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PURPOSE: A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. METHODS: Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24 h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. RESULTS: The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24 h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. CONCLUSIONS: An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.
Assuntos
Bupivacaína , Lipossomos , Liberação Controlada de Fármacos , Preparações de Ação Retardada , Reprodutibilidade dos TestesRESUMO
Extracellular small RNAs (sRNAs) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDLs) in mice were enriched with multiple classes of sRNAs derived from the endogenous transcriptome, but also from exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and nonhost species, the profiles of which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and that these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL binds to tDRs and other single-stranded sRNAs with strong affinity but did not bind to double-stranded RNA or DNA. Furthermore, we show that natural and synthetic RNA modifications influenced tDR binding to HDL. We demonstrate that reconstituted HDL bound to tDRs only in the presence of apoA-I, and purified apoA-I alone were able to bind sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, we conclude that HDL binds to single-stranded sRNAs likely through nonionic interactions with apoA-I. These results highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA interactions for therapeutic approaches to prevent or treat disease.
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Lipoproteínas HDL , Pequeno RNA não Traduzido , Animais , Apolipoproteína A-I/metabolismo , Aterosclerose , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Camundongos , Fosfatidilcolinas , Pequeno RNA não Traduzido/químicaRESUMO
Phosphatidylserine (PS) is an anionic phospholipid component in endogenous high-density lipoprotein (HDL). With the intrinsic anti-inflammatory effects of PS and the correlation between PS content and HDL functions, it was hypothesized that incorporating PS would enhance the therapeutic effects of HDL mimetic particles. To test this hypothesis, a series of synthetic high-density lipoproteins (sHDLs) were prepared with an apolipoprotein A-I (ApoA-1) mimetic peptide, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-glycero-3-phospho-l-serine (POPS). Incorporating PS was found to improve the particle stability of sHDLs. Moreover, increasing the PS content in sHDLs enhanced the anti-inflammatory effects on lipopolysaccharide-activated macrophages and endothelial cells. The incorporation of PS had no negative impact on cholesterol efflux capacity, in vivo cholesterol mobilization, and did not affect the pharmacokinetic profiles of sHDLs. Such results suggest the therapeutic potential of PS-containing sHDLs for inflammation resolution in atherosclerosis and other inflammatory diseases.
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Células Endoteliais , Lipoproteínas HDL , Lipoproteínas HDL/química , Células Endoteliais/metabolismo , Colesterol/química , Fosfolipídeos , Anti-Inflamatórios/farmacologiaRESUMO
Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.
Assuntos
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas HDL/química , Apolipoproteína A-I/farmacologia , Apolipoproteína A-I/química , Peptídeos/farmacologia , Peptídeos/química , Colesterol/química , Transporte BiológicoRESUMO
Synthetic high-density lipoprotein (sHDL) and rapamycin (Rap) have both been shown to be potential treatments for age-related macular degeneration (AMD). The low aqueous solubility of Rap, however, limits its therapeutic utility. Here we used an Apolipoprotein A-I mimetic peptide and phospholipid-based sHDL for the intravitreal delivery of Rap. By incorporation of Rap in sHDL nanoparticles (sHDL-Rap), we achieve 125-fold increase in drug aqueous concentration. When applied in vitro to retinal pigment epithelium cells, sHDL-Rap exhibited the abilities to efflux cholesterol, neutralize endotoxin, and suppress NF-κB activation. As an mTOR inhibitor, Rap induced autophagy and inhibited NF-κB-mediated pro-inflammatory signaling. Additionally, a greater reduction in lipofuscin accumulation and increased anti-inflammatory effects were achieved by sHDL-Rap relative to free drug or sHDL alone. In vivo studies demonstrated that sHDL reached the target retina pigment epithelium (RPE) layer following intravitreal administration in rats. These results suggest that sHDL-Rap holds potential as a treatment for AMD.
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Degeneração Macular , Sistemas de Liberação de Fármacos por Nanopartículas , Animais , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , NF-kappa B/metabolismo , Sistemas de Liberação de Fármacos por Nanopartículas/química , Sistemas de Liberação de Fármacos por Nanopartículas/farmacologia , Nanopartículas/química , Ratos , Epitélio Pigmentado da Retina/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêuticoRESUMO
As a cell-penetrating peptide, polyarginine is widely used in drug delivery systems based on its membrane permeation ability. Previously, we developed the mPEG-PLA-b-polyarginine(R15) triblock copolymer, which exhibited a high siRNA delivery efficiency both in vitro and in vivo. As a continued effort, here the amphiphilic diblock polymer PCL-R15 was synthesized as a simplified model to further elucidate the structure-activity relationship of arginine-based amphiphilic polymers as siRNA delivery systems, and the cellular trafficking mechanisms of the PCL-R15/siRNA nanoplexes were investigated to understand the interaction patterns between the nanoplexes and cells. Compared to the R15/siRNA complexes, the introduction of PCL moiety was found to result in the stronger interactions with cells and the enhanced transfection efficiency after the formation of condensed nanoplexes. Caveolae-mediated endocytosis and clathrin-mediated endocytosis were major routes for the internalization of PCL-R15/siRNA nanoplexes. The intracellular release of siRNA from nanoplexes was confirmed by fluorescence resonance energy transfer assay. It was also noticed that the internalized PCL-R15/siRNA nanoplexes were transported through digestive routes and trapped in lysosomes, which may be the bottleneck for efficient siRNA delivery of PCL-R15/siRNA nanoplexes. This study investigated the relationship between the polymer structure of PCL-R15 and the cellular interaction patterns, which may render implications on the rational design of polyarginine-based siRNA delivery systems.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/química , Peptídeos/química , Poliésteres/química , RNA Interferente Pequeno/administração & dosagem , Transporte Biológico , Células HeLa , Humanos , Nanopartículas/metabolismo , Poliésteres/metabolismo , RNA Interferente Pequeno/químicaRESUMO
Colon carcinomas comprise over two-thirds of all colorectal cancers with an overall 5-year survival rate of 64%, which rapidly decreases to 14% when the cancer becomes metastatic. Depending on the stage of colon carcinoma at diagnosis, patients can undergo surgery to attempt complete tumor resection or move directly to chemotherapy with one or a combination of drugs. As with most cancers, colon carcinomas do not always respond to chemotherapies, so targeted therapies and immunotherapies have been developed to aid chemotherapy. We report the development of a local combination therapy for colon carcinoma whereby chemo- and immunotherapeutic entities are delivered intratumorally to maximize efficacy and minimize off-target side effects. A hydrophobic chemotherapeutic agent, docetaxel (DTX), and cholesterol-modified Toll-like receptor 9 (TLR9) agonist CpG (cho-CpG) oligonucleotide are co-loaded in synthetic HDL (sHDL) nanodiscs. In vivo survival analysis of MC-38 tumor-bearing mice treated intratumorally with DTX-sHDL/CpG (median survival; MS = 43 days) showed significant improvement in overall survival compared to mice treated with single agents, free DTX (MS = 23 days, p < 0.0001) or DTX-sHDL (MS = 28 days, p < 0.0001). Two of seven mice treated with DTX-sHDL/CpG experienced complete tumor regression. None of the mice experienced any systemic toxicity as indicated by body weight maintenance and normal serum enzyme and protein levels. In summary, we have demonstrated that chemo- and immunotherapies can be co-loaded into sHDLs, delivered locally to the tumor, and can be used to improve survival outcomes significantly compared to chemotherapy alone.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Docetaxel/química , Lipoproteínas HDL/química , Nanopartículas/química , Oligodesoxirribonucleotídeos/química , Animais , Linhagem Celular Tumoral , Docetaxel/uso terapêutico , Feminino , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/uso terapêutico , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismoRESUMO
Apical periodontitis (AP) is a dental-driven condition caused by pathogens and their toxins infecting the inner portion of the tooth (i.e., dental pulp tissue), resulting in inflammation and apical bone resorption affecting 50% of the worldwide population, with more than 15 million root canals performed annually in the United States. Current treatment involves cleaning and decontaminating the infected tissue with chemo-mechanical approaches and materials introduced years ago, such as calcium hydroxide, zinc oxide-eugenol, or even formalin products. Here, we present, for the first time, a nanotherapeutics based on using synthetic high-density lipoprotein (sHDL) as an innovative and safe strategy to manage dental bone inflammation. sHDL application in concentrations ranging from 25 µg to 100 µg/mL decreases nuclear factor Kappa B (NF-κB) activation promoted by an inflammatory stimulus (lipopolysaccharide, LPS). Moreover, sHDL at 500 µg/mL concentration markedly decreases in vitro osteoclastogenesis (P < 0.001), and inhibits IL-1α (P = 0.027), TNF-α (P = 0.004), and IL-6 (P < 0.001) production in an inflammatory state. Notably, sHDL strongly dampens the Toll-Like Receptor signaling pathway facing LPS stimulation, mainly by downregulating at least 3-fold the pro-inflammatory genes, such as Il1b, Il1a, Il6, Ptgs2, and Tnf. In vivo, the lipoprotein nanoparticle applied after NaOCl reduced bone resorption volume to (1.3 ± 0.05) mm3 and attenuated the inflammatory reaction after treatment to (1 090 ± 184) cells compared to non-treated animals that had (2.9 ± 0.6) mm3 (P = 0.012 3) and (2 443 ± 931) cells (P = 0.004), thus highlighting its promising clinical potential as an alternative therapeutic for managing dental bone inflammation.
Assuntos
Lipoproteínas HDL , NF-kappa B , Periodontite Periapical , Animais , Periodontite Periapical/terapia , Camundongos , Lipopolissacarídeos , Osteogênese/efeitos dos fármacos , Humanos , Osteoclastos/efeitos dos fármacos , NanopartículasRESUMO
This study presents the utilization of a novel, highly lipophilic nitric oxide (NO) donor molecule, S-nitroso-1-adamantanethiol (SNAT), for developing an NO-emitting polymer surface aimed at preventing thrombus formation and bacterial infection in extracorporeal circuits (ECCs). S-nitroso-1-adamantanethiol, a tertiary nitrosothiol-bearing adamantane species, was synthesized, characterized, and used to impregnate polyvinyl chloride (PVC) tubing for subsequent in vivo evaluation. The impregnation process with SNAT preserved the original mechanical strength of the PVC. In vitro assessments revealed sustained NO release from the SNAT-impregnated PVC tubing (iSNAT), surpassing or matching endothelial NO release levels for up to 42 days. The initial NO release remained stable even after 1 year of storage at -20°C. The compatibility of iSNAT with various sterilization techniques (OPA Plus, hydrogen peroxide, EtO) was tested. Acute in vivo experiments in a rabbit model demonstrated significantly reduced thrombus formation in iSNAT ECCs compared with controls, indicating the feasibility of iSNAT to mitigate coagulation system activation and potentially eliminate the need for systemic anticoagulation. Moreover, iSNAT showed substantial inhibition of microbial biofilm formation, highlighting its dual functionality. These findings underscore the promising utility of iSNAT for long-term ECC applications, offering a multifaceted approach to enhancing biocompatibility and minimizing complications.
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Exparel is a bupivacaine multivesicular liposomes (MVLs) formulation developed based on the DepoFoam technology. The complex composition and the unique structure of MVLs pose challenges to the development and assessment of generic versions. In the present work, we developed a panel of analytical methods to characterize Exparel with respect to particle size, drug and lipid content, residual solvents, and pH. In addition, an accelerated in vitro drug release assay was developed using a rotator-facilitated, sample-and-separate experimental setup. The proposed method could achieve over 80% of bupivacaine release within 24 h, which could potentially be used for formulation comparison and quality control purposes. The batch-to-batch variability of Exparel was examined by the established analytical methods. Four different batches of Exparel showed good batch-to-batch consistency in drug content, particle size, pH, and in vitro drug release kinetics. However, slight variation in lipid contents were observed.
Assuntos
Bupivacaína , Lipossomos , Lipossomos/química , Preparações de Ação Retardada , Liberação Controlada de Fármacos , LipídeosRESUMO
Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment.
Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature.
Assuntos
Lipopolissacarídeos , Fosfolipídeos , Animais , Fosfolipídeos/química , Dimiristoilfosfatidilcolina , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêuticoRESUMO
Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.
Assuntos
Quimiometria , Fígado , Fígado/diagnóstico por imagem , Diagnóstico por Imagem , Hepatócitos , OrganoidesRESUMO
Atherosclerosis progression is driven by an imbalance of cholesterol and unresolved local inflammation in the arteries. The administration of recombinant apolipoprotein A-I (ApoA-I)-based high-density lipoprotein (HDL) nanoparticles has been used to reduce the size of atheroma and rescue inflammatory response in clinical studies. Because of the difficulty in producing large quantities of recombinant ApoA-I, here, we describe the preparation of phospholipid-based, ApoA-I-free micelles that structurally and functionally resemble HDL nanoparticles. Micelles were prepared using various phosphatidylcholine (PC) lipids combined with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2k) to form nanoparticles of 15-30 nm in diameter. The impacts of PC composition and PEGylation on the anti-inflammatory activity, cholesterol efflux capacity, and cholesterol crystal dissolution potential of micelles were investigated in vitro. The effects of micelle composition on pharmacokinetics and cholesterol mobilization ability were evaluated in vivo in Sprague Dawley rats. The study shows that the composition of HDL-mimicking micelles impacts their overall atheroprotective properties and supports further investigation of micelles as a therapeutic for the treatment of atherosclerosis.
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Endothelial inflammation is an important pathophysiological driving force in various acute and chronic inflammatory diseases. High-density lipoproteins (HDLs) play critical roles in regulating endothelial functions and resolving endothelial inflammation. In the present study, we developed synthetic HDLs (sHDLs) which actively target inflamed endothelium through conjugating vascular cell adhesion protein 1 (VCAM-1) specific VHPK peptide. The active targeting of VHPK-sHDLs was confirmed in vitro on TNF-α activated endothelial cells. VHPK-sHDLs presented potent anti-inflammatory efficacies in vitro through the reduction of proinflammatory cytokine production and inhibition of leukocyte adhesion to activated endothelium. VHPK-sHDLs showed increased binding on inflamed vessels and alleviated LPS-induced lung inflammation in vivo. The activated endothelium-targeted sHDLs may be further optimized to resolve endothelial inflammation in various inflammatory diseases.
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Sepsis is a major health issue with mortality exceeding 30% and few treatment options. We found that high-density lipoprotein cholesterol (HDL-C) abundance was reduced by 45% in septic patients compared to that in nonseptic patients. Furthermore, HDL-C abundance in nonsurviving septic patients was substantially lower than in those patients who survived. We therefore hypothesized that replenishing HDL might be a therapeutic approach for treating sepsis and found that supplementing HDL with synthetic HDL (sHDL) provided protection against sepsis in mice. In mice subjected to cecal ligation and puncture (CLP), infusing the sHDL ETC-642 increased plasma HDL-C amounts and improved the 7-day survival rate. Septic mice treated with sHDL showed improved kidney function and reduced inflammation, as indicated by marked decreases in the plasma concentrations of blood urea nitrogen (BUN) and the cytokines interleukin-6 (IL-6) and IL-10, respectively. We found that sHDL inhibited the ability of the endotoxins LPS and LPA to activate inflammatory pathways in RAW264.7 cells and HEK-Blue cells expressing the receptors TLR4 or TLR2 and NF-κB reporters. In addition, sHDL inhibited the activation of HUVECs by LPS, LTA, and TNF-α. Together, these data indicate that sHDL treatment protects mice from sepsis in multiple ways and that it might be an effective therapy for patients with sepsis.
Assuntos
Sepse , Animais , Citocinas/metabolismo , Humanos , Interleucina-6/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Sepse/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. METHODS: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. FINDINGS: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. INTERPRETATION: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. FUNDING: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.
Assuntos
Anti-Inflamatórios/administração & dosagem , Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Lipoproteínas HDL/administração & dosagem , Macrófagos/metabolismo , Fosfolipídeos/administração & dosagem , Animais , Anti-Inflamatórios/farmacologia , Aterosclerose/genética , Aterosclerose/metabolismo , Linhagem Celular , Colesterol/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Lipoproteínas HDL/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Fosfolipídeos/química , Fosfolipídeos/farmacologia , Cultura Primária de Células , Células THP-1RESUMO
Liver X nuclear receptor (LXR) agonists are promising anti-atherosclerotic agents that increase the expression of cholesterol transporters on atheroma macrophages leading to increased efflux of cholesterol to endogenous high-density lipoprotein (HDL) acceptors. HDL subsequently delivers effluxed cholesterol to the liver by the process of reverse cholesterol transport, resulting in reduction of atherosclerotic plaques. However, LXR agonists administration triggers undesirable liver steatosis and hypertriglyceridemia due to increased fatty acid and sterol synthesis. LXR-induced liver toxicity, poor drug aqueous solubility and low levels of endogenous HDL acceptors in target patient populations limit the clinical translation of LXR agonists. Here, we propose a dual-antiatherogenic strategy for administration of the LXR agonist, T0901317 (T1317), by encapsulating in synthetic HDL (sHDL) nanoparticles. sHDL had been clinically proven to serve as cholesterol acceptors, resulting in plaque reduction in atherosclerosis patients. In addition, the hydrophobic core and endogenous atheroma-targeting ability of sHDL allow for encapsulation of water-insoluble drugs and their subsequent delivery to atheroma. Several compositions of sHDL were tested to optimize both T1317 encapsulation efficiency and ability of T1317-sHDL to efflux cholesterol. Optimized T1317-sHDL exhibited more efficient cholesterol efflux from macrophages and enhanced atheroma-targeting relative to free drug. Most importantly, in an apolipoprotein E deficient (ApoE-/-) atherosclerosis progression murine model, T1317-sHDL showed superior inhibition of atherogenesis and reduced hypertriglyceridemia side effects in comparison to the free drug and blank sHDL. The T1317-sHDL pharmacological efficacy was observed at doses lower than those previously described for LXR agents, which may have additional safety benefits. In addition, the established clinical manufacturing, safety and efficacy of blank sHDL nanoparticles used in this study could facilitate future clinical translation of LXR-loaded sHDLs.
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Aterosclerose , Preparações Farmacêuticas , Animais , Colesterol , Humanos , Lipoproteínas HDL , Receptores X do Fígado , CamundongosRESUMO
Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.
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Reprogramação Celular , Glioma/terapia , Glutaratos/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Quimiorradioterapia , Mutação com Ganho de Função , Glioma/genética , Glioma/imunologia , Glioma/patologia , Humanos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/imunologia , Camundongos , Temozolomida/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/imunologiaRESUMO
Systemic lupus erythematosus (SLE) patients exhibit accelerated development of atherosclerosis and increased incidents of cardiovascular disease (CVD) that cannot be explained by traditional risk factors alone. Accumulating evidence suggests that reduced levels of high-density lipoproteins (HDLs), along with altered HDL composition and function, may contribute to the accelerated atherosclerosis in SLE patients. Normally, HDLs play various atheroprotective roles through facilitating cholesterol efflux, inhibiting vascular inflammation, and scavenging oxidative species. However, systemic inflammation, oxidative stress, and autoimmunity in SLE patients induce changes in HDL size distribution and proteomic and lipidomic signatures. These compositional changes in HDLs result in the formation of proinflammatory, dysfunctional HDL. These lupus-altered HDLs have impaired antiatherogenic function with reduced cholesterol efflux capacities, impaired antioxidation abilities, and diminished antiinflammatory properties. In fact, dysfunctional HDL may promote atherogenesis by inducing inflammation. Thus, dysfunctional HDLs could be an important biomarker of accelerated atherosclerosis in lupus. Additionally, HDL-targeted therapies, especially infusion of reconstituted HDLs, may serve as a potential therapeutic intervention for SLE patients with CVD.
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Aterosclerose/metabolismo , Dislipidemias/metabolismo , Lipoproteínas HDL/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Aterosclerose/epidemiologia , Aterosclerose/imunologia , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Dislipidemias/epidemiologia , Dislipidemias/imunologia , Humanos , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/epidemiologia , Lúpus Eritematoso Sistêmico/imunologia , Estresse Oxidativo/imunologiaRESUMO
Dialysis methods are frequently used to determine the in vitro drug release kinetics of nanoparticle drug delivery systems. However, the need for the released drug to diffuse through the dialysis membrane delays its appearance in the sampling compartment. Thus, the apparent drug release data outside the dialysis bag typically does not match the desired release kinetics inside the bag adjacent to the nanocarriers. To address this issue, here we describe a simple approach to determine the actual drug release kinetics from nano drug carriers inside the dialysis bag from the experimental data measured from the sampling compartment. First, a calibration experiment is carried out to determine the diffusion barrier properties of the dialysis membranes. The apparent drug release profile of the nanocarrier is then determined using the dialysis method, and a mathematical model is applied to determine the actual drug release kinetics from the experimental data. The model was tested on DOXIL® (doxorubicin liposomes), and an excellent agreement was found between the predicted and measured drug concentration inside the dialysis membranes. By taking the barrier effects of dialysis membranes into consideration, our model independent of drug carrier not only enables the proper interpretation of the data from dialysis studies but also helps to evaluate the dialysis methodology applied to in vitro drug release assays.