Lysine acylation in superoxide dismutase-1 electrostatically inhibits formation of fibrils with prion-like seeding.

The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

BACKGROUND: Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. METHODS: Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). RESULTS: Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. CONCLUSIONS: The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. GENERAL SIGNIFICANCE: The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R2 = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R2 = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies.

Directly lighting up RNA G-quadruplexes from test tubes to living human cells.

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Gibbs Energy of Superoxide Dismutase Heterodimerization Accounts for Variable Survival in Amyotrophic Lateral Sclerosis.

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and ΔG of heterodimerization (ΔGHet) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and ΔGHet for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The ΔGHet of each ALS variant SOD1 correlated with patient survival time after diagnosis (R(2) = 0.98), with more favorable ΔGHet correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered ΔGHet by <1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

Concurrent chemoradiotherapy plus adjuvant chemotherapy versus concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma : A matched-pair multicenter analysis of outcomes.

PURPOSE: The benefit of adjuvant chemotherapy (AC) in locoregionally advanced nasopharyngeal carcinoma (NPC) is controversial. This study compared concurrent chemoradiotherapy plus AC (CCRT/AC) with CCRT. METHODS: Pair-matched analysis based on eight clinicopathological features of 244 patients treated with platinum-based CCRT/AC or CCRT alone was performed. Survival outcomes were assessed using the Kaplan-Meier method and log-rank test. Toxicities and response rates were compared using Fisher's exact test. RESULTS: Four-year overall survival, progression-free survival, distant failure-free survival, and locoregional failure-free survival were 72 %, 61 %, 71 %, and 81 %, respectively, for the CCRT arm, compared to 74 % (hazard ratio, HR 0.89; 95 % confidence interval, CI 0.64-1.23; P = 0.474), 62 % (HR 0.91, 95 % CI 0.68-1.20, P = 0.489), 73 % (HR 0.84, 95 % CI 0.59-1.18, P = 0.316), and 84 % (HR 0.84, 95 % CI 0.52-1.24, P = 0.323), respectively, for the CCRT/AC arm. Cox multivariate regression analysis demonstrated AC was not an independent prognostic factor. Overall, there was a higher incidence of grade 3-4 toxicities in the CCRT/AC arm. The most common grade 3-4 adverse events in the CCRT/AC arm were vomiting (27 %), nausea (43 %), leukopenia/neutropenia (23 %), thrombocytopenia (8.8 %), and anemia (6.2 %). CONCLUSION: Addition of AC to CCRT increased toxicities but did not improve survival in locoregionally advanced NPC.

Arresting amyloid with coulomb's law: acetylation of ALS-linked SOD1 by aspirin impedes aggregation.

Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzyme's Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.

ROS generated during early reperfusion contribute to intermittent hypobaric hypoxia-afforded cardioprotection against postischemia-induced Ca(2+) overload and contractile dysfunction via the JAK2/STAT3 pathway.

Moderate enhanced reactive oxygen species (ROS) during early reperfusion trigger the cardioprotection against ischemia/reperfusion (I/R) injury, while the mechanism is largely unknown. Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) contributes to the cardioprotection but whether it is activated by ROS and how it regulates Ca(2+) homeostasis remain unclear. Here we investigated whether the ROS generated during early reperfusion protect the heart/cardiomyocyte against I/R-induced Ca(2+) overload and contractile dysfunction via the activation of JAK2/STAT3 signaling pathway by using a cardioprotective model of intermittent hypobaric hypoxia (IHH) preconditioning. IHH improved the postischemic recovery of myocardial contractile performance in isolated rat I/R hearts as well as Ca(2+) homeostasis and cell contraction in simulated I/R cardiomyocytes. Meanwhile, IHH enhanced I/R-increased STAT3 phosphorylation at tyrosine 705 in the nucleus and reversed I/R-suppressed STAT3 phosphorylation at serine 727 in the nucleus and mitochondria during reperfusion. Moreover, IHH improved I/R-suppressed sarcoplasmic reticulum (SR) Ca(2+)-ATPase 2 (SERCA2) activity, enhanced I/R-increased Bcl-2 expression, and promoted the co-localization and interaction of Bcl-2 with SERCA2 during reperfusion. These effects were abolished by scavenging ROS with N-(2-mercaptopropionyl)-glycine (2-MPG) and/or by inhibiting JAK2 with AG490 during the early reperfusion. Furthermore, IHH-improved postischemic SERCA2 activity and Ca(2+) homeostasis as well as cell contraction were reversed after Bcl-2 knockdown by short hairpin RNA. In addition, the reversal of the I/R-suppressed mitochondrial membrane potential by IHH was abolished by 2-MPG and AG490. These results indicate that during early reperfusion the ROS/JAK2/STAT3 pathways play a crucial role in (i) the IHH-maintained intracellular Ca(2+) homeostasis via the improvement of postischemic SERCA2 activity through the increase of SR Bcl-2 and its interaction with SERCA2; and (ii) the IHH-improved mitochondrial function.

A newly identified G-quadruplex as a potential target regulating Bcl-2 expression.

BACKGROUND: A new G-quadruplex structure located in the B-cell CLL/lymphoma 2 (Bcl-2) P1 promoter and its physiological function related to Bcl-2 transcription have been studied to find a potential anticancer therapeutic target. METHODS: Absorption, polyacrylamide gel electrophoresis, fluorescence, circular dichroism, and nuclear magnetic resonance spectra have been employed to determine G-quadruplex structure and the interaction between G-quadruplex and phenanthrolin-dicarboxylate. Real time polymerase chain reaction and luciferase assay were done to assess the physiological function of the G-quadruplex structure. RESULTS: The UV-melting and polyacrylamide gel electrophoresis studies show that the p32 DNA sequence forms an intramolecular G-quadruplex structure. Circular dichroism and nuclear magnetic resonance spectra indicate that the G-quadruplex is a hybrid-type structure with four G-tetrads. Fluorescence spectra show that a phenanthroline derivative has a higher binding affinity for p32 G-quadruplex than duplex. Further circular dichroism and nuclear magnetic resonance studies indicate that the phenanthroline derivative can regulate p32 G-quadruplex conformation. Real time polymerase chain reaction and luciferase assays show that the phenanthroline derivative has down-modulated Bcl-2 transcription activity in a concentration-dependent manner. However, no such effect was observed when p32 G-quadruplex was denatured through base mutation. CONCLUSION: The newly identified G-quadruplex located in the P1 promoter of Bcl-2 oncogene is intimately related with Bcl-2 transcription activity, which may be a promising anticancer therapeutic target. GENERAL SIGNIFICANCE: The newly identified G-quadruplex in the Bcl-2 P1 promoter may be a novel anticancer therapeutic target.

Visual detection of mercury(II) based on recognition of the G-quadruplex conformational transition by a cyanine dye supramolecule.

A supramolecular probe for visual detection of mercury (Hg) has been designed by using a cyanine dye and AS1411 G-quadruplexes, which exhibits an obvious color change from red to blue in response to an increased level of Hg(2+). The supramolecular probe exhibits high selectivity and sensitivity towards Hg(2+) and is promising for the detection of environmental samples with the naked eye.

Metal-ion-specific screening of charge effects in protein amide H/D exchange and the Hofmeister series.

In this study, protein charge ladders and mass spectrometry were used to quantify how metal cations in the Hofmeister series (Na(+), K(+), Li(+), Mg(2+), and Ca(2+)) permute the effects of lysine acetylation on the rate of amide H/D exchange in a representative protein (myoglobin, Mb). The successive acetylation of up to 18 Lys-ε-NH3(+) groups in Mb caused a linear decrease in its global rate of amide H/D exchange (as measured by mass spectrometry), despite also decreasing the thermostability of Mb by >10 °C. The ability of a metal cation to screen kinetic electrostatic effects during H/D exchange-and to abolish the protective effect of acetylation against H/D exchange-was found to depend on the position of the cation in the Hofmeister series. Na(+) and K(+) cations did not fully equalize the rates of H/D exchange among each "rung" of the charge ladder, whereas Mg(2+) and Ca(2+) did equalize rates without eliminating the hydrophobic core of the protein (i.e., without unfolding Mb); Li(+) exhibited intermediate effects. The ability of Mg(2+) and Ca(2+) to completely screen electrostatic effects associated with the H/D exchange of charge isomers of Mb suggests that Mg(2+) or Ca(2+) (but not Na(+) or K(+)) can be used to quantify the magnitude by which electrostatic charge contributes to the observed rates of amide H/D exchange in proteins.

Screening oral drugs for their interactions with the intestinal transportome via porcine tissue explants and machine learning.

In vitro systems that accurately model in vivo conditions in the gastrointestinal tract may aid the development of oral drugs with greater bioavailability. Here we show that the interaction profiles between drugs and intestinal drug transporters can be obtained by modulating transporter expression in intact porcine tissue explants via the ultrasound-mediated delivery of small interfering RNAs and that the interaction profiles can be classified via a random forest model trained on the drug-transporter relationships. For 24 drugs with well-characterized drug-transporter interactions, the model achieved 100% concordance. For 28 clinical drugs and 22 investigational drugs, the model identified 58 unknown drug-transporter interactions, 7 of which (out of 8 tested) corresponded to drug-pharmacokinetic measurements in mice. We also validated the model's predictions for interactions between doxycycline and four drugs (warfarin, tacrolimus, digoxin and levetiracetam) through an ex vivo perfusion assay and the analysis of pharmacologic data from patients. Screening drugs for their interactions with the intestinal transportome via tissue explants and machine learning may help to expedite drug development and the evaluation of drug safety.

Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations.

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ~2-8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ~2-fold (p < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (p > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ~23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparagine--an analytically elusive, sub-Dalton modification--represents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1.

The characterization of the concentration of the single-walled carbon nanotubes in aqueous dispersion by UV-Vis-NIR absorption spectroscopy.

Current and future applications of single-wall carbon nanotubes (SWCNTs) depend on the dispersion of the SWCNTs in aqueous solution and their quantitation. The concentration of SWCNTs is an important indicator to evaluate the dispersibility of the surfactant-dispersed SWCNTs suspension. Due to the complexity of the SWCNTs suspension, it is necessary to determine both the total concentration of the dispersed SWCNTs and the concentration of individually dispersed SWCNTs in aqueous suspensions, and these were evaluated through the absorbance and the resonance ratios of UV-Vis-NIR absorption spectra, respectively. However, there is no specific and reliable position assigned for either calculation of the absorbance or the resonance ratio of the UV-Vis-NIR absorption spectrum. In this paper, different ranges of wavelengths for these two parameters were studied. From this, we concluded that the wavelength range between 300 nm and 600 nm should be the most suitable for evaluation of the total concentration of dispersed SWCNTs in the suspension; also, wavelengths below 800 nm should be most suitable for evaluation of the concentration of individually dispersed SWCNTs in the suspension. Moreover, these wavelength ranges are verified by accurate dilution experiments.

Machine Learning Uncovers Food- and Excipient-Drug Interactions.

Inactive ingredients and generally recognized as safe compounds are regarded by the US Food and Drug Administration (FDA) as benign for human consumption within specified dose ranges, but a growing body of research has revealed that many inactive ingredients might have unknown biological effects at these concentrations and might alter treatment outcomes. To speed up such discoveries, we apply state-of-the-art machine learning to delineate currently unknown biological effects of inactive ingredients-focusing on P-glycoprotein (P-gp) and uridine diphosphate-glucuronosyltransferase-2B7 (UGT2B7), two proteins that impact the pharmacokinetics of approximately 20% of FDA-approved drugs. Our platform identifies vitamin A palmitate and abietic acid as inhibitors of P-gp and UGT2B7, respectively; in silico, in vitro, ex vivo, and in vivo validations support these interactions. Our predictive framework can elucidate biological effects of commonly consumed chemical matter with implications on food- and excipient-drug interactions and functional drug formulation development.

Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver.

Lipid-like nanoparticles (LNPs) have potential as non-viral delivery systems for mRNA therapies. However, repeated administrations of LNPs may lead to accumulation of delivery materials and associated toxicity. To address this challenge, we have developed biodegradable lipids which improve LNPs clearance and reduce toxicity. We modify the backbone structure of Dlin-MC3-DMA by introducing alkyne and ester groups into the lipid tails. We evaluate the performance of these lipids when co-formulated with other amine containing lipid-like materials. We demonstrate that these formulations synergistically facilitate robust mRNA delivery with improved tolerability after single and repeated administrations. We further identify albumin-associated macropinocytosis and endocytosis as an ApoE-independent LNP cellular uptake pathway in the liver. Separately, the inclusion of alkyne lipids significantly increases membrane fusion to enhance mRNA release, leading to synergistic improvement of mRNA delivery. We believe that the rational design of LNPs with multiple amine-lipids increases the material space for mRNA delivery.

Heparin-Coated Albumin Nanoparticles for Drug Combination in Targeting Inflamed Intestine.

Targeting areas of inflammation offers potential therapeutic and diagnostic benefits by maximizing drug and imaging marker on-target effects while minimizing systemic exposure that can be associated with adverse side effects. This strategy is particularly beneficial in the management of inflammatory bowel disease (IBD). Here an inflammation-targeting (IT) approach based on heparin-coated human serum albumin nanoparticles (HEP-HSA NPs) that utilize the increased intestinal permeability and changes in electrostatic interaction at the site of intestinal inflammation is described. Using small-molecule and biologic drugs as a model for drug combination, the HEP-HSA NPs demonstrate the capacity to load both drugs simultaneously; the dual-drug loaded HEP-HSA NPs exhibit a higher anti-inflammatory effect than both of the single-drug loaded NPs in vitro and selectively bind to inflamed intestine after enema administration in vivo in a murine model of colitis. Importantly, analyses of the physicochemical characteristics and targeting capacities of these NPs indicate that HEP coating modulates NP binding to the inflamed intestine, providing a foundation for future IT-NP formulation development.

Gastrointestinal synthetic epithelial linings.

Epithelial tissues line the organs of the body, providing an initial protective barrier as well as a surface for nutrient and drug absorption. Here, we identified enzymatic components present in the gastrointestinal epithelium that can serve as selective means for tissue-directed polymerization. We focused on the small intestine, given its role in drug and nutrient absorption and identified catalase as an essential enzyme with the potential to catalyze polymerization and growth of synthetic biomaterial layers. We demonstrated that the polymerization of dopamine by catalase yields strong tissue adhesion. We characterized the mechanism and specificity of the polymerization in segments of the gastrointestinal tracts of pigs and humans ex vivo. Moreover, we demonstrated proof of concept for application of these gastrointestinal synthetic epithelial linings for drug delivery, enzymatic immobilization for digestive supplementation, and nutritional modulation through transient barrier formation in pigs. This catalase-based approach to in situ biomaterial generation may have broad indications for gastrointestinal applications.

Robotically handled whole-tissue culture system for the screening of oral drug formulations.

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.

Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation.

Therapeutic messenger RNA vaccines enable delivery of whole antigens, which can be advantageous over peptide vaccines. However, optimal efficacy requires both intracellular delivery, to allow antigen translation, and appropriate immune activation. Here, we developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation. Using a three-dimensional multi-component reaction system, we synthesized and evaluated the vaccine potential of over 1,000 lipid formulations. The top candidate formulations induced a robust immune response, and were able to inhibit tumor growth and prolong survival in melanoma and human papillomavirus E7 in vivo tumor models. The top-performing lipids share a common structure: an unsaturated lipid tail, a dihydroimidazole linker and cyclic amine head groups. These formulations induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway, rather than through Toll-like receptors, and result in limited systemic cytokine expression and enhanced anti-tumor efficacy.