Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Order from Disorder with Intrinsically Disordered Peptide Amphiphiles.

Amphiphilic molecules and their self-assembled structures have long been the target of extensive research due to their potential applications in fields ranging from materials design to biomedical and cosmetic applications. Increasing demands for functional complexity have been met with challenges in biochemical engineering, driving researchers to innovate in the design of new amphiphiles. An emerging class of molecules, namely, peptide amphiphiles, combines key advantages and circumvents some of the disadvantages of conventional phospholipids and block copolymers. Herein, we present new peptide amphiphiles composed of an intrinsically disordered peptide conjugated to two variants of hydrophobic dendritic domains. These molecules, termed intrinsically disordered peptide amphiphiles (IDPA), exhibit a sharp pH-induced micellar phase-transition from low-dispersity spheres to extremely elongated worm-like micelles. We present an experimental characterization of the transition and propose a theoretical model to describe the pH-response. We also present the potential of the shape transition to serve as a mechanism for the design of a cargo hold-and-release application. Such amphiphilic systems demonstrate the power of tailoring the interactions between disordered peptides for various stimuli-responsive biomedical applications.

Lipid Nanoparticle RBD-hFc mRNA Vaccine Protects hACE2 Transgenic Mice against a Lethal SARS-CoV-2 Infection.

The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

Multidomain drug delivery systems of ß-casein micelles for the local oral co-administration of antiretroviral combinations.

The antiretroviral (ARV) cocktailrevolved the treatment of the human immunodeficiency virus (HIV) infection. Drug combinations have been also tested to treat other infectious diseases, including the recentcoronavirus disease 2019 (COVID-19) outbreak. To simplify administration fixed-dose combinationshave been introduced, however, oral anti-HIV therapy still struggles with low oral bioavailability of many ARVs.This work investigated the co-encapsulation of two clinically relevant ARV combinations,tipranavir (TPV):efavirenz (EFV) anddarunavir (DRV):efavirenz (EFV):ritonavir (RTV),within the core of ß-casein (bCN) micelles. Encapsulation efficiency in both systems was ~100%. Cryo-transmission electron microscopy and dynamic light scattering of the ARV-loaded colloidaldispersions indicatefull preservation of the spherical morphology, and x-ray diffraction confirm that the encapsulated drugs are amorphous. To prolong the physicochemical stabilitythe formulations were freeze-driedwithout cryo/lyoprotectant, and successfully redispersed, with minor changes in morphology.Then, theARV-loaded micelles were encapsulated within microparticles of Eudragit® L100, which prevented enzymatic degradation and minimized drug release under gastric-like pH conditionsin vitro. At intestinal pH, the coating polymer dissolved and released the nanocarriers and content. Overall, our results confirm the promise of this flexible and modular technology platform for oral delivery of fixed dose combinations.

Structure and characterisation of hydroxyethylcellulose-silica nanoparticles.

Functionalising nanoparticles with polymers has gained much interest in recent years, as it aids colloidal stability and manipulation of surface properties. Here, polymer-coated thiolated silica nanoparticles were synthesised by self-condensation of 3-mercaptopropyltrimethoxysilane in the presence of hydroxyethylcellulose. These nanoparticles were characterised by dynamic light scattering, small angle neutron scattering, Nanoparticle Tracking Analysis, Raman spectroscopy, FT-IR spectroscopy, thermogravimetric analysis, Ellman's assay, transmission electron microscopy and cryo-transmission electron microscopy. It was found that increasing the amount of hydroxyethylcellulose in the reaction mixture increased the nanoparticle size and reduced the number of thiol groups on their surface. Additionally, by utilising small angle neutron scattering and dynamic light scattering, it was demonstrated that higher concentrations of polymer in the reaction mixture (0.5-2% w/v) resulted in the formation of aggregates, whereby several silica nanoparticles are bridged together with macromolecules of hydroxyethylcellulose. A correlation was identified between the aggregate size and number of particles per aggregate based on size discrepancies observed between DLS and SANS measurements. This information makes it possible to control the size of aggregates during a simple one-pot synthesis; a prospect highly desirable in the design of potential drug delivery systems.

Ferritin is secreted via 2 distinct nonclassical vesicular pathways.

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

Poly(glycoamidoamine) Brushes Formulated Nanomaterials for Systemic siRNA and mRNA Delivery in Vivo.

Safe and effective delivery is required for siRNA and mRNA-based therapeutics to reach their potential. Here, we report on the development of poly(glycoamidoamine) brush nanoparticles as delivery vehicles for siRNA and mRNA. These polymers were capable of significant delivery of siRNA against FVII and mRNA-encoding erythropoietin (EPO) in mice. Importantly, these nanoparticles were well-tolerated at their effective dose based on analysis of tissue histology, systemic cytokine levels, and liver enzyme chemistry. The polymer brush nanoparticles reported here are promising for therapeutic applications.

Membrane tethering and nucleotide-dependent conformational changes drive mitochondrial genome maintenance (Mgm1) protein-mediated membrane fusion.

Cellular membrane remodeling events such as mitochondrial dynamics, vesicle budding, and cell division rely on the large GTPases of the dynamin superfamily. Dynamins have long been characterized as fission molecules; however, how they mediate membrane fusion is largely unknown. Here we have characterized by cryo-electron microscopy and in vitro liposome fusion assays how the mitochondrial dynamin Mgm1 may mediate membrane fusion. Using cryo-EM, we first demonstrate that the Mgm1 complex is able to tether opposing membranes to a gap of ∼15 nm, the size of mitochondrial cristae folds. We further show that the Mgm1 oligomer undergoes a dramatic GTP-dependent conformational change suggesting that s-Mgm1 interactions could overcome repelling forces at fusion sites and that ultrastructural changes could promote the fusion of opposing membranes. Together our findings provide mechanistic details of the two known in vivo functions of Mgm1, membrane fusion and cristae maintenance, and more generally shed light onto how dynamins may function as fusion proteins.

Stalk domain of the dynamin-like MxA GTPase protein mediates membrane binding and liposome tubulation via the unstructured L4 loop.

The human MxA protein is an interferon-induced large GTPase with antiviral activity against a wide range of viruses, including influenza viruses. Recent structural data demonstrated that MxA oligomerizes into multimeric filamentous or ring-like structures by virtue of its stalk domain. Here, we show that negatively charged lipid membranes support MxA self-assembly. Like dynamin, MxA assembled around spherical liposomes inducing liposome tubulation. Cryo-transmission electron microscopy revealed that MxA oligomers around liposomes have a "T-bar" shape similar to dynamin. Moreover, biochemical assays indicated that the unstructured L4 loop of the MxA stalk serves as the lipid-binding moiety, and mutational analysis of L4 revealed that a stretch of four lysine residues is critical for binding. The orientation of the MxA molecule within the membrane-associated oligomer is in agreement with the proposed topology of MxA oligomers based on crystallographic data. Although oligomerization of wild-type MxA around liposomes led to the creation of helically decorated tubes similar to those formed by dynamin, this lipid interaction did not stimulate GTPase activity, in sharp contrast to the assembly-stimulated nucleotide hydrolysis observed with dynamin. Moreover, MxA readily self-assembles into rings at physiological conditions, as opposed to dynamin which self-assembles only at low salt conditions or onto lipids. Thus, the present results indicate that the oligomeric structures formed by MxA critically differ from those of dynamin.