Lipid shape and packing are key for optimal design of pH-sensitive mRNA lipid nanoparticles.

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo-the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson-Boltzmann theory to capture these phenomena.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery.

mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.

High-Throughput Automation of Endosomolytic Polymers for mRNA Delivery.

In recent years, there has been an increasing interest in designing delivery systems to enhance the efficacy of RNA-based therapeutics. Here, we have synthesized copolymers comprised of dimethylaminoethyl methacrylate (DMAEMA) or diethylaminoethyl methacrylate (DEAEMA) copolymerized with alkyl methacrylate monomers ranging from 2 to 12 carbons, and developed a high throughput workflow for rapid investigation of their applicability for mRNA delivery. The structure activity relationship revealed that the mRNA encapsulation efficiency is improved by increasing the cationic density and use of shorter alkyl side chains (2-6 carbons). Minimal cytotoxicity was observed when using DEAEMA-co-BMA (EB) polyplexes up to 18 h after dosing, independent of a poly(ethylene glycol) (PEG) first block. The lowest molecular weight polymer (EB10,250) performed best, exhibiting greater transfection than polyethyenimine (PEI) based upon the number of cells transfected and mean intensity. Conventional investigations into the performance of polymeric materials for mRNA delivery is quite tedious, consequently limiting the number of materials and formulation conditions that can be studied. The high throughput approach presented here can accelerate the screening of polymeric systems and paves the way for expanding this generalizable approach to assess various materials for mRNA delivery.

Recent advances in polymeric materials for the delivery of RNA therapeutics.

Introduction: The delivery of nucleic acid therapeutics through non-viral carriers face multiple biological barriers that reduce their therapeutic efficiency. Despite great progress, there remains a significant technological gap that continues to limit clinical translation of these nanocarriers. A number of polymeric materials are being exploited to efficiently deliver nucleic acids and achieve therapeutic effects. Areas covered: We discuss the recent advances in the polymeric materials for the delivery of nucleic acid therapeutics. We examine the use of common polymer architectures and highlight the challenges that exist for their development from bench side to clinic. We also provide an overview of the most notable improvements made to circumvent such challenges, including structural modification and stimuli-responsive approaches, for safe and effective nucleic acid delivery. Expert opinion: It has become apparent that a universal carrier that follows 'one-size' fits all model cannot be expected for delivery of all nucleic acid therapeutics. Carriers need to be designed to exhibit sensitivity and specificity toward individual targets diseases/indications, and relevant subcellular compartments, each of which possess their own unique challenges. The ability to devise synthetic methods that control the molecular architecture enables the future development that allow for the construction of 'intelligent' designs.

Impact of Cationic Charge Density and PEGylated Poly(Amino Acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 2: DNA Protection, Stability, Cytotoxicity, and Transfection Efficiency.

The impact of the molecular architecture on the transfection efficiency of PEGylated poly(amino acid) block copolymers was investigated for PEG-b-p(l-Lys)x -b-p(l-Leu)y , PEG-b-p(l-Leu)x -b-p(l-Lys)y , and PEG-b-p((l-Leu)x -co-(l-Lys)y ). The block lengths of p(l-Lys) and p(l-Leu) were varied between 10, 20, and 40; and 10 and 20, respectively, to study the influence of the ionic/hydrophobic balance. The results show that ABC triblock copolymers form smaller and more stable polyplexes with plasmid DNA than AB diblock copolymers-as verified by long-term aggregation and ethidium bromide exclusion studies-protect the DNA more effectively against nucleases, and provide better transfection efficiencies, as indicated by total protein as well as luciferase expression. More detailed studies revealed that triblock copolymers with p(l-Leu) forming the C-block were most efficient in DNA complexation with a 2.3 times higher transfection rate. Furthermore, increasing the cationic character by increasing the p(l-Lys) chain length led to up to 25% higher transfection but at the same time induced some cytotoxicity. Diblock copolymers, where the amino acid-building blocks exist as a random copolymer, bind more loosely with DNA leading to less compact and less stable aggregates with lower transfection efficiencies.

Impact of Cationic Charge Density and PEGylated Poly(amino acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 1: Synthesis, Self-Assembly, and DNA Complexation.

The interaction of PEGylated poly(amino acid)s with their biological targets depends on their chemical nature and spatial arrangement of their building blocks. The synthesis, self-assembly, and DNA complexation of ABC terblock copolymers consisting of poly(ethylene glycol), (PEG), poly(l-lysine), and poly(l-leucine), are reported. Block copolymers are produced by a metal-free, living ring-opening polymerization of respective amino acid N-carboxyanhydrides using amino-terminated PEG as macroinitiator: (PEG-b-p(l-Lys)x -b-p(l-Leu)y , PEG-b-p(l-Leu)x -b-p(l-Lys)y , and PEG-b-p((l-Lys)x -co-p(l-Leu)y ). Sizes of self-assembled nanoparticles depend on the formation method. The nanoprecipitation method proves useful for copolymers with the poly(l-lysine) block protected as trifluoroacetate, effective diameters range between 92 and 132 nm, while direct dissolution in distilled water is suitable for the deprotected copolymers, yielding effective diameters between 52 and 173 nm. Critical micelle concentration (CMC) analyses corroborate particle size analyses and show a distinct impact of the molecular architecture; the lowest CMC (8 µg mL-1 ) is observed when the poly(l-leucine) segment forms the C-block and the hydrophilic, disassembly driving poly(l-lysine) segment is short. DNA complexation, evaluated by gel motility and RiboGreen analyses, depends strongly on the molecular architecture. A more efficient DNA complexation is observed when poly(l-lysine) and poly(l-leucine) form individual blocks as opposed to them forming a copolymer.

Synthesis and Application of Aurophilic Poly(Cysteine) and Poly(Cysteine)-Containing Copolymers.

The redox capacity, as well as the aurophilicity of the terminal thiol side groups, in poly(Cysteine) lend a unique characteristic to this poly(amino acid) or polypeptide. There are two major application fields for this polymer: (i) biomedical applications in drug delivery and surface modification of biomedical devices and (ii) as coating for electrodes to enhance their electrochemical sensitivity. The intended application determines the synthetic route for p(Cysteine). Polymers to be used in biomedical applications are typically polymerized from the cysteine N-carboxyanhydride by a ring-opening polymerization, where the thiol group needs to be protected during the polymerization. Advances in this methodology have led to conditions under which the polymerization progresses as living polymerization, which allows for a strict control of the molecular architecture, molecular weight and polydispersity and the formation of block copolymers, which eventually could display polyphilic properties. Poly(Cysteine) used as electrode coating is typically polymerized onto the electrode by cyclic voltammetry, which actually produces a continuous, pinhole-free film on the electrode via the formation of covalent bonds between the amino group of Cysteine and the carbon of the electrode. This resulting coating is chemically very different from the well-defined poly(Cysteine) obtained by ring-opening polymerizations. Based on the structure of cysteine a significant degree of cross-linking within the coating deposited by cyclic voltammetry can be assumed. This manuscript provides a detailed discussion of the ring-opening polymerization of cysteine, a brief consideration of the role of glutathione, a key cysteine-containing tripeptide, and examples for the utilization of poly(Cysteine) and poly(Cysteine)-containing copolymers, in both, the biomedical as well as electrochemical realm.

Versatile cationic lipids for siRNA delivery.

Exploitation of the RNA interference (RNAi) pathway offers the promise of new and effective therapies for a wide variety of diseases. Clinical development of new drugs based on this platform technology is still limited, however, by a lack of safe and efficient delivery systems. Here we report the development of a class of structurally versatile cationic lipopolyamines designed specifically for delivery of siRNA which show high levels of target transcript knockdown in a range of cell types in vitro. A primary benefit of these lipids is the ease with which they may be covalently modified by the addition of functional molecules. For in vivo applications one of the core lipids (Staramine) was modified with methoxypolyethylene glycols (mPEGs) of varying lengths. Upon systemic administration, PEGylated Staramine nanoparticles containing siRNA targeting the caveolin-1 (Cav-1) transcript caused a reduction of the Cav-1 transcript of up to 60%, depending on the mPEG length, specifically in lung tissue after 48h compared to treatment with non-silencing siRNA. In addition, modification with mPEG reduced toxicity associated with intravenous administration. The ability to produce a high level of target gene knockdown in the lung with minimal toxicity demonstrates the potential of these lipopolyamines for use in developing RNAi therapeutics for pulmonary disease.