Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery.

CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain.

Antibacterial Countermeasures via Metal-Organic Framework-Supported Sustained Therapeutic Release.

Long-term antimicrobial therapies are necessary to treat infections caused by virulent intracellular pathogens, including biothreat agents. Current treatment plans include injectable therapeutics given multiple times daily over a period for up to 8 weeks. Here, we present a metal-organic framework (MOF), zeolitic imidazolate framework-8 (ZIF-8), as a robust platform to support the sustained release of ceftazidime, an important antimicrobial agent for many critical bacterial infections. Detailed material characterization confirms the successful encapsulation of ceftazidime within the ZIF-8 matrix, indicating sustained drug release for up to a week. The antibacterial properties of ceftazidime@ZIF-8 particles were confirmed against Escherichia coli, chosen here as a representative of Gram-negative bacteria infection model in a proof-of-concept study. Further, we showed that this material system is compatible with macrophage and lung epithelial cell lines, relevant targets for antibacterial therapy for pulmonary and intracellular infections. A promising methodology to enhance the treatment of intracellular infections is to deliver the antibiotic cargo intracellularly. Importantly, this is the first study to unequivocally demonstrate direct MOF particle internalization using confocal microscopy via 3D reconstructions of z-stacks, taking advantage of the intrinsic emission properties of ZIF-8. This is an important development as it circumvents the need to use any staining dyes and addresses current methodology limitations concerning false impression of cargo uptake in the event of the carrier particle breakdown within biological media.

Mesoporous Silica Nanoparticle-Supported Lipid Bilayers (Protocells) for Active Targeting and Delivery to Individual Leukemia Cells.

Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases.

Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses-using the rotationally constrained carboxylate in R-naproxen-led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and efficacy in the treatment of several epithelial cancer types on account of established human toxicity profiles and novel activities against Rho-family GTPases.

A Pan-GTPase Inhibitor as a Molecular Probe.

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein.

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7-RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead 'locks' the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein-protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein-protein interactions, which can be relevant to drug discovery and development.

Re-examining the size/charge paradigm: differing in vivo characteristics of size- and charge-matched mesoporous silica nanoparticles.

The combination of nanoparticle (NP) size, charge, and surface chemistry (e.g., extent of modification with polyethylene glycol (PEG)) is accepted as a key determinant of NP/cellular interactions. However, the influence of spatial arrangement and accessibility of the charged molecules on the NP surface vis-à-vis the average surface charge (zeta (ζ) potential) is incompletely understood. Here we demonstrate that two types of mesoporous silica nanoparticles (MSNP) that are matched in terms of primary and hydrodynamic particle size, shape, pore structure, colloidal stability, and ζ potential, but differ in surface chemistry, viz. the spatial arrangement and relative exposure of surface amines, have profoundly different interactions with cells and tissues when evaluated in vitro and in vivo. While both particles are ∼50 nm in diameter, PEGylated, and positively charged (ζ = +40 mV), PEG-PEI (MSNPs modified with exposed polyamines), but not PEG-NMe3(+) (MSNP modified with distributed, obstructed amines) rapidly bind serum proteins, diverse cells types in vitro, and endothelial and white blood cells in vivo (ex ovo chick embryo model). This finding helps elucidate the relative role of surface exposure of charged molecules vs ζ potential in otherwise physicochemically matched MSNP and highlights protein corona neutrality as an important design consideration when synthesizing cationic NPs for biological applications.

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.