Multi-layered stimuli responsive DNA micelles for the stepwise controlled release of small molecules.

Controllable release of multiple distinct cargoes from a nanomaterial is crucial to a variety of therapeutic and catalytic applications. In this study, we describe a DNA functionalized multi-layered surface crosslinked micelle (mlSCM) consisting of individually degradable layers. The DNA modified mlSCM has the ability to encapsulate separate small molecule cargo in distinct compartments within the nanocapsule, separated by chemical crosslinkers. Through a multistep self-assembly process, we show physical separation of internalized cargo as evidenced by electron microscopy, along with observation of chemical control over release, and chemical reaction conditions, as seen by fluorescence spectroscopy and a high-performance liquid chromatography mass spectrometry assay. Additionally, we evaluated the ability of these DNA crosslinked micelles to co-release two separate cargoes into the same cellular environment through an in vitro confocal microscopy assay. We show individualized targeting of two distinct but related dyes for the detection of ATP and mitochondria. The colocalization of these dyes indicates that unique locations and signals related to cellular respiration can be identified using a single mlSCM. Through these studies we ultimately show that the mlSCM has a tailorable design with the potential to be applied to numerous applications, ranging from sensing to drug delivery.

Chimeric siRNA-DNA Surfactants for the Enhanced Delivery and Sustained Cytotoxicity of a Gold(III) Metallodrug.

Using a recently developed nucleic acid delivery platform, we demonstrate the effective delivery of metallodrug [AuIIIBr2(SSC-Inp-OEt)] (AP228; Inp = isonipecotic moiety), a hydrophobic, low solubility gold complex cytotoxic to cancer cells. It is shown that AP228 is delivered more effectively into HeLa cells using micellular surfactant assemblies compared to that of a more polar derivative [AuIIIBr2(SSC-Inp-GlcN1)] (AP209; GlcN1 = (α,ß)-d-glucosamino moiety). When AP228 is codelivered with siRNA targeting Bcl-2, a key regulator of apoptosis, the overall cytotoxic therapeutic effects of the drug are maximized. The optimized delivery and distribution of the compound is monitored by both fluorescence microscopy and inductively coupled plasma mass spectrometry. We show that codelivery of the AP228 and Bcl-2 targeting siRNA results in a substantial increase in drug efficacy, wherein the cytotoxic therapeutic effects of the drug are maximized, reducing the IC50 from 760 nM to 11 nM. This hybrid small molecule drug and therapeutic nucleic acid delivery vehicle is shown to enable both the improved solubility and uptake of the gold(III) metallodrugs and the delivery of chemically unmodified siRNA, resulting in enhanced cytotoxic effects.

Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release.

Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

Ribozyme-Spherical Nucleic Acids.

Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant RNA such as small interfering RNA (siRNA) and microRNA (miRNA). Herein, a synthetic strategy that makes use of the spherical nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported. The properties of this novel ribozyme-SNA are characterized in the context of the targeted knockdown of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein involved in chemotherapeutic resistance of solid tumors, foremost glioblastoma multiforme (GBM). Data showing the direct cleavage of full-length MGMT mRNA, knockdown of MGMT protein, and increased sensitization of GBM cells to therapy-mediated apoptosis, independent of transfection agents, provide compelling evidence for the promising properties of this new chemical architecture.

Tunable DNA cleavage by intercalating peptidoconjugates.

The properties of a novel family of peptide-based DNA-cleavage agents are described. Examination of the DNA-cleavage activities of a systematic series of peptide-intercalator conjugates revealed trends that show a strong dependence on peptide sequence. Conjugates differing by a single residue displayed reactivities that varied over a wide range. The cleavage activity was modulated by the electrostatic or steric qualities of individual amino acids. Isomeric conjugates that differed in the position of the tether also exhibited different reactivities. The mechanism of DNA cleavage for these compounds was also probed and was determined to involve hydrogen-atom abstraction from the DNA backbone. Previous studies of these compounds indicated that amino acid peroxides were the active agents in the cleavage reaction; in this report, the chemistry underlying the reaction is characterized. The results reported provide insight into how peptide sequences can be manipulated to produce biomimetic compounds.