Search

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs to deliver therapeutic proteins or vaccinate against infectious diseases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg−1, or intravenously (IV), 2 mg kg−1, and then IV administer modmRNA-LNP, 0.32 mg kg−1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver by 95-fold and 52-fold, respectively. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion and TLR3 −/− and TLR4−/− knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed., Graphical abstract Unlabelled Image

Red blood cell (RBC) hitchhiking is a method of drug delivery that can increase drug concentration in target organs by orders of magnitude. In RBC hitchhiking, drug-loaded nanoparticles (NPs) are adsorbed onto red blood cells and then injected intravascularly, which causes the NPs to transfer to cells of the capillaries in the downstream organ. RBC hitchhiking has been demonstrated in multiple species and multiple organs. For example, RBC-hitchhiking NPs localized at unprecedented levels in the brain when using intra-arterial catheters, such as those in place immediately after mechanical thrombectomy for acute ischemic stroke. RBC hitchhiking has been successfully employed in numerous preclinical models of disease, ranging from pulmonary embolism to cancer metastasis. In addition to summarizing the versatility of RBC hitchhiking, we also describe studies into the surprisingly complex mechanisms of RBC hitchhiking as well as outline future studies to further improve RBC hitchhiking's clinical utility.

Background Despite current resuscitation strategies, circulatory shock and organ injury after cardiac surgery occur in 25–40% of patients. Goal-directed resuscitation after cardiac surgery has generated significant interest, but clinical practice to normalize hemodynamic variables including mean arterial pressure, cardiac filling pressures, and cardiac output may not reverse microcirculation abnormalities and do not address cellular dysoxia. Recent advances in technology have made it possible to measure critical components of oxygen delivery and oxygen utilization systems in live human tissues and blood cells. The MicroRESUS study will be the first study to measure microcirculatory and mitochondrial function in patients with circulatory shock and link these findings with clinical outcomes. Methods and analysis This will be a prospective, observational study that includes patients undergoing elective cardiovascular surgery with cardiopulmonary bypass (CPB). Microcirculation will be quantified with sublingual incident dark field videomicroscopy. Mitochondrial respiration will be measured by performing a substrate–uncoupler–inhibitor titration protocol with high resolution respirometry on peripheral blood mononuclear cells at baseline and serial timepoints during resuscitation and at recovery as a possible liquid biomarker. Plasma samples will be preserved for future analysis to examine endothelial injury and other mechanisms of microcirculatory dysfunction. Thirty-day ventilator and vasopressor-free days (VVFDs) will be measured as a primary outcome, along with sequential organ failure assessment scores, and other clinical parameters to determine if changes in microcirculation and mitochondrial respiration are more strongly associated with clinical outcomes compared to traditional resuscitation targets. Discussion This will be the first prospective study to examine both microcirculatory and mitochondrial function in human patients with circulatory shock undergoing cardiac bypass and address a key mechanistic knowledge gap in the cardiovascular literature. The results of this study will direct future research efforts and therapeutic development for patients with shock.

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.

Introduction Wearable biosensors have the potential to monitor physiological change associated with opioid overdose among people who use drugs. Methods We enrolled 16 individuals who reported ≥4 daily opioid use events within the previous 30 day. Each was assigned a wearable biosensor that measured respiratory rate (RR) and actigraphy every 15 seconds for 5 days and also completed a daily interview assessing drug use. We describe the volume of RR data collected, how it varied by participant characteristics and drug use over time using repeated measures one-way ANOVA, episodes of acute respiratory depression (≤5 breaths/minute), and self-reported overdose experiences. Results We captured 1626.4 hours of RR data, an average of 21.7 daily hours/participant over follow-up. Individuals with longer injection careers and those engaging in polydrug use captured significantly fewer total hours of respiratory data over follow-up compared to those with shorter injections careers (94.7 vs. 119.9 hours, p = 0.04) and injecting fentanyl exclusively (98.7 vs. 119.5 hours, p = 0.008), respectively. There were 385 drug use events reported over follow-up. There were no episodes of acute respiratory depression which corresponded with participant reports of overdose experiences. Discussion Our preliminary findings suggest that using a wearable biosensor to monitor physiological changes associated with opioid use was feasible. However, more sensitive biosensors that facilitate triangulation of multiple physiological data points and larger studies of longer duration are needed.

Drug delivery research pursues many types of carriers including proteins and other macromolecules, natural and synthetic polymeric structures, nanocarriers of diverse compositions and cells. In particular, liposomes and lipid nanoparticles represent arguably the most advanced and popular human-made nanocarriers, already in multiple clinical applications. On the other hand, red blood cells (RBCs) represent attractive natural carriers for the vascular route, featuring at least two distinct compartments for loading pharmacological cargoes, namely inner space enclosed by the plasma membrane and the outer surface of this membrane. Historically, studies of liposomal drug delivery systems (DDS) astronomically outnumbered and surpassed the RBC-based DDS. Nevertheless, these two types of carriers have different profile of advantages and disadvantages. Recent studies showed that RBC-based drug carriers indeed may feature unique pharmacokinetic and biodistribution characteristics favorably changing benefit/risk ratio of some cargo agents. Furthermore, RBC carriage cardinally alters behavior and effect of nanocarriers in the bloodstream, so called RBC hitchhiking (RBC-HH). This article represents an attempt for the comparative analysis of liposomal vs RBC drug delivery, culminating with design of hybrid DDSs enabling mutual collaborative advantages such as RBC-HH and camouflaging nanoparticles by RBC membrane. Finally, we discuss the key current challenges faced by these and other RBC-based DDSs including the issue of potential unintended and adverse effect and contingency measures to ameliorate this and other concerns.

Graphical abstract Targeted drug delivery and nanomedicine hold the potential promise of delivering drugs solely to target organs or cell types, thus decreasing off-target side effects and improving efficacy. However, nano-scale drug carriers face several barriers to this goal, with one of the most formidable being the complement cascade. Complement proteins, especially C3, opsonize not just the microbes they evolved to contain, but also nanocarriers. This results in multiple problems, including marking the nanocarriers for clearance by leukocytes, likely fouling of the targeting moieties on nanocarriers, and release of toxins which produce deleterious local and systemic effects. Here, we review how complement achieves its blockade of nanomedicine, which nanocarrier materials properties best avoid complement, and current and future strategies to control complement to unleash nanomedicine’s potential.

Background The incidence of opioid-related overdose deaths has been rising for 30 years and has been further exacerbated amidst the COVID-19 pandemic. Naloxone can reverse opioid overdose, lower death rates, and enable a transition to medication for opioid use disorder. Though current formulations for community use of naloxone have been shown to be safe and effective public health interventions, they rely on bystander presence. We sought to understand the preferences and minimum necessary conditions for wearing a device capable of sensing and reversing opioid overdose among people who regularly use opioids. Methods We conducted a combined cross-sectional survey and semi-structured interview at a respite center, shelter, and syringe exchange drop-in program in Philadelphia, Pennsylvania, USA, during the COVID-19 pandemic in August and September 2020. The primary aim was to explore the proportion of participants who would use a wearable device to detect and reverse overdose. Preferences regarding designs and functionalities were collected via a questionnaire with items having Likert-based response options and a semi-structured interview intended to elicit feedback on prototype designs. Independent variables included demographics, opioid use habits, and previous experience with overdose. Results A total of 97 adults with an opioid use history of at least 3 months were interviewed. A majority of survey participants (76%) reported a willingness to use a device capable of detecting an overdose and automatically administering a reversal agent upon initial survey. When reflecting on the prototype, most respondents (75.5%) reported that they would wear the device always or most of the time. Respondents indicated discreetness and comfort as important factors that increased their chance of uptake. Respondents suggested that people experiencing homelessness and those with low tolerance for opioids would be in greatest need of the device. Conclusions The majority of people sampled with a history of opioid use in an urban setting were interested in having access to a device capable of detecting and reversing an opioid overdose. Participants emphasized privacy and comfort as the most important factors influencing their willingness to use such a device. Trial registration NCT04530591.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases., Unwanted uptake in the liver and limited accumulation in target organs is a major obstacle to targeted drug delivery. Here, the authors report on the hitchhiking of nanocarriers on red blood cells and the targeted upstream delivery to different target organs in mice, pigs and ex vivo human lungs.

Humoral and cellular host defense mechanisms including diverse phagocytes, leukocytes, and immune cells have evolved over millions of years to protect the body from microbes and other external and internal threats. These policing forces recognize engineered sub-micron drug delivery systems (DDS) as such a threat, and react accordingly. This leads to impediment of the therapeutic action, extensively studied and discussed in the literature. Here, we focus on side effects of DDS interactions with host defenses. We argue that for nanomedicine to reach its clinical potential, the field must redouble its efforts in understanding the interaction between drug delivery systems and the host defenses, so that we can engineer safer interventions with the greatest potential for clinical success.

The ubiquity and potency of antibiotics may give the false impression that infection is a solved problem. Unfortunately, even bacterial infections, the target of antibiotics, remain a major cause of illness and death. Several major unmet needs persist: biofilms, such as those on implanted hardware, largely resist antibiotics; the inflammatory host response to infection often produces more damage than the infection itself; and systemic antibiotics often decimate the gut microbiome, which can predispose to additional infections and even predispose to non-infectious diseases. Additionally, there is an increasing threat from multi-drug resistant microorganisms, though market forces may continue to inhibit innovation in this realm. These numerous unmet infection-related needs provide attractive goals for innovation of targeted drug delivery technologies, especially those of nanomedicine. Here we review several of those innovations in pre-clinical development, the two such therapies which have made it to clinical use, and the opportunities for further technology development for treating infections.

After dozens of clinical trials, there are still no Food and Drug Administration–approved drugs that improve mortality in acute respiratory distress syndrome (ARDS). These poor results may be caused in part by three unique pharmacological challenges presented by ARDS: (1) Patients with ARDS are fragile because of concomitant multiple organ dysfunction, so they do not tolerate off-target side effects of drugs; (2) inhaled drug delivery is impeded by the column of proteinaceous fluid covering the injured alveoli; and (3) ARDS is heterogeneous in its underlying pathophysiology, so targeting one pathway is unlikely to improve most patients. To address these three pharmacological problems, I present the development of pulmonary endothelium–targeted liposomes (PELs). PELs are approximately 100-nm drug carriers coated with antibodies that bind to the pulmonary capillary endothelium. In model organisms, intravenously injected PELs strongly concentrate drugs in alveoli, even in animal models displaying severe, spatially heterogeneous pathology similar to severe ARDS. By concentrating drugs in inflamed alveoli, PELs solve pharmacological challenge (1) above. By being obligate intravenous medications, they solve challenge (2). Finally, because PELs can be loaded with at least three drugs, they can solve challenge (3) with combination drug therapy. My colleagues and I are currently testing PELs loaded with numerous candidate drugs in mouse models of ARDS, and we are testing drug distribution in live pigs and ex vivo human lungs. We aim to use such preclinical validation to move PELs into a partnership with industry, and then to patients.

Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular traps (NETs), are involved in multiple pathological processes, and NET formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity toward intact NS but not to individual NS components, indicating that 2C5 could potentially target NS in NETs. In this study, NETs were generated in vitro using neutrophils and HL-60 cells differentiated into granulocyte-like cells. The specificity of 2C5 toward NETs was evaluated by ELISA, which showed that it binds to NETs with the specificity similar to that for purified nucleohistone substrate. Immunofluorescence showed that 2C5 stains NETs in both static and perfused microfluidic cell cultures, even after NET compaction. Modification of liposomes with 2C5 dramatically enhanced liposome association with NETs. Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies.

The bloodstream is the main transporting pathway for drug delivery systems (DDS) from the site of administration to the intended site of action. In many cases, components of the vascular system represent therapeutic targets. Endothelial cells, which line the luminal surface of the vasculature, play a tripartite role of the key target, barrier, or victim of nanomedicines in the bloodstream. Circulating DDS may accumulate in the vascular areas of interest and in off-target areas via mechanisms bypassing specific molecular recognition, but using ligands of specific vascular determinant molecules enables a degree of precision, efficacy, and specificity of delivery unattainable by non-affinity DDS. Three decades of research efforts have focused on specific vascular targeting, which have yielded a multitude of DDS, many of which are currently undergoing a translational phase of development for biomedical applications, including interventions in the cardiovascular, pulmonary, and central nervous systems, regulation of endothelial functions, host defense, and permeation of vascular barriers. We discuss the design of endothelial-targeted nanocarriers, factors underlying their interactions with cells and tissues, and describe examples of their investigational use in models of acute vascular inflammation with an eye on translational challenges., Graphical abstract Unlabelled Image

Systemic administration of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside-modified mRNA-LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM-1. Systemic (intravenous) administration of Ab/LNP-mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ~200-fold and 25-fold elevation of mRNA delivery and protein expression in the lungs compared to non-targeted counterparts. Unlike hepatic delivery of LNP-mRNA, Ab/LNP-mRNA is independent of apolipoprotein E. Vascular re-targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver.

Targeted drug delivery aims to improve therapeutic effects and enable mechanisms that are not feasible for untargeted agents (e.g., due to impermeable biological barriers). To achieve targeting, a drug or its carrier should possess properties providing specific accumulation from circulation at the desired site. There are several examples of systems-inspired approaches that have been applied to achieve this goal. First, proteomics analysis of plasma membrane fraction of the vascular endothelium has identified a series of target molecules and their ligands (e.g., antibodies) that deliver conjugated cargoes to well-defined vascular cells and subcellular compartments. Second, selection of ligands binding to cells of interest using phage display libraries in vitro and in vivo has provided peptides and polypeptides that bind to normal and pathologically altered cells. Finally, large-scale high-throughput combinatorial synthesis and selection of lipid- and polymer-based nanocarriers varying their chemical components has yielded a series of carriers accumulating in diverse organs and delivering RNA interference agents to diverse cells. Together, these approaches offer a basis for systems-based design and selection of targets, targeting molecules, and targeting vehicles. Current studies focus on expanding the arsenal of these and alternative targeting strategies, devising drug delivery systems capitalizing on these strategies and evaluation of their benefit/risk ratio in adequate animal models of human diseases. These efforts, combined with better understanding of mechanisms and unintended consequences of these targeted interventions, need to be ultimately translated into industrial development and the clinical domain.

Attachment of nanoparticles (NPs) to the surface of carrier red blood cells (RBCs) profoundly alters their interactions with the host organism, decelerating NP clearance from the bloodstream while enabling NP transfer from the RBC surface to the vascular cells. These changes in pharmacokinetics of NPs imposed by carrier RBCs are favorable for many drug delivery purposes. On the other hand, understanding effects of NPs on the carrier RBCs is vital for successful translation of this novel drug delivery paradigm. Here, using two types of distinct nanoparticles (polystyrene (PSNP) and lysozyme-dextran nanogels (LDNG)) we assessed potential adverse and sensitizing effects of surface adsorption of NPs on mouse and human RBCs. At similar NP loadings (approx. 50 particles per RBC), adsorption of PSNPs, but not LDNGs, induces RBCs agglutination and sensitizes RBCs to damage by osmotic, mechanical and oxidative stress. PSNPs, but not LDNGs, increase RBC stiffening and surface exposure of phosphatidylserine, both known to accelerate RBC clearance in vivo. Therefore, NP properties and loading amounts have a profound impact on RBCs. Furthermore, LDNGs appear conducive to nanoparticle drug delivery using carrier RBCs.

Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal studies provide the basis for the challenging translation endothelial nanomedicine into the clinical domain.

INTRODUCTION: Even though pulmonary diseases are among the leading causes of morbidity and mortality in the world, exceedingly few life-prolonging therapies have been developed for these maladies. Relief may finally come from nanomedicine and targeted drug delivery. AREAS COVERED: Here, we focus on four conditions for which the pulmonary endothelium plays a pivotal role: acute respiratory distress syndrome, primary graft dysfunction occurring immediately after lung transplantation, pulmonary arterial hypertension and pulmonary embolism. For each of these diseases, we first evaluate the targeted drug delivery approaches that have been tested in animals. Then we suggest a ‘need specification’ for each disease: a list of criteria (e.g., macroscale delivery method, stability, etc.) that nanomedicine agents must meet in order to warrant human clinical trials and investment from industry. EXPERT OPINION: For the diseases profiled here, numerous nanomedicine agents have shown promise in animal models. However, to maximize the chances of creating products that reach patients, nanomedicine engineers and clinicians must work together and use each disease’s need specification to guide the design of practical and effective nanomedicine agents.

Pathological cardiac hypertrophy is associated with an increased risk of heart failure and cardiovascular mortality. Calcium (Ca(2+)) -regulated gene expression is essential for the induction of hypertrophy, but it is not known how myocytes distinguish between the Ca(2+) signals that regulate contraction and those that lead to cardiac hypertrophy. We used in vitro neonatal rat ventricular myocytes to perform an RNA interference (RNAi) screen for ion channels that mediate Ca(2+)-dependent gene expression in response to hypertrophic stimuli. We identified several ion channels that are linked to hypertrophic gene expression, including transient receptor potential C3 (TrpC3). RNAi-mediated knockdown of TrpC3 decreases expression of hypertrophy-associated genes such as the A- and B-type natriuretic peptides (ANP and BNP) in response to numerous hypertrophic stimuli, while TrpC3 overexpression increases BNP expression. Furthermore, stimuli that induce hypertrophy dramatically increase TrpC3 mRNA levels. Importantly, whereas TrpC3-knockdown strongly reduces gene expression associated with hypertrophy, it has a negligible effect on cell size and on myocyte beating. These results suggest that Ca(2+) influx through TrpC3 channels increases transcription of genes associated with hypertrophy but does not regulate the signaling pathways that control cell size or contraction. Thus TrpC3 may represent an important therapeutic target for the treatment of cardiac hypertrophy and heart failure.