized by human brain cancer cells, released siRNA to the cytosol via environmentally-triggered degradation, and gene silencing was obtained both in vitro and in vivo. This study opens new frontiers for the in vitro evaluation and engineering of nanomedicines for delivery to the brain, and reports a systemically administered biodegradable nanocarrier for oligonucleotide delivery to treat glioma.

Cancer-selective nanoparticles for combinatorial siRNA delivery to primary human GBM in vitro and in vivo.

Novel treatments for glioblastoma (GBM) are urgently needed, particularly those which can simultaneously target GBM cells' ability to grow and migrate. Herein, we describe a synthetic, bioreducible, biodegradable polymer that can package and deliver hundreds of siRNA molecules into a single nanoparticle, facilitating combination therapy against multiple GBM-promoting targets. We demonstrate that siRNA delivery with these polymeric nanoparticles is cancer-selective, thereby avoiding potential side effects in healthy cells. We show that we can deliver siRNAs targeting several anti-GBM genes (Robo1, YAP1, NKCC1, EGFR, and survivin) simultaneously and within the same nanoparticles. Robo1 (roundabout homolog 1) siRNA delivery by biodegradable particles was found to trigger GBM cell death, as did non-viral delivery of NKCC1, EGFR, and survivin siRNA. Most importantly, combining several anti-GBM siRNAs into a nanoparticle formulation leads to high GBM cell death, reduces GBM migration in vitro, and reduces tumor burden over time following intratumoral administration. We show that certain genes, like survivin and EGFR, are important for GBM survival, while NKCC1, is more crucial for cancer cell migration. This represents a powerful platform technology with the potential to serve as a multimodal therapeutic for cancer.

Bioreducible Polymeric Nanoparticles Containing Multiplexed Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival.

Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly(ß-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice.

Gene Delivery Particle Engineering Strategies for Shape-dependent Targeting of Cells and Tissues.

BACKGROUND: Successful gene delivery requires overcoming both systemic and intracellular obstacles before the nucleic acid cargo can successfully reach its tissue and subcellular target location. Materials & Methods: Non-viral mechanisms to enable targeting while avoiding off-target delivery have arisen via biological, chemical, and physical engineering strategies. DISCUSSION: Herein we will discuss the physical parameters in particle design that promote tissue- and cell-targeted delivery of genetic cargo. We will discuss systemic concerns, such as circulation, tissue localization, and clearance, as well as cell-scale obstacles, such as cellular uptake and nucleic acid packaging. CONCLUSION: In particular, we will focus on engineering particle shape and size in order to enhance delivery and promote precise targeting. We will also address methods to program or change particle shape in situ using environmentally triggered cues.

Polymeric nanoparticles as cancer-specific DNA delivery vectors to human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most deadly cancer in the US, with a meager 5-year survival rate of <20%. Such unfavorable numbers are closely related to the heterogeneity of the disease and the unsatisfactory therapies currently used to manage patients with invasive HCC. Outside of the clinic, gene therapy research is evolving to overcome the poor responses and toxicity associated with standard treatments. The inadequacy of gene delivery vectors, including poor intracellular delivery and cell specificity, are major barriers in the gene therapy field. Herein, we described a non-viral strategy for effective and cancer-specific DNA delivery to human HCC using biodegradable poly(beta-amino ester) (PBAE) nanoparticles (NPs). Varied PBAE NP formulations were evaluated for transfection efficacy and cytotoxicity to a range of human HCC cells as well as healthy human hepatocytes. To address HCC heterogeneity, nine different sources of human HCC cells were utilized. The polymeric NPs composed of 2-((3-aminopropyl)amino) ethanol end-modified poly(1,5-pentanediol diacrylate-co-3-amino-1-propanol) ('536') at a 25 polymer-to-DNA weight-to-weight ratio led to high transfection efficacy to all of the liver cancer lines, but not to hepatocytes. Each individual HCC line had a significantly higher percentage of exogenous gene expression than the healthy liver cells (P<0.01). Notably, this biodegradable end-modified PBAE gene delivery vector was not cytotoxic and maintained the viability of hepatocytes above 80%. In a HCC/hepatocyte co-culture model, in which cancerous and healthy cells share the same micro-environment, 536 25 w/w NPs specifically transfected cancer cells. PBAE NP administration to a subcutaneous HCC mouse model, established with one of the human lines tested in vitro, confirmed effective DNA transfection in vivo. PBAE-based NPs enabled high and preferential DNA delivery to HCC cells, sparing healthy hepatocytes. These biodegradable and liver cancer-selective NPs are a promising technology to deliver therapeutic genes to liver cancer.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics.

INTRODUCTION: The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors. AREAS COVERED: In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery. EXPERT OPINION: The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic.

Non-virally engineered human adipose mesenchymal stem cells produce BMP4, target brain tumors, and extend survival.

There is a need for enabling non-viral nanobiotechnology to allow safe and effective gene therapy and cell therapy, which can be utilized to treat devastating diseases such as brain cancer. Human adipose-derived mesenchymal stem cells (hAMSCs) display high anti-glioma tropism and represent a promising delivery vehicle for targeted brain tumor therapy. In this study, we demonstrate that non-viral, biodegradable polymeric nanoparticles (NPs) can be used to engineer hAMSCs with higher efficacy (75% of cells) than leading commercially available reagents and high cell viability. To accomplish this, we engineered a poly(beta-amino ester) (PBAE) polymer structure to transfect hAMSCs with significantly higher efficacy than Lipofectamine™ 2000. We then assessed the ability of NP-engineered hAMSCs to deliver bone morphogenetic protein 4 (BMP4), which has been shown to have a novel therapeutic effect by targeting human brain tumor initiating cells (BTIC), a source of cancer recurrence, in a human primary malignant glioma model. We demonstrated that hAMSCs genetically engineered with polymeric nanoparticles containing BMP4 plasmid DNA (BMP4/NP-hAMSCs) secrete BMP4 growth factor while maintaining their multipotency and preserving their migration and invasion capacities. We also showed that this approach can overcome a central challenge for brain therapeutics, overcoming the blood brain barrier, by demonstrating that NP-engineered hAMSCs can migrate to the brain and penetrate the brain tumor after both intranasal and systemic intravenous administration. Critically, athymic rats bearing human primary BTIC-derived tumors and treated intranasally with BMP4/NP-hAMSCs showed significantly improved survival compared to those treated with control GFP/NP-hAMCSs. This study demonstrates that synthetic polymeric nanoparticles are a safe and effective approach for stem cell-based cancer-targeting therapies.

Bioreducible Poly(Beta-Amino Ester)s for Intracellular Delivery of SiRNA.

RNA interference (RNAi) is a powerful tool to target and knockdown gene expression in a sequence-specific manner. RNAi can be achieved by the intracellular introduction of SiRNA; however, intracellular SiRNA delivery remains a challenging obstacle. Herein we describe the use of bioreducible nanoparticles formed using poly(beta-amino ester)s (PBAEs) for safe and efficient SiRNA delivery. Methods for polymer synthesis, nanoparticle formation, and SiRNA delivery using these particles are described. A template protocol for nanoparticle screening is presented and can be used to quickly optimize SiRNA delivery for novel applications.

Exploring the role of polymer structure on intracellular nucleic acid delivery via polymeric nanoparticles.

Intracellular nucleic acid delivery has the potential to treat many genetically-based diseases, however, gene delivery safety and efficacy remains a challenging obstacle. One promising approach is the use of polymers to form polymeric nanoparticles with nucleic acids that have led to exciting advances in non-viral gene delivery. Understanding the successes and failures of gene delivery polymers and structures is the key to engineering optimal polymers for gene delivery in the future. This article discusses the polymer structural features that enable effective intracellular delivery of DNA and RNA, including protection of nucleic acid cargo, cellular uptake, endosomal escape, vector unpacking, and delivery to the intracellular site of activity. The chemical properties that aid in each step of intracellular nucleic acid delivery are described and specific structures of note are highlighted. Understanding the chemical design parameters of polymeric nucleic acid delivery nanoparticles is important to achieving the goal of safe and effective non-viral genetic nanomedicine.

Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRß within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.

Bioreducible cationic polymer-based nanoparticles for efficient and environmentally triggered cytoplasmic siRNA delivery to primary human brain cancer cells.

siRNA nanomedicines can potentially treat many human diseases, but safe and effective delivery remains a challenge. DNA delivery polymers such as poly(ß-amino ester)s (PBAEs) generally cannot effectively deliver siRNA and require chemical modification to enable siRNA encapsulation and delivery. An optimal siRNA delivery nanomaterial needs to be able to bind and self-assemble with siRNA molecules that are shorter and stiffer than plasmid DNA in order to form stable nanoparticles, and needs to promote efficient siRNA release upon entry to the cytoplasm. To address these concerns, we designed, synthesized, and characterized an array of bioreducible PBAEs that self-assemble with siRNA in aqueous conditions to form nanoparticles of approximately 100 nm and that exhibit environmentally triggered siRNA release upon entering the reducing environment of the cytosol. By tuning polymer properties, including bioreducibility and hydrophobicity, we were able to fabricate polymeric nanoparticles capable of efficient gene knockdown (91 ± 1%) in primary human glioblastoma cells without significant cytotoxicity (6 ± 12%). We were also able to achieve significantly higher knockdown using these polymers with a low dose of 5 nM siRNA (76 ± 14%) compared to commercially available reagent Lipofectamine 2000 with a 4-fold higher dose of 20 nM siRNA (40 ± 7%). These bioreducible PBAEs also enabled 63 ± 16% gene knockdown using an extremely low 1 nM siRNA dose and showed preferential transfection of glioblastoma cells versus noncancer neural progenitor cells, highlighting their potential as efficient and tumor-specific carriers for siRNA-based nanomedicine.

Bioengineered nanoparticles for siRNA delivery.

Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of nonprotein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery.

A bioreducible linear poly(ß-amino ester) for siRNA delivery.

Described here is the synthesis and characterization of a novel, bioreducible linear poly(ß-amino ester) designed to condense siRNA into nanoparticles and efficiently release it upon entering the cytoplasm. Delivery of siRNA using this polymer achieved near-complete knockdown of a fluorescent marker gene in primary human glioblastoma cells with no cytotoxicity.

Effects of base polymer hydrophobicity and end-group modification on polymeric gene delivery.

A new 320-member polymer library of end-modified poly(ß-amino ester)s was synthesized. This library was chosen such that small differences to the structures of component backbone, side-chain, and end-group monomers could be systematically and simultaneously evaluated. The in vitro transfection efficacy and cytotoxicity of DNA nanoparticles formed from this library were assessed. This library approach not only enabled us to synthesize and test a large variety of structures rapidly but also provided us with a robust data set to analyze for the effect of small structural permutations to polymer chain structure. Small changes to the side chains, backbones, and end groups within this polymer library produced dramatic results, with transfection efficacy of CMV-Luc varying over 4 orders in a 96-well plate format. Increasing hydrophobicity of the base polymer backbone and side chain tended to increase transfection efficacy, but the most hydrophobic side chains and backbones showed the least requirement for a hydrophobic pair. Optimal PBAE formulations were superior to commercially available nonviral alternatives FuGENE HD and Lipofectamine 2000, enabling ~3-fold increased luminescence (2.2 × 10(6) RLU/well vs 8.1 × 10(5) RLU/well) and 2-fold increased transfection percentage (76.7% vs 42.9%) as measured by flow cytometry with comparable or reduced toxicity.