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As a leading global cause of musculoskeletal-related disability, osteoarthritis (OA) represents a public health urgency. Understanding of disease pathogenesis has advanced substantially in the past decade, yet no disease-modifying therapeutics have advanced to the clinic. To address this challenge, the CARE-AP (Cartilage Repair strategies to alleviate Arthritis Pain) collaborative research team was convened to bring together relevant multidisciplinary expertise and perspectives from across the VA research community nationwide. The first CARE-AP Annual Research Symposium took place (virtually) in February 2022 with roughly 90 participants. A number of innovative and therapeutic strategies were discussed, including siRNA approaches coupled with novel nanoparticle-based delivery systems, cellular engineering approaches to develop reparative cells that can probe the joint environment and respond to disease-specific cues, and novel biofabrication techniques to improve tissue engineering and effect "biological joint replacement." In addition, challenges and advances in rehabilitation approaches, imaging outcomes, and clinical studies were presented, which were integrated into a framework of recommendations for running "preclinical trials" to improve successful clinical translation.
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Short-interfering RNA (siRNA) has gained significant interest for treatment of neurological diseases by providing the capacity to achieve sustained inhibition of nearly any gene target. Yet, achieving efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle. We herein describe a lipid-siRNA conjugate that, following delivery into the cerebrospinal fluid (CSF), is transported effectively through perivascular spaces, enabling broad dispersion within CSF compartments and through the CNS parenchyma. We provide a detailed examination of the temporal kinetics of gene silencing, highlighting potent knockdown for up to five months from a single injection without detectable toxicity. Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.
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We report the development of a nanotechnology to co-deliver chemocoxib A with a reactive oxygen species (ROS)-activatable and COX-2 targeted pro-fluorescent probe, fluorocoxib Q (FQ) enabling real time visualization of COX-2 and CA drug delivery into solid cancers, using a di-block PPS 135 - b -POEGA 17 copolymer, selected for its intrinsic responsiveness to elevated reactive oxygen species (ROS), a key trait of the tumor microenvironment. FQ and CA were synthesized independently, then co-encapsulated within micellar PPS 135 - b -POEGA 17 co-polymeric nanoparticles (FQ-CA-NPs), and were assessed for cargo concentration, hydrodynamic diameter, zeta potential, and ROS-dependent cargo release. The uptake of FQ-CA-NPs in mouse mammary cancer cells and cargo release was assessed by fluorescence microscopy. Intravenous delivery of FQ-CA-NPs to mice harboring orthotopic mammary tumors, followed by vital optimal imaging, was used to assess delivery to tumors in vivo . The CA-FQ-NPs exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (σ) of -1.59 ± 0.3 mV. Fluorescence microscopy showed ROS-dependent cargo release by FQ-CA-NPs in 4T1 cells, decreasing growth of 4T1 breast cancer cells, but not affecting growth of primary human mammary epithelial cells (HMECs). NP-derived fluorescence was detected in mammary tumors, but not in healthy organs. Tumor LC-MS/MS analysis identified both CA (2.38 nmol/g tumor tissue) and FQ (0.115 nmol/g tumor tissue), confirming the FQ-mediated image guidance of CA delivery in solid tumors. Thus, co-encapsulation of FQ and CA into micellar nanoparticles (FQ-CA-NPs) enabled ROS-sensitive drug release and COX-2-targeted visualization of solid tumors.
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The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.
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Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , RNA Interferente Pequeno , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Inativação Gênica , Lipídeos/química , Albuminas/genéticaRESUMO
Objective: Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration. Methods: An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables "CII-synNotch" mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFß3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-κB reporter assay. Results: CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFß3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes. Conclusion: This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.
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Osteoarthritis (OA) is a chronic, debilitating disease that substantially impairs the quality of life of affected individuals. The underlying mechanisms of OA are diverse and are becoming increasingly understood at the systemic, tissue, cellular and gene levels. However, the pharmacological therapies available remain limited, owing to drug delivery barriers, and consist mainly of broadly immunosuppressive regimens, such as corticosteroids, that provide only short-term palliative benefits and do not alter disease progression. Engineered RNA-based and cell-based therapies developed with synthetic chemistry and biology tools provide promise for future OA treatments with durable, efficacious mechanisms of action that can specifically target the underlying drivers of pathology. This Review highlights emerging classes of RNA-based technologies that hold potential for OA therapies, including small interfering RNA for gene silencing, microRNA and anti-microRNA for multi-gene regulation, mRNA for gene supplementation, and RNA-guided gene-editing platforms such as CRISPR-Cas9. Various cell-engineering strategies are also examined that potentiate disease-dependent, spatiotemporally regulated production of therapeutic molecules, and a conceptual framework is presented for their application as OA treatments. In summary, this Review highlights modern genetic medicines that have been clinically approved for other diseases, in addition to emerging genome and cellular engineering approaches, with the goal of emphasizing their potential as transformative OA treatments.
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Sistemas CRISPR-Cas , Osteoartrite , Humanos , RNA , Qualidade de Vida , Edição de Genes , Osteoartrite/genética , Osteoartrite/terapiaRESUMO
Nanoparticle (NP) supra-assembly offers unique opportunities to tune macroscopic hydrogels' mechanical strength, material degradation, and drug delivery properties. Here, synthetic, reactive oxygen species (ROS)-responsive NPs are physically crosslinked with hyaluronic acid (HA) through guest-host chemistry to create shear-thinning NP/HA hydrogels. A library of triblock copolymers composed of poly(propylene sulfide)-bl-poly(N,N-dimethylacrylamide)-bl-poly(N,N-dimethylacrylamide-co-N-(1-adamantyl)acrylamide) are synthesized with varied triblock architectures and adamantane grafting densities and then self-assembled into NPs displaying adamantane on their corona. Self-assembled NPs are mixed with ß-cyclodextrin grafted HA to yield eighteen NP/HA hydrogel formulations. The NP/HA hydrogel platform demonstrates superior mechanical strength to HA-only hydrogels, susceptibility to oxidative/enzymatic degradation, and inherent cell-protective, antioxidant function. The performance of NP/HA hydrogels is shown to be affected by triblock architecture, guest/host grafting densities, and HA composition. In particular, the length of the hydrophilic second block and adamantane grafting density of self-assembled NPs significantly impacts hydrogel mechanical properties and shear-thinning behavior, while ROS-reactivity of poly(propylene sulfide) protects cells from cytotoxic ROS and reduces oxidative degradation of HA compared to HA-only hydrogels. This work provides insight into polymer structure-function considerations for designing hybrid NP/HA hydrogels and identifies antioxidant, shear-thinning hydrogels as promising injectable delivery platforms for small molecule drugs and therapeutic cells.
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The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loadingâ>40% cargo by volumeâand highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.
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Sistemas CRISPR-Cas , Nanopartículas , Camundongos , Animais , Sistemas CRISPR-Cas/genética , Silício , Porosidade , PolímerosRESUMO
Reperfusion after ischemia causes additional cellular damage, known as reperfusion injury, for which there is still no effective remedy. Poloxamer (P)188, a tri-block copolymer-based cell membrane stabilizer (CCMS), has been shown to provide protection against hypoxia/reoxygenation (HR) injury in various models by reducing membrane leakage and apoptosis and improving mitochondrial function. Interestingly, substituting one of its hydrophilic poly-ethylene oxide (PEO) blocks with a (t)ert-butyl terminus added to the hydrophobic poly-propylene oxide (PPO) block yields a di-block compound (PEO-PPOt) that interacts better with the cell membrane lipid bi-layer and exhibits greater cellular protection than the gold standard tri-block P188 (PEO75-PPO30-PEO75). For this study, we custom-made three different new di-blocks (PEO113-PPO10t, PEO226-PPO18t and PEO113-PPO20t) to systemically examine the effects of the length of each polymer block on cellular protection in comparison to P188. Cellular protection was assessed by cell viability, lactate dehydrogenase release, and uptake of FM1-43 in mouse artery endothelial cells (ECs) following HR injury. We found that di-block CCMS were able to provide the same or better EC protection than P188. Our study provides the first direct evidence that custom-made di-block CCMS can be superior to P188 in improving EC membrane protection, raising their potential in treating cardiac reperfusion injury.
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Vasos Coronários , Traumatismo por Reperfusão , Camundongos , Animais , Células Endoteliais , Membrana Celular , Polietilenoglicóis/farmacologia , Polietilenoglicóis/química , Polímeros/farmacologiaRESUMO
Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.
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The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.
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Galectina 2 , Indolamina-Pirrol 2,3,-Dioxigenase , Animais , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Progressão da DoençaRESUMO
Gene silencing with siRNA nanoparticles (si-NPs) is promising but still clinically unrealized for inhibition of tumor driver genes. Ternary si-NPs containing siRNA, a single block NP core-forming polymer poly[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] (DMAEMA-co-BMA, 50B), and an NP surface-forming diblock polymer 20 kDa poly(ethylene glycol)-block-50B (20kPEG-50B) have the potential to improve silencing activity in tumors due to the participation of both 50B and 20kPEG-50B in siRNA electrostatic loading and endosome disruptive activity. Functionally, single block 50B provides more potent endosomolytic activity, while 20kPEG-50B colloidally stabilizes the si-NPs. Here, we systematically explored the role of the molecular weight (MW) of the core polymer and of the core:surface polymer ratio on ternary si-NP performance. A library of ternary si-NPs was formulated with variation in the MW of the 50B polymer and in the ratio of the core and surface forming polymeric components. Increasing 50B core polymer MW and ratio improved si-NP in vitro gene silencing potency, endosome disruptive activity, and stability, but these features also correlated with cytotoxicity. Concomitant optimization of 50B size and ratio resulted in the identification of lead ternary si-NPs 50B4-DP100, 50B8-DP100, and 50B12-DP25, with potent activity and minimal toxicity. Following intravenous treatment in vivo, all lead si-NPs displayed negligible toxicological effects and enhanced pharmacokinetics and tumor gene silencing relative to more canonical binary si-NPs. Critically, a single 1 mg/kg intravenous injection of 50B8-DP100 si-NPs silenced the tumor driver gene Rictor at the protein level by 80% in an orthotopic breast tumor model. 50B8-DP100 si-NPs delivering siRictor were assessed for therapeutic efficacy in an orthotopic HCC70 mammary tumor model. This formulation significantly inhibited tumor growth compared to siControl-NP treatment. 50B8-DP100 si-NPs were also evaluated for safety and were well-tolerated following a multi-dose treatment scheme. This work provides new insight on ternary si-NP structure-function relationships and identifies core polymer optimization strategies that can yield safe si-NP formulations with potent oncogene silencing.
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Nanopartículas , Polímeros , RNA Interferente Pequeno , Linhagem Celular Tumoral , Inativação GênicaRESUMO
Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.
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The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.
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This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (â¼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass- and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantlyâand differently from PEGâwithout any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.
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Polietilenoglicóis , Polímeros , Ratos , Camundongos , Humanos , Animais , Polietilenoglicóis/metabolismo , Polímeros/farmacologia , Glicerol , Espécies Reativas de Oxigênio , Ovalbumina , Estabilidade ProteicaRESUMO
Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.
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Fator de Crescimento Insulin-Like I , Nanopartículas , Animais , Cobaias , Gravidez , Feminino , Humanos , Fator de Crescimento Insulin-Like I/genética , Placenta/metabolismo , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Transgenes , Nutrientes , GlucoseRESUMO
Cyclooxygenase-2 (COX-2) expression is up-regulated in inflammatory tissues and many premalignant and malignant tumors. Assessment of COX-2 protein in vivo, therefore, promises to be a powerful strategy to distinguish pathologic cells from normal cells in a complex disease setting. Herein, we report the first redox-activatable COX-2 probe, fluorocoxib Q (FQ), for in vivo molecular imaging of pathogenesis. FQ inhibits COX-2 selectively in purified enzyme and cell-based assays. FQ exhibits extremely low fluorescence and displays time- and concentration-dependent fluorescence enhancement upon exposure to a redox environment. FQ enters the cells freely and binds to the COX-2 enzyme. FQ exhibits high circulation half-life and metabolic stability sufficient for target site accumulation and demonstrates COX-2-targeted uptake and retention in cancer cells and pathologic tissues. Once taken up, it undergoes redox-mediated transformation into a fluorescent compound fluorocoxib Q-H that results in high signal-to-noise contrast and differentiates pathologic tissues from non-pathologic tissues for real-time in vivo imaging.
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Inibidores de Ciclo-Oxigenase 2 , Neoplasias , Animais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Corantes Fluorescentes/química , OxirreduçãoRESUMO
Introduction: Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, therapeutic potential of siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA. Methods: A Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach was used to obtain modified RNA-binding aptamers, which were then fused directly to siRNA via in vitro transcription. Molecular and pharmacokinetic properties of the aptamer-siRNA chimeras were subsequently measured in vitro and in vivo. Results: In vitro assays show that albumin-binding aptamers are stable in serum while maintaining potent gene knockdown capabilities in the chimera format. In vivo, the absolute circulation half-life of the best-performing aptamer-siRNA chimera (Clone 1) was 1.6-fold higher than a scrambled aptamer chimera control. Conclusions: Aptamer-siRNA chimeras exhibit improved bioavailability without compromising biological activity. Hence, this albumin-binding aptamer-siRNA chimera approach may be a promising strategy for drug delivery applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00718-y.
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Porous, resorbable biomaterials can serve as temporary scaffolds that support cell infiltration, tissue formation, and remodeling of nonhealing skin wounds. Synthetic biomaterials are less expensive to manufacture than biologic dressings and can achieve a broader range of physiochemical properties, but opportunities remain to tailor these materials for ideal host immune and regenerative responses. Polyesters are a well-established class of synthetic biomaterials; however, acidic degradation products released by their hydrolysis can cause poorly controlled autocatalytic degradation. Here, we systemically explored reactive oxygen species (ROS)-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity for skin wound healing. The most hydrophilic PTK-UR variant, with seven ethylene glycol (EG7) repeats flanking each side of a thioketal bond, exhibited the highest ROS reactivity and promoted optimal tissue infiltration, extracellular matrix (ECM) deposition, and reepithelialization in porcine skin wounds. EG7 induced lower foreign body response, greater recruitment of regenerative immune cell populations, and resolution of type 1 inflammation compared to more hydrophobic PTK-UR scaffolds. Porcine wounds treated with EG7 PTK-UR foams had greater ECM production, vascularization, and resolution of proinflammatory immune cells compared to polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM)-treated wounds and greater early vascular perfusion and similar wound resurfacing relative to clinical gold standard Integra Bilayer Wound Matrix (BWM). In a porcine ischemic flap excisional wound model, EG7 PTK-UR treatment led to higher wound healing scores driven by lower inflammation and higher reepithelialization compared to NovoSorb BTM. PTK-UR foams warrant further investigation as synthetic biomaterials for wound healing applications.
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Materiais Biocompatíveis , Cicatrização , Animais , Bandagens , Materiais Biocompatíveis/farmacologia , Inflamação , Poliésteres , Espécies Reativas de Oxigênio , Pele , SuínosRESUMO
Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP-treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype-associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.