Metal-Phenolic-Mediated Assembly of Functional Small Molecules into Nanoparticles: Assembly and Bioapplications.

Small molecules, including therapeutic drugs and tracer molecules, play a vital role in biological processing, disease treatment and diagnosis, and have inspired various nanobiotechnology approaches to realize their biological function, particularly in drug delivery. Desirable features of a delivery system for functional small molecules (FSMs) include high biocompatibility, high loading capacity, and simple manufacturing processes, without the need for chemical modification of the FSM itself. Herein, we report a simple and versatile approach, based on metal-phenolic-mediated assembly, for assembling FSMs into nanoparticles (i.e., FSM-MPN NPs) under aqueous and ambient conditions. We demonstrate loading of anticancer drugs, latency reversal agents, and fluorophores at up to ~80 % that is mostly facilitated by π and hydrophobic interactions between the FSM and nanoparticle components. Secondary nanoparticle engineering involving coating with a polyphenol-antibody thin film or sequential co-loading of multiple FSMs enables cancer cell targeting and combination delivery, respectively. Incorporating fluorophores into FSM-MPN NPs enables the visualization of biodistribution at different time points, revealing that most of these NPs are retained in the kidney and heart 24 h post intravenous administration. This work provides a viable pathway for the rational design of small molecule nanoparticle delivery platforms for diverse biological applications.

Direct Assembly of Metal-Phenolic Network Nanoparticles for Biomedical Applications.

Coordination assembly offers a versatile means to developing advanced materials for various applications. However, current strategies for assembling metal-organic networks into nanoparticles (NPs) often face challenges such as the use of toxic organic solvents, cytotoxicity because of synthetic organic ligands, and complex synthesis procedures. Herein, we directly assemble metal-organic networks into NPs using metal ions and polyphenols (i.e., metal-phenolic networks (MPNs)) in aqueous solutions without templating or seeding agents. We demonstrate the role of buffers (e.g., phosphate buffer) in governing NP formation and the engineering of the NP physicochemical properties (e.g., tunable sizes from 50 to 270 nm) by altering the assembly conditions. A library of MPN NPs is prepared using natural polyphenols and various metal ions. Diverse functional cargos, including anticancer drugs and proteins with different molecular weights and isoelectric points, are readily loaded within the NPs for various applications (e.g., biocatalysis, therapeutic delivery) by direct mixing, without surface modification, owing to the strong affinity of polyphenols to various guest molecules. This study provides insights into the assembly mechanism of metal-organic complexes into NPs and offers a simple strategy to engineer nanosized materials with desired properties for diverse biotechnological applications.

Developing the supraparticle technology for round window-mediated drug administration into the cochlea.

The semi-permeable round window membrane (RWM) is the gateway to the cochlea. Although the RWM is considered a minimally invasive and clinically accepted route for localised drug delivery to the cochlea, overcoming this barrier is challenging, hindering development of effective therapies for hearing loss. Neurotrophin 3 (NT3) is an emerging treatment option for hearing loss, but its therapeutic effect relies on sustained delivery across the RWM into the cochlea. Silica supraparticles (SPs) are drug delivery carriers capable of providing long-term NT3 delivery, when injected directly into the guinea pig cochlea. However, for clinical translation, a RWM delivery approach is desirable. Here, we aimed to test approaches to improve the longevity and biodistribution of NT3 inside the cochlea after RWM implantation of SPs in guinea pigs and cats. Three approaches were tested (i) coating the SPs to slow drug release (ii) improving the retention of SPs on the RWM using a clinically approved gel formulation and (iii) permeabilising the RWM with hyaluronic acid. A radioactive tracer (iodine 125: 125I) tagged to NT3 (125I NT3) was loaded into the SPs to characterise drug pharmacokinetics in vitro and in vivo. The neurotrophin-loaded SPs were coated using a chitosan and alginate layer-by-layer coating strategy, named as '(Chi/Alg)SPs', to promote long term drug release. The guinea pigs were implanted with 5× 125I NT3 loaded (Chi/Alg) SPs on the RWM, while cats were implanted with 30× (Chi/Alg) SPs. A cohort of animals were also implanted with SPs (controls). We found that the NT3 loaded (Chi/Alg)SPs exhibited a more linear release profile compared to NT3 loaded SPs alone. The 125I NT3 loaded (Chi/Alg)SPs in fibrin sealant had efficient drug loading (~5 µg of NT3 loaded per SP that weights ~50 µg) and elution capacities (~49% over one month) in vitro. Compared to the SPs in fibrin sealant, the (Chi/Alg)SPs in fibrin sealant had a significantly slower 125I NT3 drug release profile over the first 7 days in vitro (~12% for (Chi/Alg) SPs in fibrin sealant vs ~43% for SPs in fibrin sealant). One-month post-implantation of (Chi/Alg) SPs, gamma count measurements revealed an average of 0.3 µg NT3 remained in the guinea pig cochlea, while for the cat, 1.3 µg remained. Histological analysis of cochlear tissue revealed presence of a 125I NT3 signal localised in the basilar membrane of the lower basal turn in some cochleae after 4 weeks in guinea pigs and 8 weeks in cats. Comparatively, and in contrast to the in vitro release data, implantation of the SPs presented better NT3 retention and distribution inside the cochlea in both the guinea pigs and cats. No significant difference in drug entry was observed upon acute treatment of the RWM with hyaluronic acid. Collectively, our findings indicate that SPs and (Chi/Alg)SPs can facilitate drug transfer across the RWM, with detectable levels inside the cat cochlea even after 8 weeks with the intracochlear approach. This is the first study to examine neurotrophin pharmacokinetics in the cochlea for such an extended period of times in these two animal species. Whilst promising, we note that outcomes between animals were variable, and opposing results were found between in vitro and in vivo release studies. These findings have important clinical ramifications, emphasising the need to understand the physical properties and mechanics of this complex barrier in parallel with the development of therapies for hearing loss.

Layer-by-Layer Particles Deliver Epigenetic Silencing siRNA to HIV-1 Latent Reservoir Cell Types.

For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation.

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.

mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts.

Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultrarare monogenic disease that arises due to loss-of-function mutations in the NPC1 gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic NPC1 mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that "GC3" codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately 1000-fold more potent than wild-type, unmodified mRNA in a luciferase reporter assay and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wild-type levels and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 µm2 reduction compared to Lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by NPC1 mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.

Nebulization of siRNA for inhalation therapy based on a microfluidic surface acoustic wave platform.

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.

Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs.

Engineered nanoparticles for the encapsulation of bioactive agents hold promise to improve disease diagnosis, prevention and therapy. To advance this field and enable clinical translation, the rational design of nanoparticles with controlled functionalities and a robust understanding of nanoparticle-cell interactions in the complex biological milieu are of paramount importance. Herein, a simple platform obtained through the nanocomplexation of glycogen nanoparticles and albumin is introduced for the delivery of chemotherapeutics in complex multicellular 2D and 3D systems. We found that the dendrimer-like structure of aminated glycogen nanoparticles is key to controlling the multivalent coordination and phase separation of albumin molecules to form stable glycogen-albumin nanocomplexes. The pH-responsive glycogen scaffold conferred the nanocomplexes the ability to undergo partial endosomal escape in tumour, stromal and immune cells while albumin enabled nanocomplexes to cross endothelial cells and carry therapeutic agents. Limited interactions of nanocomplexes with T cells, B cells and natural killer cells derived from human blood were observed. The nanocomplexes can accommodate chemotherapeutic drugs and release them in multicellular 2D and 3D constructs. The drugs loaded on the nanocomplexes retained their cytotoxic activity, which is comparable with the activity of the free drugs. Cancer cells were found to be more sensitive to the drugs in the presence of stromal and immune cells. Penetration and cytotoxicity of the drug-loaded nanocomplexes in tumour mimicking tissues were validated using a 3D multicellular-collagen construct in a perfusion bioreactor. The results highlight a simple and potentially scalable strategy for engineering nanocomplexes made entirely of biological macromolecules with potential use for drug delivery.

Nanoscale probing and imaging of HIV-1 RNA in cells with a chimeric LNA-DNA sensor.

Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network.

Luminescent Metal-Phenolic Networks for Multicolor Particle Labeling.

The development of fluorescence labeling techniques has attracted widespread interest in various fields, including biomedical science as it can facilitate high-resolution imaging and the spatiotemporal understanding of various biological processes. We report a supramolecular fluorescence labeling strategy using luminescent metal-phenolic networks (MPNs) constructed from metal ions, phenolic ligands, and common and commercially available dyes. The rapid labeling process (<5 min) produces ultrathin coatings (≈10 nm) on diverse particles (e.g., organic, inorganic, and biological entities) with customized luminescence (e.g., red, blue, multichromatic, and white light) simply through the selection of fluorophores. The fluorescent coatings are stable at pH values from 1 to 8 and in complex biological media owing to the dominant π interactions between the dyes and MPNs. These coatings exhibit negligible cytotoxicity and their strong fluorescence is retained even when internalized into intracellular compartments. This strategy is expected to provide a versatile approach for fluorescence labeling with potential in diverse fields across the physical and life sciences.

A Focus on "Bio" in Bio-Nanoscience: The Impact of Biological Factors on Nanomaterial Interactions.

Bio-nanoscience research encompasses studies on the interactions of nanomaterials with biological structures or what is commonly referred to as the biointerface. Fundamental studies on the influence of nanomaterial properties, including size, shape, composition, and charge, on the interaction with the biointerface have been central in bio-nanoscience to assess nanomaterial efficacy and safety for a range of biomedical applications. However, the state of the cells, tissues, or biological models can also influence the behavior of nanomaterials at the biointerface and their intracellular processing. Focusing on the "bio" in bio-nano, this review discusses the impact of biological properties at the cellular, tissue, and whole organism level that influences nanomaterial behavior, including cell type, cell cycle, tumor physiology, and disease states. Understanding how the biological factors can be addressed or exploited to enhance nanomaterial accumulation and uptake can guide the design of better and suitable models to improve the outcomes of materials in nanomedicine.

Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood.

Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.

Programmable Phototaxis of Metal-Phenolic Particle Microswimmers.

Light-driven directional motion is common in nature but remains a challenge for synthetic microparticles, particularly regarding collective motion on a macroscopic scale. Successfully engineering microparticles with light-driven collective motion could lead to breakthroughs in drug delivery, contaminant sensing, environmental remediation, and artificial life. Herein, metal-phenolic particle microswimmers capable of autonomously sensing and swimming toward an external light source are reported, with the speed regulated by the wavelength and intensity of illumination. These microswimmers can travel macroscopic distances (centimeters) and can remain illuminated for hours without degradation of motility. Experimental and theoretical analyses demonstrate that motion is generated through chemical transformations of the organic component of the metal-phenolic complex. Furthermore, cargos with specific spectral absorption profiles can be loaded into the particles and endow the particle microswimmers with activated motion corresponding to these spectral characteristics. The programmable nature of the light navigation, tunable size of the particles, and versatility of cargo loading demonstrate the versatility of these metal-phenolic particle microswimmers.

In Vivo T Cell-Targeting Nanoparticle Drug Delivery Systems: Considerations for Rational Design.

T cells play an important role in immunity and repair and are implicated in diseases, including blood cancers, viral infections, and inflammation, making them attractive targets for the treatment and prevention of diseases. Over recent years, the advent of nanomedicine has shown an increase in studies that use nanoparticles as carriers to deliver therapeutic cargo to T cells for ex vivo and in vivo applications. Nanoparticle-based delivery has several advantages, including the ability to load and protect a variety of drugs, control drug release, improve drug pharmacokinetics and biodistribution, and site- or cell-specific targeting. However, the delivery of nanoparticles to T cells remains a major technological challenge, which is primarily due to the nonphagocytic nature of T cells. In this review, we discuss the physiological barriers to effective T cell targeting and describe the different approaches used to deliver cargo-loaded nanoparticles to T cells for the treatment of disease such as T cell lymphoma and human immunodeficiency virus (HIV). In particular, engineering strategies that aim to improve nanoparticle internalization by T cells, including ligand-based targeting, will be highlighted. These nanoparticle engineering approaches are expected to inspire the development of effective nanomaterials that can target or manipulate the function of T cells for the treatment of T cell-related diseases.

Acoustofection: High-Frequency Vibrational Membrane Permeabilization for Intracellular siRNA Delivery into Nonadherent Cells.

The internalization of therapeutic molecules into cells-a critical step in enabling a suite of autologous ex vivo gene and cell therapies-is highly regulated by the lipid barrier imposed by the cell membrane. Strategies to increase the efficiency of delivering these exogenous payloads into the cell, while maintaining the integrity of both the therapeutic molecules to be delivered as well as the host cells they are delivered to, are therefore required. This is especially the case for suspension cells that are particularly difficult to transfect. In this work, we show that it is possible to enhance the uptake of short interfering RNA (siRNA) into nonadherent Jurkat and HuT 78 cells with a rapid poration-free method involving high-frequency (MHz order) acoustic excitation. The 2-fold enhancement in gene knockdown is almost comparable with that obtained with conventional nucleofection, which is among the most widely used intracellular delivery methods, but with considerably higher cell viabilities (>91% compared to approximately 76%) owing to the absence of pore formation. The rapid and effective delivery afforded by the platform, together with its low cost and scalability, therefore renders it a potent tool in the cell engineering pipeline.

Microemulsion-Assisted Templating of Metal-Stabilized Poly(ethylene glycol) Nanoparticles.

Poly(ethylene glycol) (PEG) is well known to endow nanoparticles (NPs) with low-fouling and stealth-like properties that can reduce immune system clearance in vivo, making PEG-based NPs (particularly sub-100 nm) of interest for diverse biomedical applications. However, the preparation of sub-100 nm PEG NPs with controllable size and morphology is challenging. Herein, we report a strategy based on the noncovalent coordination between PEG-polyphenolic ligands (PEG-gallol) and transition metal ions using a water-in-oil microemulsion phase to synthesize sub-100 nm PEG NPs with tunable size and morphology. The metal-phenolic coordination drives the self-assembly of the PEG-gallol/metal NPs: complexation between MnII and PEG-gallol within the microemulsions yields a series of metal-stabilized PEG NPs, including 30-50 nm solid and hollow NPs, depending on the MnII/gallol feed ratio. Variations in size and morphology are attributed to the changes in hydrophobicity of the PEG-gallol/MnII complexes at varying MnII/gallol ratios based on contact angle measurements. Small-angle X-ray scattering analysis, which is used to monitor the particle size and intermolecular interactions during NP evolution, reveals that ionic interactions are the dominant driving force in the formation of the PEG-gallol/MnII NPs. pH and cytotoxicity studies, and the low-fouling properties of the PEG-gallol/MnII NPs confirm their high biocompatibility and functionality, suggesting that PEG polyphenol-metal NPs are promising systems for biomedical applications.

RNAi therapeutics: an antiviral strategy for human infections.

Gene silencing induced by RNAi represents a promising antiviral development strategy. This review will summarise the current state of RNAi therapeutics for treating acute and chronic human virus infections. The gene silencing pathways exploited by RNAi therapeutics will be described and include both classic RNAi, inducing cytoplasmic mRNA degradation post-transcription and novel RNAi, mediating epigenetic modifications at the transcription level in the nucleus. Finally, the challenge of delivering gene modifications via RNAi will be discussed, along with the unique characteristics of respiratory versus systemic administration routes to highlight recent advances and future potential of RNAi antiviral treatment strategies.

Distribution of Particles in Human Stem Cell-Derived 3D Neuronal Cell Models: Effect of Particle Size, Charge, and Density.

Neurodegenerative diseases are generally characterized by a progressive loss of neuronal subpopulations, with no available cure to date. One of the main reasons for the limited clinical outcomes of new drug formulations is the lack of appropriate in vitro human cell models for research and validation. Stem cell technologies provide an opportunity to address this challenge by using patient-derived cells as a platform to test various drug formulations, including particle-based drug carriers. The therapeutic efficacy of drug delivery systems relies on efficient cellular uptake of the carrier and can be dependent on its size, shape, and surface chemistry. Although considerable efforts have been made to understand the effects of the physiochemical properties of particles on two-dimensional cell culture models, little is known of their effect in three-dimensional (3D) cell models of neurodegenerative diseases. Herein, we investigated the role of particle size (235-1000 nm), charge (cationic and anionic), and density (1.05 and 1.8 g cm-3) on the interactions of particles with human embryonic stem cell-derived 3D cell cultures of sensory neurons, called sensory neurospheres (sNSP). Templated layer-by-layer particles, with silica or polystyrene cores, and self-assembled glycogen/DNA polyplexes were used. Particles with sizes <280 nm effectively penetrated sNSP. Additionally, effective plasmid DNA delivery was observed up to 6 days post-transfection with glycogen/DNA polyplexes. The findings provide guidance in nanoparticle design for therapies aimed at neurodegenerative diseases, in particular Friedreich's ataxia, whereby sensory neurons are predominantly affected. They also demonstrate the application of 3D models of human sensory neurons in preclinical drug development.

Template-Mediated Assembly of DNA into Microcapsules for Immunological Modulation.

There is a need for effective vaccine delivery systems and vaccine adjuvants without extraneous excipients that can compromise or minimize their efficacy. Vaccine adjuvants cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) can effectively activate immune responses to secrete cytokines. However, CpG ODNs are not stable in serum due to enzymatic cleavage and are difficult to transport through cell membranes. Herein, DNA microcapsules made of CpG ODNs arranged into 3D nanostructures are developed to improve the serum stability and immunostimulatory effect of CpG. The DNA microcapsules allow encapsulation and co-delivery of cargoes, including glycogen. The DNA capsules, with >4 million copies of CpG motifs per capsule, are internalized in cells and accumulate in endosomes, where the Toll-like receptor 9 is engaged by CpG. The capsules induce up to 10-fold and 20-fold increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion, respectively, in RAW264.7 cells compared with CpG ODNs. Furthermore, the microcapsules stimulate TNF-α and IL-6 secretion in a concentration- and time-dependent manner. The immunostimulatory activity of the capsules correlates to their intracellular trafficking, endosomal confinement, and degradation, assessed by confocal and super-resolution microscopy. These DNA capsules can serve as both adjuvants to stimulate an immune reaction and vehicles to encapsulate vaccine peptides/genes to achieve synergistic immune effects.

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia.

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.