Stephany Y. Tzeng

Biodegradable Polymeric Nanoparticles Show High Efficacy and Specificity at DNA Delivery to Human Glioblastoma in Vitro and in Vivo

Current glioblastoma therapies are insufficient to prevent tumor recurrence and eventual death. Here, we describe a method to treat malignant glioma by nonviral DNA delivery using biodegradable poly(β-amino ester)s (PBAEs), with a focus on the brain tumor initiating cells (BTICs), the tumor cell population believed to be responsible for the formation of new tumors and resistance to many conventional therapies. We show transfection efficacy of >60% and low biomaterial-mediated cytotoxicity in primary human BTICs in vitro even when the BTICs are grown as 3-D oncospheres. Intriguingly, we find that these polymeric nanoparticles show intrinsic specificity for nonviral transfection of primary human BTICs over primary healthy human neural progenitor cells and that this specificity is not due to differences in cellular growth rate or total cellular uptake of nanoparticles. Moreover, we demonstrate that biodegradable PBAE/DNA nanoparticles can be fabricated, lyophilized, and then stored for at least 2 years without losing efficacy, increasing the translational relevance of this technology. Using lyophilized nanoparticles, we show transgene expression by tumor cells after intratumoral injection into an orthotopic murine model of human glioblastoma. PBAE/DNA nanoparticles were more effective than naked DNA at exogenous gene expression in vivo, and tumor cells were transfected more effectively than noninvaded brain parenchyma in vivo. This work shows the potential of nonviral gene delivery tools to target human brain tumors.

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.

The Effect and Role of Carbon Atoms in Poly(β-amino ester)s for DNA Binding and Gene Delivery

Polymeric vectors for gene delivery are a promising alternative for clinical applications, as they are generally safer than viral counterparts. Our objective was to further our mechanistic understanding of polymer structure-function relationships to allow the rational design of new biomaterials. Utilizing poly(β-amino ester)s (PBAEs), we investigated polymer-DNA binding by systematically varying the polymer molecular weight, adding single carbons to the backbone and side chain of the monomers that constitute the polymers, and varying the type of polymer end group. We then sought to correlate how PBAE binding affects the polyplex diameter and ζ potential, the transfection efficacy, and its associated cytotoxicity in human breast and brain cancer cells in vitro. Among other trends, we observed in both cell lines that the PBAE-DNA binding constant is biphasic with the transfection efficacy and that the optimal values of the binding constant with respect to the transfection efficacy are in the range (1-6) × 10(4) M(-1). A binding constant in this range is necessary but not sufficient for effective transfection.

Cystamine-terminated poly(beta-amino ester)s for siRNA delivery to human mesenchymal stem cells and enhancement of osteogenic differentiation

Enhancing human mesenchymal stem cell (hMSC) differentiation via RNA interference (RNAi) could provide an effective way of controlling cell fate for tissue engineering, but a safe and effective delivery vehicle must first be developed. Here, we evaluated an array of synthetic end-modified poly(beta-amino ester) (PBAE)-based nanoparticles to optimize siRNA delivery into hMSCs. In general, cystamine-terminated polymers caused the most knockdown, with the best polymer achieving 91% knockdown 20 days post-transfection. Binding studies revealed that the cystamine-terminated polymer bound siRNA tightly at lower weight ratios of polymer to siRNA but then efficiently released siRNA upon exposure to a reducing environment, suggesting that this class of PBAEs can form tight initial interactions with its cargo and then cause efficient, environmentally-triggered release in the cytoplasm. Finally, we tested a functional application of this system by transfecting hMSCs with siRNA against an inhibitor of osteogenesis, B-cell lymphoma (Bcl)-like protein 2 (BCL2L2). This resulted in enhanced osteogenesis over 4 weeks as evidenced by Alizarin Red S staining and calcium quantification. The bioreducible PBAE/siRNA nanoparticles developed here can provide a means of safe and effective control of hMSC differentiation for a wide variety of applications.

Therapeutic nanomedicine for brain cancer

Malignant brain cancer treatment is limited by a number of barriers, including the blood-brain barrier, transport within the brain interstitium, difficulties in delivering therapeutics specifically to tumor cells, the highly invasive quality of gliomas and drug resistance. As a result, the prognosis for patients with high-grade gliomas is poor and has improved little in recent years. Nanomedicine approaches have been developed in the laboratory, with some technologies being translated to the clinic, in order to address these needs. This review discusses the obstacles to effective treatment that are currently faced in the field, as well as various nanomedicine techniques that have been used or are being explored to overcome them, with a focus on liposomal and polymeric nanoparticles.

Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer.

Polymeric materials can be used to deliver nucleic acids such as DNA plasmids and siRNA, but often have low efficacy in human cells. To improve gene delivery, an array of over 70 hydrolytically degradable and bioreducible poly(beta-amino ester)s are synthesized and the properties of over 200 nanoparticle formulations fabricated from these biomaterials are evaluated. The effect of different polymer structures on the delivery of nucleic acids of different structures and sizes, including siRNA, linear DNA, and circular DNAs (1.8-26 kb), is evaluated. Significantly, leading hydrolytically degradable polymeric nanoparticles deliver DNA to 90 ± 2% of primary human glioblastoma cells with <10% nonspecific cytotoxicity, better than leading commercially available reagents (p < 0.01). Bioreducible polymeric nanoparticles optimized for siRNA delivery cause up to 85 ± 0.6% knockdown in these cells as well while maintaining high viability. From a single dose, knockdown is higher than for Lipofectamine 2000 (p < 0.01) and persisted for one month. Polymer molecular weight is a driving factor of transfection efficacy for some polymer structures (correlation of r(2) = 0.63), but has no influence on transfection for other structures (r(2) = 0.01). Polymers with a reducible cystamine functional group dramatically improve siRNA delivery by facilitating quick release while generally decreasing DNA delivery compared with non-reducible counterparts (p < 0.01). Other material properties facilitate DNA delivery compared with siRNA delivery or increase delivery of both DNA and siRNA.

Degradable Polymer-Coated Gold Nanoparticles for Co-Delivery of DNA and siRNA

Gold nanoparticles have utility for in vitro, ex vivo and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable the simultaneous co-delivery of DNA and short interfering RNA (siRNA). To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells.

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.

Drug delivery strategies for therapeutic angiogenesis and antiangiogenesis.

Introduction: Angiogenesis is essential to human biology and of great clinical significance. Excessive or reduced angiogenesis can result in, or exacerbate, several disease states, including tumor formation, exudative age-related macular degeneration (AMD) and ischemia. Innovative drug delivery systems can increase the effectiveness of therapies used to treat angiogenesis-related diseases. Areas covered: This paper reviews the basic biology of angiogenesis, including current knowledge about its disruption in diseases, with the focus on cancer and AMD. Anti- and proangiogenic drugs available for clinical use or in development are also discussed, as well as experimental drug delivery systems that can potentially improve these therapies to enhance or reduce angiogenesis in a more controlled manner. Expert opinion: Laboratory and clinical results have shown pro- or antiangiogenic drug delivery strategies to be effective in drastically slowing disease progression. Further research in this area will increase the efficacy, specificity and duration of these therapies. Future directions with composite drug delivery systems may make possible targeting of multiple factors for synergistic effects.

Synthetic poly(ester amine) and poly(amido amine) nanoparticles for efficient DNA and siRNA delivery to human endothelial cells

Biodegradable poly(ester amine) (PEA)-based and poly(amido amine) (PAA)-based nanoparticles were developed for efficient in vitro siRNA delivery to human umbilical vein endothelial cells (HUVECs). They were screened, characterized, and compared with traditionally studied DNA-containing particles. Several of the polymeric nanoparticles tested were found to be effective for delivering functional siRNA to green fluorescent protein (GFP) + HUVECs, achieving 60%–75% GFP knockdown while maintaining high viability. While PEAs have been used previously to form polyplexes or nanoparticles for DNA delivery, highly effective siRNA delivery in hard-to-transfect human cell types has not been previously reported. PEAs and linear nondendrimeric PAAs were also found to be effective for DNA delivery to HUVECs using GFP-encoding plasmid DNA (up to 50%–60% transfection efficiency). PEAs and PAAs can be separated into groups that form polymeric nanoparticles effective for siRNA delivery, for DNA delivery, or for both.