A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

Bubble Forming Films for Spatial Selective Cell Killing.

Photodynamic and photothermal cell killing at the surface of tissues finds applications in medicine. However, a lack of control over heat dissipation following a treatment with light might damage surrounding tissues. A new strategy to kill cells at the surface of tissues is reported. Polymeric films are designed in which iron oxide nanoparticles are embedded as photosensitizers. Irradiation of the films with pulsed laser light generates water vapor bubbles at the surface of the films. It is found that "bubble-films" can kill cells in close proximity to the films due to mechanical forces which arise when the bubbles collapse. Local irradiation of bubble-films allows for spatial selective single cell killing. As nanosurgery becomes attractive in ophthalmology to remove superficial tumors, bubble-films are applied on the cornea and it is found that irradiation of the bubble-films allows spatial and selective killing of corneal cells. As i) the photosensitizer is embedded in the films, which reduces its uptake by cells and spreading into tissues and ii) the bubble-films can be removed from the tissue after laser treatment, while iii) a low laser fluence is sufficient to generate vapor bubbles, it is foreseen that bubble-films might become promising for safe resection of superficial tumors.

Comparison of MRI Properties between Multimeric DOTAGA and DO3A Gadolinium-Dendron Conjugates.

The inherent lack of sensitivity of MRI needs the development of new Gd contrast agents in order to extend the application of this technique to cellular imaging. For this purpose, two multimeric MR contrast agents obtained by peptidic coupling between an amido amine dendron and GdDOTAGA chelates (premetalation strategy, G1-4GdDOTAGA) or DO3A derivatives which then were postmetalated (G1-4GdDO3A) have been prepared. By comparison to the monomers, an increase of longitudinal relaxivity has been observed for both structures. Especially for G1-4GdDO3A, a marked increase is observed between 20 and 60 MHz. This structure differs from G1-4GdDOTAGA by an increased rigidity due to the aromatic linker between each chelate and the organic framework. This has the effect of limiting local rotational movements, which has a positive impact on relaxivity.

The obstacle course to the inner retina: Hyaluronic acid-coated lipoplexes cross the vitreous but fail to overcome the inner limiting membrane.

Considerable research over the last few years has revealed dysregulation of growth factors in various retinal diseases, such as glaucoma, diabetic retinopathy and photoreceptor degenerations. The use of messengerRNA (mRNA) to transiently overexpress a specific factor could compensate for this imbalance. However, a critical challenge of this approach lies in the ability to efficiently deliver mRNA molecules to the retinal target cells. In this study we found that intravitreal (IVT) injection is an attractive approach to deliver mRNA to the retina, providing two critical barriers can be overcome: the vitreous and the inner limiting membrane (ILM). We demonstrated that the vitreous is indeed a major hurdle in the delivery of the cationic mRNA-complexes to retinal cells, both in terms of vitreal mobility and cellular uptake. To improve their intravitreal mobility and avoid unwanted extracellular interactions, we evaluated the use of hyaluronic acid (HA) as an electrostatic coating strategy. This HA-coating provided the complexes with a negative surface charge, markedly enhancing their mobility in the vitreous humor, without reducing their cellular internalization and transfection efficiency. However, although this coating strategy allows the mRNA-complexes to successfully overcome the vitreal barrier, the majority of the particles accumulated at the ILM. This study therefore underscores the crucial barrier function of the ILM toward non-viral retinal gene delivery and the need to smartly design mRNA-carriers able to surmount the vitreous as well as the ILM.