Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles

Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffersHEPES, Tris, and phosphate-buffered salinewith a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze–thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage

Additional file 1: of Facile Synthesis of Ligand-Free Iridium Nanoparticles and Their In Vitro Biocompatibility

Hemolytic assay—The blood compatibility of IrNPs (0-500 μM) was evaluated by monitoring hemolysis of red blood cells. No significant hemolytic activity was observed until the highest concentration of 500 μM is reached. Triton-X-100 served as a positive control. (PDF 110 kb

Drug induced micellization into ultra-high capacity and stable curcumin nanoformulations: Comparing in vitro 2D and 3D-tumor model of triple-negative breast cancer

This manuscript describes a ultra-high loaded nanoformulation of curcumin. This compound is extremely water insoluble but could be dissolved using poly(2-oxazoline)/poly(2-oxazine) based polymer amphiphiles. The resulting formulations were thoroughly characterized in solution and solid form by NMR, dynamic light scattering, electron microscopy, HPLC zeta potential measurements, XRD, respectively. Biological activity was ensured and compared in 2D and 3D cell culture

Rational Design of a Biomimetic Cell Penetrating Peptide Library

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both <i>in vitro</i> and <i>in vivo</i> and correlated with the peptide physical and chemical properties. <i>In vitro</i>, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. <i>In vivo</i>, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the <i>in vivo</i> delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA

Intracellular delivery of mRNA holds great potential for vaccine− and therapeutic discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, <i>p</i> < 0.005) and in vivo (over 2-fold, <i>p</i> < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity

Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation

The discovery of potent new materials for in vivo delivery of nucleic acids depends upon successful formulation of the active molecules into a dosage form suitable for the physiological environment. Because of the inefficiencies of current formulation methods, materials are usually first evaluated for in vitro delivery efficacy as simple ionic complexes with the nucleic acids (lipoplexes). The predictive value of such assays, however, has never been systematically studied. Here, for the first time, by developing a microfluidic method that allowed the rapid preparation of high-quality siRNA-containing lipid nanoparticles (LNPs) for a large number of materials, we have shown that gene silencing assays employing lipoplexes result in a high rate of false negatives (∼90%) that can largely be avoided through formulation. Seven novel materials with in vivo gene silencing potencies of >90% at a dose of 1.0 mg/kg in mice were discovered. This method will facilitate the discovery of next-generation reagents for LNP-mediated nucleic acid delivery

Neutrophil extracellular traps.

<p>Representative z-stacked immunofluorescence images showing neutrophil elastase and DNA/histone-H1 on the surface of microcapsules. Alginate microcapsules were retrieved 1–2 weeks following implantation, while Polystyrene and PMMA microcapsules were retrieved 3 days following implantation. Images are representative of at least 2 independent experiments with total n ≥ 5 mice, and imaging of multiple retrieved microcapsules from each mouse. Scale bar = 100 μm.</p

Flow cytometry schematics.

<p>Representative flow cytometry contour plots describing the gating scheme used to identify different immune cell subsets (isolated 2 weeks followed alginate microcapsule implantation) in the peritoneal cavity. All single cells retrieved from the peritoneal cavity were run through flow cytometry.</p

Neutrophil Function.

<p>(A)–Confirmation of neutrophil phagocytic capacity. Fluorescent nanoparticles (~190 nm polystyrene nanoparticles) were injected intraperitoneally, 1 week following alginate microcapsule implantation. <i>Left</i>–Representative flow cytometry histograms generated following gating on Ly6G⁺ cells showing nanoparticles (NP) associated with neutrophils. Grey histograms are fluorescence intensities in control mice that have not been injected with nanoparticles. <i>Right</i>–Quantification of the NP uptake histograms, showing a large increase in NP MFI 3 hours post NP injection that drops over time. Data are representative of at least 1 independent experiment with total n ≥ 4. (B)–Multiplex luminex assay to measure chemokines and cytokines secreted by neutrophils. Neutrophils were isolated using a magnetic bead based negative selection technique, followed by <i>ex vivo</i> overnight culture. Higher amounts of key inflammatory cytokines and chemokines are secreted by peritoneal cavity but not bone marrow neutrophils. B.D.L. = below detectable levels. ** and *** indicate p<0.01 and p<0.001, respectively, using a two-tailed Student's t test with Welch's correction (for samples where the levels of cytokine/chemokine are above detectable levels). # indicates p<0.01 using a two-tailed Fisher's exact test, for samples where the levels of cytokine/chemokine were below detectable levels. Data presented are based on n = 6.</p

Increased neutrophil presence in peritoneal exudate following microcapsule implantation.

<p>(A)–Representative flow cytometry contour plots showing percentages of neutrophils (CD11b⁺ Ly6G⁺) in the peritoneal exudate of mice implanted with microcapsules made of different materials. (B)–Counts of neutrophils in the peritoneal exudate 2 weeks following implantation of microcapsules made of different materials compared to control untreated and mock treated mice. <b>C</b>–Counts of monocyte/macrophage (CD11b⁺ Ly6G^- CD11c^-), dendritic cells (CD11b⁺ CD11c⁺), B cells (CD19⁺), and T cells (TCRβ⁺) in the peritoneal exudate 2 weeks following implantation of microcapsules made of different materials compared to control untreated and mock treated mice. Mock treatment entailed performing a laparotomy and injecting sterile saline (sham surgery). *** indicates p<0.001, using one-way ANOVA followed by Bonferroni post-test comparing specific sample to mock or untreated. Data are representative of at least 2 independent experiments with total n ≥ 5.</p