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1.
Mol Ther ; 32(5): 1344-1358, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38454606

RESUMEN

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.


Asunto(s)
Barrera Hematoencefálica , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico , Liposomas , Nanopartículas , Molécula 1 de Adhesión Celular Vascular , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Animales , Ratones , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Nanopartículas/química , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Lípidos/química , Sistemas de Liberación de Medicamentos/métodos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Humanos
2.
Nano Lett ; 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38598417

RESUMEN

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.

3.
Crit Care Med ; 2024 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-38578158

RESUMEN

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.

4.
Langmuir ; 40(16): 8365-8372, 2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38600821

RESUMEN

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.

5.
Microvasc Res ; 150: 104595, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37619889

RESUMEN

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.


Asunto(s)
Procedimientos Quirúrgicos Cardíacos , Molécula 1 de Adhesión Intercelular , Adulto , Humanos , Selectina E , Microcirculación , Molécula 1 de Adhesión Celular Vascular , Células Endoteliales , Procedimientos Quirúrgicos Cardíacos/efectos adversos
6.
Proc Natl Acad Sci U S A ; 117(7): 3405-3414, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32005712

RESUMEN

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.


Asunto(s)
Anticuerpos/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Encefalitis/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Nanomedicina/métodos , Animales , Barrera Hematoencefálica/inmunología , Encefalitis/genética , Encefalitis/inmunología , Endotelio Vascular/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Ratones , Receptores de Transferrina/genética , Receptores de Transferrina/inmunología , Trombomodulina/genética , Trombomodulina/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunología
7.
Am J Physiol Lung Cell Mol Physiol ; 322(6): L866-L872, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35438574

RESUMEN

Imatinib, a tyrosine kinase inhibitor, attenuates pulmonary edema and inflammation in lung injury. However, the physiological effects of this drug and their impact on outcomes are poorly characterized. Using serial computed tomography (CT), we tested the hypothesis that imatinib reduces injury severity and improves survival in ventilated rats. Hydrochloric acid (HCl) was instilled in the trachea (pH 1.5, 2.5 mL/kg) of anesthetized, intubated supine rats. Animals were randomized (n = 17 each group) to receive intraperitoneal imatinib or vehicle immediately prior to HCl. All rats then received mechanical ventilation. CT was performed hourly for 4 h. Images were quantitatively analyzed to assess the progression of radiological abnormalities. Injury severity was confirmed via hourly blood gases, serum biomarkers, bronchoalveolar lavage (BAL), and histopathology. Serial blood drug levels were measured in a subset of rats. Imatinib reduced mortality while delaying functional and radiological injury progression: out of 17 rats per condition, 2 control vs. 8 imatinib-treated rats survived until the end of the experiment (P = 0.02). Imatinib attenuated edema after lung injury (P < 0.05), and survival time in both groups was negatively correlated with increased lung mass (R2 = 0.70) as well as other physiological and CT parameters. Capillary leak (BAL protein concentration) was significantly lower in the treated group (P = 0.04). Peak drug concentration was reached after 70 min, and the drug half-life was 150 min. Imatinib decreased both mortality and lung injury severity in mechanically ventilated rats. Pharmacological inhibition of edema could be used during mechanical ventilation to improve the severity and outcome of lung injury.


Asunto(s)
Lesión Pulmonar , Edema Pulmonar , Animales , Ácido Clorhídrico , Mesilato de Imatinib/farmacología , Pulmón/patología , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/patología , Edema Pulmonar/patología , Ratas , Respiración Artificial
8.
Annu Rev Biomed Eng ; 23: 225-248, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-33788581

RESUMEN

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.


Asunto(s)
Isquemia Encefálica , Nanopartículas , Accidente Cerebrovascular , Sistemas de Liberación de Medicamentos , Eritrocitos , Humanos
9.
Bioconjug Chem ; 33(7): 1286-1294, 2022 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-35710322

RESUMEN

Engineering drug delivery systems for prolonged pharmacokinetics (PK) has been an ongoing pursuit for nearly 50 years. The gold standard for PK enhancement is the coating of nanoparticles with polymers, namely polyethylene glycol (PEGylation), which has been applied in several clinically used products. In the present work, we utilize the longest circulating and most abundant component of blood─the erythrocyte─to improve the PK behavior of liposomes. Antibody-mediated coupling of liposomes to erythrocytes was tested in vitro to identify a loading dose that did not adversely impact the carrier cells. Injection of erythrocyte targeting liposomes into mice resulted in a ∼2-fold improvement in the area under the blood concentration versus time profile versus PEGylated liposomes and a redistribution from the plasma into the cellular fraction of blood. These results suggest that in vivo targeting of erythrocytes is a viable strategy to improve liposome PK relative to current, clinically viable strategies.


Asunto(s)
Liposomas , Polietilenglicoles , Animales , Sistemas de Liberación de Medicamentos , Eritrocitos , Liposomas/farmacocinética , Ratones , Polietilenglicoles/farmacocinética , Polímeros
10.
Nanomedicine ; 13(4): 1495-1506, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28065731

RESUMEN

Inflamed organs display marked spatial heterogeneity of inflammation, with patches of inflamed tissue adjacent to healthy tissue. To investigate how nanocarriers (NCs) distribute between such patches, we created a mouse model that recapitulates the spatial heterogeneity of the inflammatory lung disease ARDS. NCs targeting the epitope PECAM strongly accumulated in the lungs, but were shunted away from inflamed lung regions due to hypoxic vasoconstriction (HVC). In contrast, ICAM-targeted NCs, which had lower whole-lung uptake than PECAM/NCs in inflamed lungs, displayed markedly higher NC levels in inflamed regions than PECAM/NCs, due to increased regional ICAM. Regional HVC, epitope expression, and capillary leak were sufficient to predict intra-organ of distribution of NCs, antibodies, and drugs. Importantly, these effects were not observable with traditional spatially-uniform models of ARDS, nor when examining only whole-organ uptake. This study underscores how examining NCs' intra-organ distribution in spatially heterogeneous animal models can guide rational NC design.


Asunto(s)
Portadores de Fármacos/farmacocinética , Epítopos/inmunología , Inflamación/patología , Pulmón/patología , Nanopartículas/química , Animales , Anticuerpos/química , Portadores de Fármacos/química , Epítopos/química , Hipoxia/fisiopatología , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patología , Vasoconstricción
11.
Am J Physiol Lung Cell Mol Physiol ; 311(6): L1062-L1075, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27694472

RESUMEN

Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.


Asunto(s)
Lesión Pulmonar Aguda/patología , Células Epiteliales Alveolares/patología , Epitelio/patología , Neutrófilos/patología , Regeneración , Ácidos , Lesión Pulmonar Aguda/inducido químicamente , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Animales , Anticuerpos/farmacología , Líquido del Lavado Bronquioalveolar , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Epitelio/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/deficiencia , Factor Estimulante de Colonias de Granulocitos/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Proteómica , Regeneración/efectos de los fármacos , Síndrome de Dificultad Respiratoria/patología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos
12.
13.
Redox Biol ; 73: 103185, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38759419

RESUMEN

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.


Asunto(s)
Antioxidantes , Isquemia Encefálica , Nanomedicina , Estrés Oxidativo , Especies Reactivas de Oxígeno , Daño por Reperfusión , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Antioxidantes/administración & dosificación , Humanos , Nanomedicina/métodos , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Animales , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación
14.
J Colloid Interface Sci ; 664: 1042-1055, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38522178

RESUMEN

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.


Asunto(s)
Membrana Dobles de Lípidos , Dióxido de Silicio , Membrana Dobles de Lípidos/química , Dióxido de Silicio/química , Polietilenglicoles/química , Inmunoglobulina G
15.
bioRxiv ; 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38915627

RESUMEN

Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading DNA would provide additional advantages such as long-term expression and availability of promoter sequences. However, here we show that plasmid DNA (pDNA) delivery via LNPs (pDNA-LNPs) induces acute inflammation in naïve mice which we find is primarily driven by the cGAS-STING pathway. Inspired by DNA viruses that inhibit this pathway for replication, we co-loaded endogenous lipids that inhibit STING into pDNA-LNPs. Specifically, loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorates serious inflammatory responses in vivo enabling prolonged transgene expression (at least 1 month). Additionally, we demonstrate the ability to iteratively optimize NOA-pDNA-LNPs' expression by performing a small LNP formulation screen, driving up expression 50-fold in vitro. Thus, NOA-pDNA-LNPs, and pDNA-LNPs co-loaded with other bioactive molecules, will provide a major new tool in the genetic medicine toolbox, leveraging the power of DNA's long-term and promoter-controlled expression.

16.
PLoS One ; 19(6): e0297451, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38857220

RESUMEN

Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.


Asunto(s)
Barrera Hematoencefálica , Lesiones Traumáticas del Encéfalo , Nanopartículas , Molécula 1 de Adhesión Celular Vascular , Animales , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Ratones , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Nanopartículas/química , Liposomas , Masculino , Sistemas de Liberación de Medicamentos , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos
17.
Adv Mater ; 36(26): e2312026, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38394670

RESUMEN

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.


Asunto(s)
Coagulación Sanguínea , Lípidos , Pulmón , Nanopartículas , Trombosis , Nanopartículas/química , Pulmón/metabolismo , Animales , Coagulación Sanguínea/efectos de los fármacos , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Lípidos/química , Trombina/metabolismo , Trombina/química , Humanos , Fibrinógeno/química , Fibrinógeno/metabolismo , Ratones
18.
bioRxiv ; 2024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38659905

RESUMEN

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.

19.
ACS Nano ; 18(22): 13983-13999, 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38767983

RESUMEN

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.


Asunto(s)
Nanomedicina , Humanos , Portadores de Fármacos/química , Liposomas/química , Nanopartículas/química , Estados Unidos
20.
Int J Pharm ; 645: 123369, 2023 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-37696344

RESUMEN

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.


Asunto(s)
Liposomas , Oximetría , Animales , Ratones , Signos Vitales , Caminata/fisiología , Frecuencia Cardíaca
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