Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

Lipid Nanoparticles for Nucleic Acid Delivery to Endothelial Cells.

Endothelial cells play critical roles in circulatory homeostasis and are also the gateway to the major organs of the body. Dysfunction, injury, and gene expression profiles of these cells can cause, or are caused by, prevalent chronic diseases such as diabetes, cardiovascular disease, and cancer. Modulation of gene expression within endothelial cells could therefore be therapeutically strategic in treating longstanding disease challenges. Lipid nanoparticles (LNP) have emerged as potent, scalable, and tunable carrier systems for delivering nucleic acids, making them attractive vehicles for gene delivery to endothelial cells. Here, we discuss the functions of endothelial cells and highlight some receptors that are upregulated during health and disease. Examples and applications of DNA, mRNA, circRNA, saRNA, siRNA, shRNA, miRNA, and ASO delivery to endothelial cells and their targets are reviewed, as well as LNP composition and morphology, formulation strategies, target proteins, and biomechanical factors that modulate endothelial cell targeting. Finally, we discuss FDA-approved LNPs as well as LNPs that have been tested in clinical trials and their challenges, and provide some perspectives as to how to surmount those challenges.

Nanofibrillar Patches of Commensal Skin Bacteria.

We demonstrate entrapment of the commensal skin bacteria Staphylococcus epidermidis in mats composed of soft nanotubes made by membrane-templated layer-by-layer (LbL) assembly. When cultured in broth, the resulting nanofibrillar patches efficiently delay the escape of bacteria and their planktonic growth, while displaying high steady-state metabolic activity. Additionally, the material properties and metabolic activity can be further tuned by postprocessing the patches with additional polysaccharide LbL layers. These patches offer a promising methodology for the fabrication of bacterial skin dressings for the treatment of skin dysbiosis while preventing adverse effects due to bacterial proliferation.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Photothermally targeted thermosensitive polymer-masked nanoparticles.

The targeted delivery of therapeutic cargos using noninvasive stimuli has the potential to improve efficacy and reduce off-target effects (toxicity). Here, we demonstrate a targeting mechanism that uses a thermoresponsive copolymer to mask a peptide ligand that binds a widely distributed receptor (integrin ß1) on the surface of silica core-gold shell nanoparticles. The nanoparticles convert NIR light into heat, which causes the copolymer to collapse, exposing the ligand peptide, allowing cell binding. The use of NIR light could allow targeting of plasmonic nanoparticles deep within tissues. This approach could be extended to a variety of applications including photothermal therapy and drug delivery.

Enhanced photothermal effect of plasmonic nanoparticles coated with reduced graphene oxide.

We report plasmonic gold nanoshells and nanorods coated with reduced graphene oxide that produce an enhanced photothermal effect when stimulated by near-infrared (NIR) light. Electrostatic interactions between nanosized graphene oxide and gold nanoparticles followed by in situ chemical reduction generated reduced graphene oxide-coated nanoparticles; the coating was demonstrated using Raman and HR-TEM. Reduced graphene oxide-coated gold nanoparticles showed enhanced photothermal effect compared to noncoated or nonreduced graphene oxide-coated gold nanoparticles. Reduced graphene oxide-coated gold nanoparticles killed cells more rapidly than did noncoated or nonreduced graphene oxide-coated gold nanoparticles.

A Machine Learning-optimized system for on demand, pulsatile, photo- and chemo-therapeutic treatment using near-infrared responsive MoS 2 -based microparticles in a breast cancer model.

Cancer therapy research is of high interest because of the persistence and mortality of the disease and the side effects of traditional therapeutic methods, while often multimodal treatments are necessary based on the patient's needs. The development of less invasive modalities for recurring treatment cycles is thus of critical significance. Herein, a light-activatable microparticle system was developed for localized, pulsatile delivery of anticancer drugs with simultaneous thermal ablation, by applying controlled ON-OFF thermal cycles using near-infrared laser irradiation. The system is composed of poly(caprolactone) microparticles of 200 µm size with incorporated molybdenum disulfide (MoS 2 ) nanosheets as the photothermal agent and hydrophilic doxorubicin or hydrophobic violacein, as model drugs. Upon irradiation the nanosheets heat up to ≥50 °C leading to polymer matrix melting and release of the drug. MoS 2 nanosheets exhibit high photothermal conversion efficiency and allow for application of low power laser irradiation for the system activation. A Machine Learning algorithm was applied to acquire optimal laser operation conditions; 0.4 W/cm 2 laser power at 808 nm, 3-cycle irradiation, for 3 cumulative minutes. In a mouse subcutaneous model of 4T1 triple-negative breast cancer, 25 microparticles were intratumorally administered and after 3-cycle laser treatment the system conferred synergistic phototherapeutic and chemotherapeutic effect. Our on-demand, pulsatile synergistic treatment resulted in increased median survival up to 40 days post start of treatment compared to untreated mice, with complete eradication of the tumors at the primary site. Such a system could have potential for patients in need of recurring cycles of treatment on subcutaneous tumors.

Engineered 3D Immuno-Glial-Neurovascular Human Brain Model.

Patient-specific, human-based cellular models that integrate biomimetic BBB, immune, and myelinated neuron components are critically needed to enable translationally relevant and accelerated discovery of neurological disease mechanisms and interventions. By engineering a brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo- like hallmarks. As proof of principle, here we utilized the miBrain to model Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types in a modular system with unique utility for elucidating cell-type specific contributions to pathogenesis. We here harness this feature to identify that risk factor APOE4 in astrocytes promotes tau pathogenesis and neuronal dysregulation through crosstalk with microglia. One-Sentence Summary: A novel patient-specific brain model with BBB, neuronal, immune, and glial components was developed, characterized, and harnessed to model Alzheimer's Disease-associated pathologies and APOE4 genetic risk.

Engineering kinetics of TLR7/8 agonist release from bottlebrush prodrugs enables tumor-focused immune stimulation.

Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 "bottlebrush prodrugs" (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.

Synergistic silencing: combinations of lipid-like materials for efficacious siRNA delivery.

Despite the promise of RNA interference (RNAi) therapeutics, progress toward the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. Nearly all siRNA delivery vehicles developed to date employ a single cationic or ionizable material. In order to increase the material space available for development of siRNA delivery therapeutics, this study examined the possibility of using binary combinations of ionizable lipid-like materials to synergistically achieve gene silencing. Interestingly, it was found that ineffective single lipid-like materials could be formulated together in a single delivery vehicle to induce near-complete knockdown of firefly luciferase and factor VII in HeLa cells and in mice, respectively. Microscopy experiments suggested that synergistic action resulted when combining materials that respectively mediated cellular uptake and endosomal escape, two important steps in the delivery process. Together, the data indicate that formulating lipid-like materials in combination can significantly improve siRNA delivery outcomes while increasing the material space available for therapeutic development. It is anticipated that this binary formulation strategy could be applicable to any siRNA delivery material in any target cell population that utilizes the two-step endosomal delivery pathway.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Controlled delivery of gold nanoparticle-coupled miRNA therapeutics via an injectable self-healing hydrogel.

Differential expression of microRNAs (miRNAs) plays a role in many diseases, including cancer and cardiovascular diseases. Potentially, miRNAs could be targeted with miRNA-therapeutics. Sustained delivery of these therapeutics remains challenging. This study couples miR-mimics to PEG-peptide gold nanoparticles (AuNP) and loads these AuNP-miRNAs in an injectable, shear thinning, self-assembling polymer nanoparticle (PNP) hydrogel drug delivery platform to improve delivery. Spherical AuNPs coated with fluorescently labelled miR-214 are loaded into an HPMC-PEG-b-PLA PNP hydrogel. Release of AuNP/miRNAs is quantified, AuNP-miR-214 functionality is shown in vitro in HEK293 cells, and AuNP-miRNAs are tracked in a 3D bioprinted human model of calcific aortic valve disease (CAVD). Lastly, biodistribution of PNP-AuNP-miR-67 is assessed after subcutaneous injection in C57BL/6 mice. AuNP-miRNA release from the PNP hydrogel in vitro demonstrates a linear pattern over 5 days up to 20%. AuNP-miR-214 transfection in HEK293 results in 33% decrease of Luciferase reporter activity. In the CAVD model, AuNP-miR-214 are tracked into the cytoplasm of human aortic valve interstitial cells. Lastly, 11 days after subcutaneous injection, AuNP-miR-67 predominantly clears via the liver and kidneys, and fluorescence levels are again comparable to control animals. Thus, the PNP-AuNP-miRNA drug delivery platform provides linear release of functional miRNAs in vitro and has potential for in vivo applications.

A New Approach for Microfabrication of Printed Circuit Boards with Ultrafine Traces.

The advances in micro/nanofabrication techniques have enabled miniaturization of printed circuit boards (PCBs) for various applications such as portable devices, smart sensors, and IoTs, to name a few. PCBs provide electrical connectivity between the components as well as mechanical support. Down-scaling of PCBs is crucial for miniaturization of large systems and devices. Currently, microtraces down to 25 µm can be microfabricated with the current microfabrication processes at an industrial scale. In the present work, we report a new approach for microfabrication of PCBs with trace widths down to 3 µm on commercially available PCB substrates. We used electroplating/electroetching, sputtering, and photolithography to achieve these fine trace sizes. The proposed fabrication technique can be used in microelectronics, system on chip, MEMS, and miniaturized circuits and systems in general.

Amino alcohol-based degradable poly(ester amide) elastomers.

Currently available synthetic biodegradable elastomers are primarily composed of crosslinked aliphatic polyesters, which suffer from deficiencies including (1) high crosslink densities, which results in exceedingly high stiffness, (2) rapid degradation upon implantation, or (3) limited chemical moieties for chemical modification. Herein, we have developed poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate)s, a new class of synthetic, biodegradable elastomeric poly(ester amide)s composed of crosslinked networks based on an amino alcohol. These crosslinked networks feature tensile Young's modulus on the order of 1MPa and reversable elongations up to 92%. These polymers exhibit in vitro and in vivo biocompatibility. These polymers have projected degradation half-lives up to 20 months in vivo.

The use of charge-coupled polymeric microparticles and micromagnets for modulating the bioavailability of orally delivered macromolecules.

Protein drugs have low bioavailability after oral administration, which is due in part to fast transit of the drugs or drug delivery vehicles through the gastrointestinal tract. Increasing the time that the drugs spend in the intestine after dosing would allow for greater absorption and increased bioavailability. We developed a formulation strategy that can be used to prolong intestinal retention of drug delivery vehicles without substantial alterations to current polymeric encapsulation strategies. A model drug, insulin, was encapsulated in negatively charged poly(lactic-co-glycolic acid) (PLGA) microparticles, and the microparticles were subsequently mixed with positively charged micromagnets, whose size will prevent them from being absorbed. Stable complexes formed through electrostatic interaction. The complexes were effectively immobilized in vitro in a model of the mouse small intestine by application of an external magnetic field. Mice that were gavaged with radio-labeled complexes and fitted with a magnetic belt retained 32.5% of the (125)I-insulin in the small intestine compared with 5.4% for the control group 6h after administration (p=0.005). Furthermore, mice similarly gavaged with complexes encapsulating insulin (120 Units/kg) exhibited long-term glucose reduction in the groups with magnetic belts. The corresponding bioavailability of insulin was 5.11% compared with 0.87% for the control group (p=0.007).

Intracellular delivery of core-shell fluorescent silica nanoparticles.

Highly fluorescent core-shell silica nanoparticles made by the modified Stöber process (C dots) are promising as tools for sensing and imaging subcellular agents and structures but will only be useful if they can be easily delivered to the cytoplasm of the subject cells. This work shows that C dots can be electrostatically coated with cationic polymers, changing their surface charge and enabling them to escape from endosomes and enter the cytoplasm and nucleus. As an example of cellular delivery, we demonstrate that these particles can also be complexed with DNA and mediate and trace DNA delivery and gene expression.

Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine).

Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50-60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines.