Kristen L. Kozielski

Polymeric Nanoparticles for Nonviral Gene Therapy Extend Brain Tumor Survival in Vivo

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

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

A new 320-member polymer library of end-modified poly(β-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.

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

Described here is the synthesis and characterization of a novel, bioreducible linear poly(β-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.

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 PDGFRβ 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. Stem Cells 2015;33:2773–2784

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.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics

ABSTRACT 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.

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.

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.

SiRNA nanomedicine: The promise of bioreducible materials

RNA interference (RNAi) presents an exciting opportunity for the treatment of a wide range of diseases refractory to conventional treatment strategies. However, a major barrier to clinical translation of nucleic acid-based therapies has been their delivery. Medical devices are required to safely and efficiently shuttle sensitive RNA cargos through the body to inside the cytoplasm of target cells. Polymeric nanoparticles containing environmentally triggered, bioreducible linkages represent a promising class of medical devices to achieve these delivery goals. Here, we discuss the obstacles to achieving successful RNAi using these devices, the early development and recent optimization of this technology, and the future prospects for the field. The use of RNAi to modulate gene expression has gained attention in the field of gene and drug delivery for many different applications, ranging from basic research to clinically driven design. This editorial will focus on siRNA, generally 21–25 nucleotide double-stranded RNA, whose intracellular processing can silence or cleave mRNA transcripts and lead to decreased expression of specific genes [1]. The RNAi pathway, originally discovered in Caenorhabditis elegans [2], was subsequently found to be active in many different organisms [3] and was first used largely as a tool to understand the function of various genes by observing the effect of the gene’s knockdown. To introduce siRNA to cells in culture, initial studies mainly relied on such methods as direct microinjection of double stranded RNA [4–6] or mechanical agitation of cultures in the presence of dsRNA [7]. Once inside the cell, the long dsRNA is cleaved by the ribo nuclease Dicer into smaller dsRNA, known as siRNA. The siRNA molecules, in combination with the RNA-induced silencing complex, direct the complex to mRNA transcripts complementary to the siRNA guide strand to prevent mRNA translation to protein (for a more detailed review, see Hannon [8]). This sequence-specific targeting ability greatly increases the range of pathways and processes that can be targeted by siRNA as compared to conventional drugs, as long as the sequence of the gene of interest is known. For example, manipulation of gene expression has been explored for in vitro differentiation of stem cells in the field of regenerative medicine [9,10], and aberrant gene expression levels can cause or contribute to the progression of many diseases [11,12]. However, to take full advantage of siRNA as a therapeutic or a tool for cellular engineering, an effective method of delivering siRNA to cells, both in vitro and in vivo, must be developed. As for intracellular delivery of other biologics and small-molecule drugs, nanosized devices have been a focus of research in this area. Barriers to nanoparticle-based siRNA delivery that must be overcome are outlined in Figure 1, and include compaction or encapsulation of nucleic acid, cellular uptake, avoidance of or escape from degradation in the endosomal pathway and release of siRNA from the device for incorporation into the RNA-induced silencing complex. Cationic polymers such siRNA nanomedicine: the promise of bioreducible materials

Electrophoresis of cell membrane heparan sulfate regulates galvanotaxis in glial cells

ABSTRACT Endogenous electric fields modulate many physiological processes by promoting directional migration, a process known as galvanotaxis. Despite the importance of galvanotaxis in development and disease, the mechanism by which cells sense and migrate directionally in an electric field remains unknown. Here, we show that electrophoresis of cell surface heparan sulfate (HS) critically regulates this process. HS was found to be localized at the anode-facing side in fetal neural progenitor cells (fNPCs), fNPC-derived astrocytes and brain tumor-initiating cells (BTICs), regardless of their direction of galvanotaxis. Enzymatic removal of HS and other sulfated glycosaminoglycans significantly abolished or reversed the cathodic response seen in fNPCs and BTICs. Furthermore, Slit2, a chemorepulsive ligand, was identified to be colocalized with HS in forming a ligand gradient across cellular membranes. Using both imaging and genetic modification, we propose a novel mechanism for galvanotaxis in which electrophoretic localization of HS establishes cell polarity by functioning as a co-receptor and provides repulsive guidance through Slit-Robo signaling. Highlighted Article: Cell surface heparan sulfate is a novel electric field sensor that regulates the galvanotaxis of glial cells through electrophoretic polarization and its function as a co-receptor for chemo-repulsive ligands such as Slit2.