Developing targeted antioxidant nanomedicines for ischemic penumbra: Novel strategies in treating brain ischemia-reperfusion injury.

During cerebral ischemia-reperfusion conditions, the excessive reactive oxygen species in the ischemic penumbra region, resulting in neuronal oxidative stress, constitute the main pathological mechanism behind ischemia-reperfusion damage. Swiftly reinstating blood perfusion in the ischemic penumbra zone and suppressing neuronal oxidative injury are key to effective treatment. Presently, antioxidants in clinical use suffer from low bioavailability, a singular mechanism of action, and substantial side effects, severely restricting their therapeutic impact and widespread clinical usage. Recently, nanomedicines, owing to their controllable size and shape and surface modifiability, have demonstrated good application potential in biomedicine, potentially breaking through the bottleneck in developing neuroprotective drugs for ischemic strokes. This manuscript intends to clarify the mechanisms of cerebral ischemia-reperfusion injury and provides a comprehensive review of the design and synthesis of antioxidant nanomedicines, their action mechanisms and applications in reversing neuronal oxidative damage, thus presenting novel approaches for ischemic stroke prevention and treatment.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Combination of Physicochemical Tropism and Affinity Moiety Targeting of Lipid Nanoparticles Enhances Organ Targeting.

Two camps have emerged for targeting nanoparticles to specific organs and cell types: affinity moiety targeting and physicochemical tropism. Here we directly compare and combine both using intravenous (IV) lipid nanoparticles (LNPs) designed to target the lungs. We utilized PECAM antibodies as affinity moieties and cationic lipids for physicochemical tropism. These methods yield nearly identical lung uptake, but aPECAM LNPs show higher endothelial specificity. LNPs combining these targeting methods had >2-fold higher lung uptake than either method alone and markedly enhanced epithelial uptake. To determine if lung uptake is because the lungs are the first organ downstream of IV injection, we compared IV vs intra-arterial (IA) injection into the carotid artery, finding that IA combined-targeting LNPs achieve 35% of the injected dose per gram (%ID/g) in the first-pass organ, the brain, among the highest reported. Thus, combining the affinity moiety and physicochemical strategies provides benefits that neither targeting method achieves alone.

A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography.

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 µg or less of purified products, this method can purify ∼100-1000 µg of DNA nanostructures in less than 30 min, with the overall collection yield of 50-70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

Anaerobic Lactate Production Is Associated With Decreased Microcirculatory Blood Flow and Decreased Mitochondrial Respiration Following Cardiovascular Surgery With Cardiopulmonary Bypass.

OBJECTIVES: Quantify the relationship between perioperative anaerobic lactate production, microcirculatory blood flow, and mitochondrial respiration in patients after cardiovascular surgery with cardiopulmonary bypass. DESIGN: Serial measurements of lactate-pyruvate ratio (LPR), microcirculatory blood flow, plasma tricarboxylic acid cycle cycle intermediates, and mitochondrial respiration were compared between patients with a normal peak lactate (≤ 2 mmol/L) and a high peak lactate (≥ 4 mmol/L) in the first 6 hours after surgery. Regression analysis was performed to quantify the relationship between clinically relevant hemodynamic variables, lactate, LPR, and microcirculatory blood flow. SETTING: This was a single-center, prospective observational study conducted in an academic cardiovascular ICU. PATIENTS: One hundred thirty-two patients undergoing elective cardiovascular surgery with cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients with a high postoperative lactate were found to have a higher LPR compared with patients with a normal postoperative lactate (14.4 ± 2.5 vs. 11.7 ± 3.4; p = 0.005). Linear regression analysis found a significant, negative relationship between LPR and microcirculatory flow index (r = -0.225; ß = -0.037; p = 0.001 and proportion of perfused vessels: r = -0.17; ß = -0.468; p = 0.009). There was not a significant relationship between absolute plasma lactate and microcirculation variables. Last, mitochondrial complex I and complex II oxidative phosphorylation were reduced in patients with high postoperative lactate levels compared with patients with normal lactate (22.6 ± 6.2 vs. 14.5 ± 7.4 pmol O2/s/106 cells; p = 0.002). CONCLUSIONS: Increased anaerobic lactate production, estimated by LPR, has a negative relationship with microcirculatory blood flow after cardiovascular surgery. This relationship does not persist when measuring lactate alone. In addition, decreased mitochondrial respiration is associated with increased lactate after cardiovascular surgery. These findings suggest that high lactate levels after cardiovascular surgery, even in the setting of normal hemodynamics, are not simply a type B phenomenon as previously suggested.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Controlling spatial distribution of functional lipids in a supported lipid bilayer prepared from vesicles.

Conjugating biomolecules, such as antibodies, to bioconjugate moieties on lipid surfaces is a powerful tool for engineering the surface of diverse biomaterials, including cells and nanoparticles. We developed supported lipid bilayers (SLBs) presenting well-defined spatial distributions of functional moieties as models for precisely engineered functional biomolecular-lipid surfaces. We used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to determine how vesicles containing a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG-N3) form SLBs as a function of the lipid phase transition temperature (Tm). Above the DPPC Tm, DPPC/DSPE-PEG-N3 vesicles form SLBs with functional azide moieties on SiO2 substrates via vesicle fusion. Below this Tm, DPPC/DSPE-PEG-N3 vesicles attach to SiO2 intact. Intact DPPC/DSPE-PEG-N3 vesicles on the SiO2 surfaces fuse and rupture to form SLBs when temperature is brought above the DPPC Tm. AFM studies show uniform and complete DPPC/DSPE-PEG-N3 SLB coverage of SiO2 surfaces for different DSPE-PEG-N3 concentrations. As the DSPE-PEG-N3 concentration increases from 0.01 to 6 mol%, the intermolecular spacing of DSPE-PEG-N3 in the SLBs decreases from 4.6 to 1.0 nm. The PEG moiety undergoes a mushroom to brush transition as DSPE-PEG-N3 concentration varies from 0.1 to 2.0 mol%. Via copper-free click reaction, IgG was conjugated to SLB surfaces with 4.6 nm or 1.3 nm inter-DSPE-PEG-N3 spacing. QCM-D and AFM data show; 1) uniform and complete IgG layers of similar mass and thickness on the two types of SLB; 2) a higher-viscosity/less rigid IgG layer on the SLB with 4.6 nm inter-DSPE-PEG-N3 spacing. Our studies provide a blueprint for SLBs modeling spatial control of functional macromolecules on lipid surfaces, including surfaces of lipid nanoparticles and cells.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up or down-regulate any protein of interest. LNPs have mostly been targeted to specific cell types or organs by physicochemical targeting in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. Here lung-tropic LNPs are examined, whose organ tropism derives from containing either a cationic or ionizable lipid conferring a positive zeta potential. Surprisingly, these LNPs are found to induce massive thrombosis. Such thrombosis is shown in the lungs and other organs, and it is shown that it is greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles, and even by lung-tropic ionizable lipids that do not have a permanent cationic charge. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen, which then activates platelets and thrombin. Based on these mechanisms, multiple solutions are engineered that enable positively charged LNPs to target the lungs while ameliorating thrombosis. The findings illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

Automated measurement of distance-walked as a "sixth vital sign" for detecting infusion reactions during preclinical testing.

Infusion reactions are a major risk for advanced therapeutics (e.g., engineered proteins nanoparticles, etc.), which can trigger the complement cascade, anaphylaxis, and other life-threatening immune responses. However, during the early phases of development, it is uncommon to assess for infusion reactions, given the labor involved in measuring multiple physiological parameters in rodents. Therefore, we sought to develop an automated quantification of rodent locomotion to serve as a sensitive screening tool for infusion reactions, with minimal added labor-time for each experiment. Here we present the detailed methods for building a motion tracking cage for mice, requiring ∼$100 of materials, ∼2 h to build and set up completely, and employing freely available software (DeepLabCut). The distance-walked after injection was first shown to have the predicted effects for stimulants (caffeine), sedatives (ketamine), and toxins (lipopolysaccharide). Additionally, the distance-walked more sensitively detected the effects of these compounds than did pulse oximetry-based measurements of the classical vital signs of heart rate, respiratory rate, and blood oxygen saturation. Finally, we examined a nanomedicine formulation that has been in preclinical development, liposomes targeted to the cell adhesion molecule ICAM. While this formulation has been studied across dozens of publications, it has not previously been noted to produce an infusion reaction. However, the automated motion tracking cage showed that ICAM-liposomes markedly reduce the distance-walked, which we confirmed by measuring the other vital signs. Importantly, the motion tracking cage added < 5 min of labor time per 5-mouse condition, while pulse oximetry with a neck cuff (by far the most stable oximetry signal in mice) required âˆ¼ 100 min of labor time. Thus, automated measurement of distance-walked can indeed serve as a "sixth vital sign" for detecting infusion reactions during preclinical testing. Additionally, the device to measure distance-walked is easy and cheap to build and requires negligible labor time for each experiment, enabling distance-walked to be recorded in nearly every infusion experiment.

Low postoperative perfused vessel density is associated with increased soluble endothelial cell adhesion molecules during circulatory shock after cardiac surgery.

INTRODUCTION: Microcirculatory dysfunction after cardiovascular surgery is associated with significant morbidity and worse clinical outcomes. Abnormal capillary blood flow can occur from multiple causes, including cytokine-mediated vascular endothelial injury, microthrombosis, and an inadequate balance between vasoconstriction and vasodilation. In response to proinflammatory cytokines, endothelial cells produce cellular adhesion molecules (CAMs) which regulate leukocyte adhesion, vascular permeability, and thus can mediate tissue injury. The relationship between changes in microcirculatory flow during circulatory shock and circulating adhesion molecules is unclear. The objective of this study was to compare changes in plasma soluble endothelial cell adhesion molecules (VCAM-1, ICAM-1, and E-Selectin) in patients with functional derangements in microcirculatory blood flow after cardiovascular surgery. METHODS: Adult patients undergoing elective cardiac surgery requiring cardiopulmonary bypass who exhibited postoperative shock were enrolled in the study. Sublingual microcirculation imaging was performed prior to surgery and within 2 h of ICU admission. Blood samples were taken at the time of microcirculation imaging for biomarker analysis. Plasma soluble VCAM-1, ICAM-1, and E-selectin in addition to plasma cytokines (IL-6, IL-8, and IL-10) were measured by commercially available enzyme-linked immunoassay. RESULTS: Of 83 patients with postoperative shock who were evaluated, 40 patients with clinical shock had a postoperative perfused vessel density (PVD) >1 SD above (High PVD group = 28.5 ± 2.3 mm/mm2, n = 20) or below (Low PVD = 15.5 ± 2.0 mm/mm2, n = 20) the mean postoperative PVD and were included in the final analysis. Patient groups were well matched for comorbidities, surgical, and postoperative details. Overall, there was an increase in postoperative plasma VCAM-1 and E-Selectin compared to preoperative levels, but there was no difference between circulating ICAM-1. When grouped by postoperative microcirculation, patients with poor microcirculation were found to have increased circulating VCAM-1 (2413 ± 1144 vs. 844 ± 786 ng/mL; p < 0.0001) and E-Selectin (242 ± 119 vs. 87 ± 86 ng/mL; p < 0.0001) compared to patients with increased microcirculatory blood flow. Microcirculatory flow was not associated with a difference in plasma soluble ICAM-1 (394 ± 190 vs. 441 ± 256; p = 0.52). CONCLUSIONS: Poor postoperative microcirculatory blood flow in patients with circulatory shock after cardiac surgery is associated with increased plasma soluble VCAM-1 and E-Selectin, indicating increased endothelial injury and activation compared to patients with a high postoperative microcirculatory blood flow. Circulating endothelial cell adhesion molecules may be a useful plasma biomarker to identify abnormal microcirculatory blood flow in patients with shock.

Does acupuncture have advantages in the rehabilitation of vascular mild cognitive impairment? A systematic review and meta-analysis.

Background: Vascular mild cognitive impairment (VMCI) is a common impairment caused by vascular factors. VMCI often occurs after stroke, and it is the main clinical manifestation of long-term disability. Many patients are treated with acupuncture in combination with other therapies. However, evidence regarding the effectiveness of this treatment regimen is lacking. Aims: This meta-analysis aimed to evaluate the efficacy of acupuncture therapy for treating VMCI. Methods: This systematic review was conducted in accordance with the preferred reporting and meta-analysis guidelines. The CNKI, Wanfang, VIP, CBM, Cochrane Library, PubMed and Embase databases were searched from inception to August 20, 2022. After two researchers independently screened the literature, they extracted the data and evaluated the risk of bias in the included studies. Revman 5.3 software was used for the meta-analysis. Summary of review: Thirty-two randomized controlled trials (RCTs) were included. The overall effective rate of acupuncture for treating VMCI was 3.06, 95% CI [2.39, 3.91], (P < 0.05). Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), Barthel Index and Activities of Daily Living (ADLs) scores significantly differed between the treatment and control groups, with weighted mean differences (WMDs) [95% CI] (P value) of 1.97 [1.44, 2.49] (P < 0.05), 2.02 [1.50, 2.54] (P < 0.05), 5.54 [3.81, 7.28] (P < 0.05), and 3.43 [2.53, 4.33] (P < 0.05), respectively. The overall effective rate of electroacupuncture (EA) for treating VMCI was better than that of the control group (RR = 2.25, 95% CI, [1.13, 4.50], P < 0.05). MoCA, MMSE, Barthel index and ADL scores differed significantly between the treatment and control groups, with WMDs [95% CI] (P value) of 1.79 [1.20, 2.38] (P < 0.05), 1.45 [0.87, 2.03] (P < 0.05), 5.78 [2.38, 9.18] (P < 0.05), and 3.15 [2.15, 4.15] (P < 0.05), respectively. Acupuncture alone and combined with drug therapy were thus superior to drug therapy alone for improving cognitive function. EA also has potential advantages. Conclusions: Acupuncture combined with another therapy is better than other therapies alone, such as simple drug therapy, for treating VMCI. However, variations in study duration (4-12 weeks) limit us from drawing any definitive conclusions about long-term effects. Therefore, more RCTs with rigorous designs and reasonable treatment and follow-up durations are needed.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up- or down-regulate any protein of interest. LNPs have been targeted to specific cell types or organs by physicochemical targeting, in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. In a popular approach, physicochemical targeting is accomplished by formulating with charged lipids. Negatively charged lipids localize LNPs to the spleen, and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. The mechanism depends on the LNPs binding to fibrinogen and inducing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs while not inducing thrombosis. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

Targeting lipid nanoparticles to the blood brain barrier to ameliorate acute ischemic stroke.

After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS. Graphical abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.

Mechanisms by Which Liposomes Improve Inhaled Drug Delivery for Alveolar Diseases.

Diseases of the pulmonary alveolus, such as pulmonary fibrosis, are leading causes of morbidity and mortality, but exceedingly few drugs are developed for them. A major reason for this gap is that after inhalation, drugs are quickly whisked away from alveoli due to their high perfusion. To solve this problem, the mechanisms by which nano-scale drug carriers dramatically improve lung pharmacokinetics using an inhalable liposome formulation containing nintedanib, an antifibrotic for pulmonary fibrosis, are studied. Direct instillation of liposomes in murine lung increases nintedanib's total lung delivery over time by 8000-fold and lung half life by tenfold, compared to oral nintedanib. Counterintuitively, it is shown that pulmonary surfactant neither lyses nor aggregates the liposomes. Instead, each lung compartment (alveolar fluid, alveolar leukocytes, and parenchyma) elutes liposomes over 24 h, likely serving as "drug depots." After deposition in the surfactant layer, liposomes are transferred over 3-6 h to alveolar leukocytes (which take up a surprisingly minor 1-5% of total lung dose instilled) in a nonsaturable fashion. Further, all cell layers of the lung parenchyma take up liposomes. These and other mechanisms elucidated here should guide engineering of future inhaled nanomedicine for alveolar diseases.

Meta-analysis of material properties influencing nanoparticle plasma pharmacokinetics.

Thorough characterization of the plasma pharmacokinetics (PK) is a critical step in clinical development of novel therapeutics and is routinely performed for small molecules and biologics. However, there is a paucity of even basic characterization of PK for nanoparticle-based drug delivery systems. This has led to untested generalizations about how nanoparticle properties govern PK. Here, we present a meta-analysis of 100 nanoparticle formulations following IV administration in mice to identify any correlations between four PK parameters derived by non-compartmental analysis (NCA) and four cardinal properties of nanoparticles: PEGylation, zeta potential, size, and material. There was a statistically significant difference between the PK of particles stratified by nanoparticle properties. However, single linear regression between these properties and PK parameters showed poor predictability (r2 < 0.10 for all analyses), while multivariate regressions showed improved predictability (r2 > 0.38, except for t1/2). This suggests that no single nanoparticle property alone is even moderately predictive of PK, while the combination of multiple nanoparticle features does provide moderate predictive power. Improved reporting of nanoparticle properties will enable more accurate comparison between nanoformulations and will enhance our ability to predict in vivo behavior and design optimal nanoparticles.

Targeted drug delivery to the brain endothelium dominates over passive delivery via vascular leak in experimental intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most common causes of fatal stroke, yet has no specific drug therapies. Many attempts at passive intravenous (IV) delivery in ICH have failed to deliver drugs to the salvageable area around the hemorrhage. The passive delivery method assumes vascular leak through the ruptured blood-brain barrier will allow drug accumulation in the brain. Here we tested this assumption using intrastriatal injection of collagenase, a well-established experimental model of ICH. Fitting with hematoma expansion in clinical ICH, we showed that collagenase-induced blood leak drops significantly by 4 h after ICH onset and is gone by 24 h. We observed passive-leak brain accumulation also declines rapidly over ∼4 h for 3 model IV therapeutics (non-targeted IgG; a protein therapeutic; PEGylated nanoparticles). We compared these passive leak results with targeted brain delivery by IV monoclonal antibodies (mAbs) that actively bind vascular endothelium (anti-VCAM, anti-PECAM, anti-ICAM). Even at early time points after ICH induction, where there is high vascular leak, brain accumulation via passive leak is dwarfed by brain accumulation of endothelial-targeted agents: At 4 h after injury, anti-PECAM mAbs accumulate at 8-fold higher levels in the brain vs. non-immune IgG; anti-VCAM nanoparticles (NPs) deliver a protein therapeutic (superoxide dismutase, SOD) at 4.5-fold higher levels than the carrier-free therapeutic at 24 h after injury. These data suggest that relying on passive vascular leak provides inefficient delivery of therapeutics even at early time points after ICH, and that a better strategy might be targeted delivery to the brain endothelium, which serves as the gateway for the immune attack on the peri-hemorrhage inflamed brain region.

Topical nitroglycerin to detect reversible microcirculatory dysfunction in patients with circulatory shock after cardiovascular surgery: an observational study.

Persistent abnormalities in microcirculatory function are associated with poor clinical outcomes in patients with circulatory shock. We sought to identify patients with acutely reversible microcirculatory dysfunction using a low-dose topical nitroglycerin solution and handheld videomicroscopy during circulatory shock after cardiac surgery. Forty subjects were enrolled for the study, including 20 preoperative control and 20 post-operative patients with shock. To test whether microcirculatory dysfunction is acutely reversible during shock, the sublingual microcirculation was imaged with incident dark field microscopy before and after the application of 0.1 mL of a 1% nitroglycerin solution (1 mg/mL). Compared to the control group, patients with shock had a higher microcirculation heterogeneity index (MHI 0.33 vs. 0.12, p < 0.001) and a lower microvascular flow index (MFI 2.57 vs. 2.91, p < 0.001), total vessel density (TVD 22.47 vs. 25.90 mm/mm2, p = 0.005), proportion of perfused vessels (PPV 90.76 vs. 95.89%, p < 0.001) and perfused vessel density (PVD 20.44 vs. 24.81 mm/mm2, p < 0.001). After the nitroglycerin challenge, patients with shock had an increase in MFI (2.57 vs. 2.97, p < 0.001), TVD (22.47 vs. 27.51 mm/mm2, p < 0.009), PPV (90.76 vs. 95.91%, p < 0.001), PVD (20.44 vs. 26.41 mm/mm2, p < 0.001), venular RBC velocity (402.2 vs. 693.9 µm/s, p < 0.0004), and a decrease in MHI (0.33 vs. 0.04, p < 0.001. Thirteen of 20 patients showed a pharmacodynamic response, defined as an increase in PVD > 1.8 SD from shock baseline. Hemodynamics and vasoactive doses did not change during the 30-min study period. Our findings suggest a topical nitroglycerin challenge with handheld videomicroscopy can safely assess for localized recruitment of the microcirculatory blood flow in patients with circulatory shock and may be a useful test to identify nitroglycerin responsiveness.

Protocol for the MicroRESUS study: The impact of circulatory shock and resuscitation on microcirculatory function and mitochondrial respiration after cardiovascular surgery.

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.

Nanoparticle-Induced Augmentation of Neutrophils' Phagocytosis of Bacteria.

Despite the power of antibiotics, bacterial infections remain a major killer, due to antibiotic resistance and hosts with dysregulated immune systems. We and others have been developing drug-loaded nanoparticles that home to the sites of infection and inflammation via engineered tropism for neutrophils, the first-responder leukocytes in bacterial infections. Here, we examined how a member of a broad class of neutrophil-tropic nanoparticles affects neutrophil behavior, specifically questioning whether the nanoparticles attenuate an important function, bacterial phagocytosis. We found these nanoparticles actually augment phagocytosis of non-opsonized bacteria, increasing it by ∼50%. We showed this augmentation of phagocytosis is likely co-opting an evolved response, as opsonized bacteria also augment phagocytosis of non-opsonized bacteria. Enhancing phagocytosis of non-opsonized bacteria may prove particularly beneficial in two clinical situations: in hypocomplementemic patients (meaning low levels of the main bacterial opsonins, complement proteins, seen in conditions such as neonatal sepsis and liver failure) or for bacteria that are largely resistant to complement opsonization (e.g., Neisseria). Additionally, we observe that; 1) prior treatment with bacteria augments neutrophil uptake of neutrophil-tropic nanoparticles; 2) neutrophil-tropic nanoparticles colocalize with bacteria inside of neutrophils. The observation that neutrophil-tropic nanoparticles enhance neutrophil phagocytosis and localize with bacteria inside neutrophils suggests that these nanoparticles will serve as useful carriers for drugs to ameliorate bacterial diseases.