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Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.
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Nanopartículas em Multicamadas , Engenharia Tecidual , Materiais Biocompatíveis , Sistemas de Liberação de Medicamentos , PolieletrólitosRESUMO
BACKGROUND: Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB is prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) with standard half-life or extended half-life. Unmet needs remain regarding the development of non-invasive administration routes for coagulation factors. The aim of this study was to evaluate the effectiveness of intranasal delivery (IND) of rFIX and rFIX fused to Fc fragment (rFIX-Fc) in mice. METHODS: Drops of rFIX and rFIX-Fc were deposited in the nostrils of wild-type, FcRn knock-out, FcRn humanized, and FIX knock-out mice. rFIX mucosal uptake was evaluated by measuring plasma FIX antigen and FIX activity (FIX:C) levels, and by performing histologic analysis of the nasal mucosa following IND. RESULTS: After IND, both rFIX and rFIX-Fc were equally delivered to the blood compartment, irrespective of the mouse strain studied, mostly through a passive mechanism of transportation across the mucosal barrier, independent of FcRn receptor. Both plasma FIX antigen and FIX:C activity levels increased following IND in FIX knock-out mice. CONCLUSION: This proof-of-concept study describes evidence supporting the nasal route as an alternative to FIX i.v. infusion for the treatment of HB.
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Hemofilia A , Hemofilia B , Camundongos , Animais , Fator IX/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Camundongos Knockout , Hemofilia A/tratamento farmacológico , Proteínas Recombinantes/uso terapêuticoRESUMO
Vaccine technology is still facing challenges regarding some infectious diseases, which can be addressed by innovative drug delivery systems. In particular, nanoparticle-based vaccines combined with new types of adjuvants are actively explored as a platform for improving the efficacy and durability of immune protection. Here, biodegradable nanoparticles carrying an antigenic model of HIV were formulated with two combinations of poloxamers, 188/407, presenting or not presenting gelling properties, respectively. The study aimed to determine the influence of poloxamers (as a thermosensitive hydrogel or a liquid solution) on the adaptive immune response in mice. The results showed that poloxamer-based formulations were physically stable and did not induce any toxicity using a mouse dendritic cell line. Then, whole-body biodistribution studies using a fluorescent formulation highlighted that the presence of poloxamers influenced positively the dissemination profile by dragging nanoparticles through the lymphatic system until the draining and distant lymph nodes. The strong induction of specific IgG and germinal centers in distant lymph nodes in presence of poloxamers suggested that such adjuvants are promising components in vaccine development.
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Poloxâmero , Vacinas , Poloxâmero/metabolismo , Adjuvantes de Vacinas , Distribuição Tecidual , Antígenos , Linfonodos/metabolismo , Adjuvantes Imunológicos/química , Células DendríticasRESUMO
mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.
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Among mucosal administration routes for vaccines, the sublingual route has been proven capable of inducing a potent systemic and mucosal immune response. However, the absence of a simple and compliant delivery system and the lack of robust mucosal adjuvants impede the development of sublingual vaccines. Here, we describe a mucoadhesive patch made of a layer-by-layer assembly of polysaccharides, chitosan, and hyaluronic acid. The mucoadhesive patch was covered by adjuvanted nanoparticles carrying viral proteins. We showed that the nanoparticles effectively cross the outer layers of the sublingual mucosa to reach the epithelium. Furthermore, the encapsulated adjuvants, 3M-052 and mifamurtide, targeting toll-like receptor (TLR) 7/8 and nucleotide-binding oligomerization domain-2 (NOD2), respectively, remain fully active after encapsulation into nanoparticles and exhibit a cytokine/chemokine signature similar to the mucosal gold-standard adjuvant, the cholera toxin. However, the particulate adjuvants induced more moderate levels of proinflammatory interleukin (IL)-6 and keratinocyte chemoattractant (KC), suggesting a controlled activation of the innate immune response.
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Adjuvantes Imunológicos , Imunidade nas Mucosas , Animais , Camundongos , Administração Sublingual , Adjuvantes Imunológicos/farmacologia , Vacinas de Subunidades Antigênicas , Adjuvantes Farmacêuticos , Mucosa , Camundongos Endogâmicos BALB CRESUMO
Polymeric and/or lipid platforms are promising tools for nucleic acid delivery into cells. We previously reported a lipid-polymer nanocarrier, named LipoParticles, consisting of polylactic acid nanoparticles surrounded by cationic lipids, and allowing the addition of mRNA and cationic LAH4-1 peptide at their surface. Although this mRNA platform has shown promising results in vitro in terms of mRNA delivery and translation, the bulk method used to prepare LipoParticles relies on a multistep and time-consuming procedure. Here, we developed an automated process using a microfluidic system to prepare LipoParticles, and we compared it to the bulk method in terms of morphology, physicochemical properties, and ability to vectorize and deliver mRNA in vitro. LipoParticles prepared by microfluidic presented a smaller size and more regular spherical shape than bulk method ones. In addition, we showed that the total lipid content in LipoParticles was dependent on the method of preparation, influencing their ability to complex mRNA. LipoParticles decorated with two mRNA/LAHA-L1 ratios (1/20, 1/5) could efficiently transfect mouse DC2.4 cells except for the automated 1/5 assay. Moreover, the 1/5 mRNA/LAHA-L1 ratio drastically reduced cell toxicity observed in 1/20 ratio assays. Altogether, this study showed that homogeneous LipoParticles can be produced by microfluidics, which represents a promising platform to transport functional mRNA into cells.
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Respiratory viral infections have been a leading cause of morbidity and mortality worldwide. Despite massive advancements in the virology field, no specific treatment exists for most respiratory viral infections. Approved therapies against respiratory viruses rely almost exclusively on synthetic drugs that have potential side effects, restricting their use. This review aims to present natural marine sulfated polysaccharides possessing promising antiviral activity against respiratory viruses that could be a safe alternative to synthetic broad-spectrum antiviral drugs. The antiviral properties of marine sulfated polysaccharides are presented according to their mechanism of action on different types and strains of respiratory viruses, and the potential limits of their use are discussed.
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The sublingual mucosa is an appealing route for drug administration. However, in the context of increased use of therapeutic proteins, development of protein delivery systems that will protect the protein bioactivity is needed. As proteins are fragile and complex molecules, current sublingual formulations of proteins are in liquid dosage. Yet, protein dilution and short residence time at the sublingual mucosa are the main barriers for the control of the dose that is delivered. In this work, a simple delivery scaffold based on the assembly of two polysaccharides, chitosan and hyaluronic acid, is presented. The natural polymers were assembled by the Layer-by-Layer methodology to produce a mucoadhesive and oro-dispersible freestanding membrane, shown to be innocuous for epithelial human cells. The functionalization of the membrane with proteins led to the production of a bioactive patch with efficient loading and release of proteins, and suitable mechanical properties for manipulation. Sublingual administration of the patch in mouse evidenced the absence of inflammation and an extended time of contact between the model protein ovalbumin and the mucosa compared to liquid formulation. The delivery of fluorescent ovalbumin in mouse sublingual mucosa demonstrated the penetration of the protein in the epithelium 10 min after the patch administration. Moreover, a migration assay with a chemokine incorporated into the patch showed no decrease in bioactivity of the loaded protein after enzymatic release. This study therefore provides a promising strategy to develop a sublingual protein delivery system. STATEMENT OF SIGNIFICANCE: Although the oral route is largely used for drug delivery, it has limitations for the delivery of proteins that can be degraded by pH or gastric enzymes. The sublingual route therefore appears as an interesting approach for protein administration. In this work, a simple delivery scaffold is presented based on the assembly of two polysaccharides by the Layer-by-Layer methodology to produce a mucoadhesive patch. The produced patch allowed efficient loading and release of proteins, as well as protection of their bioactivity. An extended time of contact between the protein and the mucosa compared to liquid formulation was highlighted in mouse model. This study provides a promising strategy to develop a sublingual protein delivery system.
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Mucosa Bucal , Polímeros , Administração Sublingual , Animais , Sistemas de Liberação de Medicamentos , Camundongos , ProteínasRESUMO
Messenger RNA-based vaccines have the potential to trigger robust cytotoxic immune responses, which are essential for fighting cancer and infectious diseases like HIV. Dendritic Cells (DCs) are choice targets for mRNA-based vaccine strategies, as they link innate and adaptive immune responses and are major regulators of cytotoxic and humoral adaptive responses. However, efficient delivery of antigen-coding mRNAs into DC cytosol has been highly challenging. In this study, we developed an alternative to lipid-based mRNA delivery systems, using poly(lactic acid) nanoparticles (PLA-NPs) and cationic cell-penetrating peptides as mRNA condensing agent. The formulations are assembled in two steps: (1) formation of a polyplex between mRNAs and amphipathic cationic peptides (RALA, LAH4 or LAH4-L1), and (2) adsorption of polyplexes onto PLA-NPs. LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes are taken up by DCs via phagocytosis and clathrin-dependent endocytosis, and induce strong protein expression in DCs in vitro. They modulate DC innate immune response by activating both endosome and cytosolic Pattern Recognition Receptors (PRRs), and induce markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature. Thus, LAH4-L1/mRNA and PLA-NP/LAH4-L1/mRNA represent a promising platform for ex vivo treatment and mRNA vaccine development.
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Peptídeos Penetradores de Células/química , Células Dendríticas/metabolismo , Nanopartículas/química , Poliésteres/química , Animais , Endocitose/fisiologia , Humanos , Fagocitose/fisiologia , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
Reconstructing functional skin after a wound remains a challenge due to the complexity of healing. In this regard, biocompatible nanoparticles (NPs) carrying and releasing bioactive drugs in a controlled and sustained manner may significantly improve the efficacy of wound therapies compared with current treatments. Topical administration of drug-loaded NPs allows optimal delivery to the dermis and improves product efficacy. Furthermore, associating NPs with scaffolds represents a new concept of 'dressing'. Experimental in vivo, ex vivo, and in vitro models have been developed in preclinical assays to evaluate the beneficial effects of nanoparticulate dressings. Drug-loaded NPs are promising tools for innovative wound healing treatment, especially with regard to their multifunctional properties.
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Bandagens , Portadores de Fármacos , Nanopartículas , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/terapia , Administração Tópica , Animais , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Humanos , Nanopartículas/química , Nanopartículas/uso terapêuticoRESUMO
BACKGROUND: Satellite cells are quiescent resident muscle stem cells that present an important potential to regenerate damaged tissue. However, this potential is diminished once they are removed from their niche environment in vivo, prohibiting the long-term study and genetic investigation of these cells. This study therefore aimed to provide a novel biomaterial platform for the in-vitro culture of human satellite cells that maintains their stem-like quiescent state, an important step for cell therapeutic studies. METHODS: Human muscle satellite cells were isolated from two donors and cultured on soft biopolymeric films of controlled stiffness. Cell adhesive phenotype, maintenance of satellite cell quiescence and capacity for gene manipulation were investigated using FACS, western blotting, fluorescence microscopy and electron microscopy. RESULTS: About 85% of satellite cells cultured in vitro on soft biopolymer films for 3 days maintained expression of the quiescence marker Pax7, as compared with 60% on stiffer films and 50% on tissue culture plastic. The soft biopolymeric films allowed satellite cell culture for up to 6 days without renewing the media. These cells retained their stem-like properties, as evidenced by the expression of stem cell markers and reduced expression of differentiated markers. In addition, 95% of cells grown on these soft biopolymeric films were in the G0/G1 stage of the cell cycle, as opposed to those grown on plastic that became activated and began to proliferate and differentiate. CONCLUSIONS: Our study identifies a new biomaterial made of a biopolymer thin film for the maintenance of the quiescence state of muscle satellite cells. These cells could be activated at any point simply by replating them onto a plastic culture dish. Furthermore, these cells could be genetically manipulated by viral transduction, showing that this biomaterial may be further used for therapeutic strategies.
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Células-Tronco Adultas/citologia , Proliferação de Células , Cultura Primária de Células/métodos , Células Satélites de Músculo Esquelético/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Biopolímeros/farmacologia , Diferenciação Celular , Células Cultivadas , Meios de Cultura/química , Humanos , Masculino , Pessoa de Meia-Idade , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/fisiologia , Alicerces Teciduais/químicaRESUMO
Vaccines have successfully eradicated a large number of diseases. However, some infectious diseases (such as HIV, Chlamydia trachomatis or Bacillus anthracis) keep spreading since there is no vaccine to prevent them. One way to overcome this issue is the development of new adjuvant formulations which are able to induce the appropriate immune response without sacrificing safety. Lymph nodes are the site of lymphocyte priming by antigen-presenting cells and subsequent adaptive immune response, and are a promising target for vaccine formulations. In this review, we describe the properties of different polymer-based (e.g., poly lactic-co-glycolic acid, poly lactic acid ) particulate adjuvants as innovative systems, capable of co-delivering immunopotentiators and antigens. We point out how these nanoparticles enhance the delivery of antigens, and how their physicochemical properties modify their uptake by antigen-presenting cells and their migration into lymph nodes. We describe why polymeric nanoparticles increase the persistence into lymph nodes and promote a mature immune response. We also emphasize how nanodelivery directs the response to a specific antigen and allows the induction of a cytotoxic immune response, essential for the fight against intracellular pathogens or cancer. Finally, we highlight the interest of the association between polymer-based vaccines and immunopotentiators, which can potentiate the effect of the molecule by directing it to the appropriate compartment and reducing its toxicity.
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Introduced in the '90s by Prof. Moehwald, Lvov, and Decher, the layer-by-layer (LbL) assembly of polyelectrolytes has become a popular technique to engineer various types of objects such as films, capsules and free standing membranes, with an unprecedented control at the nanometer and micrometer scales. The LbL technique allows to engineer biofunctional surface coatings, which may be dedicated to biomedical applications in vivo but also to fundamental studies and diagnosis in vitro. Initially mostly developed as 2D coatings and hollow capsules, the range of complex objects created by the LbL technique has greatly expanded in the past 10 years. In this Review, the aim is to highlight the recent progress in the field of LbL films for biomedical applications and to discuss the various ways to spatially and temporally control the biochemical and mechanical properties of multilayers. In particular, three major developments of LbL films are discussed: 1) the new methods and templates to engineer LbL films and control cellular processes from adhesion to differentiation, 2) the major ways to achieve temporal control by chemical, biological and physical triggers and, 3) the combinations of LbL technique, cells and scaffolds for repairing 3D tissues, including cardio-vascular devices, bone implants and neuro-prosthetic devices.
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Materiais Biocompatíveis , Eletrólitos , Microscopia de Fluorescência , Propriedades de SuperfícieRESUMO
Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined â¼50 µm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1 month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.
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Membranas Artificiais , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos/citologia , Osseointegração/efeitos dos fármacos , Polissacarídeos/farmacologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Mioblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Imagem com Lapso de Tempo , Fator de Crescimento Transformador beta/farmacologia , Microtomografia por Raio-XRESUMO
In the cellular microenvironment, growth factor gradients are crucial in dictating cell fate. Towards developing materials that capture the native microenvironment we engineered biomimetic films that present gradients of matrix-bound bone morphogenetic proteins (BMP-2 and BMP-7). To this end layer-by-layer films composed of poly(L-lysine) and hyaluronan were combined in a simple microfluidic device enabling spatially controlled growth factor diffusion along the film. Linear long-range gradients of both BMPs induced the trans-differentiation of C2C12 myoblasts towards the osteogenic lineage in a dose dependent manner with a different signature for each BMP. The osteogenic marker alkaline phosphatase (ALP) increased in a linear manner for BMP-7 and non-linearly for BMP-2. Moreover, an increased expression of the myogenic marker troponin T was observed with decreasing matrix-bound BMP concentration, providing a substrate that it is both osteo- and myo-inductive. Lastly, dual parallel matrix-bound gradients of BMP-2 and -7 revealed a complete saturation of the ALP signal. This suggested an additive or synergistic effect of the two BMPs. This simple technology allows for determining quickly and efficiently the optimal concentration of matrix-bound growth factors, as well as for investigating the presentation of multiple growth factors in their solid-phase and in a spatially controlled manner.
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Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Membranas Artificiais , Mioblastos/efeitos dos fármacos , Polímeros/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ácido Hialurônico/química , Camundongos , Técnicas Analíticas Microfluídicas , Mioblastos/citologia , Mioblastos/metabolismo , Polilisina/química , Polímeros/farmacologiaRESUMO
Free-standing films have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce a thick membrane and to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any postprocessing step. The dry thickness was varied over a large range from 4 to 35 µm. A 2-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free-standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side.
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Alginatos/química , Quitosana/química , Eletrólitos/química , Membranas Artificiais , Mioblastos/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Dextranos , Corantes Fluorescentes , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio , Camundongos , Peso Molecular , Mioblastos/citologia , Permeabilidade , Polipropilenos , Poliestirenos , Alicerces Teciduais , MolhabilidadeRESUMO
Photo-cross-linkable polyelectrolyte films, whose nanomechanical properties can be varied under UV light illumination, were prepared from poly(l-lysine) (PLL) and a hyaluronan derivative modified with photoreactive vinylbenzyl groups (HAVB). The adhesion and the growth of two model bacteria, namely Escherichia coli and Lactococcus lactis , were studied on non-cross-linked and cross-linked films to investigate how the film stiffness influences the bacterial behavior. While the Gram positive L. lactis was shown to grow slowly on both films, independently of their rigidity, the Gram negative E. coli exhibited a more rapid growth on non-cross-linked softer films compared to the stiffer ones. Experiments performed on photopatterned films showing both soft and stiff regions, confirmed a faster development of E. coli colonies on softer regions. Interestingly, this behavior is opposite to the one reported before for mammalian cells. Therefore, the photo-cross-linked (PLL/HAVB) films are interesting coatings for tissue engineering since they promote the growth of mammalian cells while limiting the bacterial colonization.
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Aderência Bacteriana , Escherichia coli/crescimento & desenvolvimento , Lactococcus lactis/crescimento & desenvolvimento , Polímeros/química , Reagentes de Ligações Cruzadas , Eletrólitos/química , Escherichia coli/metabolismo , Ácido Hialurônico/química , Lactococcus lactis/metabolismo , Polilisina/química , Polímeros/metabolismoRESUMO
In vivo, cells are sensitive to the stiffness of their micro-environment and especially to the spatial organization of the stiffness. In vitro studies of this phenomenon can help to better understand the mechanisms of the cell response to spatial variations of the matrix stiffness. In this work, we design polelyelectrolyte multilayer films made of poly(L-lysine) and a photo-reactive hyaluronan derivative. These films can be photo-crosslinked through a photomask to create spatial patterns of rigidity. Quartz substrates incorporating a chromium mask are prepared to expose selectively the film to UV light (in a physiological buffer), without any direct contact between the photomask and the soft film. We show that these micropatterns are chemically homogeneous and flat, without any preferential adsorption of adhesive proteins. Three groups of pattern geometries differing by their shape (circles or lines), size (form 2 to 100 µm) or interspacing distance between the motifs are used to study the adhesion and spatial organization of myoblast cells. On large circular micropatterns, the cells form large assemblies that are confined to the stiffest parts. Conversely, when the size of the rigidity patterns is subcellular, the cells respond by forming protrusions. Finally, on linear micropatterns of rigidity, myoblasts align and their nuclei drastically elongate in specific conditions. These results pave the way for the study of the different steps of myoblast fusion in response to matrix rigidity in well-defined geometrical conditions.