Daniel K. Bonner

Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

The synthetic tuning of clickable pH responsive cationic polypeptides and block copolypeptides

A series of pH responsive synthetic polypeptides has been developed based on an N-carboxyanhydride ring opening polymerization combined with a facile and versatile click chemistry. Poly(γ-propargyl L-glutamate) (PPLG) homopolymers and poly(ethylene glycol-b-γ-propargyl L-glutamate) (PEG-b-PPLG) block copolymers were substituted with various amine moieties that range in pKa and hydrophobicity, providing the basis for a library of new synthetic structures that can be tuned for specific interactions and responsive behaviors. These amine-functionalized polypeptides have the ability to change solubility, or reversibly self-assemble into micelles with changes in the degree of ionization; they also adopt an α-helical structure at biologically relevant pHs. Here we characterize the pH responsive behavior of the new polypeptides and the hydrolysis of the ester containing amine side chains. We examine the reversible micellization with block copolymers of the polypeptides and nucleic acid encapsulation that demonstrate the potential use of these materials for systemic drug and gene delivery.

Dye structure–optical property correlations in near-infrared fluorescent core-shell silica nanoparticles

In this paper we report on dye structure–optical property correlations for a range of near-infrared emitting (NIR, 650–900 nm) fluorescent dyes in a 9–14 nm diameter core-shell silica particle architecture (C dots), including Cy5, Alexa Fluor 700, DY730, Alexa Fluor 750, and DY780. For each dye an apparent per-dye enhancement in fluorescence is observed over free dye in aqueous solution ranging from 1.2× to 6.6×, highlighting the versatility of the silica encapsulation approach. For the Cy5 and DY730 dye/particle sample pairs photobleaching was undertaken and revealed that the particles photobleach slower than the dyes. For a particular NIR dye series with identical chemical backbone, DY730, DY731, DY732, and DY734, we demonstrate that with increasing number of sulfonated substituent groups the per-dye brightness enhancement decreases. Finally, we show that in special cases, like Cy5, brightness enhancement of dyes in dots over free dye may be a combination of effects from dye conjugation chemistry and immobilization in the silica matrix. These results provide powerful design criteria for next generation optical probes for applications ranging from bioimaging to security.

Intracellular Trafficking of Polyamidoamine–Poly(ethylene glycol) Block Copolymers in DNA Delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.

Crosslinked linear polyethylenimine enhances delivery of DNA to the cytoplasm.

Crosslinked polyethylenimines (PEIs) have been frequently examined over the past decade since they can maintain the transfection efficiency of commercially available, 25k branched PEI, but exhibit less cytotoxicity. The argument is often made that the degradability of such polymers, generally synthesized with either disulfide or hydrolytically degradable crosslinkers, is critical to the high efficiency and low toxicity of the system. In this work, we present a crosslinked linear PEI (xLPEI) system in which either disulfide-responsive or non-degradable linkages are incorporated. As with previous systems, strong transfection efficiency in comparison with commercial standards was achieved with low cytotoxicity. However, these properties were shown to be present when either the degradable or non-degradable crosslinker was used. Uncomplexed polymer was demonstrated to be the critical factor determining transfection efficiency for these polymers, mediating efficient endosomal escape without signs of cell membrane damage. While several crosslinked PEI systems in the literature have demonstrated the effect of the disulfide moiety, this work demonstrates that disulfide-mediated unpackaging may not be as important as conventionally thought for some PEI systems.

Large stokes-shift fluorescent silica nanoparticles with enhanced emission over free dye for single excitation multiplexing.

We describe a polycondensation reaction of silica precursors in a modified Stober-type basic ethanol solution to produce graded Stokes-shift core-shell silica nanoparticles providing bright and spectrally multiplexed sets of fluorescent tags that are excitable using a single excitation source. Dynamic light scattering and scanning electron microscopy demonstrate particle sizes in the sub-10 nm regime. Absorption matched emission comparisons between encapsulated and free dyes in aqueous solution reveal about an order of magnitude per-dye brightness enhancements that are apparent by simply looking at the solutions under laser illumination conditions.

A plug-and-play ratiometric pH-sensing nanoprobe for high-throughput investigation of endosomal escape.

An important aspect in the design of nanomaterials for delivery is an understanding of its uptake and ultimate release to the cytosol of target cells. Real-time chemical sensing using a nanoparticle-based platform affords exquisite insight into the trafficking of materials and their cargo into cells. This versatile and tunable technology provides a powerful tool to probe the mechanism of cellular entry and cytosolic delivery of a variety of materials, allowing for a simple and convenient means to screen materials towards efficient delivery of therapeutics such as nucleic acids.

Fluorescent Core-Shell Silica Nanoparticles: An Alternative Radiative Materials Platform

We report on monodisperse fluorescent core-shell silica nanoparticles (C dots) with enhanced brightness and photostability as compared to parent free dye in aqueous solution. Dots containing either tetramethylrhodamine or 7-nitrobenz-2-oxa-1,3-diazole dyes with diameters ranging from tens of nanometers to microns are discussed. The benefits of the core-shell architecture are described in terms of enhanced fluorescent yield of the fluorophores in the quasi-solid-state environment within the particle as compared with parent free dye in water. Several applications of these particles in the fields of photonics and the life sciences are discussed. Specifically, fluorescent core-shell silica nanoparticles are investigated as an active medium for photonic building blocks assembled on zinc sulfide-based seed particles. Initial assembly results for these composite raspberry structures are shown. Finally, applications in the life sciences are explored, including targeting of specific antibody receptors using these single-emission nanoparticles. We expand on single-emission core-shell architecture to incorporate environmentally-sensitive fluorophores to create quantitative ratiometric nanoscale sensors capable of interrogating chemical concentrations on the sub-cellular to molecular levels and demonstrate initial results of intracellular pH imaging. The concept of a single particle laboratory (SPL) is introduced as an active investigator of its environment.