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.

Del Nido cardioplegia versus blood cardioplegia in coronary artery bypass grafting.

Purpose: The del Nido cardioplegia (DC) has been extensively used in congenital heart surgery for over two decades and is becoming popular in adult cardiac surgery. We evaluated the efficacy and safety of DC, compared to conventional blood cardioplegia (BC), in adult patients undergoing isolated coronary artery bypass grafting (CABG). Methods: This metachronous study included a total of 2330 consecutive patients who underwent isolated CABG. The study population was divided into two groups: BC group, consisting of 1165 patients (May 2012 through December 2015); and DC (del Nido) cardioplegia group consisting of 1165 patients (January 2016 through June 2018). Propensity matching yielded 735 well-matched pairs. The propensity-matched cohorts of BC and DC were compared in terms of myocardial function outcomes and other clinical outcomes to determine the efficacy and safety of both the cardioplegic solutions. Results: There was no difference in 30-day mortality [odds ratio (OR), 0.74; 95% confidence interval (CI), 0.16-3.35, p = 0.70]. There was a significant decrease in the DC group in the postoperative events, including re-exploration rates [OR, 0.25; 95% CI, 0.118-0.568, p < 0.001], myocardial infarction [OR, 0.282; 95% CI, 0.133-0.596, p < 0.001], left ventricular dysfunction [OR, 0.60; 95% CI, 0.396-0.916, p = 0.018], and acute kidney injury (AKI) [OR, 0.255; 95% CI, 0.156-0.418, p < 0.001]. The rate of spontaneous return to sinus rhythm was significantly higher in the DC group [OR, 5.162; 95% CI, 3.701-7.198, p < 0.001]. Cardiopulmonary bypass time (95.2 ± 29.1 min vs. 82.1 ± 28.8 min, p < 0.001) and aortic cross-clamp (ACC) time (57.3 ± 19 min vs. 48.7 ± 19.0 min, p < 0.001) were higher in the DC group, but the absolute difference in ACC time was only 8 min. There was no difference in AKI requiring renal replacement therapy [OR, 0.62; 95% CI, 0.203-1.912, p = 0.40], postoperative cerebrovascular accidents [OR, 0.398; 95% CI, 0.077-2.059, p = 0.073], and postoperative ventricular arrhythmias [OR, 0.80; 95% CI, 0.456-1.916, p = 0.47]. Conclusion: This study revealed comparable clinical outcomes and effective myocardial protection with DC, compared to BC in patients undergoing isolated CABG. In addition, DC demonstrated the ease of administration with the feasibility of single-dose administration.

Trends of re-operative coronary artery bypass grafting: A two-decade experience.

BACKGROUND: The prevalence of redo coronary artery bypass grafting has markedly changed over the last two decades. We aim to assess the trends of redo coronary artery bypass grafting and clinical outcomes of redo coronary artery bypass grafting over two different periods. METHODS: This is a retrospective, metachronous, observational study of patients who underwent redo coronary artery bypass grafting between January 1998 through December 2004 and January 2005 through September 2021 who were categorized into group 1 and group 2, respectively. The trends in the prevalence of redo coronary artery bypass grafting, and 30-day outcomes were analyzed. RESULTS: A total of 261 patients underwent a redo coronary artery bypass grafting. Group 1 and group 2 included 114 and 147 patients, respectively. 62.59% of group 2 patients underwent off-pump coronary artery bypass grafting compared to 6.14% in group 1 (p < 0.01). The patients undergoing redo coronary artery bypass grafting in group 2 were significantly older (60.65 ± 7.78 years vs. 50.99 ± 7.66 years, p < 0.001) and were more likely to be hypertensive (49.66% vs. 29.82%, p < 0.001). There was no significant difference in the proportion of diabetes (p = 0.82), smoking (p = 0.24), dyslipidemia (p = 0.64), and preoperative myocardial infarction (p = 0.14). The proportion of patients who presented post-PCI was significantly higher in group 2 (27.89% vs. 10.53%, p < 0.001). There was also a shift in the usage of grafts from the left internal thoracic artery to the saphenous vein graft (p < 0.001). CONCLUSION: The annual rate of redo coronary artery bypass grafting was declining but has now reached a plateau. The current outcomes of patients undergoing redo coronary artery bypass grafting are comparable to that of the earlier times despite a higher co-morbidity burden in patients of recent times.

The Promising Therapeutic Potential of Oligonucleotides for Pulmonary Fibrotic Diseases.

INTRODUCTION: Fibrotic lung diseases represent a large subset of diseases with an unmet clinical need. Oligonucleotide therapies (ONT) are a promising therapeutic approach for the treatment of pulmonary disease as they can inhibit pathways that are otherwise difficult to target. Additionally, targeting the lung specifically with ONT is advantageous because it reduces the possibilities of systemic side effects and tolerability concerns. AREAS COVERED: This review presents the chemical basis of designing various ONTs currently known to treat fibrotic lung diseases. Further, the authors have also discussed the delivery vehicle, routes of administration, physiological barriers of the lung, and toxicity concerns with ONTs. EXPERT OPINION: ONTs provide a promising therapeutic approach for the treatment of fibrotic diseases of the lung, particularly because ONTs directly delivered to the lung show little systemic side effects compared to current therapeutic strategies. Dry powder aerosolized inhalers may be a good strategy for getting ONTs into the lung in humans. However, as of now, no dry powder ONTs have been approved for use in the clinical setting, and this challenge must be overcome for future therapies. Various delivery methods that can aid in direct targeting may also improve the use of ONTs for lung fibrotic diseases.

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.

Predictors of mortality and morbidity in total anomalous pulmonary venous connection with biventricular physiology: A 10-year Indian single centre experience of 492 patients.

Background: Surgical correction of total anomalous pulmonary venous connection (TAPVC) remains associated with significant mortality despite advances in intra-operative and postoperative management. We retrospectively analyzed 492 consecutive TAPVC patients with biventricular physiology, who were operated at our centre, with regard to predictors of mortality, morbidity, and intermediate-term outcomes. Materials and Methods: A total of 492 TAPVC patients with biventricular physiology were operated at our centre from August 2009 to November 2019. Their medical records were reviewed and were followed up during March-April 2020 for any symptoms of cardiac disease. Results: Of 492, 302 (61.38%) were healthy at follow-up, 29 (5.89%) had postoperative mortality, 23 (4.67%) had mortality during the follow-up period, and 138 (28.05%) were lost to follow up. Age <1 month and weight <2.5 kg were associated with higher mortality with odds ratios (OR) of 6.37 and 5.56, respectively. There was no difference in mortality in different types of TAPVC. Obstructed TAPVC was associated with higher mortality with OR of 3.05. Acute kidney injury requiring peritoneal dialysis and sepsis were associated with higher mortality with ORs of 10.17 and 3.29, respectively. All follow-up mortality occurred in <1 year from the index operation. Anastomotic gradients were significantly higher in patients who died. Conclusions: Although peri-operative TAPVC mortality has reduced, mortality on follow-up continues to occur and is partly due to the obstruction of pulmonary venous pathway. Meticulous follow-up holds the key in further reducing the mortality. Larger studies are needed for the identification of risk factors for pulmonary venous obstruction and its preventive strategies.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

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.

The Evolving Druggability and Developability Space: Chemically Modified New Modalities and Emerging Small Molecules.

The druggability and developability space is rapidly evolving in the post-genomic era. In the past, Lipinski's rule-of-five (Ro5) emerged and served as a guide for drug-like molecule design for oral delivery in the traditional druggable target space. In contrast, in this new era, a transition is occurring in drug discovery towards novel approaches to bind and modulate challenging biological targets that have led to transformative treatments for patients. Consequently, drugging novel targets using a variety of emerging molecular modalities, namely beyond the Ro5 (bRo5) small molecules (such as protein-protein interaction modulators, protein-targeted chimeras, or PROTACs), peptide/peptidomimetics, and nucleic acid-based modalities, have become a key focus in drug discovery. Herein, the emerging druggability and developability space is discussed side by side to build a general understanding of the potential development challenges of these novel modalities. An overview is provided on the evolving novel targets and molecular modalities, followed by a detailed analysis of the druggability aspects as well as the strategies used to progress drug candidate, and the trending chemistry and formulation strategies used to assess developability.

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 occult renal disease on the outcomes of off-pump and on-pump coronary artery bypass grafting.

PURPOSE: Occult renal disease (ORD) is a clinical condition in which glomerular filtration rate (GFR) is less than 60 ml/min/1.73 m2, while serum creatinine is ≤ 1.3 mg/dl. The aim of the study was to compare the incidence of postoperative stage I acute kidney injury (AKI) according to Acute Kidney Injury Network (AKIN) classification in patients with ORD undergoing either off-pump or on-pump coronary artery bypass grafting. METHODS: A single center prospective randomized study was conducted from March 2011 through January 2014. A total of 120 coronary artery disease (CAD) patients with ORD undergoing coronary artery bypass grafting (CABG) were randomized to either off-pump (group1, n = 62) or on-pump (group2, n = 58) CABG in 1:1 ratio by computer-generated random number table. The GFR and serum creatinine levels were measured preoperatively and postoperatively on day 1 and day 5. The primary outcome (postoperative AKI (stage I)) and secondary outcomes (AKI (stage III) requiring renal replacement therapy (RRT) death, myocardial infarction (MI), cerebrovascular accident, atrial fibrillation (AF), and re-exploration for bleeding) at 30 days were analyzed between the groups. RESULTS: There is no significant difference in baseline characteristics of patients between off-pump and on-pump group. The incidence of postoperative AKI (stage I) was similar between on-pump (20.69%) and off-pump (16.13%) groups (p = 0.51). There was no significant difference in mortality (p = 0.33), postoperative MI (p = 0.34), cerebrovascular accident (p = 1.00), re-exploration (p = 0.96), and AF (p = 0.50). The number of patients of stage III AKI requiring RRT was higher in the off-pump group (3 patients, 4.8%) and none in the on-pump group (p = 0.08). CONCLUSIONS: This study demonstrated that on-pump CABG is associated with significantly lower GFR and significantly higher serum creatinine on postoperative day 1 which return to baseline by postoperative day 5. In patients with ORD undergoing CABG, the incidence of postoperative AKI and major adverse cardiac and cerebrovascular events were similar between off-pump or on-pump CABG patients.

Synthesis of LewisX-O-Core-1 threonine: A building block for O-linked LewisX glycopeptides.

LewisX (LeX) is a branched trisaccharide Galß1→4(Fucα1→3)GlcNAc that is expressed on many cell surface glycoproteins and plays critical roles in innate and adaptive immune responses. However, efficient synthesis of glycopeptides bearing LeX remains a major limitation for structure-function studies of the LeX determinant. Here we report a total synthesis of a LeX pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted [3 + 2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked LeX glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked LeX or sialyl LewisX (sLeX).

Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo.

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer.

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.

Total synthesis and bioactivity of 18(R)-hydroxyeicosapentaenoic acid.

Resolvins are family of lipid mediators derived from omega-3 polyunsaturated fatty acids, which are generated during the resolution phase of acute inflammation. Resolvin E1 is biosynthesized from eicosapentaenoic acid via 18(R)-hydroxyeicosapentaenoic acid (18R-HEPE) in the Cox-2 and lipoxygenase mediated pathway and has proven to exhibit potent anti-inflammatory activity. We report herein the first total chemical synthesis of 18R-HEPE and demonstrate that this compound displays in vivo bioactivity by blocking neutrophil infiltration in a murine model of zymosan-induced peritonitis.

Cell surface engineering with polyelectrolyte multilayer thin films.

Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.

Synthesis of an Fmoc-threonine bearing core-2 glycan: a building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis.

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis(x) oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.

Chemoselective immobilization of peptides on abiotic and cell surfaces at controlled densities.

We report herein a new and enabling approach for decorating both abiotic and cell surfaces with the extracellular matrix IKVAV peptide in a site-specific manner using strain promoted azide-alkyne cycloaddition. A cyclooctyne-derivatized IKVAV peptide was synthesized and immobilized on the surface of pancreatic islets through strain-promoted azide-alkyne cycloaddition with cell surface azides generated by the electrostatic adsorption of a cytocompatible poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymer bearing azido groups (PP-N(3)). Both "one-pot" and sequential addition of PP-N(3) and a cyclooctyne-derivatized IKVAV peptide conjugate enabled efficient modification of the pancreatic islet surface in less than 60 min. The ability to bind peptides at controlled surface densities was demonstrated in a quantitative manner using microarrays. Additionally, the technique is remarkably rapid and highly efficient, opening new avenues for the molecular engineering of cellular interfaces and protein and peptide microarrays.

Noncovalent cell surface engineering with cationic graft copolymers.

Chemical approaches to cell surface engineering have emerged as powerful tools for resurfacing the molecular landscape of cells and tissues. Here we report a new strategy for re-engineering cell surfaces through electrostatic adsorption of appropriately structured and functionalized poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers to cellular interfaces. Grafting of methoxy terminated tetra(ethylene glycol) chains to PLL abrogated polycation cytotoxicity in a charge density and PEG dependent manner, and copolymers structured with a unique balance of grafted PEG chains and free lysine monomers adsorbed to cell surfaces without compromising viability. Structurally analogous PLL-g-PEG copolymers bearing terminally functionalized PEG grafts were used as 'cell surface active' polymeric carriers for biotin, hydrazide, and azide moieties, which selectively captured streptavidin-, aldehyde-, and cyclooctyne-labeled probes, respectively, on cell surfaces. This strategy opens new opportunities in cell surface engineering, including generation of unique cell surface motifs, rapid and combinatorial surface modification, and use of biologically complex solvents. Tailored PLL-g-PEG copolymers offer a promising and enabling tool for bio/chemically remodeling cells and tissues with broad potential in biomedical and biotechnological applications.