RESUMEN
Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.
Asunto(s)
Endosomas , ARN Interferente Pequeño/genética , CitosolRESUMEN
BACKGROUND: Irradiation with ultra-high dose rate (FLASH) has been shown to spare normal tissue without hampering tumor control in several in vivo studies. Few cell lines have been investigated in vitro, and previous results are inconsistent. Assuming that oxygen depletion accounts for the FLASH sparing effect, no sparing should appear for cells irradiated with low doses in normoxia. METHODS: Seven cancer cell lines (MDA-MB-231, MCF7, WiDr, LU-HNSCC4, HeLa [early passage and subclone]) and normal lung fibroblasts (MRC-5) were irradiated with doses ranging from 0 to 12 Gy using FLASH (≥800 Gy/s) or conventional dose rates (CONV, 14 Gy/min), with a 10 MeV electron beam from a clinical linear accelerator. Surviving fraction (SF) was determined with clonogenic assays. Three cell lines were further studied for radiation-induced DNA-damage foci using a 53BP1-marker and for cell cycle synchronization after irradiation. RESULTS: A tendency of increased survival following FLASH compared with CONV was suggested for all cell lines, with significant differences for 4/7 cell lines. The magnitude of the FLASH-sparing expressed as a dose-modifying factor at SF=0.1 was around 1.1 for 6/7 cell lines and around 1.3 for the HeLasubclone. Similar cell cycle distributions and 53BP1-foci numbers were found comparing FLASH to CONV. CONCLUSION: We have found a FLASH effect appearing at low doses under normoxic conditions for several cell lines in vitro. The magnitude of the FLASH effect differed between the cell lines, suggesting inherited biological susceptibilities for FLASH irradiation.
RESUMEN
BACKGROUND: It is time-consuming for oncologists to delineate volumes for radiotherapy treatment in computer tomography (CT) images. Automatic delineation based on image processing exists, but with varied accuracy and moderate time savings. Using convolutional neural network (CNN), delineations of volumes are faster and more accurate. We have used CTs with the annotated structure sets to train and evaluate a CNN. MATERIAL AND METHODS: The CNN is a standard segmentation network modified to minimize memory usage. We used CTs and structure sets from 75 cervical cancers and 191 anorectal cancers receiving radiation therapy at Skåne University Hospital 2014-2018. Five structures were investigated: left/right femoral heads, bladder, bowel bag, and clinical target volume of lymph nodes (CTVNs). Dice score and mean surface distance (MSD) (mm) evaluated accuracy, and one oncologist qualitatively evaluated auto-segmentations. RESULTS: Median Dice/MSD scores for anorectal cancer: 0.91-0.92/1.93-1.86 femoral heads, 0.94/2.07 bladder, and 0.83/6.80 bowel bag. Median Dice scores for cervical cancer were 0.93-0.94/1.42-1.49 femoral heads, 0.84/3.51 bladder, 0.88/5.80 bowel bag, and 0.82/3.89 CTVNs. With qualitative evaluation, performance on femoral heads and bladder auto-segmentations was mostly excellent, but CTVN auto-segmentations were not acceptable to a larger extent. DISCUSSION: It is possible to train a CNN with high overlap using structure sets as ground truth. Manually delineated pelvic volumes from structure sets do not always strictly follow volume boundaries and are sometimes inaccurately defined, which leads to similar inaccuracies in the CNN output. More data that is consistently annotated is needed to achieve higher CNN accuracy and to enable future clinical implementation.
RESUMEN
Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to â¼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.
Asunto(s)
Endosomas/metabolismo , Imagenología Tridimensional , ARN Interferente Pequeño/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Dextranos/metabolismo , Endosomas/efectos de los fármacos , Galectinas/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Células MCF-7 , Bibliotecas de Moléculas Pequeñas/farmacología , Esferoides Celulares/metabolismo , Esferoides Celulares/patologíaRESUMEN
Small interfering RNAs (siRNAs), which downregulate gene expression guided by sequence complementarity, can be used therapeutically to block the synthesis of disease-causing proteins. The main obstacle to siRNA drugs - their delivery into the target cell cytosol - has been overcome to allow suppression of liver gene expression. Here, we review the results of recent clinical trials of siRNA therapeutics, which show efficient and durable gene knockdown in the liver, with signs of promising clinical outcomes and little toxicity. We also discuss the barriers to more widespread applications that target tissues besides the liver and the most promising avenues to overcome them.
Asunto(s)
Terapia Genética/métodos , Hepatopatías , Hígado , ARN Interferente Pequeño , Animales , Humanos , Hígado/metabolismo , Hígado/patología , Hepatopatías/genética , Hepatopatías/metabolismo , Hepatopatías/patología , Hepatopatías/terapia , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM(+) cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM(+) luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM(-) mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM(+) Her2(+) and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer.
Asunto(s)
Aptámeros de Nucleótidos/administración & dosificación , Neoplasias de la Mama/patología , Neoplasias Glandulares y Epiteliales/patología , Células Madre Neoplásicas/fisiología , ARN Interferente Pequeño/administración & dosificación , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Glandulares y Epiteliales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Carga Tumoral , Quinasa Tipo Polo 1RESUMEN
A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.
Asunto(s)
Endosomas/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Proteínas Luminiscentes/farmacocinética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Células HeLa , Humanos , Lípidos/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Nanopartículas/química , ARN Interferente Pequeño/metabolismoRESUMEN
The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.
Asunto(s)
Caveolina 1/metabolismo , Endocitosis , Exosomas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Sistema de Señalización de MAP Quinasas , Microdominios de Membrana/metabolismo , Animales , Transporte Biológico , Butadienos/farmacología , Células CHO , Células COS , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Citoesqueleto/metabolismo , Endosomas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glioblastoma/metabolismo , Células HeLa , Proteínas de Choque Térmico , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Chaperonas Moleculares , Nitrilos/farmacología , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Cell-penetrating peptides (CPPs) are widely used to deliver macromolecular cargoes to intracellular sites of action. Many CPPs have been demonstrated to rely on cell surface heparan sulfate proteoglycans (HSPGs) for efficient cellular entry and delivery. In this chapter, we describe methods for the study of PG involvement in CPP uptake. We provide descriptions of how to determine whether uptake of a CPP of interest is dependent on PGs. We also provide detailed protocols for the purification of PGs by anion-exchange chromatography as well as the characterization of the HSPG core protein composition of a cell line of interest. Finally, we present methods for modulating the expression level of specific HSPG core proteins as a means to determine the core protein specificity in the uptake of a particular CPP.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Proteoglicanos/metabolismo , Animales , Células CHO , Péptidos de Penetración Celular/aislamiento & purificación , Cricetinae , Cricetulus , Etanolaminas/química , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Transporte de Proteínas , Sindecano-2/biosíntesis , Sindecano-2/deficiencia , Sindecano-2/genética , Sindecano-2/aislamiento & purificaciónRESUMEN
An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.
Asunto(s)
Proteínas HSP70 de Choque Térmico/fisiología , Sustancias Macromoleculares/metabolismo , Magnetismo , Proteínas de la Membrana/fisiología , Nanopartículas/química , Vesículas Transportadoras/metabolismo , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Línea Celular , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Células HeLa , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Immunoblotting , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Microscopía Electrónica , Interferencia de ARN , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Vesículas Transportadoras/ultraestructura , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.
Asunto(s)
Membrana Celular/metabolismo , Epítopos/química , Glipicanos/química , Proteoglicanos de Heparán Sulfato/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Sindecanos/química , Animales , Células CHO , Cricetinae , Cricetulus , Endocitosis , Antígenos VIH/química , Células HeLa , Humanos , Nanopartículas/química , Biblioteca de Péptidos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Macromolecular drugs hold great promise as novel therapeutics of several major disorders, such as cancer and cardiovascular disease. However, their use is limited by lack of efficient, safe, and specific delivery strategies. Successful development of such strategies requires interdisciplinary collaborations involving researchers with expertise on e.g., polymer chemistry, cell biology, nano technology, systems biology, advanced imaging methods, and clinical medicine. This poses obvious challenges to the scientific community, but also provides opportunities for the unexpected at the interface between different disciplines. This review summarizes recent studies of macromolecular delivery that should be of interest to researchers involved in macromolecular drug synthesis as well as in vitro and in vivo drug delivery studies.
Asunto(s)
Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/métodos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/uso terapéutico , Técnicas de Sonda MolecularRESUMEN
Cell penetrating peptides (CPPs) are currently used to deliver various macromolecular cargos to intracellular sites of action both in vitro and in vivo on an experimental basis. During the last few years, even more evidence has accumulated indicating that the main route of entry for most CPPs is through endocytosis rather than direct membrane penetration, as initially proposed. The specific endocytosis pathway utilized by CPPs is, however, still ill-defined and potentially varies depending on what CPPs, cargos, and cell lines are being studied. In this chapter, we provide detailed protocols for an initial characterization of the uptake mechanism involved in CPP-mediated delivery of DNA. Methods to both quantitatively and qualitatively study the uptake using fluorescence-assisted cell sorting (FACS) and confocal microscopy, respectively, are provided. Furthermore, methods to study the intracellular fate of the internalized cargo by co-localization studies between internalized DNA and established endosomal markers, e.g., transferrin, dextran as well as caveolin-1, are described. Finally, we provide a protocol to determine the dependence on dynamin, i.e., a central mediator of vesicle fission at the cell membrane, for DNA-peptide complex uptake using a dominant-negative construct of dynamin-2.
Asunto(s)
ADN/metabolismo , Péptidos/metabolismo , Separación Celular , Dinaminas/metabolismo , Endocitosis , Humanos , Microscopía ElectrónicaRESUMEN
Macromolecular drugs hold great promise as novel therapeutics of several major disorders, such as cancer and cardiovascular disease. However, their use is limited by lack of efficient, safe, and specific delivery strategies. Successful development of such strategies requires interdisciplinary collaborations involving researchers with expertise on, e.g., polymer chemistry, cell biology, nanotechnology, systems biology, advanced imaging methods, and clinical medicine. This not only poses obvious challenges to the scientific community but also provides opportunities for the unexpected at the interface between different disciplines. This introductory chapter summarizes and gives references to studies on macromolecular delivery that should be of interest to a broad scientific audience involved in macromolecular drug synthesis as well as in vitro and in vivo drug delivery studies.
Asunto(s)
Sistemas de Liberación de Medicamentos , Sustancias Macromoleculares/administración & dosificación , Transporte Biológico , Endocitosis , Vehículos FarmacéuticosRESUMEN
The prevailing view that eukaryotic cells are restrained from intercellular exchange of genetic information has been challenged by recent reports on nanotubes, exosomes, apoptotic bodies, and nucleic acid-binding peptides that provide novel pathways for cell-cell communication, with implications in health and disease.
Asunto(s)
Comunicación Celular/fisiología , Exosomas/metabolismo , Nanotubos/química , Péptidos/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Células Eucariotas/metabolismo , Humanos , Modelos Biológicos , Ácidos Nucleicos/metabolismo , Interferencia de ARN , TerapéuticaRESUMEN
The growth-promoting polyamines are polybasic compounds that efficiently enter cancer cells by as yet incompletely defined mechanisms. Strategies to inhibit their internalization may have important implications in the management of tumor disease. Here, we show that cellular binding and uptake of polyamines are inhibited by a single chain variable fragment anti-heparan sulfate (HS) antibody. Polyamine uptake was inhibited in a dose-dependent manner, and was associated with compensatory up-regulation of ornithine decarboxylase (ODC), i.e. the key enzyme of the polyamine biosynthesis pathway. Conversely, depletion of intracellular polyamines by the specific ODC-inhibitor alpha-difluoromethylornithine (DFMO) resulted in increased cellular binding of polyamine and anti-HS antibody. Importantly, anti-HS antibody also efficiently targeted DFMO-induced polyamine uptake, and combined polyamine biosynthesis inhibition by DFMO, and uptake inhibition by anti-HS antibody attenuated tumor cell proliferation in vitro. In conclusion, cell-surface HS proteoglycan is a relevant target for antibody-mediated inhibition of the uptake of polyamines, and polyamine-dependent cell proliferation.
Asunto(s)
Poliaminas Biogénicas/antagonistas & inhibidores , Heparitina Sulfato/inmunología , Fragmentos de Inmunoglobulinas/farmacología , Animales , Poliaminas Biogénicas/fisiología , Transporte Biológico , Células CHO , Proliferación Celular , Cricetinae , Cricetulus , Eflornitina/farmacología , Células HeLa , HumanosRESUMEN
Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis-disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.
Asunto(s)
ADN/química , Pinocitosis , Proteoglicanos/metabolismo , Animales , Transporte Biológico , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Medios de Cultivo/farmacología , Humanos , Espectrometría de Masas , Microscopía Confocal , Microscopía Fluorescente , Polielectrolitos , Polímeros/química , Proteoglicanos/químicaRESUMEN
A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Pinocitosis/fisiología , Priones/metabolismo , Androstadienos/farmacología , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Citocalasina D/farmacología , Estructura Terciaria de Proteína , Transporte de Proteínas , WortmaninaRESUMEN
Recent evidence for efficient delivery of macromolecules, such as peptides and nucleic acids, from the cell exterior to the nucleus offers the interesting possibility of developing novel treatments directed at intranuclear targets. The findings should also stimulate the search for physiological ligands that utilize similar transport mechanisms to regulate pathobiological processes. Cytokines, growth factors and their receptors, as well as morphogens have all been shown to enter the nucleus to evoke biological responses in target cells. The rational design of intracellular drug delivery vehicles requires an increased understanding of the elaborate systems that mediate cellular communication and coordination with the extracellular environment without inflicting on the integrity of the cell. This review discusses some aspects of the carriers and barriers in macromolecular transport.
Asunto(s)
Membrana Celular/efectos de los fármacos , Clatrina/fisiología , Endocitosis/fisiología , Sustancias Macromoleculares/metabolismo , Membrana Nuclear/fisiología , Matriz Nuclear/fisiología , Proteoglicanos/fisiología , Transporte Biológico/fisiología , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Endocitosis/efectos de los fármacos , Membrana Nuclear/metabolismo , Fagocitosis/fisiología , Proteoglicanos/metabolismoRESUMEN
Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37.DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans.