RESUMO
Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.
Assuntos
Nanopartículas , Ácidos Nucleicos , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Dióxido de Silício/química , Trifosfato de AdenosinaRESUMO
Peptide nucleic acids and their conjugates to peptides can self-assemble and generate complex architectures. In this work, we explored the self-assembly of PNA dimers conjugated to the dipeptide WW. Our studies suggest that the indole ring of tryptophan promotes aggregation of the conjugates. The onset of fluorescence is observed upon self-assembly. The structure of self-assembled WWgc is concentration-dependent, being spherical at low concentrations and fibrous at high concentrations. As suggested by molecular modeling studies, fibers are stabilized by stacking interactions between tryptophans and Watson-Crick hydrogen bonds between nucleobases.
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Ácidos Nucleicos Peptídicos , Triptofano , Ácidos Nucleicos Peptídicos/química , Dipeptídeos/química , Peptídeos , Modelos MolecularesRESUMO
Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA "at" is replaced by guanine-cytosine dimer "gc", disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.
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Nanoestruturas , Ácidos Nucleicos Peptídicos , Nanoestruturas/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Fenilalanina/química , Polímeros , TiminaRESUMO
BACKGROUND: Platinum nanoparticles have been demonstrated to have excellent anticancer properties. However, because of the lack of specificity they must be delivered to the tumor in amounts sufficient to reach the desired therapeutic objectives. Interestingly, exosomes are considered as excellent natural selective delivery nanotools, but until know their targeting properties have not being combined with the anticancer properties of platinum nanoparticles. RESULTS: In this work we combine the targeting capabilities of exosomes and the antitumoral properties of ultrasmall (< 2 nm) platinum nanoparticles as a novel, low toxicity alternative to the use of cisplatin. A mild methodology based on the room temperature CO-assisted in situ reduction of Pt2+ precursor was employed to preserve the integrity of exosomes, while generating ultrasmall therapeutic PtNPs directly inside the vesicles. The resulting PtNPs-loaded exosomes constitute a novel hybrid bioartificial system that was readily internalized by the target cells inducing antiproliferative response, as shown by flow cytometry and microscopy experiments in vitro. In vivo Pt-Exos showed antitumoral properties similar to that of cisplatin but with a strongly reduced or in some cases no toxic effect, highlighting the advantages of this approach and its potential for translation to the clinic. CONCLUSIONS: In this study, a nanoscale vector based on ultrasmall PtNPs and exosomes has been created exhibiting antitumoral properties comparable or higher to those of the FDA approved cisplatin. The preferential uptake of PtNPs mediated by exosomal transfer between certain cell types has been exploited to create a selective antitumoral novel bioartificial system. We have demonstrated their anticancer properties both in vitro and in vivo comparing the results obtained with the administration of equivalent amounts of cisplatin, and showing a spectacular reduction of toxicity.
Assuntos
Exossomos , Nanopartículas Metálicas , Nanopartículas , Neoplasias , Humanos , Cisplatino/farmacologia , Platina , Linhagem Celular TumoralRESUMO
The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.
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Cromatografia em Camada Fina/métodos , Exossomos/metabolismo , Fibroblastos/metabolismo , Melanoma Experimental/patologia , Fosfolipídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Melanoma Experimental/metabolismo , Camundongos , Células NIH 3T3 , Fosfolipídeos/análiseRESUMO
BACKGROUND: Exosomes are endocytic-extracellular vesicles with a diameter around 100 nm that play an essential role on the communication between cells. In fact, they have been proposed as candidates for the diagnosis and the monitoring of different pathologies (such as Parkinson, Alzheimer, diabetes, cardiac damage, infection diseases or cancer). RESULTS: In this study, magnetic nanoparticles (Fe3O4NPs) were successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. This was carried out under flow in a 1.6 mm (outer diameter) microchannel whose wall was in contact with a set of NdFeB permanent magnets, giving a high magnetic field across the channel diameter that allowed exosome separation with a high yield. To show the usefulness of the method, the direct capture of exosomes from whole blood of patients with pancreatic cancer (PC) was performed, as a proof of concept. The captured exosomes were then subjected to analysis of CA19-9, a protein often used to monitor PC patients. CONCLUSIONS: Here, we describe a new microfluidic device and the procedure for the isolation of exosomes from whole blood, without any need of previous isolation steps, thereby facilitating translation to the clinic. The results show that, for the cases analyzed, the evaluation of CA19-9 in exosomes was highly sensitive, compared to serum samples.
Assuntos
Antígenos Glicosídicos Associados a Tumores/sangue , Antígenos Glicosídicos Associados a Tumores/isolamento & purificação , Análise Química do Sangue/métodos , Exossomos/química , Neoplasias Pancreáticas/diagnóstico , Anticorpos/química , Anticorpos/metabolismo , Boro/química , Campos Eletromagnéticos , Humanos , Ferro/química , Dispositivos Lab-On-A-Chip , Nanopartículas de Magnetita/química , Neodímio/química , Propriedades de SuperfícieRESUMO
BACKGROUND: Exosomes are considered key elements for communication between cells, but very little is known about the mechanisms and selectivity of the transference processes involving exosomes released from different cells. RESULTS: In this study we have investigated the transfer of hollow gold nanoparticles (HGNs) between different cells when these HGNs were loaded within exosomes secreted by human placental mesenchymal stem cells (MSCs). These HGNs were successfully incorporated in the MSCs exosome biogenesis pathway and released as HGNs-loaded exosomes. Time-lapse microscopy and atomic emission spectroscopy allowed us to demonstrate the selective transfer of the secreted exosomes only to the cell type of origin when studying different cell types including cancer, metastatic, stem or immunological cells. CONCLUSIONS: In this study we demonstrate the selectivity of in vitro exosomal transfer between certain cell types and how this phenomenon can be exploited to develop new specific vectors for advanced therapies. Specifically, we show how this preferential uptake can be leveraged to selectively induce cell death by light-induced hyperthermia only in cells of the same type as those producing the corresponding loaded exosomes. We describe how the exosomes are preferentially transferred to some cell types but not to others, thus providing a better understanding to design selective therapies for different diseases.
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Exossomos/metabolismo , Nanopartículas Metálicas , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Endocitose , Exossomos/química , Ouro/química , Ouro/metabolismo , Temperatura Alta , Humanos , Raios Infravermelhos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestruturaRESUMO
BACKGROUND: In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. METHODS: Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. RESULTS: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. CONCLUSIONS: Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.
Assuntos
Vetores Genéticos/química , Nanopartículas Metálicas , Transfecção/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/síntese química , Ouro , Células HeLa , Humanos , Polietilenoglicóis , PolietilenoiminaRESUMO
Several studies have reported the successful use of bio-orthogonal catalyst nanoparticles (NPs) for cancer therapy. However, the delivery of the catalysts to the target tissues in vivo remains an unsolved challenge. The combination of catalytic NPs with extracellular vesicles (EVs) has been proposed as a promising approach to improve the delivery of therapeutic nanomaterials to the desired organs. In this study, we have developed a nanoscale bio-hybrid vector using a CO-mediated reduction at low temperature to generate ultrathin catalytic Pd nanosheets (PdNSs) as catalysts directly inside cancer-derived EVs. We have also compared their biodistribution with that of PEGylated PdNSs delivered by the EPR effect. Our results indicate that the accumulation of PdNSs in the tumour tissue was significantly higher when they were administered within the EVs compared to the PEGylated PdNSs. Conversely, the amount of Pd found in non-target organs (i.e., liver) was lowered. Once the Pd-based catalytic EVs were accumulated in the tumours, they enabled the activation of a paclitaxel prodrug demonstrating their ability to carry out bio-orthogonal uncaging chemistries in vivo for cancer therapy.
Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Humanos , Animais , Catálise , Camundongos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Paládio/química , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Linhagem Celular Tumoral , Distribuição Tecidual , Polietilenoglicóis/química , Nanopartículas/química , Pró-Fármacos , Camundongos NusRESUMO
Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large scale is still challenging. One of the fluids from which they could be isolated in large quantities is milk. Moreover, regeneration is a well-known property of milk. The present work seeks to optimize a method for isolating exosomes from bovine and human milk, comparing different storage conditions and different extraction protocols. We found differences in the yield extraction associated with pre-storage milk conditions and observed some differences according to the processing agent. When we removed milk fat globules and added rennet before freezing, we obtained a cleaner final fraction. In summary, we attempted to optimize a rennet-based new milk-exosome isolation method and concluded that pre-treatment, followed by freezing of samples, yielded the best exosome population.
Assuntos
Exossomos , Leite , Exossomos/metabolismo , Exossomos/química , Animais , Bovinos , Leite/química , Humanos , Leite Humano/química , Quimosina/química , Quimosina/metabolismo , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Glicolipídeos , GlicoproteínasRESUMO
Exosomes are extracellular vesicles that are involved in cell-cell communication. Considering their bioavailability and accessibility in all the body fluids (including the blood, semen, breast milk, saliva, and urine), their use has been proposed as an alternative noninvasive tool for the diagnosis, monitoring, and prognosis of several diseases, including cancer. The isolation of exosomes and their subsequent analysis are emerging as a promising technique in diagnostics and personalized medicine. The most widely employed isolation procedure is differential ultracentrifugation, but this approach is laborious, time-consuming, and expensive and with limited isolation yield. Microfluidic devices are now emerging as novel platforms for exosome isolation, which is a low cost technology and enables high purity and fast treatment of exosome isolation. Our approach describes a microfluidic device that enables inflow capture and separation from whole blood using antibody-functionalized magnetic nanoparticles. This device allows isolation of pancreatic cancer-derived exosomes from whole blood without the need of any pretreatment, resulting in a high sensitivity.
Assuntos
Exossomos , Vesículas Extracelulares , Técnicas Analíticas Microfluídicas , Neoplasias Pancreáticas , Humanos , Microfluídica , Neoplasias Pancreáticas/diagnóstico , Neoplasias PancreáticasRESUMO
The recent nanomedicine advancements have introduced a variety of smart nanoparticles in cancer treatment and diagnostics. However, their application to the clinic is still hindered by several challenges related to their biocompatibility, elimination and biodistribution. Here we propose breakable organosilica mesoporous nanoparticles, i.e. nanocages, able to efficiently incorporate cargo molecules and be coated, with different lipid compositions, to enhance their biomimetic behaviour. We exploit the electrostatic interactions between the organosilica surface and the opposite charge of the lipid mixtures in order to obtain an efficient organosilica coverage. The lipid-coated nanocages are proved to have an incredibly high hemocompatibility, significantly increased with respect to pristine nanocages, and excellent colloidal stability and biocompatibility. The cargo-loaded and lipid-coated nanocages are tested and compared in vitro on two different cancer cell lines, demonstrating the key role played by the lipid coating in mediating the internalization of the nanocages, evaluated by the enhanced and rapid cellular uptake. The efficient intracellular delivery of the therapeutic agents is then assured by the destruction of the organosilica, due to the disulfide bridges, introduced into the silica framework, that in reducing media, like the intracellular one, are reduced to thiols causing the breaking of the nanoparticles. The possibility to image and effectively kill cancer cells demonstrates the potentiality of the lipid-coated nanocages as a powerful tool in anticancer research and as a promising smart theranostic platform.
Assuntos
Biomimética , Sistemas de Liberação de Medicamentos , Distribuição Tecidual , Transporte Biológico , LipídeosRESUMO
Platinum-based chemotherapy is the first-line treatment for different cancer types, and in particular, for malignant pleural mesothelioma patients (a tumor histotype with urgent medical needs). Herein, a strategy is presented to stabilize, transport, and intracellularly release a platinumIV (PtIV ) prodrug using a breakable nanocarrier. Its reduction, and therefore activation as an anticancer drug, is promoted by the presence of glutathione in neoplastic cells that also causes the destruction of the carrier. The nanocage presents a single internal cavity in which the hydrophobic complex (Pt(dach)Cl2 (OH)2 ), (dach = R,R-diaminocyclohexane) is encapsulated. The in vitro uptake and the internalization kinetics in cancer model cells are evaluated and, using flow cytometry analysis, the successful release and activation of the Pt-based drug inside cancer cells are demonstrated. The in vitro findings are confirmed by the in vivo experiments on a mice model obtained by xenografting MPM487, a patient-derived malignant pleural mesothelioma. MPM487 confirms the well-known resistance of malignant pleural mesothelioma to cisplatin treatment while an interesting 50% reduction of tumor growth is observed when mice are treated with the PtIV , entrapped in the nanocages, at an equivalent dose of the platinum complex.
Assuntos
Antineoplásicos , Mesotelioma Maligno , Neoplasias , Animais , Camundongos , Compostos Organoplatínicos/química , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
In vivo cell tracking by non-invasive imaging technologies is needed to accelerate the clinical translation of innovative cell-based therapies. In this regard, 19F-MRI has recently gained increased attention for unbiased localization of labeled cells over time. To push forward the use of 19F-MRI for cell tracking, the development of highly performant 19F-probes is required. PLGA-based NPs containing PERFECTA, a multibranched superfluorinated molecule with an optimal MRI profile thanks to its 36 magnetically equivalent fluorine atoms, are promising 19F-MRI probes. In this work we demonstrate the importance of the surface functionalization of these NPs in relation to their interaction with the biological environment, stressing the pivotal role of the formation of the protein corona (PC) in their cellular labelling efficacy. In particular, our studies showed that the formation of PC NPs strongly promotes the cellular internalization of these NPs in microglia cells. We advocate that the formation of PC NPs in the culture medium can be a key element to be used for the optimization of cell labelling with a considerable increase of the detection sensitivity by 19F-MRI.
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Small extracellular vesicle (EV) membranes display characteristic protein-lipidic composition features that are related to their cell of origin, providing valuable clues regarding their parental cell composition and real-time state. This could be especially interesting in the case of cancer cell-derived EVs, as their membranes could serve as valuable tools in liquid biopsy applications and to detect changes in the tumor malignancy. X-Ray Photoelectron Spectroscopy (XPS) is a powerful surface analysis technique able to detect every chemical element present, being also sensitive to their chemical environment. Here we explore the use of XPS as a fast technique to characterize EV membrane composition, with possible application in cancer research. Notably, we have focused on the nitrogen environment as an indicator of the relative abundance of pyridine-type bonding, primary, secondary and tertiary amines. Specifically, we have analyzed how tumoral and healthy cells have different nitrogen chemical environments that can indicate the presence or absence of malignancy. In addition, a collection of human serum samples from cancer patients and healthy donors was also analyzed. The differential XPS analysis of EVs collected from patients confirmed that the patterns of amine evolution could be related to markers of cancer disease, opening the possibility of their use as a non-invasive blood biomarker.
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Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.
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Heterogeneous catalysis has emerged as a promising alternative for the development of new cancer therapies. In addition, regarding the tumor microenvironment as a reactor with very specific chemical features has provided a new perspective in the search for catalytic nanoarchitectures with specific action against chemical species playing a key role in tumor metabolism. One of these species is glutathione (GSH), whose depletion is the cornerstone of emerging strategies in oncology, since this metabolite plays a pivotal regulatory role as antioxidant agent, dampening the harmful effects of intracellular reactive oxidative species (ROS). Herein, we present copper-iron oxide spinel nanoparticles that exhibit a versatile and selective catalytic response to reduce GSH levels while generating ROS in a cascade reaction. We demonstrate a clear correlation between GSH depletion and apoptotic cell death in tumor cells in the presence of the copper-iron nanocatalyst. Furthermore, we also provide a novel analytical protocol, alternative to state-of-the-art commercial kits, to accurately monitoring the concentration of GSH intracellular levels in both tumor and healthy cells. We observe a selective action of the nanoparticles, with lower toxicity in healthy cell lines, whose intrinsic GSH levels are lower, and intense apoptosis in tumor cells accompanied by a fast reduction of GSH levels.
Assuntos
Nanopartículas , Neoplasias , Catálise , Linhagem Celular Tumoral , Cobre/farmacologia , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Ferro/farmacologia , Neoplasias/tratamento farmacológico , Óxidos/farmacologia , Espécies Reativas de Oxigênio , Microambiente TumoralRESUMO
The main current challenges in oncology are (1) avoiding systemic side effects in therapy, and (2) developing alternative treatment strategies for metastatic tumours. Nanomedicine was assumed to provide answers to these issues, but delivering enough therapeutic nanoparticles (NPs) to tumours still remains a huge challenge in nanomaterials-based treatments. Extracellular vesicles (EVs) play a key role in cell communication processes and can be combined with nanomaterials to improve their targeting capabilities. In this work, we leverage the ability of EVs derived from stem cells to reach tumour areas successfully, being used as delivery vehicles for nanoparticles acting as hyperthermia agents. Once small extracellular vesicles (sEVs) loaded with NIR-sensitive hollow gold NPs reached primary subcutaneous solid tumours, they were irradiated with a NIR laser and almost complete tumour remission was obtained. More interestingly, those sEV vehicles were also able to reach multinodular areas similar to those on advanced metastatic phases, eradicating most tumour growth regions in multiple cancerous nodules located in the pancreas region.
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Vesículas Extracelulares , Hipertermia Induzida , Nanopartículas , Linhagem Celular Tumoral , Nanopartículas/uso terapêutico , Células-TroncoRESUMO
Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.
Assuntos
Nanopartículas , Nanopartículas/química , Imageamento por Ressonância Magnética , Rastreamento de Células , Sobrevivência Celular , Estrutura MolecularRESUMO
The use of exosomes as selective delivery vehicles of therapeutic agents, such as drugs or hyperthermia-capable nanoparticles, is being intensely investigated on account of their preferential tropism toward their parental cells. However, the methods used to introduce a therapeutic load inside exosomes often involve disruption of their membrane, which may jeopardize their targeting capabilities, attributed to their surface integrins. On the other hand, in recent years bio-orthogonal catalysis has emerged as a new tool with a myriad of potential applications in medicine. These bio-orthogonal processes, often based on Pd-catalyzed chemistry, would benefit from systems capable of delivering the catalyst to target cells. It is therefore highly attractive to combine the targeting capabilities of exosomes and the bio-orthogonal potential of Pd nanoparticles to create new therapeutic vectors. In this protocol, we provide detailed information on an efficient procedure to achieve a high load of catalytically active Pd nanosheets inside exosomes, without disrupting their membranes. The protocol involves a multistage process in which exosomes are first harvested, subjected to impregnation with a Pd salt precursor followed by a mild reduction process using gas-phase CO, which acts as both a reducing and growth-directing agent to produce the desired nanosheets. The technology is scalable, and the protocol can be conducted by any researcher having basic biology and chemistry skills in ~3 d.