RESUMO
The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion.
RESUMO
We show distinct CH-π interactions and assembly pathways for the amphiphile N-(fluorenylmethoxycarbonyl)-galactosamine and its epimer N-(fluorenylmethoxycarbonyl)-glucosamine. These differences result in the formation of supramolecular nanofibrous systems with opposite chirality. Our results showcase the importance of the carbohydrates structural diversity for their specific biointeractions and the opportunity that their ample interactome offers for synthesis of versatile and tunable supramolecular (bio) materials.
Assuntos
Tensoativos , Estereoisomerismo , Tensoativos/química , Tensoativos/síntese química , Carboidratos/química , Galactosamina/química , Glucosamina/química , Glucosamina/análogos & derivados , Substâncias Macromoleculares/química , Substâncias Macromoleculares/síntese química , Nanofibras/químicaRESUMO
Targeted breast cancer therapies hold the potential to improve the efficiency of drug delivery to the pathology site without impacting the viability and function of healthy cells. Herein, we developed multifunctional nanocarriers that target simultaneously several downstream signaling processes in triple negative breast cancer cells. The system comprises pH sensitive CaCO3 nanoparticles (NPs) as carriers of the anticancer drug doxorubicin (DOX). The NPs were coated in a layer-by-layer (LbL) fashion using poly-l-lysine and hyaluronic acid to target receptors overexpressed in breast cancer (e.g. CD44, RHAMM). Spheroids of the triple-negative Hs578T cell line were used as a 3D model to assess the therapeutic potential of this system. Our results showed that the NPs act via a synergistic mechanism that combines Ca2+ overload causing cell calcification and DNA damage by DOX. The LbL coating was crucial for the protection of the healthy cells, i.e. it provides NPs with targeting capacity. The overall data suggests that the LbL-coated NPs loaded with DOX hold great potential for the treatment of breast cancer.
Assuntos
Carbonato de Cálcio , Doxorrubicina , Portadores de Fármacos , Nanopartículas , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Doxorrubicina/farmacologia , Doxorrubicina/química , Doxorrubicina/administração & dosagem , Nanopartículas/química , Portadores de Fármacos/química , Carbonato de Cálcio/química , Feminino , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cálcio/metabolismo , Cálcio/química , Tamanho da Partícula , Polilisina/química , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/administração & dosagem , Propriedades de Superfície , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Hialurônico/químicaRESUMO
Spinal cord injuries (SCI) have devastating physical, psychological, and psychosocial consequences for patients. One challenge of nerve tissue repair is the lack of a natural extracellular matrix (ECM) that guides the regenerating axons. Hyaluronic acid (HA) is a major ECM component and plays a fundamental role in facilitating lesion healing. Herein, we developed HA-based adhesive hydrogels by modification of HA with dopamine, a mussel-inspired compound with excellent adhesive properties in an aqueous environment. The hydrogels were loaded with the anti-inflammatory drug ibuprofen and the response of neuronal cells (SH-SY5Y) was evaluated in terms of viability, morphology, and adhesion. The obtained results suggested that the developed materials can bridge lesion gaps, guide axonal growth, and simultaneously act as a vehicle for the delivery of bioactive compounds.
Assuntos
Neuroblastoma , Traumatismos da Medula Espinal , Humanos , Ácido Hialurônico , Hidrogéis , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Neurônios/patologia , Medula Espinal/patologiaRESUMO
Block copolymer (BCP) self-assembly has emerged as a feasible method for large-scale fabrication with remarkable precision - features that are not common for most of the nanofabrication techniques. In this review, recent advancements in the molecular design of BCP along with state-of-the-art processing methodologies based on microphase separation alone or its combination with different lithography methods are presented. Furthermore, the bioapplications of the generated nanopatterns in the development of protein arrays, cell-selective surfaces, and antibacterial coatings are explored. Finally, the current challenges in the field are outlined and the potential breakthroughs that can be achieved by adopting BCP approaches already applied in the fabrication of electronic devices are discussed.
Assuntos
Antibacterianos , Eletrônica , Membrana Celular , PolímerosRESUMO
Breast cancer is resistant to conventional treatments due to the specific tumour microenvironment, the associated acidic pH and the overexpression of receptors that enhance cells tumorigenicity. Herein, we optimized the synthesis of acidic resorbable calcium carbonate (CaCO3) nanoparticles and the encapsulation of a low molecular weight model molecule (Rhodamine). The addition of ethylene glycol during the synthetic process resulted in a particle size decrease: we obtained homogeneous CaCO3 particles with an average size of 564 nm. Their negative charge enabled the assembly of layer-by-layer (LbL) coatings with surface-exposed hyaluronic acid (HA), a ligand of tumour-associated receptor CD44. The coating decreased Rhodamine release by two-fold compared to uncoated nanoparticles. We demonstrated the effect of nanoparticles on two breast cancer cell lines with different aggressiveness - SK-BR-3 and the more aggressive MDA-MB-231 - and compared them with the normal breast cell line MCF10A. CaCO3 nanoparticles (coated and uncoated) significantly decreased the metabolic activity of the breast cancer cells. The interactions between LbL-coated nanoparticles and cells depended on HA expression on the cell surface: more particles were observed on the surface of MDA-MB-231 cells, which had the thickest endogenous HA coating. We concluded that CaCO3 nanoparticles are potential candidates to carry low molecular weight chemotherapeutics and deliver them to aggressive breast cancer sites with an HA-abundant pericellular matrix.
Assuntos
Neoplasias da Mama , Nanopartículas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Carbonato de Cálcio/farmacologia , Carbonato de Cálcio/química , Células MCF-7 , Rodaminas , Nanopartículas/química , Microambiente TumoralRESUMO
Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.
Assuntos
Heparina , Protaminas , Animais , Ratos , Humanos , Heparina/efeitos adversos , Protaminas/química , Ratos Sprague-Dawley , Anticoagulantes/farmacologia , Anticoagulantes/químicaRESUMO
We applied a bottom-up approach to develop biofunctional supramolecular hydrogels from an aromatic glycodipeptide. The self-assembly of the glycopeptide was induced by either temperature manipulation (heating-cooling cycle) or solvent (DMSO to water) switch. The sol-gel transition was salt-triggered in cell culture media and resulted in gels with the same chemical compositions but different mechanical properties. Human adipose derived stem cells (hASCs) cultured on these gels under basal conditions (i.e., without differentiation factors) overexpressed neural markers, such as GFAP, Nestin, MAP2, and ßIII-tubulin, confirming the differentiation into neural lineages. The mechanical properties of the gels influenced the number and distribution of the adhered cells. A comparison with gels obtained from the nonglycosylated peptide showed that glycosylation is crucial for the biofunctionality of the hydrogels by capturing and preserving essential growth factors, e.g., FGF-2.
Assuntos
Glicopeptídeos , Hidrogéis , Humanos , Hidrogéis/farmacologia , Hidrogéis/química , Diferenciação Celular , Adipócitos , Células-Tronco , Células CultivadasRESUMO
The central nervous system (CNS) has a limited ability to regenerate after a traumatic injury or a disease due to the low capacity of the neurons to re-grow and the inhibitory environment formed in situ. Current therapies include the use of drugs and rehabilitation, which do not fully restore the CNS functions and only delay the pathology progression. Tissue engineering offers a simple and versatile solution for this problem through the use of bioconstructs that promote nerve tissue repair by bridging cavity spaces. In this approach, the choice of biomaterial is crucial. Herein, we present recent advances in the design and development of adhesive and self-healing materials that support CNS healing. The adhesive materials have the advantage of promoting recovery without the use of needles or sewing, while the self-healing materials have the capacity to restore the tissue integrity without the need for external intervention. These materials can be used alone or in combination with cells and/or bioactive agents to control the inflammation, formation of free radicals, and proteases activity. We discuss the advantages and drawbacks of different systems. The remaining challenges that can bring these materials to clinical reality are also briefly presented.
Assuntos
Adesivos , Materiais Biocompatíveis , Adesivos/uso terapêutico , Materiais Biocompatíveis/uso terapêutico , Sistema Nervoso Central/lesões , Engenharia Tecidual , NeurôniosRESUMO
The three-dimensional (3D) organization of cells affects their mobility, proliferation, and overall response to treatment. Spheroids, organoids, and microfluidic chips are used in cancer research to reproduce in vitro the complex and dynamic malignant microenvironment. Herein, single- and double-channel microfluidic devices are used to mimic the spatial organization of brain tumors and investigate the therapeutic efficacy of molecular and nano anti-cancer agents. Human glioblastoma multiforme (U87-MG) cells were cultured into a Matrigel matrix embedded within the microfluidic devices and exposed to different doses of free docetaxel (DTXL), docetaxel-loaded spherical polymeric nanoparticles (DTXL-SPN), and the aromatic N-glucoside N-(fluorenylmethoxycarbonyl)-glucosamine-6-phosphate (Fmoc-Glc6P). We observed that in the single-channel microfluidic device, brain tumor cells are more susceptible to DTXL treatment as compared to conventional cell monolayers (50-fold lower IC50 values). In the double-channel device, the cytotoxicity of free DTXL and DTXL-SPN is comparable, but significantly lowered as compared to the single-channel configuration. Finally, the administration of 500 µM Fmoc-Glc6P in the double-channel microfluidic device shows a 50 % U87-MG cell survival after only 24 h, and no deleterious effect on human astrocytes over 72 h. Concluding, the proposed microfluidic chips can be used to reproduce the 3D complex spatial arrangement of solid tumors and to assess the anti-cancer efficacy of therapeutic compounds administrated in situ or systemically.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Nanopartículas , Humanos , Docetaxel , Neoplasias Encefálicas/tratamento farmacológico , Dispositivos Lab-On-A-Chip , Microambiente TumoralRESUMO
The tumor microenvironment (TME) is a dynamic and complex matter shaped by heterogenous cancer and cancer-associated cells present at the tumor site. Hyaluronan (HA) is a major TME component that plays pro-tumorigenic and carcinogenic functions. These functions are mediated by different hyaladherins expressed by cancer and tumor-associated cells triggering downstream signaling pathways that determine cell fate and contribute to TME progression toward a carcinogenic state. Here, the interaction of HA is reviewed with several cell-surface hyaladherins-CD44, RHAMM, TLR2 and 4, LYVE-1, HARE, and layilin. The signaling pathways activated by these interactions and the respective response of different cell populations within the TME, and the modulation of the TME, are discussed. Potential cancer therapies via targeting these interactions are also briefly discussed.
Assuntos
Receptores de Hialuronatos , Neoplasias , Humanos , Receptores de Hialuronatos/metabolismo , Microambiente Tumoral , Transdução de Sinais , Membrana Celular/metabolismo , Ácido Hialurônico/metabolismoRESUMO
Dental implants, usually made of titanium, are exposed to hostile oral microflora that facilitate bacterial infections and subsequent inflammation. To mitigate these processes, we coated titanium substrates with block copolymer nanopatterns and investigated the bactericidal effect of these coatings against Gram-positive and Gram-negative bacteria. We found that the bactericidal efficacy of the coatings depends on their morphology and surface chemistry as well as on the bacterial strain: an optimal combination can lead to significant bacterial death for a short time, i.e. 90% for 90 min. Human gingival fibroblasts in contact with the nanopatterned coatings showed similar cell attachment and morphology as on bare Ti. Immunostaining assays showed similar levels of CCR7 and CD206 in macrophages cultured over the nanopatterns and bare Ti, demonstrating adequate properties for tissue integration. The nanopatterns induced a small increase in macrophage aspect ratio, which might indicate early states of M2 polarization, given the absence of CD206.
Assuntos
Implantes Dentários , Titânio , Humanos , Titânio/farmacologia , Titânio/química , Antibacterianos/farmacologia , Antibacterianos/química , Propriedades de Superfície , Bactérias Gram-Negativas , Bactérias Gram-PositivasRESUMO
We report on the synthesis of hyaluronan (HA) brush-like copolymers and their application as antagonists of tumorigenic CD44-HA interactions. HA (4.8 kDa, ca. 24 saccharides) was grafted on 2-hydrohyethyl methacrylate (HEMA) by end-on oxime ligation. The obtained copolymers were compared with low and high molecular weight HA in terms of hydrolysis kinetics in the presence of hyaluronidase (isothermal titration calorimetry) and interactions with CD44 (surface plasmon resonance). The results evidenced that the high molecular weight HA and HA-g-HEMA have a much higher affinity to CD44 than low molecular weight HA. Additionally, slower enzymatic degradation was observed for the copolymer, making it an excellent candidate for active targeting of tumorigenic CD44-HA interactions. We, therefore, investigated the effect of the copolymer on cancer cell lines with different expression of CD44 and observed an efficient declustering of CD44 that is usually associated with reduction of metastasis and drug resistance.
Assuntos
Neoplasias da Mama , Ácido Hialurônico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Hialuronoglucosaminidase/metabolismo , Metacrilatos , Oximas , Polímeros/farmacologiaRESUMO
Hyaluronan (HA) synthesis and degradation are altered during carcinogenesis leading to an increased HA content in the tumor microenvironment, which correlates with poor prognosis and treatment outcomes. The main HA receptors, CD44 and RHAMM, are also overexpressed in tumors where they activate anti-apoptotic, proliferative, invasive, and migration signaling pathways. Herein, we used a unidirectional HA gradient to investigate in a high-throughput fashion the bi-directional communication between HA and breast cancer cell lines with different surface expression of CD44 and RHAMM. We found that the expression of CD44 and RHAMM depends on the HA density: the expression of these receptors is promoted at higher HA density and RHAMM is more sensitive to these changes when compared to CD44. Blocking either CD44 or RHAMM revealed different functions on binding and recognizing HA and a compensatory expression between these two receptors that maintains protumorigenic effectors such as cortactin. STATEMENT OF SIGNIFICANCE: We show that the expression of main hyaluronan (HA) receptors CD44 and RHAMM is enhanced in a HA concentration-dependent manner. Blocking activity experiments with either RHAMM or CD44 reveal the redundancy of these two receptors towards HA recognition and activation/recruitment of protumorigenic molecular effector, cortactin. These experiments also demonstrate that cells with overexpressed RHAMM are more sensitive to HA density than CD44 positive cells. The reported results are important for the development of therapies that target the hyaluronan signaling in the tumor microenvironment.
Assuntos
Neoplasias da Mama , Proteínas da Matriz Extracelular , Receptores de Hialuronatos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Movimento Celular/fisiologia , Cortactina/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Microambiente TumoralRESUMO
We describe bioadhesive membranes developed from marine renewable biomaterials, namely chitosan and collagen extracted from fish skins. Collagen was functionalized with catechol groups (Coll-Cat) to provide the membranes with superior adhesive properties in a wet environment and blended with chitosan to improve the mechanical properties. The blended membranes were compared to chitosan and chitosan blended with unmodified collagen in terms of surface morphology, wettability, weight loss, water uptake, mechanical and adhesive properties. The metabolic activity, the viability and the morphology of L929 fibroblastic cells seeded on these membranes were also assessed. Our results show that the functionalization with catechol groups improves the adhesive and mechanical properties of the membranes and enhances cell attachment and proliferation. These data suggest that the developed marine origin-raw membranes present a potential towards the restoration of the structural and functional properties of damaged soft tissues.
Assuntos
Quitosana , Adesivos/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Catecóis/química , Quitosana/química , Colágeno/química , Membranas ArtificiaisRESUMO
Molecular gradients are common in biosystems and play an essential role in physiological and pathological processes. During carcinogenesis, for example, hyaluronan (HA) homeostasis is dysregulated by cancer cells and the altered synthesis and degradation processes result in the formation of HA gradients within the tumor microenvironment. Herein, a platform is developed to study the biological role of HA gradient in breast cancer cells. Cells with different aggressiveness and expression of CD44-the main HA receptor usually overexpressed in breast cancers, are selected for this study. The developed platform is compatible with several imaging modalities and allows assessment of cell density, morphology, CD44 expression, and cell motility in a function of HA density. Using high-throughput analysis, it is shown that cells that do not express CD44 do not change along the gradient, while CD44 positive cells respond differently to the HA gradient depending on the level of CD44 expression and HA density. This different response is associated with the activation of different signaling pathways by the CD44-HA interactions.
Assuntos
Neoplasias da Mama , Receptores de Hialuronatos , Ácido Hialurônico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
We report on the supramolecular self-assembly of tripeptides and their O-glycosylated analogues, in which the carbohydrate moiety is coupled to a central serine or threonine flanked by phenylalanine residues. The substitution of serine with threonine introduces differential side-chain interactions, which results in the formation of aggregates with different morphology. O-glycosylation decreases the aggregation propensity because of rebalancing of the π interactions. The glycopeptides form aggregates with reduced stiffness but increased thermal stability. Our results demonstrate that the designed minimalistic glycopeptides retain critical functional features of glycoproteins and therefore are promising tools for elucidation of molecular mechanisms involved in the glycoprotein interactome. They can also serve as an inspiration for the design of functional glycopeptide-based biomaterials.
Assuntos
Glicoproteínas/metabolismo , Oligopeptídeos/metabolismo , Glicoproteínas/química , Glicosilação , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Conformação Proteica , Multimerização ProteicaRESUMO
Extracellular vesicles (EVs) are particles originating from the exfoliation of the cellular membrane. They are involved in cell-to-cell and cell-to-matrix signaling, exchange of bioactive molecules, tumorigenesis and metastasis, among others. To mitigate the limited understanding of EVs transfer phenomena, we developed a simplistic model that mimics EVs and their interactions with cells and the extracellular matrix. The proposed model is a layer by layer (LbL) film built from the polycationic poly-l-lysine (PLL) and the glycosaminoglycan hyaluronic acid (HA) to provide ECM mimicry. Positively charged 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N1,N1,N14,N14-tetramethyl-N1,N14-ditetradecyltetradecane-1,14-diaminium dibromide (GS14) liposomes were embedded in this construct to act as EVs analogs. To simulate EVs carrying substances, Nile Red was loaded as a model of lipophilic cargo molecules. The integration of each component was followed by quartz crystal microbalance measurements, which confirmed the immobilization of intact liposomes on the underlying (PLL/HA)3 soft film. The release of Nile Red from liposomes either embedded in the LbL construct or exposed at its surface revealed a fast first order release. This system was validated as a model for EV/cell interactions by incubation with breast cancer cells MDA-MB-231. We observed higher internalization for embedded liposomes when compared with surface-exposed ones, showcasing that the ECM mimic layers do not constitute a barrier to liposome/cell interactions but favor them.
Assuntos
Vesículas Extracelulares , Lipossomos , Ácido Hialurônico , Técnicas de Microbalança de Cristal de QuartzoRESUMO
CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM) are the main hyaluronan (HA) receptors. They are commonly overexpressed in different cancers activating signaling pathways related to tumor progression, metastasis and chemoresistance. Besides their involvement in signal transduction via interaction with HA, currently, there is a little information about the possible crosstalk between CD44 and RHAMM and the role of HA in this process. In the present work, we used immunocytochemistry combined with Förster resonance energy transfer (FRET) microscopy and co-immunoprecipitation to elucidate the involvement of HA in CD44 and RHAMM expression, co-localization and crosstalk. We studied breast cancer cells lines with different degrees of invasiveness and expression of these receptors in the absence of exogenous HA and compared the data with the results obtained for cultures supplemented with either soluble HA or seeded on substrates with end-on immobilized HA. Our results demonstrated that cells response depends on the HA presentation: CD44/RHAMM complexation was upregulated in all cell lines upon interaction with immobilized HA, but not with its soluble form. Moreover, the results showed that the expression of both CD44 and RHAMM is regulated via interactions with HA indicating cell-specific feedback loop(s) in the signaling cascade.
Assuntos
Proteínas da Matriz Extracelular , Receptores de Hialuronatos , Ácido Hialurônico , Linhagem Celular Tumoral , Movimento Celular , Humanos , Transdução de SinaisRESUMO
Hyaluronic acid (HA) has a key role in cancer progression. The HA's molecular weight (Mw) is altered in this pathological state: increased concentration of shorter fragments due to the overexpressed hyaluronidases and ROS. Aiming to mimic this microenvironment, we developed a Layer-by-Layer (LbL) platform presenting HA of different Mws, namely 6.4, 752 and 1500 kDa, to study the influence of HA Mw on the formation of focal adhesion sites (FAs), and the involvement of paxillin and CD44 in this process. High paxillin expression and formation of FAs, via CD44, is observed for MKN45 cells seeded on LbLs presenting HA 6.4 kDa, with the activation of the ERK1/2 pathway, responsible for cell motility and tumour progression. In contrast, activation of p38 pathway, usually related with cancer latency, is observed for cells seeded on LbLs with high Mw HA, i.e. 1500 kDa. Overall, we demonstrate the suitability of the developed platform to study cancer invasiveness.