Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.

Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C3-ZIF(cell membrane type). Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C3-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C3-ZIFHELA. In vivo testing confirmed the selectivity of C3-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.

Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework.

CRISPR/Cas9 is a combined protein (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary solutions to genetic diseases. It has, however, a double delivery problem owning to the large protein size and the highly charged RNA component. In this work, we report the first example of CRISPR/Cas9 encapsulated by nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17% and enhanced endosomal escape promoted by the protonated imidazole moieties. The gene editing potential of CRISPR/Cas9 encapsulated by ZIF-8 (CC-ZIFs) is further verified by knocking down the gene expression of green fluorescent protein by 37% over 4 days. The nanoscale CC-ZIFs are biocompatible and easily scaled-up offering excellent loading capacity and controlled codelivery of intact Cas9 protein and sgRNA.

Supramolecular Self-Assembly of Histidine-Capped-Dialkoxy-Anthracene: A Visible-Light-Triggered Platform for Facile siRNA Delivery.

Supramolecular self-assembly of histidine-capped-dialkoxy-anthracene (HDA) results in the formation of light-responsive nanostructures. Single-crystal X-ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond is established between the imidazole moieties while the second involves the oxygen atom of one amide group and the hydrogen atom of a second amide group. When protonated in acidic aqueous media, HDA successfully complexes siRNA yielding spherical nanostructures. This biocompatible platform controllably delivers siRNA with high efficacy upon visible-light irradiation leading up to 90 % of gene silencing in live cells.

Microwave-Induced Chemotoxicity of Polydopamine-Coated Magnetic Nanocubes.

Polydopamine-coated FeCo nanocubes (PDFCs) were successfully synthesized and tested under microwave irradiation of 2.45 GHz frequency and 0.86 W/cm(2) power. These particles were found to be non-toxic in the absence of irradiation, but gained significant toxicity upon irradiation. Interestingly, no increase in relative heating rate was observed when the PDFCs were irradiated in solution, eliminating nanoparticle (NP)-induced thermal ablation as the source of toxicity. Based on these studies, we propose that microwave-induced redox processes generate the observed toxicity.

"Light-on" sensing of antioxidants using gold nanoclusters.

Depletion of intracellular antioxidants is linked to major cytotoxic events and cellular disorders, such as oxidative stress and multiple sclerosis. In addition to medical diagnosis, determining the concentration of antioxidants in foodstuffs, food preservatives, and cosmetics has proved to be very vital. Gold nanoclusters (Au-NCs) have a core size below 2 nm and contain several metal atoms. They have interesting photophysical properties, are readily functionalized, and are safe to use in various biomedical applications. Herein, a simple and quantitative spectroscopic method based on Au-NCs is developed to detect and image antioxidants such as ascorbic acid. The sensing mechanism is based on the fact that antioxidants can protect the fluorescence of Au-NCs against quenching by highly reactive oxygen species. Our method shows great accuracy when employed to detect the total antioxidant capacity in commercial fruit juice. Moreover, confocal fluorescence microscopy images of HeLa cells show that this approach can be successfully used to image antioxidant levels in living cells. Finally, the potential application of this "light-on" detection method in multiple logic gate fabrication was discussed using the fluorescence intensity of Au-NCs as output.

Zeolitic imidazolate frameworks activate endosomal Toll-like receptors and potentiate immunogenicity of SARS-CoV-2 spike protein trimer.

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework-8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.

A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

A Machine Learning-optimized system for on demand, pulsatile, photo- and chemo-therapeutic treatment using near-infrared responsive MoS 2 -based microparticles in a breast cancer model.

Cancer therapy research is of high interest because of the persistence and mortality of the disease and the side effects of traditional therapeutic methods, while often multimodal treatments are necessary based on the patient's needs. The development of less invasive modalities for recurring treatment cycles is thus of critical significance. Herein, a light-activatable microparticle system was developed for localized, pulsatile delivery of anticancer drugs with simultaneous thermal ablation, by applying controlled ON-OFF thermal cycles using near-infrared laser irradiation. The system is composed of poly(caprolactone) microparticles of 200 µm size with incorporated molybdenum disulfide (MoS 2 ) nanosheets as the photothermal agent and hydrophilic doxorubicin or hydrophobic violacein, as model drugs. Upon irradiation the nanosheets heat up to ≥50 °C leading to polymer matrix melting and release of the drug. MoS 2 nanosheets exhibit high photothermal conversion efficiency and allow for application of low power laser irradiation for the system activation. A Machine Learning algorithm was applied to acquire optimal laser operation conditions; 0.4 W/cm 2 laser power at 808 nm, 3-cycle irradiation, for 3 cumulative minutes. In a mouse subcutaneous model of 4T1 triple-negative breast cancer, 25 microparticles were intratumorally administered and after 3-cycle laser treatment the system conferred synergistic phototherapeutic and chemotherapeutic effect. Our on-demand, pulsatile synergistic treatment resulted in increased median survival up to 40 days post start of treatment compared to untreated mice, with complete eradication of the tumors at the primary site. Such a system could have potential for patients in need of recurring cycles of treatment on subcutaneous tumors.

Sustained and targeted delivery of checkpoint inhibitors by metal-organic frameworks for cancer immunotherapy.

The major impediments to the implementation of cancer immunotherapies are the sustained immune effect and the targeted delivery of these therapeutics, as they have life-threatening adverse effects. In this work, biomimetic metal-organic frameworks [zeolitic imidazolate frameworks (ZIFs)] are used for the controlled delivery of nivolumab (NV), a monoclonal antibody checkpoint inhibitor that was U.S. Food and Drug Administration-approved back in 2014. The sustained release behavior of NV-ZIF has shown a higher efficacy than the naked NV to activate T cells in hematological malignancies. The system was further modified by coating NV-ZIF with cancer cell membrane to enable tumor-specific targeted delivery while treating solid tumors. We envisage that such a biocompatible and biodegradable immunotherapeutic delivery system may promote the development and the translation of hybrid superstructures into smart and personalized delivery platforms.

Tunable and Linker Free Nanogaps in Core-Shell Plasmonic Nanorods for Selective and Quantitative Detection of Circulating Tumor Cells by SERS.

Controlling the size, number, and shape of nanogaps in plasmonic nanostructures is of significant importance for the development of novel quantum plasmonic devices and quantitative sensing techniques such as surface-enhanced Raman scattering (SERS). Here, we introduce a new synthetic method based on coordination interactions and galvanic replacement to prepare core-shell plasmonic nanorods with tunable enclosed nanogaps. Decorating Au nanorods with Raman reporters that strongly coordinate Ag+ ions (e.g., 4-mercaptopyridine) afforded uniform nucleation sites to form a sacrificial Ag shell. Galvanic replacement of the Ag shell by HAuCl4 resulted in Au-AgAu core-shell structure with a uniform intra-nanoparticle gap. The size (length and width) and morphology of the core-shell plasmonic nanorods as well as the nanogap size depend on the concentration of the coordination complexes formed between Ag+ ions and 4-mercaptopyridine. Moreover, encapsulating Raman reporters within the nanogaps afforded an internal standard for sensitive and quantitative SERS analysis. To test the applicability, core-shell plasmonic nanorods were functionalized with aptamers specific to circulating tumor cells such as MCF-7 (Michigan Cancer Foundation-7, breast cancer cell line). This system could selectively detect as low as 20 MCF-7 cells in a blood mimicking fluid employing SERS. The linking DNA duplex on core-shell plasmonic nanorods can also intercalate hydrophobic drug molecules such as Doxorubicin, thereby increasing the versatility of this sensing platform to include drug delivery. Our synthetic method offers the possibility of developing multifunctional SERS-active materials with a wide range of applications including biosensing, imaging, and therapy.

Engineering Hydrophobic Organosilica Nanoparticle-Doped Nanofibers for Enhanced and Fouling Resistant Membrane Distillation.

Engineering and scaling-up new materials for better water desalination are imperative to find alternative fresh water sources to meet future demands. Herein, the fabrication of hydrophobic poly(ether imide) composite nanofiber membranes doped with novel ethylene-pentafluorophenylene-based periodic mesoporous organosilica nanoparticles is reported for enhanced and fouling resistant membrane distillation. Novel organosilica nanoparticles were homogeneously incorporated into electrospun nanofiber membranes depicting a proportional increase of hydrophobicity to the particle contents. Direct contact membrane distillation experiments on the organosilica-doped membrane with only 5% doping showed an increase of flux of 140% compared to commercial membranes. The high porosity of organosilica nanoparticles was further utilized to load the eugenol antimicrobial agent which produced a dramatic enhancement of the antibiofouling properties of the membrane of 70% after 24 h.

Colloidal Gold Nanoclusters Spiked Silica Fillers in Mixed Matrix Coatings: Simultaneous Detection and Inhibition of Healthcare-Associated Infections.

Healthcare-associated infections (HAIs) are the infections that patients get while receiving medical treatment in a medical facility with bacterial HAIs being the most common. Silver and gold nanoparticles (NPs) have been successfully employed as antibacterial motifs; however, NPs leaching in addition to poor dispersion and overall reproducibility are major hurdles to further product development. In this study, the authors design and fabricate a smart antibacterial mixed-matrix membrane coating comprising colloidal lysozyme-templated gold nanoclusters as nanofillers in poly(ethylene oxide)/poly(butylene terephthalate) amphiphilic polymer matrix. Mesoporous silica nanoparticles-lysozyme functionalized gold nanoclusters disperse homogenously within the polymer matrix with no phase separation and zero NPs leaching. This mixed-matrix coating can successfully sense and inhibit bacterial contamination via a controlled release mechanism that is only triggered by bacteria. The system is coated on a common radiographic dental imaging device (photostimulable phosphor plate) that is prone to oral bacteria contamination. Variation and eventually disappearance of the red fluorescence surface under UV light signals bacterial infection. Kanamycin, an antimicrobial agent, is controllably released to instantly inhibit bacterial growth. Interestingly, the quality of the images obtained with these coated surfaces is the same as uncoated surfaces and thus the safe application of such smart coatings can be expanded to include other medical devices without compromising their utility.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

The delivery of large cargos of diameter above 15nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~534kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

Protein-gold clusters-capped mesoporous silica nanoparticles for high drug loading, autonomous gemcitabine/doxorubicin co-delivery, and in-vivo tumor imaging.

Functional nanocarriers capable of transporting high drug contents without premature leakage and to controllably deliver several drugs are needed for better cancer treatments. To address this clinical need, gold cluster bovine serum albumin (AuNC@BSA) nanogates were engineered on mesoporous silica nanoparticles (MSN) for high drug loadings and co-delivery of two different anticancer drugs. The first drug, gemcitabine (GEM, 40wt%), was loaded in positively-charged ammonium-functionalized MSN (MSN-NH3(+)). The second drug, doxorubicin (DOX, 32wt%), was bound with negatively-charged AuNC@BSA electrostatically-attached onto MSN-NH3(+), affording highly loaded pH-responsive MSN-AuNC@BSA nanocarriers. The co-delivery of DOX and GEM was achieved for the first time via an inorganic nanocarrier, possessing a zero-premature leakage behavior as well as drug loading capacities seven times higher than polymersome NPs. Besides, unlike the majority of strategies used to cap the pores of MSN, AuNC@BSA nanogates are biotools and were applied for targeted red nuclear staining and in-vivo tumor imaging. The straightforward non-covalent combination of MSN and gold-protein cluster bioconjugates thus leads to a simple, yet multifunctional nanotheranostic for the next generation of cancer treatments.

Photoresponsive Bridged Silsesquioxane Nanoparticles with Tunable Morphology for Light-Triggered Plasmid DNA Delivery.

Bridged silsesquioxane nanocomposites with tunable morphologies incorporating o-nitrophenylene-ammonium bridges are described. The systematic screening of the sol-gel parameters allowed the material to reach the nanoscale with controlled dense and hollow structures of 100-200 nm. The hybrid composition of silsesquioxanes with 50% organic content homogeneously distributed in the nanomaterials endowed them with photoresponsive properties. Light irradiation was performed to reverse the surface charge of nanoparticles from +46 to -39 mV via a photoreaction of the organic fragments within the particles, as confirmed by spectroscopic monitorings. Furthermore, such nanoparticles were applied for the first time for the on-demand delivery of plasmid DNA in HeLa cancer cells via light actuation.

Probing structural changes of self assembled i-motif DNA.

We report an i-motif structural probing system based on Thioflavin T (ThT) as a fluorescent sensor. This probe can discriminate the structural changes of RET and Rb i-motif sequences according to pH change.