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Bioorthogonal activation of pro-dyes and prodrugs using transition-metal catalysts (TMCs) provides a promising strategy for imaging and therapeutic applications. TMCs can be loaded into polymeric nanoparticles through hydrophobic encapsulation to generate polymeric nanocatalysts with enhanced solubility and stability. However, biomedical use of these nanostructures faces challenges due to unwanted tissue accumulation of nonbiodegradable nanomaterials and cytotoxicity of heavy-metal catalysts. We report here the creation of fully biodegradable nanocatalysts based on an engineered FDA-approved polymer and the naturally existing catalyst hemin. Stable nanocatalysts were generated through kinetic stabilization using flash nanoprecipitation. The therapeutic potential of these nanocatalysts was demonstrated through effective treatment of bacterial biofilms through the bioorthogonal activation of a pro-antibiotic.
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Nanopartículas , Nanoestructuras , Elementos de Transición , Polímeros/química , Nanopartículas/química , Elementos de Transición/química , Antibacterianos/farmacologíaRESUMEN
Applications involving ultrasound treatment as a therapeutic strategy have gained interest due to its enhanced tissue penetration, broad availability, and minimal invasiveness. Recently, ultrasound treatment has been utilized for applications such as controlled drug delivery, enhanced drug penetration, sonodynamic therapy for generating ROS species, and targeted tissue ablation. However, our ability to study and explore applications is limited by the lack of in vitro models that enable efficient and representative screening of ultrasound-based therapeutic strategies. There is a need for cell culture approaches that mimic the mechanical environment of native tissues, which can prevent uncontrolled cell lysis due to ultrasonic energy. We developed two-dimensional and three-dimensional collagen-based materials for culturing cells in vitro that withstand ultrasound treatment. We hypothesized that the collagen matrix mimics the extracellular matrix and absorb most of the energy from ultrasound treatment - similar to in vivo effects - thereby preventing uncontrolled cell lysis. In this study, we developed a strategy for fabricating both the 2D coatings and 3D hydrogels coatings and tested the viability of the cultured cells post different durations of ultrasound treatment.
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High internal phase emulsions (HIPEs) provide a versatile platform for encapsulating large volumes of therapeutics that are immiscible in water. A stable scaffold is obtained by polymerizing the external phase, resulting in polyHIPEs. However, fabrication of polyHIPEs usually requires using a considerable quantity of surfactants along with nonbiocompatible components, which hinders their biological applications, e.g., drug-eluting devices. We describe here a straightforward method for generating porous biomaterials by using proteins as both the emulsifier and the building blocks for the fabrication of polyHIPEs. We demonstrate the versatility of this method by using different essential oils as the internal phase. After the gelation of protein building blocks is triggered by the addition of reducing agents, a stable protein hydrogel containing essential oils can be formed. These oils can be either extracted to obtain protein-based porous scaffolds or slowly released for antimicrobial applications.
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Antiinfecciosos , Aceites Volátiles , Antiinfecciosos/farmacología , Materiales Biocompatibles , Emulsiones , Hidrogeles , Aceites Volátiles/farmacología , Porosidad , Sustancias Reductoras , Tensoactivos , AguaRESUMEN
Bacterial biofilms are a major healthcare concern resulting in refractory conditions such as chronic wounds, implant infections and failure, and multidrug-resistant infections. Aggressive and invasive strategies are employed to cure biofilm infections but are prone to long and expensive treatments, adverse side-effects, and low patient compliance. Recent strategies such as ultrasound-based therapies and antimicrobial nanomaterials have shown some promise in the effective eradication of biofilms. However, maximizing therapeutic effect while minimizing healthy tissue damage is a key challenge that needs to be addressed. Here a combination treatment involving ultrasound and antimicrobial polymeric nanoparticles (PNPs) that synergistically eradicate bacterial biofilms is reported. Ultrasound treatment rapidly disrupts biofilms and increases penetration of antimicrobial PNPs thereby enhancing their antimicrobial activity. This results in superior biofilm toxicity, while allowing for a two- to sixfold reduction in both the concentration of PNPs as well as the duration of ultrasound. Furthermore, that this reduction minimizes cytotoxicity toward fibroblast cells, while resulting in a 100- to 1000-fold reduction in bacterial concentration, is demonstrated.
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Antiinfecciosos , Nanopartículas , Humanos , Biopelículas , Antibacterianos/farmacología , Bacterias , Polímeros/farmacología , Antiinfecciosos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Current strategies for the delivery of proteins into cells face general challenges of endosomal entrapment and concomitant degradation of protein cargo. Efficient delivery directly to the cytosol overcomes this obstacle: we report here the use of biotin-streptavidin tethering to provide a modular approach to the generation of nanovectors capable of a cytosolic delivery of biotinylated proteins. This strategy uses streptavidin to organize biotinylated protein and biotinylated oligo(glutamate) peptide into modular complexes that are then electrostatically self-assembled with a cationic guanidinium-functionalized polymer. The resulting polymer-protein nanocomposites demonstrate efficient cytosolic delivery of six biotinylated protein cargos of varying size, charge, and quaternary structure. Retention of protein function was established through efficient cell killing via delivery of the chemotherapeutic enzyme granzyme A. This platform represents a versatile and modular approach to intracellular delivery through the noncovalent tethering of multiple components into a single delivery vector.
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Biotina , Nanocompuestos , Estreptavidina/química , Biotina/química , Citosol/metabolismo , Proteínas/química , Polímeros/químicaRESUMEN
PURPOSE: Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery. METHODS: Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFPE15) was assessed through diffuse cytosolic and nuclear fluorescence, and cell killing with chemotherapeutic enzyme Granzyme A (GrAE10). Efficiency was quantified between freshly prepared and lyophilized samples. RESULTS: Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFPE15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrAE10 elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability. CONCLUSION: Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrAE10. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
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Polímeros , Proteínas , Citosol/metabolismo , Endosomas/metabolismo , Liofilización , Polímeros/química , Proteínas/químicaRESUMEN
Bioorthogonal transformations are chemical reactions that use pathways which biological processes do not access. Bioorthogonal chemistry provides new approaches for imaging and therapeutic strategies, as well as tools for fundamental biology. Bioorthogonal catalysis enables the development of bioorthogonal "factories" for on-demand and in situ generation of drugs and imaging tools. Transition metal catalysts (TMCs) are widely employed as bioorthogonal catalysts due to their high efficiency and versatility. The direct application of TMCs in living systems is challenging, however, due to their limited solubility, instability in biological media and toxicity. Incorporation of TMCs into nanomaterial scaffolds can be used to enhance aqueous solubility, improve long-term stability in biological environment and minimize cytotoxicity. These nanomaterial platforms can be engineered for biomedical applications, increasing cellular uptake, directing biodistribution, and enabling active targeting. This review summarizes strategies for incorporating TMCs into nanomaterial scaffolds, demonstrating the potential and challenges of moving bioorthogonal nanocatalysts and nanozymes toward the clinic.
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Nanoestructuras , Elementos de Transición , Catálisis , Nanoestructuras/toxicidad , Distribución TisularRESUMEN
Nosocomial infections, caused by bacterial contamination of medical devices and implants, are a serious healthcare concern. We demonstrate here, the use of fluorous-cured protein nanofilm coatings for generating antimicrobial surfaces. In this approach, bacteria-repelling films are created by heat-curing proteins in fluorous media. These films are then loaded with antibiotics, with release controlled via electrostatic interactions between therapeutic and protein film building blocks to provide bactericidal surfaces. This film fabrication process is additive-free, biocompatible, biodegradable, and can be used to provide antimicrobial coatings for both three-dimensional (2D) and 3D objects for use in indwelling devices.
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Antibacterianos/farmacología , Incrustaciones Biológicas/prevención & control , Materiales Biocompatibles Revestidos/química , Preparaciones de Acción Retardada/química , Animales , Antibacterianos/química , Bovinos , Colistina/química , Colistina/farmacología , Liberación de Fármacos , Fluoresceína/química , Colorantes Fluorescentes/química , Fluorocarburos/química , Prótesis e Implantes , Pseudomonas aeruginosa/efectos de los fármacos , Rodamina 123/química , Albúmina Sérica Bovina/químicaRESUMEN
Bioorthogonal transformation of imaging and therapeutic substrates using transition metal catalysts (TMCs) provides a toolkit with diverse applications in biomedicine. Controlled localization of bioorthogonal catalysis is key for enhancing their therapeutic efficacy by minimizing off-target effects. Red blood cells (RBCs) are highly biocompatible and are susceptible to hemolysis by bacterial toxins, providing them with intrinsic targeting to bacterial infections. A hitchhiking strategy using RBCs is reported, that activates bioorthogonal catalysis at infection sites. A library of nanoparticles embedded with TMCs (nanozymes) featuring diverse functional groups with different binding ability to RBCs is generated. These engineered nanozymes bind to RBCs and subsequently release upon hemolysis by bacterial toxins, resulting in selective accumulation at the site of bacterial infections. The antimicrobial action is specific: catalytic activation of pro-antibiotics eradicated pathogenic biofilms without harming non-virulent bacterial species.
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Infecciones Bacterianas , Nanopartículas , Elementos de Transición , Infecciones Bacterianas/tratamiento farmacológico , Catálisis , Eritrocitos , HumanosRESUMEN
Titanium is widely utilized for manufacturing medical implants due to its inherent mechanical strength and biocompatibility. Recent studies have focused on developing coatings to impart unique properties to Ti implants, such as antimicrobial behavior, enhanced cell adhesion, and osteointegration. Ca- and Si-based ceramic (CS) coatings can enhance bone integration through the release of Ca and Si ions. However, high degradation rates of CS ceramics create a basic environment that reduces cell viability. Polymeric or protein-based coatings may be employed to modulate CS degradation. However, it is challenging to ensure coating stability over extended periods of time without compromising biocompatibility. In this study, we employed a fluorous-cured collagen shell as a drug-loadable scaffold around CS nanorod coatings on Ti implants. Fluorous-cured collagen coatings have enhanced mechanical and enzymatic stability and are able to regulate the release of Ca and Si ions. Furthermore, the collagen scaffold was loaded with antimicrobial peptides to impart antimicrobial activity while promoting cell adhesion. These multifunctional collagen coatings simultaneously regulate the degradation of CS ceramics and enhance antimicrobial activity, while maintaining biocompatibility.
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Antibacterianos/farmacología , Nanotubos/química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Silicatos/química , Titanio/química , Cicatrización de Heridas/efectos de los fármacos , Antibacterianos/química , Antibacterianos/toxicidad , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/toxicidad , Colágeno/química , Colágeno/toxicidad , Humanos , Pruebas de Sensibilidad Microbiana , Nanotubos/toxicidad , Osteoblastos/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Silicatos/toxicidad , Staphylococcus aureus/efectos de los fármacos , Titanio/toxicidad , HumectabilidadRESUMEN
Proteins are naturally occurring functional building blocks that are useful for the fabrication of materials. Naturally-occurring proteins are biodegradable and most are biocompatible and non-toxic, making them attractive for the fabrication of biomaterials. Moreover, the fabrication of protein-based materials can be conducted in a green and sustainable manner due to their high aqueous solubility. Consequently, the applicability of protein-based materials is limited by their aqueous and mechanical instability. This review summarizes strategies for the stabilization of protein films, highlighting their salient features and potential limitations. Applications of protein films ranging from food packaging materials, tissue engineering scaffolds, antimicrobial coatings etc. are also discussed. Finally, the need for robust and efficient fabrication strategies for translation to commercial applications as well as potential applications of protein films in the field of sensing, diagnostics and controlled release systems are discussed.
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Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.
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Citosol/metabolismo , Polímeros/química , Ingeniería de Proteínas , Proteínas/metabolismo , Animales , Línea Celular , Citosol/química , Humanos , Ratones , Estructura Molecular , Polímeros/metabolismo , Proteínas/químicaRESUMEN
Hydrodynamic approaches are important for biomedical diagnostics, chemical analysis, and a broad range of industrial applications. Size-based separation and sorting is an important tool for these applications. We report the integration of hypersound technology with patterned protein films to provide efficient sorting of microparticles based on particle charge and size. We employed a hypersonic resonator for the acoustic streaming of the fluidic system to generate microvortices that exert drag forces on the objects on the surface that are dictated by their radius of curvature. We demonstrate a size-based sorting of anionic silica particles using protein patterns and gradients fabricated using attractive cationic and repulsive anionic proteins.
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Acústica , Hidrodinámica , Fenómenos Mecánicos , Tamaño de la Partícula , Dióxido de SilicioRESUMEN
Bioorthogonal catalysis provides a promising strategy for imaging and therapeutic applications, providing controlled in situ activation of pro-dyes and prodrugs. In this work, the use of a polymeric scaffold to encapsulate transition metal catalysts (TMCs), generating bioorthogonal "polyzymes," is presented. These polyzymes enhance the stability of TMCs, protecting the catalytic centers from deactivation in biological media. The therapeutic potential of these polyzymes is demonstrated by the transformation of a nontoxic prodrug to an anticancer drug (mitoxantrone), leading to the cancer cell death in vitro.
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Antineoplásicos , Profármacos , Elementos de Transición , Catálisis , PolímerosRESUMEN
We demonstrate here the protection of biorthogonal transition metal catalysts (TMCs) in biological environments by using self-assembled monolayers on gold nanoparticles (AuNPs). Encapsulation of TMCs in this hydrophobic environment preserves catalytic activity in presence of pH conditions and complex biological media that would deactivate free catalyst. Significantly, the protection affords by these nanozymes extends to isolation of the catalyst active site, as demonstrated by the independence of rate over a wide pH range, in strong contrast to the behavior of the free catalyst.
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Complejos de Coordinación/química , Metales Pesados/química , Nanoestructuras/química , Catálisis , Complejos de Coordinación/síntesis química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Metales Pesados/aislamiento & purificación , Estructura Molecular , Tamaño de la PartículaRESUMEN
Industrial use of nanomaterials is rapidly increasing, making the effects of these materials on the environment and human health of critical concern. Standard nanotoxicity evaluation methods rely on detecting cell death or major dysfunction and will miss early signs of toxicity. In this work, the use of rapid and sensitive nanosensors that can efficiently detect subtle phenotypic changes on the cell surface following nanomaterial exposure is reported. Importantly, the method reveals significant phenotypic changes at dosages where other conventional methods show normal cellular activity. This approach holds promise in toxicological and pharmacological evaluations to ensure safer and better use of nanomaterials.
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Técnicas Biosensibles , Células , Nanopartículas , Toxicología , Técnicas Biosensibles/normas , Células/efectos de los fármacos , Monitoreo del Ambiente , Humanos , Nanopartículas/toxicidad , Toxicología/instrumentaciónRESUMEN
Bioorthogonal activation of prodrugs provides a strategy for on-demand on-site production of therapeutics. Intracellular activation provides a strategy to localize therapeutics, potentially minimizing off-target effects. To this end, nanoparticles embedded with transition metal catalysts (nanozymes) were engineered to generate either "hard" irreversible or "soft" reversible coronas in serum. The hard corona induced nanozyme aggregation, effectively inhibiting nanozyme activity, whereas only modest loss of activity was observed with the nonaggregating soft corona nanozymes. In both cases complete activity was restored by treatment with proteases. Intracellular activity mirrored this reactivation: endogenous proteases in the endosome provided intracellular activation of both nanozymes. The role of intracellular proteases in nanozyme reactivation was verified through treatment of the cells with protease inhibitors, which prevented reactivation. This study demonstrates the use of intracellular proteolysis as a strategy for localization of therapeutic generation to within cells.
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Corona de Proteínas , Elementos de Transición , Catálisis , Endosomas , ProteolisisRESUMEN
Nanocarrier-mediated protein delivery is a promising strategy for fundamental research and therapeutic applications. However, the efficacy of the current platforms for delivery into cells is limited by endosomal entrapment of delivered protein cargo with concomitantly inefficient access to the cytosol and other organelles, including the nucleus. We report here a robust, versatile polymeric-protein nanocomposite (PPNC) platform capable of efficient (≥90%) delivery of proteins to the cytosol. We synthesized a library of guanidinium-functionalized poly(oxanorborneneimide) (PONI) homopolymers with varying molecular weights to stabilize and deliver engineered proteins featuring terminal oligoglutamate "E-tags". The polymers were screened for cytosolic delivery efficiency using imaging flow cytometry with cytosolic delivery validated using confocal microscopy and activity of the delivered proteins demonstrated through functional assays. These studies indicate that the PPNC platform provides highly effective and tunable cytosolic delivery over a wide range of formulations, making them robust agents for therapeutic protein delivery.
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Portadores de Fármacos/metabolismo , Integrasas/metabolismo , Proteínas Luminiscentes/metabolismo , Ácido Poliglutámico/metabolismo , Polímeros/metabolismo , Portadores de Fármacos/síntesis química , Guanidinas/síntesis química , Guanidinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Imidas/síntesis química , Imidas/metabolismo , Nanocompuestos/química , Polímeros/síntesis química , Ingeniería de Proteínas , Proteína Fluorescente RojaRESUMEN
Collagen I (Col-I) is widely used in the fabrication of biomaterials due to its biocompatibility; however, Col-I based biomaterials are susceptible to mechanical failure during handling, which limits their applicability to biomaterials. Chemical or physical treatment can improve the mechanical properties of collagen; however, these processes can create issues of cytotoxicity or denaturation. We report here an alternative strategy to improve the stability and mechanical properties of Col-I while preserving its native structure, through thermal treatment in fluorous media. Thermal treatment of Col-I in fluorous solvent generates compact, stable films with significantly increased mechanical strength. Furthermore, the use of fluorous media significantly reduces the extent of swelling and the rate of proteolytic degradation, but it preserves the high cell biocompatibility.