RESUMEN
The porous coordination cage PCC-1 represents a new platform potentially useful for the cellular delivery of drugs with poor cell permeability and solubility. PCC-1 is a metal-organic polyhedron constructed from zinc metal ions and organic ligands through coordination bonds. PCC-1 possesses an internal cavity that is suitable for drug encapsulation. To better understand the biocompatibility of PCC-1 with human cells, the cell entry mechanism, disassembly, and toxicity of the nanocage were investigated. PCC-1 localizes in the nuclei and cytoplasm within minutes upon incubation with cells, independent of endocytosis and cargo, suggesting direct plasma membrane translocation of the nanocage carrying its guest in its internal cavity. Furthermore, the rates of cell entry correlate to extracellular concentrations, indicating that PCC-1 is likely diffusing passively through the membrane despite its relatively large size. Once inside cells, PCC-1 disintegrates into zinc metal ions and ligands over a period of several hours, each component being cleared from cells within 1 day. PCC-1 is relatively safe for cells at low micromolar concentrations but becomes inhibitory to cell proliferation and toxic above a concentration or incubation time threshold. However, cells surviving these conditions can return to homeostasis 3-5 days after exposure. Overall, these findings demonstrate that PCC-1 enters live cells by crossing biological membranes spontaneously. This should prove useful to deliver drugs that lack this capacity on their own, provided that the dosage and exposure time are controlled to avoid toxicity.
Asunto(s)
Sistemas de Liberación de Medicamentos , Internalización del Virus , Humanos , Membrana Celular/metabolismo , Metales/metabolismo , Compuestos Orgánicos/metabolismo , Zinc/metabolismo , Iones/metabolismoRESUMEN
Delivery tools, including cell-penetrating peptides (CPPs), are often inefficient due to a combination of poor endocytosis and endosomal escape. Aspects that impact the delivery of CPPs are typically characterized using tissue culture models. One problem of using cell culture is that cell culture protocols have the potential to contribute to endosomal uptake and endosomal release of CPPs. Hence, a systematic study to identify which aspects of cell culturing techniques impact the endocytic uptake of a typical CPP, the TMR-TAT peptide (peptide sequence derived from HIV1-TAT with the N-terminus labeled with tetramethylrhodamine), was conducted. Aspects of cell culturing protocols previously found to generally modulate endocytosis, such as cell density, washing steps, and cell aging, did not affect TMR-TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. To establish a range of endocytosis achievable by different cell culture protocols, TMR-TAT uptake was compared among protocols. These protocols led to changes in uptake of more than 13-fold, indicating that differences in cell culturing techniques have a cumulative effect on CPP uptake. Taken together this study highlights how different protocols can influence the amount of endocytic uptake of TMR-TAT. Additionally, parameters that can be exploited to improve CPP accumulation in endosomes were identified. The protocols identified herein have the potential to be paired with other delivery enhancing strategies to improve overall delivery efficiency of CPPs. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00591-1.
RESUMEN
Cell-penetrating peptides (CPPs) are promising tools for the intracellular delivery of various biological payloads. However, the impact of payload conjugation on the cell-penetrating activity of CPPs is poorly understood. This study focused on dfTAT, a modified version of the HIV-TAT peptide with enhanced endosomal escape activity, to explore how different payloads affect its cell-penetrating activity. We systematically examined dfTAT conjugated with the SnoopTag/SnoopCatcher pair and found that while smaller payloads such as short peptides do not significantly impair dfTAT's cell delivery activity, larger payloads markedly reduce both its endocytic uptake and endosomal escape efficiency. Our results highlight the role of the payload size and bulk in limiting CPP-mediated delivery. While further research is needed to understand the molecular underpinnings of these effects, our findings pave the way for developing more effective CPP-based delivery systems.
Asunto(s)
Péptidos de Penetración Celular , Endosomas , Endosomas/metabolismo , Péptidos de Penetración Celular/química , Transporte BiológicoRESUMEN
Amyloid formation is a hallmark of many medical diseases including diabetes type 2, Alzheimer's and Parkinson diseases. Under these pathological conditions, misfolded proteins self-assemble forming oligomers and fibrils, structurally heterogeneous aggregates that exhibit a large variety of shapes and forms. A growing body of evidence points to drastic changes in the lipid profile in organs affected by amyloidogenic diseases. In this study, we investigated the extent to which individual phospho- and sphingolipids, as well as their mixtures can impact insulin aggregation. Our results show that lipids and their mixtures uniquely alter rates of insulin aggregation simultaneously changing the secondary structure of protein aggregates that are grown in their presence. These structurally different protein-lipid aggregates impact cell viability to different extent while using distinct mechanisms of toxicity. These findings suggest that irreversible changes in lipid profiles of organs may trigger formation of toxic protein species that in turn are responsible for the onset and progression of amyloidogenic diseases.
Asunto(s)
Insulina , Enfermedad de Parkinson , Humanos , Amiloide/química , Amiloide/metabolismo , Estructura Secundaria de Proteína , LípidosRESUMEN
We report on the successful delivery of the Cre recombinase enzyme in the neural cells of mice in vivo by simple coinjection with peptides derived from HIV-TAT. Cre delivery activates the expression of a reporter gene in both neurons and astrocytes of the cortex without tissue damage and with a transduction efficiency that parallels or exceeds that of a commonly used adeno-associated virus. Our data indicate that the delivery peptides mediate efficient endosomal leakage and cytosolic escape in cells that have endocytosed Cre. The peptides, therefore, act in trans and do not require conjugation to the payload, greatly simplifying sample preparation. Moreover, the delivery peptides are exclusively composed of natural amino acids and are consequently readily degradable and processed by cells. We envision that this approach will be beneficial to applications that require the transient introduction of proteins into cells in vivo.
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Dependovirus , Péptidos , Aminoácidos , Animales , Sistema Nervioso Central , Dependovirus/genética , Genes Reporteros , Ratones , Péptidos/químicaRESUMEN
To deliver useful biological payloads into the cytosolic space of cells, cell-penetrating peptides have to cross biological membranes. The molecular features that control or enhance this activity remain unclear. Herein, a dimeric template of the arginine-rich HIV TAT CPP was used to establish the effect of incorporating groups and residues of various chemical structures and properties. A positive correlation is established between the relative hydrophobicity of these additional moieties and the ability of the CPP conjugates to deliver a peptidic probe into live cells. CPP conjugates with low hydrophobicity lead to no detectable delivery activity, while CPPs containing groups of increasing hydrophobicity achieve intracellular delivery at low micromolar concentrations. Notably, the chemical structures of the hydrophobic groups do not appear to play a role in overall cell penetration activity. The cell penetration activity detected is consistent with endosomal escape. Leakage assays with lipid bilayer of endosomal membrane composition also establish a positive correlation between hydrophobicity and membrane permeation. Overall, these results indicate that the presence of a relatively hydrophobic moiety, regardless of structure, is required in a CPP structure to enhance its cell penetration. It also indicates that simple modifications, including fluorophores used for cell imaging or small payloads, modulate the activity of CPPs and that a given CPP-conjugate may be unique in its membrane permeation properties.
Asunto(s)
Péptidos de Penetración Celular , Arginina/metabolismo , Péptidos de Penetración Celular/química , Endosomas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismoRESUMEN
This paper reports a smart intracellular nanocarrier for sustainable and controlled drug release in non-invasive neuroregeneration. The nanocarrier is composed by superparamagnetic iron oxide-gold (SPIO-Au) core-shell nanoparticles (NPs) conjugated with porous coordination cages (PCCs) through the thiol-containing molecules as bridges. The negatively charged PCC-2 and positively charged PCC-3 are compared for intracellular targeting. Both types result in intracellular targeting via direct penetration across cellular membranes. However, the pyrene (Py)-PEG-SH bridge enabled functionalization of SPIO-Au NPs with PCC-3 exhibits higher interaction with PC-12 neuron-like cells, compared with the rhodamine B (RhB)-PEG-SH bridge enabled case and the stand-alone SPIO-Au NPs. With neglectable toxicities to PC-12 cells, the proposed SPIO-Au-RhB(Py)-PCC-2(3) nanocarriers exhibit effective drug loading capacity of retinoic acid (RA) at 13.505 µg/mg of RA/NPs within 24 h. A controlled release of RA is achieved by using a low-intensity 525 nm LED light (100% compared to 40% for control group within 96 h).
Asunto(s)
Portadores de Fármacos , Compuestos Férricos , Oro , Nanopartículas , Regeneración Nerviosa/efectos de los fármacos , Tretinoina , Animales , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Compuestos Férricos/química , Compuestos Férricos/farmacocinética , Compuestos Férricos/farmacología , Oro/química , Oro/farmacocinética , Oro/farmacología , Nanopartículas/química , Nanopartículas/uso terapéutico , Células PC12 , Porosidad , Ratas , Tretinoina/química , Tretinoina/farmacocinética , Tretinoina/farmacologíaRESUMEN
Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Here, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of CPP's membrane leakage activity. These results therefore support a model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Endosomas/metabolismo , Lisofosfolípidos/metabolismo , Monoglicéridos/metabolismo , Animales , Línea Celular , Péptidos de Penetración Celular/química , Cricetulus , Endosomas/química , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lisofosfolípidos/química , Monoglicéridos/químicaRESUMEN
Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.
Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Colorantes Fluorescentes , Galectina 3/metabolismo , Galectinas/metabolismo , VIH/química , Humanos , Ratones , Rodaminas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Expressed protein ligation allows for the attachment of a chemically labeled peptide to the N- or C-terminus of a recombinant protein. In this book chapter, the practical considerations involved in using this protein engineering technology are described. In particular, approaches used to design optimal ligation sites are discussed. In addition, several methods used to generate the reactive fragments required for EPL are highlighted in practical details. Finally, strategies that one can implement to achieve efficient ligation reactions are presented.
Asunto(s)
Clonación Molecular/métodos , Ingeniería de Proteínas/métodos , Aminoácidos/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ésteres/química , Expresión Génica , Inteínas , Péptidos/síntesis química , Péptidos/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Sulfhidrilo/químicaRESUMEN
Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Citosol/metabolismo , Endosomas/metabolismo , Sustancias Macromoleculares/metabolismo , Citosol/efectos de los fármacos , Endosomas/efectos de los fármacos , Humanos , Pirazinas/farmacología , Piridinas/farmacologíaRESUMEN
Our work on the complexation of fluoride anions using group 15 Lewis acids has led us to investigate the use of these main group compounds as anion transporters. In this paper, we report on the anion transport properties of tetraarylstibonium and tetraarylbismuthonium cations of the general formula [Ph3PnAr]+ with Pn = Sb or Bi and with Ar = phenyl, naphthyl, anthryl, or pyrenyl. Using EYPC-based large unilamellar vesicles, we show that these main group cations transport hydroxide, fluoride and chloride anions across phospholipid bilayers. A comparison of the properties of [Ph3SbAnt]+ and [Ph3BiAnt]+ (Ant = 9-anthryl) illustrates the favorable role played by the Lewis acidity of the central pnictogen element with respect to the anion transport. Finally, we show that [Ph3SbAnt]+ accelerates the fluoride-induced hemolysis of human red blood cells, an effect that we assign to the transporter-facilitated influx of toxic fluoride anions.
RESUMEN
The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell-based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.
Asunto(s)
Bioensayo/métodos , Membrana Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Ingeniería de Proteínas/métodos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Técnicas Citológicas/métodos , Semivida , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Ubiquitina/metabolismo , Ubiquitina/farmacologíaRESUMEN
Cell delivery reagents often exploit the endocytic pathway as a route of cell entry. Once endocytosed, these reagents must overcome endosomal entrapment to ensure the release of their macromolecular cargo into the cytosol of cells. In this review, we describe several examples of prototypical synthetic reagents that are capable of endosomal escape and examine their mechanisms of action, their efficiencies, and their effects on cells. Although these delivery systems are chemically distinct, some commonalities in how they interact with cellular membranes can be inferred. This, in turn, sheds some light on the process of endosomal escape, and may help guide the development and optimization of next-generation delivery tools.
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Citosol/metabolismo , Portadores de Fármacos/metabolismo , Endosomas/metabolismo , Ácidos Nucleicos/administración & dosificación , Proteínas/administración & dosificación , Animales , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Endocitosis , Humanos , Lípidos/química , Ácidos Nucleicos/farmacocinética , Péptidos/química , Péptidos/metabolismo , Polímeros/química , Polímeros/metabolismo , Proteínas/farmacocinéticaRESUMEN
Understanding the key factors for successful subcellular compartment targeting for cargo delivery systems is of great interest in a variety of fields such as bionanotechnology, cell biology, and nanotherapies. However, the fundamental basis for intracellular transportation with these systems has thus far rarely been discussed. As a cargo vector, porous coordination cages (PCCs) have great potential for use in cancer nanotherapy and to elucidate fundamental insight regarding subcellular compartment targeting. Herein, it is shown that the transportation of PCC cargo vectors though various subcellular barriers of the mammalian cell can be manipulated by tuning the vector's electronic property and surface affinity. It is found that the PCCs become selectively aggregated at the cell membrane, the cytoplasm, or the nucleus, respectively. When a DNA topoisomerase inhibitor is delivered into the nucleus by a neutral and lipophilic PCC, the anticancer efficacy is dramatically improved. The findings shed light to tune the interactions at the "bio-nano" interface. This study provides a key strategy for future work in targeting specific cell organelles for cell imaging, cargo delivery, and therapy. This research also offers key insight into the engineering of nanoscopic materials for furnishing cell organelle-specificity.
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Sistemas de Liberación de Medicamentos/métodos , Animales , Biotecnología/métodos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Porosidad , Inhibidores de TopoisomerasaRESUMEN
Cell-penetrating peptides (CPPs) are typically prone to endocytic uptake into human cells. However, they are often inefficient at escaping from endosomes, which limits their ability to deliver cargos into cells. This review highlights the efforts that our laboratory has devoted toward developing CPPs that can mediate the leakage of endosomal membranes, and consequently gain better access to the intracellular milieu. In particular, we have identified a CPP named dimeric fluorescent TAT (dfTAT) with high endosomolytic activity. We describe how we have used this reagent and its analogs to develop efficient cytosolic delivery protocols and learn about molecular and cellular parameters that control the cell permeation process. Specifically, we discuss how late endosomes represent exploitable gateways for intracellular entry. We also describe how certain features in CPPs, including guanidinium content, charge density, multimerization, chirality, and susceptibility to degradation modulate the activity that these peptidic agents take toward endosomal membranes and cytosolic egress.
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Péptidos de Penetración Celular/química , Endosomas/metabolismo , Sustancias Macromoleculares/farmacología , Guanidina , Humanos , Sustancias Macromoleculares/químicaRESUMEN
Prodrug activation, by exogenously administered enzymes, for cancer therapy is an approach to achieve better selectivity and less systemic toxicity than conventional chemotherapy. However, the short half-lives of the activating enzymes in the bloodstream has limited its success. Demonstrated here is that a tyrosinase-MOF nanoreactor activates the prodrug paracetamol in cancer cells in a long-lasting manner. By generating reactive oxygen species (ROS) and depleting glutathione (GSH), the product of the enzymatic conversion of paracetamol is toxic to drug-resistant cancer cells. Tyrosinase-MOF nanoreactors cause significant cell death in the presence of paracetamol for up to three days after being internalized by cells, while free enzymes totally lose activity in a few hours. Thus, enzyme-MOF nanocomposites are envisioned to be novel persistent platforms for various biomedical applications.
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Acetaminofén/farmacología , Antineoplásicos/farmacología , Estructuras Metalorgánicas/metabolismo , Monofenol Monooxigenasa/metabolismo , Nanopartículas/metabolismo , Acetaminofén/química , Acetaminofén/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Estructuras Metalorgánicas/química , Modelos Moleculares , Estructura Molecular , Monofenol Monooxigenasa/química , Nanopartículas/química , Tamaño de la Partícula , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP-containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane-disrupting and cell-penetrating activities.
Asunto(s)
Endosomas/metabolismo , Lípidos/química , Péptidos/metabolismo , Línea Celular Tumoral , Citosol/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismoRESUMEN
The linearmycin family of polyketides was originally classified as antifungal metabolites. However, in addition to antifungal activity, we previously found that linearmycins cause cellular lysis and colony degradation of the Gram-positive bacterium Bacillus subtilis. We recently showed that Streptomyces sp. strain Mg1 incorporates linearmycins into extracellular vesicles, which are capable of lysing B. subtilis. However, the mechanism of linearmycin-induced lysis was hitherto unexplored. Therefore, we sought to determine how linearmycin-laden vesicles cause lysis. In this study, we found that linearmycins inhibited the growth of all Gram-positive bacteria that we tested, but lysis was limited to some Bacillus species. Next, we found that linearmycin-induced lysis occurred even when cellular metabolism and growth were inhibited, which suggested that linearmycins possess the intrinsic capacity to lyse cells, unlike cell-wall targeting antibiotics. We showed that linearmycin exposure caused changes consistent with rapid depolarization of the B. subtilis cytoplasmic membrane, which was correlated with a loss of viability. Finally, using liposomes as in vitro membrane models, we demonstrated that linearmycins are capable of disrupting lipid bilayers without any other cellular components. Taken together, our results strongly indicate that the cytoplasmic membrane is the direct antibacterial target of linearmycins.
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Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Policétidos/farmacología , Antibacterianos/aislamiento & purificación , Antifúngicos , Bacillus/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Membrana Dobles de Lípidos , Liposomas , Potenciales de la Membrana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Policétidos/aislamiento & purificaciónRESUMEN
Enhancing or restoring enzymatic function in cells is highly desirable in applications ranging from ex vivo cellular manipulations to enzyme replacement therapies in humans. However, because enzymes degrade in biological milieus, achieving long-term enzymatic activities can be challenging. Herein we report on the in cellulo properties of nanofactories that consist of antioxidative enzymes encapsulated in metal-organic frameworks (MOFs). We demonstrate that, while free enzymes display weak activities for only a short duration, these efficient nanofactories protect human cells from toxic reactive oxygen species for up to a week. Remarkably, these results are obtained in spite of the nanofactories being localized in lysosomes, acidic organelles that contain a variety of proteases. The long-term persistence of the nanofactories is attributed to the chemical stability of MOF in low pH environment and to the protease resistance provided by the protective cage formed by the MOF around the encapsulated enzymes.