Ionic Liquid Coating-Driven Nanoparticle Delivery to the Brain: Applications for NeuroHIV.

Delivering cargo to the central nervous system (CNS) remains a pharmacological challenge. For infectious diseases such as HIV, the CNS acts as a latent reservoir that is inadequately managed by systemic antiretrovirals (ARTs). ARTs thus cannot eradicate HIV, and given CNS infection, patients experience neurological deficits collectively referred to as "neuroHIV". Herein, the development of bioinspired ionic liquid-coated nanoparticles (IL-NPs) for in situ hitchhiking on red blood cells (RBCs) is reported, which enables 48% brain delivery of intracarotid arterial- infused cargo. Moreover, IL choline trans-2-hexenoate (CA2HA 1:2) demonstrates preferential accumulation in parenchymal microglia over endothelial cells post-delivery. This study further demonstrates successful loading of abacavir (ABC), an ART that is challenging to encapsulate, into IL-NPs, and verifies retention of antiviral efficacy in vitro. IL-NPs are not cytotoxic to primary human peripheral blood mononuclear cells (PBMCs) and the CA2HA 1:2 coating itself confers notable anti-viremic capacity. In addition, in vitro cell culture assays show markedly increased uptake of IL-NPs into neural cells compared to bare PLGA nanoparticles. This work debuts bioinspired ionic liquids as promising nanoparticle coatings to assist CNS biodistribution and has the potential to revolutionize the delivery of cargos (i.e., drugs, viral vectors) through compartmental barriers such as the blood-brain-barrier (BBB).

Ionic liquid-coated lipid nanoparticles increase siRNA uptake into CNS targets.

Lipidoid nanoparticles (LNPs) have transformed the field of drug delivery and are clinically used for the delivery of nucleic acids to liver and muscle targets. Post-intravenous administration, LNPs are naturally directed to the liver due to the adsorption of plasma proteins like apolipoprotein E. In the present work, we have re-engineered LNPs with ionic liquids (ILs) to reduce plasma protein adsorption and potentially increase the accumulation of LNPs in hard-to-deliver central nervous system (CNS) targets such as brain endothelial cells (BECs) and neurons. We have developed two approaches to re-engineer LNPs using a choline trans-2-hexenoate IL: first, we have optimized an IL-coating process using the standard LNP formulation and in the second approach, we have incorporated ILs into the LNPs by replacing the PEG-lipid component in the standard formulation using ILs. IL-coated as well as IL-incorporated LNPs were colloidally stable with morphologies similar to the standard LNPs. IL-coated LNPs showed superior uptake into mouse BECs and neurons and demonstrated reduced mouse plasma protein adsorption compared to the standard LNPs. Overall, our results (1) demonstrate the feasibility of re-engineering the clinically approved LNP platform with highly tunable biomaterials like ILs for the delivery of therapeutics to CNS targets like BECs and neurons and (2) suggest that the surface properties of LNPs play a critical role in altering their affinity to and uptake into hard-to-deliver cell types.

NIR-II emissive donor-acceptor-donor fluorophores for dual fluorescence bioimaging and photothermal therapy applications.

Fluorescence bioimaging with near-infrared II (NIR-II) emissive organic fluorophores has proven to be a viable noninvasive diagnostic technique. However, there is still the need for the development of fluorophores that possess increased stability as well as functionalities that impart stimuli responsiveness. Through strategic design, we can synthesize fluorophores that possess not only NIR-II optical profiles but also pH-sensitivity and the ability to generate heat upon irradiation. In this work, we employ a donor-acceptor-donor (D-A-D) design to synthesize a series of NIR-II fluorophores. Here we use thienothiadiazole (TTD) as the acceptor, 3-hexylthiophene (HexT) as the π-spacer and vary the alkyl amine donor units: N,N-dimethylaniline (DMA), phenylpiperidine (Pip), and phenylmorpholine (Morp). Spectroscopic analysis shows that all three derivatives exhibit emission in the NIR-II region with λemimax ranging from 1030 to 1075 nm. Upon irradiation, the fluorophores exhibited noticeable heat generation through non-radiative processes. The ability to generate heat indicates that these fluorophores will act as theranostic (combination therapeutic and diagnostic) agents in which simultaneous visualization and treatment can be performed. Additionally, biosensing capabilities were supported by changes in the absorbance properties while under acidic conditions as a result of protonation of the alkyl amine donor units. The fluorophores also show minimal toxicity in a human mammary cell line and with murine red blood cells. Overall, initial results indicate viable NIR-II materials for multiple biomedical applications.

Imidazolium-based zwitterionic liquid-modified PEG-PLGA nanoparticles as a potential intravenous drug delivery carrier.

Zwitterionic-based systems offer promise as next-generation drug delivery biomaterials capable of enhancing nanoparticle (NP) stimuli-responsiveness, biorecognition, and biocompatibility. Further, imidazole-functionalized amphiphilic zwitterions are able to readily bind to various biological macromolecules, enabling antifouling properties for enhanced drug delivery efficacy and bio-targeting. Herein, we describe structurally tuned zwitterionic imidazole-based ionic liquid (ZIL)-coated PEG-PLGA nanoparticles made with sonicated nanoprecipitation. Upon ZIL surface modification, the hydrodynamic radius increased by nearly 20 nm, and the surface charge significantly shifted closer to neutral. 1H NMR spectra suggests that the amount of ZIL on the nanoparticle surface is controlled by the structure of the ZIL and that the assembly occurs as a result of non-covalent interactions of ZIL-coated nanoparticle with the polymer surface. These nanoparticle-zwitterionic liquid (ZIL) constructs demonstrate selective affinity towards red blood cells in whole mouse blood and show relatively low human hemolysis at ∼5%. Additionally, we observe higher nanoparticle accumulation of ZIL-NPs compared with unmodified NP controls in human triple-negative breast cancer cells (MDA-MB-231). Furthermore, although the ZIL shows similar protein adsorption by SDS-PAGE, LC-MS/MS protein analysis data demonstrate a difference in the relative abundance and depletion of proteins in mouse and human serum. Hence, we show that ZIL-coated nanoparticles provide a new potential platform to enhance RBC-based drug delivery systems for cancer treatments.

Selective Blood Cell Hitchhiking in Whole Blood with Ionic Liquid-Coated PLGA Nanoparticles to Redirect Biodistribution After Intravenous Injection.

Less than 5% of intravenously-injected nanoparticles (NPs) reach destined sites in the body due to opsonization and immune-based clearance in vascular circulation. By hitchhiking in situ onto specific blood components post-injection, NPs can selectively target tissue sites for unprecedentedly high drug delivery rates. Choline carboxylate ionic liquids (ILs) are biocompatible liquid salts <100X composed of bulky asymmetric cations and anions. This class of ILs has been previously shown to significantly extend circulation time and redirect biodistribution in BALB/c mice post-IV injection via hitchhiking on red blood cell (RBC) membranes. Herein, we synthesized & screened 60 choline carboxylic acid-based ILs to coat PLGA NPs and present the impact of structurally engineering the coordinated anion identity to selectively interface and hitchhike lymphocytes, monocytes, granulocytes, platelets, and RBCs in whole mouse blood for in situ targeted drug delivery. Furthermore, we find this nanoparticle platform to be biocompatible (non-cytotoxic), translate to human whole blood by resisting serum uptake and maintaining modest hitchhiking, and also significantly extend circulation retention over 24 hours in BALB/c healthy adult mice after IV injection. Because of their altered circulation profiles, we additionally observe dramatically different organ accumulation profiles compared to bare PLGA NPs. This study establishes an initial breakthrough platform for a modular and transformative targeting technology to hitchhike onto blood components with high efficacy and safety in the bloodstream post-IV administration.

Development of ionic liquid-coated PLGA nanoparticles for applications in intravenous drug delivery.

Polymeric nanoparticles (NPs) are a promising platform for medical applications in drug delivery. However, their use as drug carriers is limited by biological (e.g., immunological) barriers after intravenous administration. Ionic liquids (ILs), formed from bulky asymmetric cations and anions, have a wide variety of physical internal and external interfacing properties. When assembled on polymeric NPs as biomaterial coatings, these external-interfacing properties can be tuned to extend their circulation half-life when intravenously injected, as well as drive biodistribution to sites of interest for selective organ accumulation. In our work, we are particularly interested in optimizing IL coatings to enable red blood cell hitchhiking in whole blood. In this protocol, we describe the preparation and physicochemical and biological characterization of choline carboxylate IL-coated polymeric NPs. The procedure is divided into five stages: (1) synthesis and characterization of choline-based ILs (1 week); (2) bare poly(lactic-co-glycolic acid) (50:50, acid terminated) Resomer 504H (PLGA) NP assembly, modified from previously established protocols, with dye encapsulation (7 h); (3) modification of the bare particles with IL coating (3 h); (4) physicochemical characterization of both PLGA and IL-PLGA NPs by dynamic light scattering, 1H nuclear magnetic resonance spectroscopy, and transmission electron microscopy (1 week); (5) ex vivo evaluation of intravenous biocompatibility (including serum-protein resistance and hemolysis) and red blood cell hitchhiking in whole BALB/c mouse blood via fluorescence-activated cell sorting (1 week). With practice and technique refinement, this protocol is accessible to late-stage graduate students and early-stage postdoctoral scientists.

Lipid nanoparticle-mediated drug delivery to the brain.

Lipid nanoparticles (LNPs) have revolutionized the field of drug delivery through their applications in siRNA delivery to the liver (Onpattro) and their use in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While LNPs have been extensively studied for the delivery of RNA drugs to muscle and liver targets, their potential to deliver drugs to challenging tissue targets such as the brain remains underexplored. Multiple brain disorders currently lack safe and effective therapies and therefore repurposing LNPs could potentially be a game changer for improving drug delivery to cellular targets both at and across the blood-brain barrier (BBB). In this review, we will discuss (1) the rationale and factors involved in optimizing LNPs for brain delivery, (2) ionic liquid-coated LNPs as a potential approach for increasing LNP accumulation in the brain tissue and (3) considerations, open questions and potential opportunities in the development of LNPs for delivery to the brain.

Topical Administration of an Apoptosis Inducer Mitigates Bleomycin-Induced Skin Fibrosis.

Pathological fibrosis is distinguished from physiological wound healing by persistent myofibroblast activation, suggesting that therapies that induce myofibroblast apoptosis selectively could prevent progression and potentially reverse the established fibrosis, such as for scleroderma (a heterogeneous autoimmune disease characterized by multiorgan fibrosis). Navitoclax (NAVI) is a BCL-2/BCL-xL inhibitor with antifibrotic properties and has been investigated as a potential therapeutic for fibrosis. NAVI makes myofibroblasts particularly vulnerable to apoptosis. However, despite NAVI's significant potency, clinical translation of BCL-2 inhibitors, NAVI in this case, is hindered due to the risk of thrombocytopenia. Therefore, in this work, we utilized a newly developed ionic liquid formulation of NAVI for direct topical application to the skin, thereby avoiding systemic circulation and off-target-mediated side effects. The ionic liquid composed of choline and octanoic acid (COA) at a 1:2 molar ionic ratio increases skin diffusion and transportation of NAVI and maintains their retention within the dermis for a prolonged duration. Topical administration of NAVI-mediated BCL-xL and BCL-2 inhibition results in the transition of myofibroblast to fibroblast and ameliorates pre-existing fibrosis, as demonstrated in a scleroderma mouse model. We have observed a significant reduction of α-SMA and collagen, which are known as fibrosis marker proteins, as a result of the inhibition of anti-apoptotic proteins BCL-2/BCL-xL. Overall, our findings show that COA-assisted topical delivery of NAVI upregulates apoptosis specific to myofibroblasts, with minimal presence of the drug in the systemic circulation, resulting in an accelerated therapeutic effect with no discernible drug-associated toxicity.

Ionic Liquid Coating-Driven Nanoparticle Delivery to the Brain: Applications for NeuroHIV.

Delivering cargo to the central nervous system (CNS) remains a pharmacological challenge. For infectious diseases such as HIV, the CNS acts as a latent reservoir that is inadequately managed by systemic antiretrovirals (ARTs). ARTs thus cannot eradicate HIV, and given CNS infection, patients experience an array of neurological deficits that are collectively referred to as 'neuroHIV'. Herein we report the development of bioinspired ionic liquid-coated nanoparticles (IL-NPs) for in situ hitchhiking on red blood cells (RBCs), which enabled 48% delivery of intravenously infused cargo to the brain. Moreover, the ionic liquid (IL) choline trans-2-hexenoate (CA2HA 1:2) demonstrated preferential accumulation in parenchymal microglia over endothelial cells post-delivery. We further demonstrate the successful loading of abacavir (ABC), an ART that is challenging to encapsulate, into the IL-coated NPs and verify the retention of antiviral efficacy in vitro. IL-NPs were not cytotoxic to primary human peripheral blood mononuclear cells (PBMCs) and the CA2HA 1:2 coating conferred notable anti-viremic capacity on its own. In addition, in vitro cell culture assays showed markedly increased uptake of IL-coated nanoparticles into neuronal cells compared to bare nanoparticles. This work debuts bioinspired ionic liquids as promising nanoparticle coatings to assist CNS biodistribution and has the potential to revolutionize the delivery of cargos (i.e., drugs, viral vectors) through compartmental barriers such as the blood-brain-barrier (BBB), illustrated in the graphical abstract below.

ROS-Responsive Glycopolymeric Nanoparticles for Enhanced Drug Delivery to Macrophages.

Macrophages play a diverse, key role in many pathologies, including inflammatory diseases, cardiovascular diseases, and cancer. However, many therapeutic strategies targeting macrophages suffer from systemic off-target toxicity resulting in notoriously narrow therapeutic windows. To address this shortcoming, the development of poly(propylene sulfide)-b-poly(methacrylamidoglucopyranose) [PPS-b-PMAG] diblock copolymer-based nanoparticles (PMAG NPs) capable of targeting macrophages and releasing drug in the presence of reactive oxygen species (ROS) is reported. PMAG NPs have desirable physicochemical properties for systemic drug delivery, including slightly negative surface charge, ≈100 nm diameter, and hemo-compatibility. Additionally, due to the presence of PPS in the NP core, PMAG NPs release drug cargo preferentially in the presence of ROS. Importantly, PMAG NPs display high cytocompatibility and are taken up by macrophages in cell culture at a rate ≈18-fold higher than PEGMA NPs-NPs composed of PPS-b-poly(oligoethylene glycol methacrylate). Computational studies indicate that PMAG NPs likely bind with glucose transporters such as GLUT 1/3 on the macrophage cell surface to facilitate high levels of internalization. Collectively, this study introduces glycopolymeric NPs that are uniquely capable of both receptor-ligand targeting to macrophages and ROS-dependent drug release and that can be useful in many immunotherapeutic settings.

Improved nanoformulation and bio-functionalization of linear-dendritic block copolymers with biocompatible ionic liquids.

Linear-dendritic block copolymers (LDBCs) have emerged as promising materials for drug delivery applications, with their hybrid structure exploiting advantageous properties of both linear and dendritic polymers. LDBCs have promising encapsulation efficiencies that can be used to encapsulate both hydrophobic and hydrophilic dyes for bioimaging, cancer therapeutics, and small biomolecules. Additionally, LDBCS can be readily functionalized with varying terminal groups for more efficient targeted delivery. However, depending on structural composition and surface properties, LDBCs also exhibit high dispersities (D), poor shelf-life, and potentially high cytotoxicity to non-target interfacing blood cells during intravenous drug delivery. Here, we show that choline carboxylic acid-based ionic liquids (ILs) electrostatically solvate LDBCs by direct dissolution and form stable and biocompatible IL-integrated LDBC nano-assemblies. These nano-assemblies are endowed with red blood cell-hitchhiking capabilities and show altered cellular uptake behavior ex vivo. When modified with choline and trans-2-hexenoic acid, IL-LDBC dispersity dropped by half compared to bare LDBCs, and showed a significant shift of the cationic surface charge towards neutrality. Proton nuclear magnetic resonance spectroscopy evidenced twice the total amount of IL on the LDBCs relative to an established IL-linear PLGA platform. Transmission electron microscopy suggested the formation of a nanoparticle surface coating, which acted as a protective agent against RBC hemolysis, reducing hemolysis from 73% (LDBC) to 25% (IL-LDBC). However, dramatically different uptake behavior of IL-LDBCs vs. IL-PLGA NPs in RAW 264.7 macrophage cells suggests a different conformational IL-NP surface assembly on the linear versus the linear-dendritic nanoparticles. These results suggest that by controlling the physical chemistry of polymer-IL interactions and assembly on the nanoscale, biological function can be tailored toward the development of more effective and more precisely targeted therapies.

Protein-avoidant ionic liquid (PAIL)-coated nanoparticles to increase bloodstream circulation and drive biodistribution.

The rapid clearance of intravenously administered nanoparticles (NPs) from the bloodstream is a major unsolved problem in nanomedicine. Here, we describe the first use of biocompatible protein-avoidant ionic liquids (PAILs) as NP surface modifiers to reduce opsonization. An ionic liquid choline hexenoate, selected for its aversion to serum proteins, was used to stably coat the surface of poly(lactic-co-glycolic acid) (PLGA) NPs. Compared with bare PLGA and poly(ethylene glycol)-coated PLGA particles, the PAIL-PLGA NPs showed resistance to protein adsorption in vitro and greater retention in blood of mice at 24 hours. Choline hexenoate redirected biodistribution of NPs, with preferential accumulation in the lungs with 50% of the administered dose accumulating in the lungs and <5% in the liver. Lung accumulation was attributed to spontaneous attachment of the PAIL-coated NPs on red blood cells in vivo. Overall, ionic liquids are a promising class of materials for NP modification for biomedical applications.

Urological complications of obesity and diabetes in males and females of three mouse models: temporal manifestations.

Diabetic bladder dysfunction is a frequent complication of diabetes. Although many mouse models of diabetes now exist, there has been little systematic effort to characterize them for the timing of onset and severity of bladder dysfunction. We monitored metabolic status and tested bladder function by void spot assay and limited anesthetized cystometry in both male and female mice of three models of obesity and diabetes: a type 1 diabetes model (the Akita mouse) and two type 2 diabetes models [the diet-induced obese (DIO) model and the ob/ob mouse]. Akita mice had insulin pellets implanted subcutaneously every 3 mo to mimic poorly controlled type 1 diabetes in humans. Mice were hyperglycemic by 48 days after implants. Female mice exhibited no bladder dysfunction at any age up to 20 mo and gained weight normally. In contrast, by 7 mo, male Akita mice developed a profound polyuria and failed to show normal weight gain. There were no observable signs of bladder dysfunction in either sex. DIO mice on high/low-fat diets for 16 mo exhibited mild hyperglycemia in female mice (not in male mice), mild weight gain, and no evidence of bladder dysfunction. Ob/ob mice were followed for 8 mo and became extremely obese. Male and female mice were glucose intolerant, insulin intolerant, and hyperinsulinemic at 4 mo. By 8 mo, their metabolic status had improved but was still abnormal. Urine volume increased in male mice but not in female mice. Bladder dysfunction was observed in the spotting patterns of female mice at 4 and 6 mo of age, resolving by 8 mo. We conclude there are dramatic sex-related differences in lower urinary tract function in these models. Male Akita mice may be a good model for polyuria-related bladder remodeling, whereas female ob/ob mice may better mimic storage problems related to loss of outlet control in a setting of type 2 diabetes complicated by obesity.

Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss.

Hearing loss affects more than two-thirds of the elderly population, and more than 17% of all adults in the U.S. Sensorineural hearing loss related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs, stimulating intense interest in therapies to regenerate synaptic function. 7,8-Dihydroxyflavone (DHF) is a selective and potent agonist of tropomyosin receptor kinase B (TrkB) and protects the neuron from apoptosis. Despite evidence that TrkB agonists can promote survival of SGNs, local delivery of drugs such as DHF to the inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached SGNs. The results suggest that bisphosphonate-TrkB agonist conjugates have promise as a novel approach to targeted delivery of drugs to treat sensorineural hearing loss.