Nanoscale metal-organic frameworks for the delivery of nucleic acids to cancer cells.

Therapeutic nucleic acids (TNAs) are gaining increasing interest in the treatment of severe diseases including viral infections, inherited disorders, and cancers. However, the efficacy of intracellularly functioning TNAs is also reliant upon their delivery into the cellular environment, as unmodified nucleic acids are unable to cross the cell membrane mainly due to charge repulsion. Here we show that TNAs can be effectively delivered into the cellular environment using engineered nanoscale metal-organic frameworks (nanoMOFs), with the additional ability to tailor which cells receive the therapeutic cargo determined by the functional moieties grafted onto the nanoMOF's surface. This study paves the way to integrate the highly ordered programmable nucleic acids into larger-scale functionalized nanoassemblies.

Controlled Organization of Inorganic Materials Using Biological Molecules for Activating Therapeutic Functionalities.

Precise control over the assembly of biocompatible three-dimensional (3D) nanostructures would allow for programmed interactions within the cellular environment. Nucleic acids can be used as programmable crosslinkers to direct the assembly of quantum dots (QDs) and tuned to demonstrate different interparticle binding strategies. Morphologies of self-assembled QDs are evaluated via gel electrophoresis, transmission electron microscopy, small-angle X-ray scattering, and dissipative particle dynamics simulations, with all results being in good agreement. The controlled assembly of 3D QD organizations is demonstrated in cells via the colocalized emission of multiple assembled QDs, and their immunorecognition is assessed via enzyme-linked immunosorbent assays. RNA interference inducers are also embedded into the interparticle binding strategy to be released in human cells only upon QD assembly, which is demonstrated by specific gene silencing. The programmability and intracellular activity of QD assemblies offer a strategy for nucleic acids to imbue the structure and therapeutic function into the formation of complex networks of nanostructures, while the photoluminescent properties of the material allow for optical tracking in cells in vitro.

Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier.

Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.

Combination of Nucleic Acid and Mesoporous Silica Nanoparticles: Optimization and Therapeutic Performance In Vitro.

Programmable nucleic acid nanoparticles (NANPs) with precisely controlled functional compositions can regulate the conditional activation of various biological pathways and responses in human cells. However, the intracellular delivery of NANPs alone is hindered by their susceptibility to nuclease activity and inefficient crossing of biological membranes. In this work, we optimized the internalization and therapeutic performance of several representative NANPs delivered with mesoporous silica nanoparticles (MSNPs) tailored for efficient electrostatic association with NANPs. We compared the immunostimulatory properties of different NA-MS-NP complexes formed with globular, planar, and fibrous NANPs and demonstrated the maximum immunostimulation for globular NANPs. As a proof of concept, we assessed the specific gene silencing by NA-MS-NP complexes functionalized with siRNA targeting green fluorescent protein expressed in triple-negative human breast cancer cells. We showed that the fibrous NANPs have the highest silencing efficiency when compared to globular or planar counterparts. Finally, we confirmed the multimodal ability of MSNPs to co-deliver a chemotherapy drug, doxorubicin, and NANPs targeting apoptosis regulator gene BCL2 in triple-negative breast cancer and melanoma cell lines. Overall, the combination of NANPs and MSNPs may become a new promising approach to efficiently treat cancer and other diseases via the simultaneous targeting of various pathways.

A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (Nanps) for controlled gene silencing, immunostimulation, and biodistribution.

Programmable nucleic acid nanoparticles (NANPs) provide controlled coordination of therapeutic nucleic acids (TNAs) and other biological functionalities. Beyond multivalence, recent reports demonstrate that NANP technology can also elicit a specific immune response, adding another layer of customizability to this innovative approach. While the delivery of nucleic acids remains a challenge, new carriers are introduced and tested continuously. Polymeric platforms have proven to be efficient in shielding nucleic acid cargos from nuclease degradation while promoting their delivery and intracellular release. Here, we venture beyond the delivery of conventional TNAs and combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine with functionalized NANPs. Furthermore, we compare several representative NANPs to assess how their overall structures influence their delivery with the same carrier. An extensive study of various formulations both in vitro and in vivo reveals differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution, with fibrous NANPs advancing for TNA delivery.

Aptamers as Modular Components of Therapeutic Nucleic Acid Nanotechnology.

Nucleic acids play a central role in all domains of life, either as genetic blueprints or as regulators of various biochemical pathways. The chemical makeup of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), generally represented by a sequence of four monomers, also provides precise instructions for folding and higher-order assembly of these biopolymers that, in turn, dictate biological functions. The sequence-based specific 3D structures of nucleic acids led to the development of the directed evolution of oligonucleotides, SELEX (systematic evolution of ligands by exponential enrichment), against a chosen target molecule. Among the variety of functions, selected oligonucleotides named aptamers also allow targeting of cell-specific receptors with antibody-like precision and can deliver functional RNAs without a transfection agent. The advancements in the field of customizable nucleic acid nanoparticles (NANPs) opened avenues for the design of nanoassemblies utilizing aptamers for triggering or blocking cell signaling pathways or using aptamer-receptor combinations to activate therapeutic functionalities. A recent selection of fluorescent aptamers enables real-time tracking of NANP formation and interactions. The aptamers are anticipated to contribute to the future development of technologies, enabling an efficient assembly of functional NANPs in mammalian cells or in vivo. These research topics are of top importance for the field of therapeutic nucleic acid nanotechnology with the promises to scale up mass production of NANPs suitable for biomedical applications, to control the intracellular organization of biological materials to enhance the efficiency of biochemical pathways, and to enhance the therapeutic potential of NANP-based therapeutics while minimizing undesired side effects and toxicities.

Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface.

RNA is an attractive biopolymer for engineering self-assembling materials suitable for biomedical applications. Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduce gaps into the double-stranded regions of the RNA rings. Molecular dynamic simulations are used to assess the dynamic behavior and the changes in the flexibility of novel designs. The changes suggested by simulations, however, cannot be clearly confirmed by the conventional techniques such as nondenaturing polyacrylamide gel electrophoresis (native-PAGE) and dynamic light scattering (DLS). Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of new assemblies. To address these deficiencies, we introduce a computer-assisted quantification strategy. This strategy is based on an algorithmic atomic force microscopy (AFM)-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validate this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis are anticipated to provide a broad spectrum approach for the future development of nucleic acid-based nanotechnology.

Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

Fluorescence Blinking as an Output Signal for Biosensing.

We demonstrate the first biosensing strategy that relies on quantum dot (QD) fluorescence blinking to report the presence of a target molecule. Unlike other biosensors that utilize QDs, our method does not require the analyte to induce any fluorescence intensity or color changes, making it readily applicable to a wide range of target species. Instead, our approach relies on the understanding that blinking, a single particle phenomenon, is obscured when several QDs lie within the detection volume of a confocal microscope. If QDs are engineered to aggregate when they encounter a particular target molecule, the observation of quasi-continuous emission should indicate its presence. As proof of concept, we programmed DNAs to drive rapid isothermal assembly of QDs in the presence of a target strand (oncogene K-ras). The assemblies, confirmed by various gel techniques, contained multiple QDs and were readily distinguished from free QDs by the absence of blinking.