RESUMEN
Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.
Asunto(s)
ADN , Nanotubos , Arcilla , Neuroglobina , ARN Mensajero/genética , Nanotubos/químicaRESUMEN
Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups' research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.
RESUMEN
G-quadruplex (G4) oligonucleotides are higher-order DNA and RNA secondary structures of enormous relevance due to their implication in several biological processes and pathological states in different organisms. Strategies aiming at modulating human G4 structures and their interrelated functions are first-line approaches in modern research aiming at finding new potential anticancer treatments or G4-based aptamers for various biomedical and biotechnological applications. Plants offer a cornucopia of phytocompounds that, in many cases, are effective in binding and modulating the thermal stability of G4s and, on the other hand, contain almost unexplored G4 motifs in their genome that could inspire new biotechnological strategies. Herein, we describe some G4 structures found in plants, summarizing the existing knowledge of their functions and biological role. Moreover, we review some of the most promising G4 ligands isolated from vegetal sources and report on the known relationships between such phytochemicals and G4-mediated biological processes that make them potential leads in the pharmaceutical sector.
RESUMEN
The development of new strategies for enhancing drug delivery to the brain represents a major challenge in treating cerebral diseases. In this paper, we report on the synthesis and structural characterization of a biocompatible nanoparticle (NP) made up of poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) co-polymer (namely PELGA) functionalized with the membranotropic peptide gH625 (gH) and the iron-mimicking peptide CRTIGPSVC (CRT) for transport across the blood-brain barrier (BBB). gH possesses a high translocation potency of the cell membrane. Conversely, CRT selectively recognizes the brain endothelium, which interacts with transferrin (Tf) and its receptor (TfR) through a non-canonical ligand-directed mechanism. We hypothesize that the delivery across the BBB of PELGA NPs should be efficiently enhanced by the NP functionalization with both gH and CRT. Synthesis of peptides and their conjugation to the PLGA as well as NP physical-chemical characterization are performed. Moreover, NP uptake, co-localization, adhesion under dynamic conditions, and permeation across in vitro BBB model are evaluated as a function of gH/CRT functionalization ratio. Results establish that the cooperative effect of CRT and gH may change the intra-cellular distribution of NPs and strengthen NP delivery across the BBB at the functionalization ratio 33% gHâ»66% CRT.
Asunto(s)
Cerebelo/citología , Portadores de Fármacos/química , Endotelio/química , Nanopartículas/química , Péptidos/química , Polímeros/síntesis química , Animales , Materiales Biocompatibles/química , Barrera Hematoencefálica/química , Barrera Hematoencefálica/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/química , Cerebelo/metabolismo , Diseño de Fármacos , Endotelio/citología , Endotelio/metabolismo , Lactatos/química , Ratones , Péptidos/metabolismo , Polietilenglicoles/química , Ácido Poliglicólico/química , Polímeros/química , Receptores de Transferrina/metabolismo , Transferrina/metabolismoRESUMEN
The blood brain barrier (BBB) represents a challenge in the development of new nano-delivery systems able to reach the central nervous system (CNS). In order to test the efficacy of these nanocarriers, it is fundamental to use in vitro models that resemble the in vivo cell culture conditions. Here, we demonstrate for the first time the ability of a membranotropic peptide, namely gH625, to transport a cargo-acting as a shuttle-across the BBB layer under flow conditions that mimic the blood flow rate. To this aim, a BBB microfluidic device was designed based on a transparent polyester porous membrane sandwiched between a top and a bottom overlying channel made of poly(methyl methacrylate) (PMMA). Our data clearly indicate that this microfluidic system allows the growth of brain endothelial bEnd.3 cells and the formation of a confluent layer at 7 days of culture that hinders the passage of nanoparticles compared to porous membrane alone. The device was validated at a 5 µL/min working flow rate, where the capability of the model to remain intact after nanoparticle passage was shown. Very interestingly, the decoration with the gH625 peptide enhances the adhesion of nanoparticles to the endothelial layer and the BBB crossing in flow conditions, thus confirming the efficacy of the gH625 as a delivery platform to the brain. Biotechnol. Bioeng. 2017;114: 1087-1095. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo , Péptidos/metabolismo , Animales , Transporte Biológico , Línea Celular , Diseño de Equipo , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Péptidos/químicaRESUMEN
Nowadays, microfluidic channels of a few tens of micrometers are required and widely used in many fields, especially for surface-processing applications and miniaturization of biological assays. Herein, we selected micromilling as a low-cost technology and proposed an approach capable of overcoming its limitations; in fact, microstructures below 20-30 µm in depth are difficult to obtain, and the manufacturing error is rather high, as it is inversely proportional to the depth. Indeed, the proposed method uses a confined dehydration process of a patterned gelatin substrate fabricated via replica molding onto a micromilled poly(methyl methacrylate) substrate to produce a gelatin master with demonstrated final micrometric features down to 3 µm for the channel depth and, in specific configurations, down to 5 µm for the channel width. Finally, we demonstrated the ability to flux liquids in miniaturized microfluidic devices and fabricated and tested-as an example-micrometric microstructures arrays connected via microchannels for biological assays.
Asunto(s)
Bioensayo/instrumentación , Bioensayo/métodos , Desecación , Gelatina/química , Dispositivos Laboratorio en un ChipRESUMEN
Square microchannels are easy to fabricate by means of micromachining or lithographic techniques. However, in vitro vascular microcapillaries--as well as plug production and microparticle alignment--require mainly circular microchannels that can be used also in applications based on open microchannels. Nowadays, a simple, low cost, and versatile method to fabricate circular microchannels is still missing. Here, we report on a fast, inexpensive, flexible and reproducible method to fabricate circular microchannels by coupling spin coating with micromilled square microchannels. The proposed method is based on the balance between the displacement of liquid PDMS induced by centrifugal forces and the surface tension that tends to keep the liquid accumulated especially in the corners, which become therefore rounded. To show the versatility of the described experimental study we prepared a variety of rounded microchannels, including branched and PMMA-PDMS hybrid configuration microchannels. Finally, an endothelial cell layer was formed by culturing brain endothelial bEnd.3 cells inside the proposed circular microchannels. Results demonstrated a more successful adhesion, growth, and homogeneous distribution of the cells along the circular microchannel than those observed in the square microchannel used as a control.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Técnicas Electroquímicas/métodos , Células Endoteliales/citología , Microfluídica/instrumentación , Polímeros/síntesis química , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/economía , Proliferación Celular , Técnicas Electroquímicas/economía , Ratones , Microfluídica/métodos , Polímeros/química , Polímeros/economíaRESUMEN
A major issue in chemotherapy is the lack of specificity of many antitumor drugs, which cause severe side effects and an impaired therapeutic response. Here we report on the design and characterization of model tumor activated prodrug-conjugated polystyrene (PS) nanoparticles (TAP-NPs) for the release of doxorubicin (Dox) triggered by matrix metalloprotease-2 (MMP2) enzyme, which is overexpressed in the extracellular matrix of tumors. In particular, TAP-NPs were produced by attaching Dox to poly(ethylene glycol) (PEG) through two MMP2-cleavable enzymes. The resulting adduct was then tethered to PS NPs. Results showed that Dox release was actually triggered by MMP2 cleavage and was dependent on enzyme concentration, with a plateau around 20 nM. Furthermore, significant cell cytotoxicity was observed towards three cell lines only in the presence of MMP2, but not in cells without enzyme pre-treatment, even after NP internalization by cells. These findings indicate the potential of TAP-NPs as suitable nanocarriers for an on demand, tumor--specific delivery of antitumor drugs after the response to an endogenous stimulus. Further advancements will focus on the translation of this production technology to biodegradable systems for the safe transport of cytotoxic drug to tumor tissues.