RESUMEN
Proteoglycans play a crucial role in proper tissue morphology and function throughout the body that is defined by a combination of their core protein and the attached glycosaminoglycan chains. Although they serve a myriad of roles, the functions of extracellular proteoglycans can be generally sorted into four categories: modulation of tissue mechanical properties, regulation and protection of the extracellular matrix, sequestering of proteins, and regulation of cell signaling. The loss of proteoglycans can result in significant tissue dysfunction, ranging from poor mechanical properties to uncontrolled inflammation. Because of the key roles they play in proper tissue function and due to their complex synthesis, the past two decades have seen significant research into the development of proteoglycan mimetic molecules to recapitulate the function of proteoglycans for therapeutic and tissue engineering applications. These strategies have ranged from semisynthetic graft copolymers to recombinant proteoglycan domains synthesized by genetically engineered cells. In this review, we highlight some of the important functions of extracellular proteoglycans, as well as the strategies developed to recapitulate these functions.
Asunto(s)
Proteoglicanos , Ingeniería de Tejidos , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanos/metabolismoRESUMEN
Hydrogels have been widely investigated in biomedical fields due to their similar physical and biochemical properties to the extracellular matrix (ECM). Collagen and hyaluronic acid (HA) are the main components of the ECM in many tissues. As a result, hydrogels prepared from collagen and HA hold inherent advantages in mimicking the structure and function of the native ECM. Numerous studies have focused on the development of collagen and HA hydrogels and their biomedical applications. In this extensive review, we provide a summary and analysis of the sources, features, and modifications of collagen and HA. Specifically, we highlight the fabrication, properties, and potential biomedical applications as well as promising commercialization of hydrogels based on these two natural polymers.
RESUMEN
Glycosaminoglycans (GAGs), such as hyaluronic acid (HA) and chondroitin sulfate (CS), have seen widespread adoption as components of tissue engineering scaffolds because of their potent bioactive properties and ease of chemical modification. However, modification of the biopolymers will impair biological recognition of the GAG and reduce the bioactive properties of the material. In this work, we studied how the degree of thiolation of HA and CS, along with other key hydrogel design parameters, affected the physical and bioactive properties of the bulk hydrogel. Although properties, such as the HA molecular weight, did not have a major effect, increasing the degree of thiolation of both HA and CS decreased their biorecognition in experimental analogues for cell/matrix remodeling and binding. Furthermore, combining HA and CS into dual polymer network hydrogels also modulated the physical and bioactive properties, as seen with differences in gel stiffness, degradation rate, and encapsulated cell viability.
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Glicosaminoglicanos , Hidrogeles , Sulfatos de Condroitina , Ácido Hialurónico , Polímeros , Ingeniería de TejidosRESUMEN
Biomaterials that provide signals present in the native extracellular matrix have been proposed as scaffolds to support improved cartilage regeneration. This study harnesses the biological activity of collagen type II and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends. The collagen blend hydrogels were able to incorporate both types of collagen and retained chondroitin sulfate and hyaluronic acid. Cryo-scanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%). The complex modulus was larger for the 3:1 gels (G* = 5.0 Pa) compared to the 1:1 gels (G* = 1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Regeneración/efectos de los fármacos , Ingeniería de Tejidos , Materiales Biocompatibles/química , Materiales Biocompatibles/uso terapéutico , Cartílago Articular/crecimiento & desarrollo , Condrocitos/efectos de los fármacos , Sulfatos de Condroitina/química , Colágeno Tipo I/administración & dosificación , Colágeno Tipo I/química , Colágeno Tipo II/administración & dosificación , Colágeno Tipo II/química , Humanos , Ácido Hialurónico/química , Hidrogeles/administración & dosificación , Hidrogeles/química , Andamios del Tejido/químicaRESUMEN
Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.
Asunto(s)
Antiinflamatorios/administración & dosificación , Cartílago/patología , Nanopartículas , Cartílago/efectos de los fármacos , Preparaciones de Acción Retardada/administración & dosificación , Portadores de Fármacos , Inflamación/tratamiento farmacológico , PéptidosRESUMEN
Pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are mediators in the development of many inflammatory diseases. To demonstrate that macrophages take up and respond to thermosensitive nanoparticle drug carriers, we synthesized PEGylated poly(N-isopropylacrylamide-2-acrylamido-2-methyl-1-propanesulfonate) particles cross-linked with degradable disulfide (N,N'-bis(acryloyl)cystamine) (NGPEGSS). An anti-inflammatory peptide (KAFAK) was loaded and released from the thermosensitive nanoparticles and shown to suppress levels of TNF-α and IL-6 production in macrophages. Cellular uptake of fluorescent, thermosensitive, and degradable nanoparticles and therapeutic efficacy of free KAFAK peptide compared to that of KAFAK loaded in PEGylated degradable thermosensitive nanoparticles were examined. The data suggests that the degradable, thermosensitive nanoparticles loaded with KAFAK may be an effective tool to treat inflammatory diseases.
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Antiinflamatorios/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Interleucina-6/metabolismo , Nanopartículas/química , Factor de Necrosis Tumoral alfa/metabolismo , Acrilamidas/química , Alcanosulfonatos/química , Animales , Antiinflamatorios/farmacología , Línea Celular , Péptidos de Penetración Celular/farmacología , Reactivos de Enlaces Cruzados/química , Calor , Interleucina-6/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Polietilenglicoles/química , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: Corticosteroids may be beneficial in treating vocal fold scarring. Current drug delivery methods do not permit controlled corticosteroid release. Here we investigate the effects of poly-lactic-co-glycolic acid (PLGA) microparticles loaded with the corticosteroid dexamethasone in reducing collagen synthesis and inflammation in vocal fold fibroblasts treated with and without TGF-ß1. STUDY DESIGN: Experimental, in vitro study. METHODS: PLGA microparticles of differing molecular weight and terminating moieties were synthesized using a hydrogel template method. The release of dexamethasone was characterized from these microparticles over 4 days. Based on the release studies, ester-terminated low molecular weight PLGA microparticles were loaded with dexamethasone and applied to TGF-ß1 treated vocal fold fibroblasts for 4 days. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays (ELISAs) were used to assess the effects of released dexamethasone on collagen synthesis and inflammatory mediators. RESULTS: COL3A1 and COL1A2 were significantly down-regulated after exposure to ester-terminated low molecular weight PLGA microparticles loaded with dexamethasone. The loaded microparticles also reduced interleukin-6 synthesis. CONCLUSION: These data show promise in using a PLGA microparticle-based delivery system to control dexamethasone release over 4 days. Our findings lay the groundwork for developing more effective treatments for vocal fold scarring.
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Dexametasona/administración & dosificación , Ácido Láctico , Ácido Poliglicólico , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Disfunción de los Pliegues Vocales/tratamiento farmacológico , Pliegues Vocales/patología , Materiales Biocompatibles , Células Cultivadas , Cicatriz/tratamiento farmacológico , Cicatriz/metabolismo , Cicatriz/patología , Colágeno/biosíntesis , Colágeno/genética , Citocinas/biosíntesis , Citocinas/genética , Preparaciones de Acción Retardada , Portadores de Fármacos , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glucocorticoides/administración & dosificación , Humanos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta1/metabolismo , Disfunción de los Pliegues Vocales/metabolismo , Disfunción de los Pliegues Vocales/patología , Pliegues Vocales/efectos de los fármacos , Pliegues Vocales/metabolismoRESUMEN
The seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus, and methicillin-resistant Staphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis compared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Proteínas Recombinantes/farmacología , Staphylococcus epidermidis/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/efectos adversos , Biopelículas/efectos de los fármacos , Línea Celular Tumoral , Farmacorresistencia Bacteriana Múltiple , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Lisostafina/farmacología , Cloruro de Magnesio/química , Pruebas de Sensibilidad Microbiana , Proteínas Recombinantes/efectos adversos , Cloruro de Sodio/química , Resistencia a la VancomicinaRESUMEN
Following balloon injury, smooth muscle cells (SMCs) serve as targets for many of the pro-inflammatory and pro-fibrotic factors, including platelet-derived growth factor (PDGF) and interferon-γ (IFN-γ) released from activated inflammatory cells and platelets. Previously, our lab designed a mimic of the proteoglycan decorin, termed DS-SILY20, that suppressed vascular SMC proliferation, migration, and protein synthesis in vitro, and injured vessels treated with DS-SILY20 demonstrated reduced hyperplasia in vivo. Here we characterize the effects of DS-SILY20 on modulating PDGF and IFN-γ stimulation in both proliferative and quiescent human SMCs to further evaluate the potential impact of DS-SILY20-SMC interaction on restenosis. Nanomolar dissociation constants were observed between DS-SILY20 and both PDGF and IFN-γ. PDGF significantly increased migration, proliferation, and protein and cytokine expression, as well as increased ERK-1/2 and p38 MAPK phosphorylation in both quiescent and proliferative cultures. However, DS-SILY20 inhibited these increases, presumably through sequestration of the PDGF. Consistent with the complex responses seen with IFN-γ in SMC physiology in the literature, the response of SMC cultures to IFN-γ was variable and complex. However, where increased activity was seen with IFN-γ, DS-SILY20 attenuated this activity. Overall, the results suggest that DS-SILY20 would be an ideal alternative to traditional therapeutics used and may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.
Asunto(s)
Decorina/fisiología , Interferón gamma/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Decorina/farmacología , Humanos , Datos de Secuencia Molecular , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
Cardiovascular disease remains one of the largest contributors to death worldwide. Improvements in cardiovascular technology leading to the current generation of drug-eluting stents, bioresorbable stents, and drug-eluting balloons, coupled with advances in antirestenotic therapeutics developed by pharmaceutical community, have had a profound impact on quality of life and longevity. However, these procedures and devices contribute to both short- and long-term complications. Thus, room for improvement and development of new, alternative strategies exists. Two major approaches have been investigated to improve outcomes following percutaneous coronary intervention including perivascular delivery and luminal paving. For both approaches, polymers play a major role as controlled research vehicles, carriers for cells, and antithrombotic coatings. With improvements in catheter delivery devices and increases in our understanding of the biology of healthy and diseased vessels, the time is ripe for development of novel macromolecular coatings that can protect the vessel lumen following balloon angioplasty and promote healthy vascular healing.
Asunto(s)
Reestenosis Coronaria , Stents Liberadores de Fármacos , Fibrinolíticos/uso terapéutico , Intervención Coronaria Percutánea/efectos adversos , Complicaciones Posoperatorias , Trombosis , Túnica Íntima , Reestenosis Coronaria/etiología , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/patología , Reestenosis Coronaria/prevención & control , Humanos , Hiperplasia , Complicaciones Posoperatorias/metabolismo , Complicaciones Posoperatorias/patología , Complicaciones Posoperatorias/prevención & control , Trombosis/etiología , Trombosis/metabolismo , Trombosis/patología , Trombosis/prevención & control , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
The popularity of Glycosaminoglycans (GAGs) in drug delivery systems has grown as their innate ability to sequester and release charged molecules makes them adept in the controlled release of therapeutics. However, peptide therapeutics have been relegated to synthetic, polymeric systems, despite their high specificity and efficacy as therapeutics because they are rapidly degraded in vivo when not encapsulated. We present a GAG-based nanoparticle system for the easy encapsulation of cationic peptides, which offers control over particle diameter, peptide release behavior, and swelling behavior, as well as protection from proteolytic degradation, using a singular, organic polymer and no covalent linkages. These nanoparticles can encapsulate cargo with a particle diameter range spanning 130-220 nm and can be tuned to release cargo over a pH range of 4.5 to neutral through the modulation of the degree of sulfation and the molecular weight of the GAG. This particle system also confers better in vitro performance than the unencapsulated peptide via protection from enzymatic degradation. This method provides a facile way to protect therapeutic peptides via the inclusion of the presented binding sequence and can likely be expanded to larger, more diverse cargo as well, abrogating the complexity of previously demonstrated systems while offering broader tunability.
RESUMEN
Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.
Asunto(s)
Condrocitos , Sulfatos de Condroitina , Ácido Hialurónico , Hidrogeles , Inflamación , Hidrogeles/química , Hidrogeles/farmacología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Sulfatos de Condroitina/farmacología , Sulfatos de Condroitina/química , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/patología , Animales , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Citocinas/metabolismo , Agrecanos/metabolismo , Ingeniería de Tejidos/métodos , Osteoartritis/patología , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismoRESUMEN
Collagen (Col) is commonly used as a natural biomaterial for biomedical applications. Although Col I is the most prevalent col type employed, many collagen types work together in vivo to confer function and biological activity. Thus, blending collagen types can better recapitulate many native environments. This work investigates how hydrogel properties can be tuned through blending collagen types (col I/II and col I/III) and by varying polymerization temperatures. Col I/II results in poorly developed fibril networks, which softened the gels, especially at lower polymerization temperatures. Conversely, col I/III hydrogels exhibit well-connected fibril networks with localized areas of fine fibrils and result in stiffer hydrogels. A decreased molecular mass recovery rate is observed in blended hydrogels. The altered fibril morphologies, mechanical properties, and biological signals of the blended gels can be leveraged to alter cell responses and can be used as models for different tissue types (e.g., healthy vs fibrotic tissue). Furthermore, the biomimetic hydrogel properties are a tool that can be used to modulate the transport of drugs, nutrients, and wastes in tissue engineering applications.
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Despite advances in breast cancer treatment, there remains a need for local management of noninvasive, low-grade ductal carcinoma in situ (DCIS). These focal lesions are well suited for local intraductal treatment. Intraductal administration supported target site drug retention, improved efficacy, and reduced systemic exposure. Here, we used a poly(N-isopropyl acrylamide, pNIPAM) nanoparticle delivery system loaded with cytotoxic piplartine and an MAPKAP Kinase 2 inhibitor (YARA) for this purpose. For tumor environment targeting, a collagen-binding peptide SILY (RRANAALKAGELYKSILYGSG-hydrazide) was attached to pNIPAM nanoparticles, and the nanoparticle diameter, zeta potential, drug loading, and release were assessed. The system was evaluated for cytotoxicity in a 2D cell culture and 3D spheroids. In vivo efficacy was evaluated using a chemical carcinogenesis model in female Sprague-Dawley rats. Nanoparticle delivery significantly reduced the IC50 of piplartine (4.9 times) compared to the drug in solution. The combination of piplartine and YARA in nanoparticles further reduced the piplartine IC50 (~15 times). Treatment with these nanoparticles decreased the in vivo tumor incidence (5.2 times). Notably, the concentration of piplartine in mammary glands treated with nanoparticles (35.3 ± 22.4 µg/mL) was substantially higher than in plasma (0.7 ± 0.05 µg/mL), demonstrating targeted drug retention. These results indicate that our nanocarrier system effectively reduced tumor development with low systemic exposure.
RESUMEN
Since extracellular vesicles (EVs) have emerged as a promising drug delivery system, diverse methods have been used to load them with active pharmaceutical ingredients (API) in preclinical and clinical studies. However, there is yet to be an engineered EV formulation approved for human use, a barrier driven in part by the intrinsic heterogeneity of EVs. API loading is rarely assessed in the context of single vesicle measurements of physicochemical properties but is likely administered in a heterogeneous fashion to the detriment of a consistent product. Here, we applied a suite of single-particle resolution methods to determine the loading of rhodamine 6G (R6G) surrogate cargo mimicking hydrophilic small molecule drugs across four common API loading methods: sonication, electroporation, freeze-thaw cycling and passive incubation. Loading efficiencies and alterations in the physical properties of EVs were assessed, as well as co-localization with common EV-associated tetraspanins (i.e., CD63, CD81 and CD9) for insight into EV subpopulations. Sonication had the highest loading efficiency, yet significantly decreased particle yield, while electroporation led to the greatest number of loaded API particles, albeit at a lower efficiency. Moreover, results were often inconsistent between repeated runs within a given method, demonstrating the difficulty in developing a rigorous loading method that consistently loaded EVs across their heterogeneous subpopulations. This work highlights the significance of how chosen quantification metrics can impact apparent conclusions and the importance of single-particle characterization of EV loading.
RESUMEN
Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2, or MK2), a serine/threonine kinase downstream of p38 mitogen-activated protein kinase, has been implicated in inflammation and fibrosis. Compared with pathologically normal lung tissue, significantly higher concentrations of activated MK2 are evident in lung biopsies of patients with idiopathic pulmonary fibrosis (IPF). Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust, activated MK2 expression on Day 7 (prefibrotic stage) and Day 14 (postfibrotic stage). To determine the effects of MK2 inhibition during the postinflammatory/prefibrotic and postfibrotic stages, C57BL/6 mice received intratracheal bleomycin instillation (0.025 U; Day 0), followed by PBS or the MK2 inhibitor (MK2i; 37.5 µg/kg), administered via either local (nebulized) or systemic (intraperitoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning on either Day 7 or Day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 concentrations, and modulated the local mRNA expression of profibrotic cytokine il-1ß, matrix-related genes col1a2, col3a1, and lox, and transforming growth factor-ß family members, including smad3, serpine1 (pai1), and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, the secretion of collagen Type I, fibronectin, and the activation of focal adhesion kinase, whereas activated MK2 was attenuated at optimal doses. The peptide-mediated inhibition of MK2 affects both inflammatory and fibrotic responses, and thus may offer a promising therapeutic target for IPF.
Asunto(s)
Bleomicina/efectos adversos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fibrosis Pulmonar/inducido químicamente , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/farmacología , Bleomicina/administración & dosificación , Diferenciación Celular , Colágeno Tipo I/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP-1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP-1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae-mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin-mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP.
Asunto(s)
Péptidos de Penetración Celular/química , Endocitosis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Péptidos/química , Inhibidores de Proteínas Quinasas/farmacología , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/síntesis química , Células Epiteliales/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Cell-penetrating anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) has the ability to suppress pro-inflammatory cytokines TNF-α and IL-6 when released from degradable and non-degradable poly(NIPAm-AMPS) nanoparticles. In vitro human macrophage model with THP1 human monocytes and ex vivo bovine knee cartilage tissue both showed a dose-dependent suppression of pro-inflammatory cytokines when treated with KAFAK-loaded poly(NIPAm-AMPS) nanoparticles. When bovine knee cartilage explants were treated with KAFAK-loaded poly(NIPAm-AMPS) nanoparticles, rapid and highly selective targeting of only damaged tissue occurred. This study has demonstrated selective targeting and therapeutic efficacy of KAFAK when released from both degradable and non-degradable poly(NIPAm-AMPS) nanoparticles in in vitro and ex vivo models. As a result, poly(NIPAm-AMPS) nanoparticles loaded with KAFAK could be a very effective tool to treat osteoarthritis. FROM THE CLINICAL EDITOR: Inflammatory arthritis remains a major medical problem with substantial socio-economic impact. Anti-inflammatory KAFAK peptide when released from degradable and non-degradable poly(NIPAm-AMPS) nanoparticles has the ability to penetrate cells and suppress pro-inflammatory cytokines, resulting in rapid and highly selective targeting of only damaged tissue in bovine knee cartilage explants. This approach may provide a very effective future tool in addressing osteoarthritis.
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Cartílago/patología , Péptidos de Penetración Celular/farmacología , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/patología , Nanopartículas/química , Temperatura , Resinas Acrílicas/química , Secuencia de Aminoácidos , Animales , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Bovinos , Línea Celular , Péptidos de Penetración Celular/química , Humanos , Interleucina-6/biosíntesis , Macrófagos/metabolismo , Datos de Secuencia Molecular , Polímeros/química , Ácidos Sulfónicos/química , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Therapeutic peptides capable of reducing inflammation via inhibition of the MAP kinase 2 pathway have the potential to reduce inflammation in atopic dermatitis by suppressing secretion of inflammatory cytokines by resident keratinocytes. One of the biggest hurdles to the use of therapeutic peptides, however, is their rapid degradation by intrinsic proteases and peptidases found in serum. Here we introduce a new nanoparticle technology that enhances and extends the bioactivity of a MAP KAP kinase 2 inhibitor peptide (MK2i) via electrostatic complexation with Dermatan sulfate (DS), a glycosaminoglycan, and explore their properties under various conditions. DS-MK2i nanoparticles can be made using electrospray ionization or sonication and vortexing with no stabilizing polymers or crosslinking. Average particle diameter, polydispersity index, and zeta potential were measured over a pH range of 2.5-11.5, in increments of 0.5, in water and at physiological ionic strength. Both particle types were shown to be shelf stable, robust, and behave differently in response to pH. They are also significantly more effective at suppressing cytokine secretion in inflamed, human keratinocytes than peptide alone in the presence of serum, providing a facile method of protecting peptides for therapeutic delivery in conditions such as atopic dermatitis, and abrogating the need for serum-starvation in in vitro testing.
Asunto(s)
Dermatitis Atópica , Nanopartículas , Humanos , Dermatitis Atópica/tratamiento farmacológico , Glicosaminoglicanos , Péptidos/química , Nanopartículas/química , InflamaciónRESUMEN
Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.